Gene_locus Report for: canar-LipB Candida antarctica (Trichosporon oryzae) (yeast) Lipase B CALB, CALipB ,Novozyme 435Comment Other strains: Pseudozyma antarctica (strain T-34) (Yeast) (Candida antarctica); Pseudozyma aphidis DSM 70725 ; Through structure homology this protein is close to other fungus Lipase_3 but it is far from all other sequence in ESTHER. Novozyme 435 (Novozyme-435 N435, N-435, Novozym 435 Novozym435 Novozyme435) is CAL-B immobilized onto a macroporous acrylic polymer resin (Lewatit VP OC 1600, Bayer) by Novozyme. Fermenta Biotech BEX 10000 ; promiscuous activity 68/96 substrates Martinez-Martinez et al. 2018 Relationship
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) There are 2 a/b hydrolases in Candida antarctica view in a: Table
Fungal-Bact_LIP : canan-lipasA Candida antarctica (Trichosporon oryzae) (yeast) CalA Lipase AWarning: This entry is a compilation of different species or line or strain with more than 90% amino acide identity. You can retrieve all strain data
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) Pseudozyma antarctica T-34: N, E. Pseudozyma aphidis DSM 70725: N, E.
Sequence
References 70 more
Capone M, Zanetti-Polzi L, Leonzi I, Spreti N, Daidone I Ref: J Biomol Struct Dyn, :1, 2022 : PubMed ESTHER: Capone_2022_J.Biomol.Struct.Dyn__1 PubMedSearch: Capone 2022 J.Biomol.Struct.Dyn 1 PubMedID: 35593533 Gene_locus related to this paper: canar-LipB Abstract Candida antarctica Lipase B (CALB) is a paradigm for the family of lipases. At pH 7, the optimal pH for catalysis, the protonation state of an aspartic acid of the active site (Asp134) could not be conclusively assigned. In fact, the pK(a) estimate provided by a widely used computational tool, namely PropKa, that predicts pKa values of ionizable groups in proteins based on the crystallographic structure, is only slightly above 7 (pKa = 7.25). This, along with the lack of an experimental evaluation, makes the assignment of its protonation state at neutral pH challenging. Here, we calculate the pK(a) of Asp134 by means of a fully atomistic multiscale computational approach based on classical molecular dynamics (MD) simulation and the perturbed matrix method (PMM), namely the MD-PMM approach. MD-PMM is able to take into account the dynamics of the system and, at the same time, to treat the deprotonation step at the quantum level. The calculations provide a pK(a) value of 8.9 +/- 1.1, hence suggesting that Asp134 in CALB should be protonated at neutral, and even at slightly basic, pH. Title: Design of 2,5-furandicarboxylic based polyesters degraded in different environmental conditions: Comprehensive experimental and theoretical study Hu H, Li J, Luo S, Tian Y, Wang J, Zhao YL, Zhang R, Zhu J Ref: J Hazard Mater, 425:127752, 2022 : PubMed ESTHER: Hu_2022_J.Hazard.Mater_425_127752 PubMedSearch: Hu 2022 J.Hazard.Mater 425 127752 PubMedID: 34906869 Gene_locus related to this paper: canar-LipB Abstract Nowadays, the promotion and application of aliphatic-aromatic copolyesters, such as poly (butylene adipate-co-terephthalate) (PBAT), are growing into a general trend. Although the structures of diacids exerted substantial impacts on degradation behavior, the underlying mechanisms have rarely been studied. In this work, 2,5-Furandicarboxylic acid was combined with succinic acid (PBSF), adipic acid (PBAF) and diglycolic acid (PBDF) to prepare three kinds of copolyesters. They showed unique degradation behaviors in buffer, enzyme environment and artificial seawater. These characteristics are closely related to the structural compositions of diacids. PBAFs displayed impressive biodegradability when catalyzed by Candida antarctica lipase B (CALB), while the more hydrophilic PBDFs exhibited faster hydrolysis in both buffer and artificial seawater. PBSFs, with hydrophobic and short segments, obtained a relatively slower rate of hydrolysis and enzymatic degradation. The reactivity sites and hydrolytic pathway were revealed by the combination of DFT calculation and Fukui function analysis. MD simulations, QM/MM optimizations and theozyme calculations showed that PBAF-CALB was prone to form a pre-reaction state, leading to the reduced energy barrier in the acylation process. This work revealed the effects of different structural features of diacids on polymer degradation and paved a way to design target biodegradable polymers in different degradation conditions. Title: Enhanced biodegradation of waste poly(ethylene terephthalate) using a reinforced plastic degrading enzyme complex Hwang DH, Lee ME, Cho BH, Oh JW, You SK, Ko YJ, Hyeon JE, Han SO Ref: Sci Total Environ, :156890, 2022 : PubMed ESTHER: Hwang_2022_Sci.Total.Environ__156890 PubMedSearch: Hwang 2022 Sci.Total.Environ 156890 PubMedID: 35753492 Gene_locus related to this paper: idesa-peth, canar-LipB Abstract Poly(ethylene terephthalate) (PET) is synthesized via a rich ester bond between terephthalate (TPA) and ethylene glycol (EG). Because of this, PET degradation takes a long time and PET accumulates in the environment. Many studies have been conducted to improve PET degrading enzyme to increase the efficiency of PET depolymerization. However, enzymatic PET decomposition is still restricted, making upcycling and recycling difficult. Here, we report a novel PET degrading complex composed of Ideonella sakaiensis PETase and Candida antarctica lipase B (CALB) that improves degradability, binding ability and enzyme stability. The reaction mechanism of chimeric PETase (cPETase) and chimeric CALB (cCALB) was confirmed by PET and bis (2-hydroxyethyl terephthalate) (BHET). cPETase generated BHET and mono (2-hydroxyethyl terephthalate (MHET) and cCALB produced terephthalate (TPA). Carbohydrate binding module 3 (CBM3) in the scaffolding protein greatly improved PET film binding affinity. Finally, the final enzyme complex demonstrated a 6.5-fold and 8.0-fold increase in the efficiency of hydrolysis from PET with either high crystalline or waste to TPA than single enzymes, respectively. This complex could effectively break down waste PET while maintaining enzyme stability and would be applied for biological upcycling of TPA. 70 less
Capone M, Zanetti-Polzi L, Leonzi I, Spreti N, Daidone I Ref: J Biomol Struct Dyn, :1, 2022 : PubMed ESTHER: Capone_2022_J.Biomol.Struct.Dyn__1 PubMedSearch: Capone 2022 J.Biomol.Struct.Dyn 1 PubMedID: 35593533 Gene_locus related to this paper: canar-LipB Abstract Candida antarctica Lipase B (CALB) is a paradigm for the family of lipases. At pH 7, the optimal pH for catalysis, the protonation state of an aspartic acid of the active site (Asp134) could not be conclusively assigned. In fact, the pK(a) estimate provided by a widely used computational tool, namely PropKa, that predicts pKa values of ionizable groups in proteins based on the crystallographic structure, is only slightly above 7 (pKa = 7.25). This, along with the lack of an experimental evaluation, makes the assignment of its protonation state at neutral pH challenging. Here, we calculate the pK(a) of Asp134 by means of a fully atomistic multiscale computational approach based on classical molecular dynamics (MD) simulation and the perturbed matrix method (PMM), namely the MD-PMM approach. MD-PMM is able to take into account the dynamics of the system and, at the same time, to treat the deprotonation step at the quantum level. The calculations provide a pK(a) value of 8.9 +/- 1.1, hence suggesting that Asp134 in CALB should be protonated at neutral, and even at slightly basic, pH. Title: Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases Galmes M, Nodling AR, He K, Luk LYP, widerek K, Moliner V Ref: Chem Sci, 13:4779, 2022 : PubMed ESTHER: Galmes_2022_Chem.Sci_13_4779 PubMedSearch: Galmes 2022 Chem.Sci 13 4779 PubMedID: 35655887 Gene_locus related to this paper: bacsu-pnbae, canar-LipB Abstract While there has been emerging interest in designing new enzymes to solve practical challenges, computer-based options to redesign catalytically active proteins are rather limited. Here, a rational QM/MM molecular dynamics strategy based on combining the best electrostatic properties of enzymes with activity in a common reaction is presented. The computational protocol has been applied to the re-design of the protein scaffold of an existing promiscuous esterase from Bacillus subtilis Bs2 to enhance its secondary amidase activity. After the alignment of Bs2 with a non-homologous amidase Candida antarctica lipase B (CALB) within rotation quaternions, a relevant spatial aspartate residue of the latter was transferred to the former as a means to favor the electrostatics of transition state formation, where a clear separation of charges takes place. Deep computational insights, however, revealed a significant conformational change caused by the amino acid replacement, provoking a shift in the pK (a) of the inserted aspartate and counteracting the anticipated catalytic effect. This prediction was experimentally confirmed with a 1.3-fold increase in activity. The good agreement between theoretical and experimental results, as well as the linear correlation between the electrostatic properties and the activation energy barriers, suggest that the presented computational-based investigation can transform in an enzyme engineering approach. Title: Design of 2,5-furandicarboxylic based polyesters degraded in different environmental conditions: Comprehensive experimental and theoretical study Hu H, Li J, Luo S, Tian Y, Wang J, Zhao YL, Zhang R, Zhu J Ref: J Hazard Mater, 425:127752, 2022 : PubMed ESTHER: Hu_2022_J.Hazard.Mater_425_127752 PubMedSearch: Hu 2022 J.Hazard.Mater 425 127752 PubMedID: 34906869 Gene_locus related to this paper: canar-LipB Abstract Nowadays, the promotion and application of aliphatic-aromatic copolyesters, such as poly (butylene adipate-co-terephthalate) (PBAT), are growing into a general trend. Although the structures of diacids exerted substantial impacts on degradation behavior, the underlying mechanisms have rarely been studied. In this work, 2,5-Furandicarboxylic acid was combined with succinic acid (PBSF), adipic acid (PBAF) and diglycolic acid (PBDF) to prepare three kinds of copolyesters. They showed unique degradation behaviors in buffer, enzyme environment and artificial seawater. These characteristics are closely related to the structural compositions of diacids. PBAFs displayed impressive biodegradability when catalyzed by Candida antarctica lipase B (CALB), while the more hydrophilic PBDFs exhibited faster hydrolysis in both buffer and artificial seawater. PBSFs, with hydrophobic and short segments, obtained a relatively slower rate of hydrolysis and enzymatic degradation. The reactivity sites and hydrolytic pathway were revealed by the combination of DFT calculation and Fukui function analysis. MD simulations, QM/MM optimizations and theozyme calculations showed that PBAF-CALB was prone to form a pre-reaction state, leading to the reduced energy barrier in the acylation process. This work revealed the effects of different structural features of diacids on polymer degradation and paved a way to design target biodegradable polymers in different degradation conditions. Title: Enhanced biodegradation of waste poly(ethylene terephthalate) using a reinforced plastic degrading enzyme complex Hwang DH, Lee ME, Cho BH, Oh JW, You SK, Ko YJ, Hyeon JE, Han SO Ref: Sci Total Environ, :156890, 2022 : PubMed ESTHER: Hwang_2022_Sci.Total.Environ__156890 PubMedSearch: Hwang 2022 Sci.Total.Environ 156890 PubMedID: 35753492 Gene_locus related to this paper: idesa-peth, canar-LipB Abstract Poly(ethylene terephthalate) (PET) is synthesized via a rich ester bond between terephthalate (TPA) and ethylene glycol (EG). Because of this, PET degradation takes a long time and PET accumulates in the environment. Many studies have been conducted to improve PET degrading enzyme to increase the efficiency of PET depolymerization. However, enzymatic PET decomposition is still restricted, making upcycling and recycling difficult. Here, we report a novel PET degrading complex composed of Ideonella sakaiensis PETase and Candida antarctica lipase B (CALB) that improves degradability, binding ability and enzyme stability. The reaction mechanism of chimeric PETase (cPETase) and chimeric CALB (cCALB) was confirmed by PET and bis (2-hydroxyethyl terephthalate) (BHET). cPETase generated BHET and mono (2-hydroxyethyl terephthalate (MHET) and cCALB produced terephthalate (TPA). Carbohydrate binding module 3 (CBM3) in the scaffolding protein greatly improved PET film binding affinity. Finally, the final enzyme complex demonstrated a 6.5-fold and 8.0-fold increase in the efficiency of hydrolysis from PET with either high crystalline or waste to TPA than single enzymes, respectively. This complex could effectively break down waste PET while maintaining enzyme stability and would be applied for biological upcycling of TPA. Title: Short-chain alcohols inactivate an immobilized industrial lipase through two different mechanisms Mangiagalli M, Ami D, de Divitiis M, Brocca S, Catelani T, Natalello A, Lotti M Ref: Biotechnol J, :e2100712, 2022 : PubMed ESTHER: Mangiagalli_2022_Biotechnol.J__e2100712 PubMedSearch: Mangiagalli 2022 Biotechnol.J e2100712 PubMedID: 35188703 Gene_locus related to this paper: canar-LipB Abstract Broadly used in biocatalysis as acyl acceptors or (co)-solvents, short-chain alcohols often cause irreversible loss of enzyme activity. Understanding the mechanisms of inactivation is a necessary step toward the optimization of biocatalytic reactions and the design of enzyme-based sustainable processes. In this work, we explored the functional and structural response of an immobilized enzyme, Novozym 435, exposed to methanol, ethanol, and tert-butanol. N-435 consists of Candida antarctica lipase B (CALB) adsorbed on polymethacrylate beads and finds application in a variety of processes involving the presence of short-chain alcohols. The nature of the N-435 material required the development of an ad hoc method of structural analysis, based on Fourier transform infrared microspectroscopy, which was complemented by catalytic activity assays and by morphological observation by transmission electron microscopy. We found that the inactivation of N-435 is highly dependent on alcohol concentration and occurs through two different mechanisms. Short-chain alcohols induce conformational changes leading to CALB aggregation, which is only partially prevented by immobilization. Moreover, alcohol modifies the texture of the solid support promoting the enzyme release. Overall, knowledge of the molecular mechanisms underlying Novozym 435 inactivation induced by short-chain alcohols promises to overcome the limitations that usually occur during industrial processes. This article is protected by copyright. All rights reserved. Title: Effect of the acyl-group length on the chemoselectivity of the lipase-catalyzed acylation of propranolol-a computational study Doerr M, Romero A, Daza MC Ref: J Mol Model, 27:198, 2021 : PubMed ESTHER: Doerr_2021_J.Mol.Model_27_198 PubMedSearch: Doerr 2021 J.Mol.Model 27 198 PubMedID: 34115202 Gene_locus related to this paper: canar-LipB Abstract The selective N-acylation of 1,2-amino alcohols has been proposed to occur through the proton shuttle mechanism. However, the O-acetylation of propranolol catalyzed by Candida antarctica lipase B is an exception. We investigated the relation between the chemoselectivity of this reaction and the acyl group length. For this purpose, we compared the acyl groups: ethanoyl, butanoyl, octanoyl, and hexadecanoyl. We studied the Michaelis complexes between serine-acylated Candida antarctica lipase B and propranolol, employing a computational approach that involved sampling Michaelis complex conformations through ensemble docking plus consensus scoring and molecular dynamics simulations. The conformations were then classified as near attack conformations for acylation of the amino or hydroxy group. The relative populations of these two classes of conformations were found to be consistent with the experimentally observed chemoselective O-acetylation. We predict that increasing the length of the hydrocarbon chain of the acyl group will cause O-acylation to be unfavorable with respect to N-acylation. The nucleophilic attack of propranolol to the acylated lipase was found to be more favorable through the classical mechanism when compared with the proton shuttle mechanism. Title: Combined Theoretical and Experimental Study to Unravel the Differences in Promiscuous Amidase Activity of Two Nonhomologous Enzymes Galmes M, Nodling AR, Luk L, Swiderek K, Moliner V Ref: ACS Catal, 11:8635, 2021 : PubMed ESTHER: Galmes_2021_ACS.Catal_11_8635 PubMedSearch: Galmes 2021 ACS.Catal 11 8635 PubMedID: 35875595 Gene_locus related to this paper: bacsu-pnbae, canar-LipB Abstract Convergent evolution has resulted in nonhomologous enzymes that contain similar active sites that catalyze the same primary and secondary reactions. Comparing how these enzymes achieve their reaction promiscuity can yield valuable insights to develop functions from the optimization of latent activities. In this work, we have focused on the promiscuous amidase activity in the esterase from Bacillus subtilis (Bs2) and compared with the same activity in the promiscuous lipase B from Candida antarctica (CALB). The study, combining multiscale quantum mechanics/molecular mechanics (QM/MM) simulations, deep machine learning approaches, and experimental characterization of Bs2 kinetics, confirms the amidase activity of Bs2 and CALB. The computational results indicate that both enzymes offer a slightly different reaction environment reflected by electrostatic effects within the active site, thus resulting in a different reaction mechanism during the acylation step. A convolutional neural network (CNN) has been used to understand the conserved amino acids among the evolved protein family and suggest that Bs2 provides a more robust protein scaffold to perform future mutagenesis studies. Results derived from this work will help reveal the origin of enzyme promiscuity, which will find applications in enzyme (re)design, particularly in creating a highly active amidase. Title: Synthesis of lipophilic arbutin ester by enzymatic transesterification in high pressure carbon dioxide Liu KJ Ref: Enzyme Microb Technol, 148:109818, 2021 : PubMed ESTHER: Liu_2021_Enzyme.Microb.Technol_148_109818 PubMedSearch: Liu 2021 Enzyme.Microb.Technol 148 109818 PubMedID: 34116761 Gene_locus related to this paper: canar-LipB Abstract In this study, a novel one-step enzymatic acylation was developed for the synthesis of hydrophobic arbutin ester, by using supercritical carbon dioxide (SC-CO(2)) as the reaction solvent. Immobilized Novozym 435 from Candida antarctica was identified as the best biocatalyst for producing arbutin palmitate through transesterification between arbutin and palmitic acid ethyl ester in SC-CO(2). A transesterification yield of 85.21 % was obtained in batch operation using palmitic acid ethyl ester as the acyl donor, hexane/propylene glycol as the co-solvent and Novozym 435 as the enzyme at 10 MPa and 60 degreesC for 20 h in SC-CO(2). The yield of arbutin palmitate increased with increasing temperature over the range of 40-60 degreesC in the current study. Operating at an arbutin/palmitic acid ethyl ester molar ratio of 5.0, the conversion of arbutin decreased, probably due to an inhibitory effect of the high concentration of palmitic acid ethyl ester on the enzyme. The 38 % original enzyme activity of Novozym 435 was maintained after being used for 3 cycles (60 h) under optimized conditions. Title: Perspectives on the Role of Enzymatic Biocatalysis for the Degradation of Plastic PET Magalhaes RP, Cunha JM, Sousa SF Ref: Int J Mol Sci, 22:11257, 2021 : PubMed ESTHER: Magalhaes_2021_Int.J.Mol.Sci_22_11257 PubMedSearch: Magalhaes 2021 Int.J.Mol.Sci 22 11257 PubMedID: 34681915 Gene_locus related to this paper: 9entr-EsEstB, 9bact-a0a2h5z9r5, 9psed-peh, 9actn-h6wx58, idesa-mheth, idesa-peth, thecd-d1a2h1, humin-cut, 9acto-d4q9n1, 9acto-f7ix06, 9bact-g9by57, bacsu-lip, bacsu-pnbae, canar-LipB, thela-m4tp71, psemy-a4y035, thefu-1831, thefu-q6a0i4, strsw-c9zcr8, fusox-CUT, sacvd-c7mve8 Abstract Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering techniques and the discovery of novel microorganisms and enzymes with degradation ability have been key. One of the most produced plastics is PET, a long chain polymer of terephthalic acid (TPA) and ethylene glycol (EG) repeating monomers. Many enzymes with PET degradation activity have been discovered, characterized, and engineered in the last few years. However, classification and integrated knowledge of these enzymes are not trivial. Therefore, in this work we present a summary of currently known PET degrading enzymes, focusing on their structural and activity characteristics, and summarizing engineering efforts to improve activity. Although several high potential enzymes have been discovered, further efforts to improve activity and thermal stability are necessary. Title: From Waste to ValueDirect Utilization of alpha-Angelica Lactone as a Nonconventional Irreversible Acylating Agent in a Chromatography-Free Lipase-Catalyzed KR Approach toward sec-Alcohols Poterala M, Borowiecki P Ref: ACS Sustainable Chem. Eng, 9:10276, 2021 : PubMed ESTHER: Poterala_2021_ACS.Sustainable.Chem.Eng_9_10276 PubMedSearch: Poterala 2021 ACS.Sustainable.Chem.Eng 9 10276 Gene_locus related to this paper: canar-LipB Abstract Although the classical enzymatic kinetic resolutions (EKRs) are among the most important reactions in biocatalysis, the requirement of application of chromatographic purification technique, which is responsible for the generation of large amounts of waste organic solvents, is its major drawback. To minimize the environmental impact of such attempts and to address the current sustainability challenges, we decided to develop a novel EKR methodology, which relies on the usage of cheap, fully renewable, nontoxic, and irreversible acyl group donor reagent, namely, -angelica lactone. The employed activated enol lactone-based acyl donor proved to be a versatile reagent enabling efficient and highly enantioselective (up to E >> 500) lipase-catalyzed resolution of a set of racemic sec-alcohols with up to >99% ee and near to quantitative isolation yields according to conversions achieved during the respective EKR procedure. Moreover, alpha-angelica lactone provided, in this case, an ability of fast and straightforward separation of the enzymatic reactions products via chromatography-free reactive liquid-liquid extraction (LLE) workup using either saturated aqueous sodium bisulfate in dimethylformamide (DMF) or Girard's P reagent in a mixture of EtOH/AcOH (90:10, v/v), respectively. The NaHSO3/DMF LLE workup and the subsequent reisolation of the respective levulinate from the aqueous layer turned out to be less efficient than a separation with Girard's P reagent. The selective LLE of optically active levulinate-Girard P-hydrazones from the respective alcohols and further hydrolysis of the respective hydrazides with diluted HClaq. could be performed with high levels of recovery of both EKR products isolated usually without loss of enantiomeric purity. Title: Finite-temperature effect in the O-acylation of (R,S)-propranolol catalyzed by Candida antarctica lipase B Valderrama DIB, Daza MC, Doerr M Ref: J Mol Graph Model, 107:107951, 2021 : PubMed ESTHER: Valderrama_2021_J.Mol.Graph.Model_107_107951 PubMedSearch: Valderrama 2021 J.Mol.Graph.Model 107 107951 PubMedID: 34111759 Gene_locus related to this paper: canar-LipB Abstract CalB is a triacylglycerol hydrolase (E.C.3.1.1.3) used in the O-acylation of the beta-adrenergic blocking agent (R,S)-propranolol. The catalytic mechanism involves two steps: enzyme acylation and enzyme deacylation. The enantioselectivity of the O-acylation of (R,S)-propranolol originates from the second step, where the acyl-enzyme transfers the acyl group to the racemic substrate. This step occurs via an initial Michaelis complex (MCC) and a tetrahedral intermediate (TI-2). To gain more insight into the molecular basis of this reaction, we performed an exhaustive conformational sampling along the reaction coordinate of the enantioselective step of the reaction (MCCTI-2EPC) applying a QM/MM MD protocol (SCC-DFTB/CHARMM) in combination with umbrella sampling and the weighted histogram analysis method. To identify finite temperature effects we compare the PMF and the potential energy pathway. It is found that the effect of the finite temperature in this reaction is a destabilization of the tetrahedral intermediate and an increase of the barrier height of its formation. This increase is higher for the S-enantiomer. Title: Exploring the Origin of Amidase Substrate Promiscuity in CALB by a Computational Approach Galmes MA, Garcia-Junceda E, Swiderek K, Moliner V Ref: ACS Catal, 10:1938, 2020 : PubMed ESTHER: Galmes_2020_ACS.Catal_10_1938 PubMedSearch: Galmes 2020 ACS.Catal 10 1938 Gene_locus related to this paper: canar-LipB Abstract Enzyme promiscuity attracts the interest of the industrial and academic sectors because of its application in the design of biocatalysts. The amidase activity of Candida antarctica lipase B (CALB) on two different substrates has been studied by theoretical quantum mechanics/molecular mechanics methods, supported by experimental kinetic measurements. The aim of the study is to understand the substrate promiscuity of CALB in this secondary reaction and the origin of its promiscuous catalytic activity. The computational results predict activation free energies in very good agreement with the kinetic data and confirm that the activity of CALB as an amidase, despite depending on the features of the amide substrate, is dictated by the electrostatic effects of the protein. The protein polarizes and activates the substrate as well as stabilizes the transition state, thus enhancing the rate constant. Our results can provide guides for future designs of biocatalysts based on electrostatic arguments. Title: Lipase-Catalyzed Reactive Extrusion: Copolymerization of sigma-Caprolactone and -Pentadecalactone Li C, Xu W, Lu Y, Gross RA Ref: Macromol Rapid Commun, 41:e2000417, 2020 : PubMed ESTHER: Li_2020_Macromol.Rapid.Commun_41_e2000417 PubMedSearch: Li 2020 Macromol.Rapid.Commun 41 e2000417 PubMedID: 33047442 Gene_locus related to this paper: canar-LipB Abstract This study assesses the use of immobilized lipase catalyst N435 during reactive extrusion (REX) versus magnetically stirred bulk and solution reaction conditions for the copolymerization of sigma-caprolactone with omega-pentadecalactone (CL/PDL 1:1 molar). N435-catalyzed REX for reaction times from 1 to 3 h results in total %-monomer conversion, M(n) , and D values increase from 92.7% to 98.8%, 36.1 to 51.3 kDa, and 1.85 to 1.96, respectively. Diad fraction analysis by quantitative (13) C NMR reveals that, after just 1 h, rapid N435-catalyzed transesterification reactions occur that give random copolyesters. In contrast, for bulk polymerization with magnetic stirring in round bottom flasks, reaction times from 1 to 3 h result in the following: M(n) increases from 12.4 to 25.6 kDa, D decreases from 2.98 to 1.87, and the randomness index increases from 0.74 and 0.86 as PDL*-PDL diads are dominant. These results highlight that REX avoids problems associated with internal batch mixing that are encountered in bulk polymerizations. In sharp contrast to a previous study of 1:1 molar PDL/delta-valerolactone (VL) copolymerizations by N435-catalyzed REX, VL %-conversion increases to just 40.1% in 1 h whereas CL reaches 94.7%. Title: Global and Kinetic Profiles of Substrate Diffusion in Candida antarctica Lipase B: Molecular Dynamics with the Markov-State Model Lu C, Peng X, Lu D, Liu Z Ref: ACS Omega, 5:9806, 2020 : PubMed ESTHER: Lu_2020_ACS.Omega_5_9806 PubMedSearch: Lu 2020 ACS.Omega 5 9806 PubMedID: 32391467 Gene_locus related to this paper: canar-LipB Abstract Profiling substrate diffusion pathways with kinetic information, which accounts for the dynamic nature of enzyme-substrate interaction, can enable molecular reengineering of enzymes and process optimization of enzymatic catalysis. Candida antarctica lipase B (CALB) is extensively used for producing various chemicals because of its rich catalytic mechanisms, broad substrate spectrum, thermal stability, and tolerance to organic solvents. In this study, an all-atom molecular dynamics (MD) combined with Markov-state models (MSMs) implemented in pyEMMA was proposed to simulate diffusion pathways of 4-nitrophenyl ester (4NPE), a commonly used substrate, from the surface into the active site of CALB. Six important metastable conformations of CALB were identified in the diffusion process, including a closed state. An induced-fit mechanism incorporating multiple pathways with molecular information was proposed, which might find unprecedented applications for the rational design of lipase for green catalysis. Title: Enzymatic Polycondensation of 1,6-Hexanediol and Diethyl Adipate: A Statistical Approach Predicting the Key-Parameters in Solution and in Bulk Nasr K, Meimoun J, Favrelle-Huret A, Winter J, Raquez JM, Zinck P Ref: Polymers (Basel), 12:, 2020 : PubMed ESTHER: Nasr_2020_Polymers.(Basel)_12_ PubMedSearch: Nasr 2020 Polymers.(Basel) 12 PubMedID: 32847050 Gene_locus related to this paper: canar-LipB Abstract Among the various catalysts that can be used for polycondensation reactions, enzymes have been gaining interest for three decades, offering a green and eco-friendly platform towards the sustainable design of renewable polyesters. However, limitations imposed by their delicate nature, render them less addressed. As a case study, we compare herein bulk and solution polycondensation of 1,6-hexanediol and diethyl adipate catalyzed by an immobilized lipase from Candida antarctica. The influence of various parameters including time, temperature, enzyme loading, and vacuum was assessed in the frame of a two-step polymerization with the help of response surface methodology, a statistical technique that investigates relations between input and output variables. Results in solution (diphenyl ether) and bulk conditions showed that a two-hour reaction time was enough to allow adequate oligomer growth for the first step conducted under atmospheric pressure at 100 degreesC. The number-average molecular weight (M(n)) achieved varied between 5000 and 12,000 g.mol(-1) after a 24 h reaction and up to 18,500 gmol(-1) after 48 h. The statistical analysis showed that vacuum was the most influential factor affecting the M(n) in diphenyl ether. In sharp contrast, enzyme loading was found to be the most influential parameter in bulk conditions. Recyclability in bulk conditions showed a constant M(n) of the polyester over three cycles, while a 17% decrease was noticed in solution. The following work finally introduced a statistical approach that can adequately predict the M(n) of poly(hexylene adipate) based on the choice of parameter levels, providing a handy tool in the synthesis of polyesters where the control of molecular weight is of importance. Title: Principles of lipid-enzyme interactions in the limbus region of the catalytic site of Candida antarctica Lipase B Silvestrini L, Cianci M Ref: Int J Biol Macromol, :, 2020 : PubMed ESTHER: Silvestrini_2020_Int.J.Biol.Macromol__ PubMedSearch: Silvestrini 2020 Int.J.Biol.Macromol PubMedID: 32380114 Gene_locus related to this paper: canar-LipB Inhibitor(s) related to this paper: Paraoxon Abstract Lipases (E.C. 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates such as triacylglycerols and phospholipids. Candida antarctica Lipase B (CALB) acts in aqueous as well as in low-water media, thus being of considerable biochemical significance with high interest also for its industrial applications. The hydrolysis reaction follows a two-step mechanism, or 'interfacial activation', with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Once positioned within the catalytic triad, substrates are then hydrolysed, and products released. However, the intermediate steps of substrate transfer from the lipidic-aqueous phase to the enzyme surface and then down to the catalytic site are still unclear. By inhibiting CALB with ethyl phosphonate and incubating with glyceryl tributyrate (2,3-di (butanoyloxy) propyl butanoate), the crystal structure of the lipid-enzyme complex, at 1.55A resolution, shows the tributyrin in the limbus region of active site. The substrate is found 10A above the catalytic Ser, with the glycerol backbone pre-aligned for further processing by key interactions via an extended water network with alpha-helix10 and alpha-helix5. The findings offer new elements to elucidate the mechanism of substrate recognition, transfer and catalysis of Candida antarctica Lipase B (CALB) and lipases in general. Title: Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution Cen Y, Singh W, Arkin M, Moody TS, Huang M, Zhou J, Wu Q, Reetz MT Ref: Nat Commun, 10:3198, 2019 : PubMed ESTHER: Cen_2019_Nat.Commun_10_3198 PubMedSearch: Cen 2019 Nat.Commun 10 3198 PubMedID: 31324776 Gene_locus related to this paper: canar-LipB Abstract Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser --> Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105(-)/His224(+) rather than a concerted process. Title: Biocatalyst CAL-B catalyzed synthesis of modified nucleosides: An overview Rajesh Kumar, Vijay Kumar, Divya Mathur, Ram Kumar, Arbind Kumar, Prasad AK Ref: Synth Syst Biotechnol, 49:1659, 2019 : PubMed ESTHER: Kumar_2019_Synth.Commun_49_1659 PubMedSearch: Kumar 2019 Synth.Commun 49 1659 Gene_locus related to this paper: canar-LipB Abstract Nucleosides are valuable biologically active compounds, which display antitumour and antiviral activities. Various types of bioactivenucleosides are designed to improve their therapeutic efficacy, However this strategy faces the difficult selectivity issues of nucleoside chemistry. Therefore, the goal of this review is to give an idea ofthe opportunities provided by biocatalyst CAL-B procedures in the preparation of different types of bioactive nucleoside compounds. The function of Candida Antarctica lipase-B (CAL-B) in organic synthesis is reviewed. This enzyme has been found to be a principally efficient and robust lipase catalyzing an unexpected variety of reactions including many different regio- chemo, and diastereo-selective synthesis. Moreover, the structure of Candida Antarctica lipase-B is an example of an enzyme for which its specificity has been predicted based on acylation and deacylation mechanism on substrates. Title: Effect of organic solvent on enzymatic degradation of cyclic PBS-based polymers by lipase N435 Li CT, Zhang M, Weng YX, Zhao D Ref: Int J Biol Macromol, 137:215, 2019 : PubMed ESTHER: Li_2019_Int.J.Biol.Macromol_137_215 PubMedSearch: Li 2019 Int.J.Biol.Macromol 137 215 PubMedID: 31255620 Gene_locus related to this paper: canar-LipB Abstract Poly(butylene succinate-co-cyclohexane dimethanol succinate) (P(BS-co-CHDMS)) and poly(butylene succinate-co-butanediol cyclohexanedicarboxylic acid) (P(BS-co-BCHDA)) were catalytically degraded by Candida antarctica lipase Novozyme 435 (N435) in CHCl3 and THF. The results indicated that the degradation rate was P(BS-co-CHDMS)>P(BS-co-BCHDA)>poly(butylene succinate) (PBS). The degradation rate of copolyesters was higher in CHCl3 than in THF, the highest degradation rate of 67% being obtained for P(BS-co-CHDMS). Hence, the CHCl3 solvent is more suitable for the enzyme-catalytic degradation of copolyesters, since the lipase can easier recognize the butylene succinate (BS-), (butanediol cyclohexanedicarboxylic acid) (BCA-), and (cyclohexane dimethanol succinate-type) (CMS-type) ester bonds in this solvent. Moreover, it can recognize the CMS-type ester bonds with a higher specificity than the (butanediol cyclohexanedicarboxylic acid type) (BCA-type) ester bonds. Molecular simulation results indicated that the structure of the lipase was stable in CHCl3 and THF. However, CHCl3 proved to be more suitable for a stable activity of the enzyme. The active pocket contains acyl-binding hydrophilic residues which are recognized by the substrate. The increase in the content of saturated cycles can increase the hydrophobicity of the substrate and thus, the amount of substrate bond to enzyme active site is increased, which facilitates the enzymatic degradation of copolyesters. Title: Kinetics and thermodynamics of lipase catalysed synthesis of propyl caprate Parikh DT, Lanjekar KJ, Rathod VK Ref: Biotechnol Lett, 41:1163, 2019 : PubMed ESTHER: Parikh_2019_Biotechnol.Lett_41_1163 PubMedSearch: Parikh 2019 Biotechnol.Lett 41 1163 PubMedID: 31463715 Gene_locus related to this paper: canar-LipB Abstract OBJECTIVE: To investigate kinetics and thermodynamics of lipase-catalyzed esterification of capric acid with 1-propyl alcohol in a solvent-free system for synthesis of propyl caprate. RESULTS: The capric acid conversion of 83.82% is achieved at temperature 60 degrees C, speed of agitation 300 rpm, molar ratio acid:alcohol 1:3, enzyme loading 2% (w/w) and molecular sieves loading 5% (w/w). The activation energy (Ea) for the reaction was determined as 37.79 kJ mol(-1). Furthermore, enthalpy (DeltaH), entropy (DeltaS) and Gibbs free energy (DeltaG) values were found out to be + 90.45 kJ mol(-1), + 278.99 J mol(-1) K(-1) and - 2.35 kJ mol(-1) respectively. CONCLUSIONS: The results showed that the lipase-catalyzed esterification exhibits an ordered bi-bi mechanism with capric acid inhibiting the reaction and forming the dead-end complex with the lipase. Under the given set of reaction conditions, the lipase catalysed esterification reaction was anticipated to be spontaneous, referring to the value of the Gibbs free energy change (DeltaG). Moreover, the esterification process was found to be endothermic, based on the values of enthalpy (DeltaH) and entropy (DeltaS). Title: Stereodivergent Protein Engineering of a Lipase To Access All Possible Stereoisomers of Chiral Esters with Two Stereocenters Xu J, Cen Y, Singh W, Fan J, Wu L, Lin X, Zhou J, Huang M, Reetz MT, Wu Q Ref: Journal of the American Chemical Society, 141:7934, 2019 : PubMed ESTHER: Xu_2019_J.Am.Chem.Soc_141_7934 PubMedSearch: Xu 2019 J.Am.Chem.Soc 141 7934 PubMedID: 31023008 Gene_locus related to this paper: canar-LipB Inhibitor(s) related to this paper: 6J1T-B7U Abstract Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants of each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultrasmall mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity. Title: Entropy connects water structure and dynamics in protein hydration layer Dahanayake JN, Mitchell-Koch KR Ref: Phys Chem Chem Phys, 20:14765, 2018 : PubMed ESTHER: Dahanayake_2018_Phys.Chem.Chem.Phys_20_14765 PubMedSearch: Dahanayake 2018 Phys.Chem.Chem.Phys 20 14765 PubMedID: 29780979 Gene_locus related to this paper: canar-LipB Abstract The enzyme Candida Antarctica lipase B (CALB) serves here as a model for understanding connections among hydration layer dynamics, solvation shell structure, and protein surface structure. The structure and dynamics of water molecules in the hydration layer were characterized for regions of the CALB surface, divided around each alpha-helix, beta-sheet, and loop structure. Heterogeneous hydration dynamics were observed around the surface of the enzyme, in line with spectroscopic observations of other proteins. Regional differences in the structure of the biomolecular hydration layer were found to be concomitant with variations in dynamics. In particular, it was seen that regions of higher density exhibit faster water dynamics. This is analogous to the behavior of bulk water, where dynamics (diffusion coefficients) are connected to water structure (density and tetrahedrality) by excess (or pair) entropy, detailed in the Rosenfeld scaling relationship. Additionally, effects of protein surface topology and hydrophobicity on water structure and dynamics were evaluated using multiregression analysis, showing that topology has a somewhat larger effect on hydration layer structure-dynamics. Concave and hydrophobic protein surfaces favor a less dense and more tetrahedral solvation layer, akin to a more ice-like structure, with slower dynamics. Results show that pairwise entropies of local hydration layers, calculated from regional radial distribution functions, scale logarithmically with local hydration dynamics. Thus, the Rosenfeld relationship describes the heterogeneous structure-dynamics of the hydration layer around the enzyme CALB. These findings raise the question of whether this may be a general principle for understanding the structure-dynamics of biomolecular solvation. Title: Comparison of Candida antarctica Lipase B Variants for Conversion of epsilon-Caprolactone in Aqueous Medium-Part 2 Hock H, Engel S, Weingarten S, Keul H, Schwaneberg U, Moller M, Bocola M Ref: Polymers (Basel), 10:, 2018 : PubMed ESTHER: Hock_2018_Polymers.(Basel)_10_524 PubMedSearch: Hock 2018 Polymers.(Basel) 10 524 PubMedID: 30966558 Gene_locus related to this paper: canar-LipB Abstract Enzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the "gold standard" Novozym((R)) 435. Title: Determinants and prediction of esterase substrate promiscuity patterns Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A and Ferrer M <32 more author(s)> Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A, Thies S, Mendez-Garcia C, Popovic A, Brown G, Chernikova TN, Garcia-Moyano A, Bjergah GE, Perez-Garcia P, Hai T, Del Pozo MV, Stokke R, Steen IH, Cui H, Xu X, Nocek BP, Alcaide M, Distaso M, Mesa V, Pelaez AI, Sanchez J, Buchholz PCF, Pleiss J, Fernandez-Guerra A, Glockner FO, Golyshina OV, Yakimov MM, Savchenko A, Jaeger KE, Yakunin AF, Streit WR, Golyshin PN, Guallar V, Ferrer M (- 32) Ref: ACS Chemical Biology, 13:225, 2018 : PubMed ESTHER: Martinez-Martinez_2018_ACS.Chem.Biol_13_225 PubMedSearch: Martinez-Martinez 2018 ACS.Chem.Biol 13 225 PubMedID: 29182315 Gene_locus related to this paper: 9zzzz-a0a2k8jn75, 9zzzz-a0a2k8jt94, 9zzzz-a0a0g3fj44, 9zzzz-a0a0g3fh10, 9zzzz-a0a0g3fh03, 9bact-a0a1s5qkj8, 9zzzz-a0a0g3feh5, 9zzzz-a0a0g3fkz4, 9zzzz-a0a0g3fh07, 9zzzz-a0a0g3fh34, 9zzzz-a0a0g3fh31, 9bact-KY458167, alcbs-q0vqa3, 9bact-a0a1s5qki8, 9zzzz-a0a0g3feq8, 9zzzz-a0a0g3feh8, 9zzzz-a0a0g3fh19, 9bact-KY203037, 9bact-a0a1s5ql22, 9bact-a0a1s5qm34, 9bact-KY203034, 9bact-r9qzg0, 9bact-a0a1s5qly8, 9zzzz-a0a0g3fkz8, 9zzzz-a0a0g3feg9, 9zzzz-KY203033, 9zzzz-a0a0g3fes4, 9zzzz-a0a0g3fh42, 9bact-a0a1s5qlx2, 9zzzz-KY483651, 9bact-a0a1s5qmh4, 9zzzz-KY203032, 9zzzz-EH87, 9zzzz-a0a0g3fei1, 9zzzz-a0a0g3fet2, 9zzzz-KY483647, 9zzzz-EH82, 9zzzz-a0a0g3fe15, 9bact-KY203031, 9bact-t1w006, 9zzzz-a0a0g3fet6, 9bact-KY458164, geoth-g8myf3, 9bact-a0a1s5ql04, 9gamm-a0a1y0ihk7, 9bact-a0a1s5qly6, 9bact-a0a1s5qkg4, 9bact-a0a1s5qkm4, 9gamm-s5tv80, 9gamm-a0a0c4zhg2, 9zzzz-t1b379, 9gamm-KY483646, 9bact-KY458160, 9zzzz-a0a0g3fj57, 9gamm-s5t8349, 9arch-KY203036, 9bact-KY458168, 9zzzz-a0a0g3fes0, 9zzzz-t1be47, 9bact-KY458159, 9zzzz-a0a0g3fh39, 9bact-t1vzd5, 9prot-EH41, 9bact-Lip114, alcbs-q0vt77, 9bact-a0a1s5qke6, 9bact-a0a1s5qkf3, 9prot-SRP030024, 9gamm-s5t532, 9bact-a0a1s5qkl2, 9bact-a0a1s5qkk8, 9zzzz-KY203030, 9zzzz-t1d4I7, 9prot-KY019260, 9bact-a0a1s5qm38, 9arch-KY458161, 9prot-KY010302, 9zzzz-a0a0g3fl25, 9actn-KY010298, 9gamm-s5u059, 9bact-a0a1s5qmi7, 9bact-KY010297, 9bact-KY483642, 9bact-a0a1s5qkj1, 9bact-KY010299, 9bact-KY483648, alcbs-q0vtl7, 9bact-a0a1s5qf1, 9bact-a0a1s5qkg0, 9bact-a0a0h4tgu6, 9bact-MilE3, 9bact-LAE6, 9alte-MGS-MT1, 9bact-r9qzf7, 9gamm-k0c6t6, alcbs-q0vl36, alcbs-q0vlq1, alcbs-q0vq49, bacsu-pnbae, canar-LipB, canan-lipasA, geost-lipas, marav-a1u5n0, pseps-i7k8x5, staep-GEHD, symth-q67mr3, altma-s5cfn7, cycsp-k0c2b8, alcbs-q0vlk5, 9bact-k7qe48, 9bact-MGS-M1, 9bact-MGS-M2, 9bact-a0a0b5kns5, 9zzzz-a0a0g3fej4, 9zzzz-a0a0g3fj60, 9zzzz-a0a0g3fej0, 9zzzz-a0a0g3fj64, 9bact-a0a0b5kc16, 9zzzz-a0a0g3feg6, 9zzzz-a0a0g3feu6 Abstract Esterases receive special attention because their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others, remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps ranking (classifying) promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence datasets. Title: Enzymatic Degradation of Poly(butylene succinate) Copolyesters Synthesized with the Use of Candida antarctica Lipase B Wcislek A, Sonseca Olalla A, McClain A, Piegat A, Sobolewski P, Puskas J, El Fray M Ref: Polymers (Basel), 10:, 2018 : PubMed ESTHER: Wcislek_2018_Polymers.(Basel)_10_ PubMedSearch: Wcislek 2018 Polymers.(Basel) 10 PubMedID: 30966722 Gene_locus related to this paper: canar-LipB Abstract Biodegradable polymers are an active area of investigation, particularly ones that can be produced from sustainable, biobased monomers, such as copolymers of poly(butylene succinate) (PBS). In this study, we examine the enzymatic degradation of poly(butylene succinate-dilinoleic succinate) (PBS-DLS) copolymers obtained by "green" enzymatic synthesis using lipase B from Candida antarctica (CALB). The copolymers differed in their hard to soft segments ratio, from 70:30 to 50:50 wt %. Enzymatic degradation was carried out on electrospun membranes (scaffolds) and compression-moulded films using lipase from Pseudomomas cepacia. Poly(epsilon-caprolactone) (PCL) was used as a reference aliphatic polyester. The degradation process was monitored gravimetrically via water uptake and mass loss. After 24 days, approx. 40% mass loss was observed for fibrous materials prepared from the PBS-DLS 70:30 copolymer, as compared to approx. 10% mass loss for PBS-DLS 50:50. Infrared spectroscopy (FTIR) and size exclusion chromatography (SEC) analysis were used to examine changes in chemical structure. Differential scanning calorimetry (DSC) and scanning light microscopy (LSM) revealed changes in degree of crystallinity, and changes in surface morphology, consistent with a surface erosion mechanism. We conclude that the obtained copolymers are suitable for tissue engineering applications thanks to tuneable degradation and lack of acidification during breakdown. Title: A novel self-activation mechanism of Candida antarctica lipase B Luan B, Zhou R Ref: Phys Chem Chem Phys, 19:15709, 2017 : PubMed ESTHER: Luan_2017_Phys.Chem.Chem.Phys_19_15709 PubMedSearch: Luan 2017 Phys.Chem.Chem.Phys 19 15709 PubMedID: 28589990 Gene_locus related to this paper: canar-LipB Abstract Candida antarctica lipase B (CalB), resembling many other lipases structure-wise, contains a flexible lid that undergoes a surprisingly large conformational change when catalyzing hydrophobic substrates (e.g. triglycerides). Despite extensive and important applications in industry, it is so far still elusive whether CalB can be activated on a hydrophobic surface, like other lipases. From large-scale all-atom molecular dynamics simulations, we discovered an open state that strikingly shows a much wider and more stable entrance to the catalytic site than the one suggested by previous crystal structures. Simulations demonstrate that in the newly found open state CalB possesses a "lid-holder" structure that intimately harbors the lid of CalB, i.e. a remarkable self-activation mechanism. To account for the unusual interfacial activation of CALB revealed in a recent experiment, we further introduce a simple model: the activation occurs only when the binding free energy between the lid and a hydrophobic surface is larger than a critical value, 4.0 kcal mol-1 that is the one between the lid and the "lid-holder". Our findings shed light on possible protein engineering of lipases to permit either self-activation with broadened catalytic targets (including water soluble ones) or surface activation with elevated activities. Title: Stereoselectivity-Tailored, Metal-Free Hydrolytic Dynamic Kinetic Resolution of Morita-Baylis-Hillman Acetates Using an Engineered Lipase-Organic Base Cocatalyst Xia B, Xu J, Xiang Z, Cen Y, Hu Y, Lin X, Wu Q Ref: ACS Catal, 7:4542, 2017 : PubMed ESTHER: Xia_2017_ACS.Catal_7_4542 PubMedSearch: Xia 2017 ACS.Catal 7 4542 Gene_locus related to this paper: canar-LipB Abstract Metal-free enantiocomplementary hydrolytic dynamic kinetic resolution of Morita-Baylis-Hillman (MBH) acetates was developed using triethylamine (TEA) as a racemization catalyst and wild-type or engineered lipase B from Candida antarctica (CALB) as stereoselectivity-determining catalyst, leading to chiral MBH alcohols with tailor-made R or S configurations on an optional basis. In the TEA-WT CALB catalysis system, WT CALB displays excellent S enantioselectivity for a series of MBH acetates tested (up to 96% ee and 98% conversion). Reversal of enantioselectivity in favor of (R)-MBH alcohols (95% ee; 95% conversion) was achieved by generating a focused site-specific mutagenesis library composed of less than 20 variants. Molecular modeling explains the origin of stereoselectivity. Title: Structural and Experimental Evidence for the Enantiomeric Recognition toward a Bulky sec-Alcohol by Candida antarctica Lipase B Park K, Kim S, Park J, Joe S, Min B, Oh J, Song J, Park SY, Park S, Lee H Ref: , 6:7458, 2016 : PubMed ESTHER: Park_2016_ACS.Catal_6_7458 PubMedSearch: Park 2016 ACS.Catal 6 7458 Gene_locus related to this paper: canar-LipB Inhibitor(s) related to this paper: Phosphonate-MSW Abstract Candida antarctica lipase B (CAL-B) exhibits remarkable enantioselectivity for various chiral sec-alcohols, and the enantioselectivity is structurally well-understood. Two substituents at the chiral center of a sec-alcohol separately bind two pockets, namely, large and medium binding pockets. It has been believed that the medium pocket is too small to accommodate a large substituent (larger than an ethyl group), and thus, bulky sec-alcohols bearing two large substituents have been regarded as a poor substrate for CAL-B. However, we found that CAL-B can catalyze the transesterification of N-Boc-protected rac-2-amino-1-phenylethanol (1a) enantioselectively with a moderate reaction rate. X-ray crystallography and computer modeling revealed that the rotation of the Leu278 side chain creates a space to accept the N-Boc-aminomethylene group of 1a. Moreover, a sec-alcohol substrate with less than one hydrogen atom at the gamma-position from the hydroxyl group is required to achieve a moderate reaction rate. On the basis of this observation, we diversified bulky N-Boc-protected rac-2-amino-1-arylethanols for the transesterifications with high enantioselectivities (E > 200). Title: Crystal and molecular structure of hexagonal form of lipase B from Candida antarctica Strzelczyk P, Bujacz GD, Kielbasinski P, Blaszczyk J Ref: Acta Biochim Pol, 63:103, 2016 : PubMed ESTHER: Strzelczyk_2016_Acta.Biochim.Pol_63_103 PubMedSearch: Strzelczyk 2016 Acta.Biochim.Pol 63 103 PubMedID: 26716135 Gene_locus related to this paper: canar-LipB Abstract During crystallization screenings of commercially available hydrolytic enzymes, the new, hexagonal crystal form of CAL-B, has been discovered and hereby reported. The NAG molecules, which were closing the glycosylation site in the orthorhombic form, in hexagonal structure make the glycosylation site open. It is unknown whether the opening and closing of the glycosylation site by the 'lid' NAG molecules, could be related to the opening and closing of the active center of the enzyme upon substrate binding and product release. Title: Lipase active site covalent anchoring of Rh(NHC) catalysts: towards chemoselective artificial metalloenzymes Basauri-Molina M, Riemersma CF, Wurdemann MA, Kleijn H, Klein Gebbink RJ Ref: Chem Commun (Camb), 51:6792, 2015 : PubMed ESTHER: Basauri-Molina_2015_Chem.Commun.(Camb)_51_6792 PubMedSearch: Basauri-Molina 2015 Chem.Commun.(Camb) 51 6792 PubMedID: 25786894 Gene_locus related to this paper: canar-LipB, fusso-cutas Inhibitor(s) related to this paper: Rh-pNP Abstract A Rh(NHC) phosphonate complex reacts with the lipases cutinase and Candida antarctica lipase B resulting in the first (soluble) artificial metalloenzymes formed by covalent active site-directed hybridization. When compared to unsupported complexes, these new robust hybrids show enhanced chemoselectivity in the (competitive) hydrogenation of olefins over ketones. Title: Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation Stauch B, Fisher SJ, Cianci M Ref: J Lipid Res, 56:2348, 2015 : PubMed ESTHER: Stauch_2015_J.Lipid.Res_56_2348 PubMedSearch: Stauch 2015 J.Lipid.Res 56 2348 PubMedID: 26447231 Gene_locus related to this paper: canar-LipB Abstract Lipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or "interfacial activation," with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 A resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases. Title: Interfacial Activation of Candida antarctica Lipase B: Combined Evidence from Experiment and Simulation Zisis T, Freddolino PL, Turunen P, van Teeseling MC, Rowan AE, Blank KG Ref: Biochemistry, 54:5969, 2015 : PubMed ESTHER: Zisis_2015_Biochemistry_54_5969 PubMedSearch: Zisis 2015 Biochemistry 54 5969 PubMedID: 26346632 Gene_locus related to this paper: canar-LipB Abstract Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Candida antarctica lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small "lid" (helix alpha5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents. Title: Theoretical Study of Primary Reaction of Pseudozyma antarctica Lipase B as the Starting Point To Understand Its Promiscuity Swiderek K, Mart S, Moliner V Ref: ACS Catal, 4:426, 2014 : PubMed ESTHER: Swiderek_2014_ACS.Catal_4_426 PubMedSearch: Swiderek 2014 ACS.Catal 4 426 Gene_locus related to this paper: canar-LipB Abstract Pseudozyma antarctica lipase B (PALB) is a serine hydrolase that catalyzes the hydrolysis of carboxylic acid esters in aqueous medium, but it has also shown catalytic activity for a plethora of reactions. This promiscuous activity has found widespread applications. In the present paper, the primary reaction of PALB, its native hydrolytic activity, has been studied using hybrid quantum mechanical/molecular mechanical (QM/MM) potentials. Free energy surfaces, obtained from QM/MM Molecular Dynamics (MD) simulations, show that the reaction takes place by means of a multistep mechanism where the first step, the activation of the carbonyl group of the substrate and the nucleophilic attack of Ser105 to the carbonyl carbon atom, presents the highest energy transition state. Our results, which are in good agreement with kinetic experimental data, suggest that the origin of the catalytic activity of the enzyme is due to favorable interactions established between the residues of the active site that create an oxyanion hole, Gln106 and Thr40, as well as the Asp187 that is capable of modulating the pKa of His224 to act as a base or an acid depending on the step of the catalytic process. Understanding the main features of PALB in catalyzing the hydrolysis reaction can be the starting point to design mutations that improve the efficiency of its secondary reactions. This is, to design an optimum biocatalyst capable of accelerating fundamental reactions in organic synthesis is the primary aim. Title: Exploring the Solid State Properties of Enzymatic Poly(amine-co-ester) Terpolymers to Expand their Applications in Gene Transfection Voevodina I, Scandola M, Zhang J, Jiang Z Ref: RSC Adv, 4:8953, 2014 : PubMed ESTHER: Voevodina_2014_RSC.Adv_4_8953 PubMedSearch: Voevodina 2014 RSC.Adv 4 8953 PubMedID: 24683469 Gene_locus related to this paper: canar-LipB Abstract Polymers bearing amino functional groups are an important class of materials capable of serving as non-viral carriers for DNA delivery to living cells. In this work biodegradable poly(amine-co-ester) terpolymers were synthesized via ring-opening and polycondensation copolymerization of lactone (sigma-caprolactone (CL), omega-dodecalactone, omega-pentadecalactone (PDL), and omega-hexadecalactone) with diethyl sebacate (DES) and N-methyldiethanolamine (MDEA) in diphenyl ether, catalyzed by Candida antarctica lipase B (CALB). All lactone-DES-MDEA terpolymers had random distributions of lactone, sebacate, MDEA repeat units in the polymer chains. PDL-DES-MDEA terpolymers were studied in the composition range from 21 mol% to 90 mol% PDL whereas the terpolymers with other lactones were investigated at a single composition (80 mol% lactone). DSC and WAXS analyses showed that all investigated terpolymers crystallize in their respective homopolylactone crystal lattice. Terpolymers with large lactones and a high lactone content melt well above room temperature and are hard solids, whereas terpolymers with small lactones (e.g. CL) or with a low lactone content melt below/around ambient temperature and are waxy/gluey materials. Given the importance of hydrophobicity in influencing gene delivery, water contact angle measurements were carried out on lactone-DES-MDEA terpolymers showing that it is possible to tune the hydrophilic-to-hydrophobic balance by varying polymer composition and size of lactone units. To demonstrate the feasibility of using solid terpolymers as nanocarriers for DNA delivery, PDL-DES-MDEA copolymers with 65-90% PDL were successfully transformed into free-standing nanoparticles with average particle size ranging from 163 to 175 nm. Our preliminary results showed that LucDNA-loaded nanoparticles of the terpolymer with 65% PDL were effective for luciferase gene transfection of HEK293 cells. Title: Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site Xie Y, An J, Yang G, Wu G, Zhang Y, Cui L, Feng Y Ref: Journal of Biological Chemistry, 289:7994, 2014 : PubMed ESTHER: Xie_2014_J.Biol.Chem_289_7994 PubMedSearch: Xie 2014 J.Biol.Chem 289 7994 PubMedID: 24448805 Gene_locus related to this paper: canar-LipB Abstract Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 A of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 degrees C and a 12 degrees C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible alpha10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. Title: Genome Sequence of the Basidiomycetous Yeast Pseudozyma antarctica T-34, a Producer of the Glycolipid Biosurfactants Mannosylerythritol Lipids Morita T, Koike H, Koyama Y, Hagiwara H, Ito E, Fukuoka T, Imura T, Machida M, Kitamoto D Ref: Genome Announc, 1:e0006413, 2013 : PubMed ESTHER: Morita_2013_Genome.Announc_1_E0006413 PubMedSearch: Morita 2013 Genome.Announc 1 E0006413 PubMedID: 23558529 Gene_locus related to this paper: canar-LipB, psea3-m9mfk8, psea3-m9lyb4, psea3-m9mhk2, psea3-m9lth4, psea3-m9mcb7, psea3-m9lv13, psea3-m9m1x3, psea3-m9mf48, psea3-m9m693, psea3-m9mbv1, psea3-m9lqi4 Abstract The basidiomycetous yeast Pseudozyma antarctica T-34 is an excellent producer of mannosylerythritol lipids (MELs), members of the multifunctional extracellular glycolipids, from various feedstocks. Here, the genome sequence of P. antarctica T-34 was determined and annotated. Analysis of the sequence might provide insights into the properties of this yeast that make it superior for use in the production of functional glycolipids, leading to the further development of P. antarctica for industrial applications. Title: Bioinformatic analysis of alpha/beta-hydrolase fold enzymes reveals subfamily-specific positions responsible for discrimination of amidase and lipase activities Suplatov DA, Besenmatter W, Svedas VK, Svendsen A Ref: Protein Engineering Des Sel, 25:689, 2012 : PubMed ESTHER: Suplatov_2012_Protein.Eng.Des.Sel_25_689 PubMedSearch: Suplatov 2012 Protein.Eng.Des.Sel 25 689 PubMedID: 23043134 Gene_locus related to this paper: canar-LipB Abstract Superfamily of alpha-beta hydrolases is one of the largest groups of structurally related enzymes with diverse catalytic functions. Bioinformatic analysis was used to study how lipase and amidase catalytic activities are implemented into the same structural framework. Subfamily-specific positions-conserved within lipases and peptidases but different between them-that were supposed to be responsible for functional discrimination have been identified. Mutations at subfamily-specific positions were used to introduce amidase activity into Candida antarctica lipase B (CALB). Molecular modeling was implemented to evaluate influence of selected residues on binding and catalytic conversion of amide substrate by corresponding library of mutants. In silico screening was applied to select reactive enzyme-substrate complexes that satisfy knowledge-based criteria of amidase catalytic activity. Selected CALB variants with substitutions at subfamily-specific positions Gly39, Thr103, Trp104, and Leu278 were produced and showed significant improvement of experimentally measured amidase activity. Based on these results, we suggest that value of subfamily-specific positions should be further explored in order to develop a systematic tool to study structure-function relationship in enzymes and to use this information for rational enzyme engineering. Title: Lipase-catalyzed simultaneous biosynthesis of biodiesel and glycerol carbonate from corn oil in dimethyl carbonate Min JY, Lee EY Ref: Biotechnol Lett, 33:1789, 2011 : PubMed ESTHER: Min_2011_Biotechnol.Lett_33_1789 PubMedSearch: Min 2011 Biotechnol.Lett 33 1789 PubMedID: 21516311 Gene_locus related to this paper: canar-LipB Abstract Biodiesel [fatty acid methyl esters (FAMEs)] and glycerol carbonate were synthesized from corn oil and dimethyl carbonate (DMC) via transesterification using lipase (Novozyme 435) in solvent-free reaction in which excess DMC was used as the substrate and reaction medium. Glycerol carbonate was also simultaneously formed from DMC and glycerol. Conversions of FAMEs and glycerol carbonate were examined in batch reactions. The FAMEs and glycerol carbonate reached 94 and 62.5% from oil and DMC (molar ratio of 1:10) with 0.2% (v/v) water and 10% (w/w) Novozyme 435 (based on oil weight) at 60 degrees C. When Novozyme 435 was washed with acetone after each reaction, more than 80% activity still remained after seven recycling. Title: Enzymatic coproduction of biodiesel and glycerol carbonate from soybean oil and dimethyl carbonate Seong PJ, Jeon BW, Lee M, Cho DH, Kim DK, Jung KS, Kim SW, Han SO, Kim YH, Park C Ref: Enzyme Microb Technol, 48:505, 2011 : PubMed ESTHER: Seong_2011_Enzyme.Microb.Technol_48_505 PubMedSearch: Seong 2011 Enzyme.Microb.Technol 48 505 PubMedID: 22113023 Gene_locus related to this paper: canar-LipB Abstract The enzymatic coproduction of biodiesel and glycerol carbonate by the transesterification of soybean oil was studied using lipase as catalyst in organic solvent. To produce biodiesel and glycerol carbonate simultaneously, experiments were designed sequentially. Enzyme screening, the molar ratio of dimethyl carbonate (DMC) to soybean oil, reaction temperature and solvent effects were investigated. The results of enzyme screening, at 100 g/L Novozym 435 (immobilized Candida antarctica lipase B), biodiesel and glycerol carbonate showed conversions of 58.7% and 50.7%, respectively. The optimal conditions were 60 degreeC, 100 g/L Novozym 435, 6.0:1 molar ratio with tert-butanol as solvent: 84.9% biodiesel and 92.0% glycerol carbonate production was achieved. Title: Covalent anchoring of a racemization catalyst to CALB-beads: towards dual immobilization of DKR catalysts Wieczorek B, Traff A, Krumlinde P, Dijkstra HP, Egmond MR, van Koten G, Backvall JE, Klein Gebbink RJ Ref: Tetrahedron Letters, 52:1601, 2011 : PubMed ESTHER: Wieczorek_2011_Tetrahedron.Lett_52_1601 PubMedSearch: Wieczorek 2011 Tetrahedron.Lett 52 1601 Gene_locus related to this paper: canar-LipB, fusso-cutas Inhibitor(s) related to this paper: ArM-Rhubidium-cofactor-8 Abstract The preparation of a heterogeneous bifunctional catalytic system, combining the catalytic properties of an organometallic catalyst (racemization) with those of an enzyme (enantioselective acylation) is described. A novel ruthenium phosphonate inhibitor was synthesized and covalently anchored to a lipase immobilized on a solid support (CALB, Novozym 435). The immobilized bifunctional catalytic system showed activity in both racemization of (S)-1-phenylethanol and selective acylation of 1-phenylethanol. Title: Biosynthesis of glycerol carbonate from glycerol by lipase in dimethyl carbonate as the solvent Lee KH, Park CH, Lee EY Ref: Bioprocess Biosyst Eng, 33:1059, 2010 : PubMed ESTHER: Lee_2010_Bioprocess.Biosyst.Eng_33_1059 PubMedSearch: Lee 2010 Bioprocess.Biosyst.Eng 33 1059 PubMedID: 20502921 Gene_locus related to this paper: canar-LipB Abstract Glycerol carbonate was synthesized from renewable glycerol and dimethyl carbonate using lipase in solvent-free reaction system in which excess dimethyl carbonate played as the reaction medium. A variety of lipases have been tested for their abilities to catalyze transesterification reaction, and Candida antartica lipase B and Novozyme 435 exhibited higher catalytic activities. The silica-coated glycerol with a 1:1 ratio was supplied to prevent two-phase formation between hydrophobic dimethyl carbonate and hydrophilic glycerol. Glycerol carbonate was successfully synthesized with more than 90% conversion from dimethyl carbonate and glycerol with a molar ratio of 10 using Novozyme 435-catalyzed transesterification at 70 degrees C. The Novozyme 435 [5% (w/w) and 20% (w/w)] and silica gel were more than four times recycled with good stability in a repeated batch operation for the solvent-free synthesis of glycerol carbonate. Title: Lipase-catalyzed esterification of (S)-naproxen ethyl ester in supercritical carbon dioxide Kwon CH, Lee JH, Kim SW, Kang JW Ref: J Microbiol Biotechnol, 19:1596, 2009 : PubMed ESTHER: Kwon_2009_J.Microbiol.Biotechnol_19_1596 PubMedSearch: Kwon 2009 J.Microbiol.Biotechnol 19 1596 PubMedID: 20075625 Gene_locus related to this paper: canar-LipB Abstract A lipase-catalyzed esterification reaction of (S)-naproxen ethyl ester by CALB (Candida antarctica lipase B) enzyme was performed in supercritical carbon dioxide. Experiments were performed in a high-pressure cell for 10 h at a stirring rate of 150 rpm over a temperature range of 313.15 to 333.15 K and a pressure range of 50 to 175 bar. The productivity of (S)-naproxen ethyl ester was compared with the result in ambient condition. The total reaction time and conversion yields of the catalyzed reaction in supercritical carbon dioxide were compared with those at ambient temperature and pressure. The experimental results show that the conversion and reaction rate were significantly improved at critical condition. The maximum conversion yield was 9.9% (216 h) at ambient condition and 68.9% (3 h) in supercritical state. The effects of varying amounts of enzyme and water were also examined and the optimum condition was found (7 g of enzyme and 2% water content). Title: Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation Qian Z, Horton JR, Cheng X, Lutz S Ref: Journal of Molecular Biology, 393:191, 2009 : PubMed ESTHER: Qian_2009_J.Mol.Biol_393_191 PubMedSearch: Qian 2009 J.Mol.Biol 393 191 PubMedID: 19683009 Gene_locus related to this paper: canar-LipB Inhibitor(s) related to this paper: 4-methylumbelliferyl-hexylphosphonate Abstract Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants' temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283 Delta 7) in the apo-form determined at 1.49 A resolution and with a bound phosphonate inhibitor at 1.69 A resolution confirmed the formation of a homodimer by swapping of the enzyme's 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover. Title: Understanding the plasticity of the alpha/beta hydrolase fold: lid swapping on the Candida antarctica lipase B results in chimeras with interesting biocatalytic properties Skjot M, De Maria L, Chatterjee R, Svendsen A, Patkar SA, Ostergaard PR, Brask J Ref: Chembiochem, 10:520, 2009 : PubMed ESTHER: Skjot_2009_Chembiochem_10_520 PubMedSearch: Skjot 2009 Chembiochem 10 520 PubMedID: 19156649 Gene_locus related to this paper: canar-LipB Abstract The best of both worlds. Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix alpha5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation. The Candida antarctica lipase B (CALB) has found very extensive use in biocatalysis reactions. Long molecular dynamics simulations of CALB in explicit aqueous solvent confirmed the high mobility of the regions lining the channel that leads into the active site, in particular, of helices alpha5 and alpha10. The simulation also confirmed the function of helix alpha5 as a lid of the lipase. Replacing it with corresponding lid regions from the CALB homologues from Neurospora crassa and Gibberella zeae resulted in two new CALB mutants. Characterization of these revealed several interesting properties, including increased hydrolytic activity on simple esters, specifically substrates with C(alpha) branching on the carboxylic side, and much increased enantioselectivity in hydrolysis of racemic ethyl 2-phenylpropanoate (E>50), which is a common structure of the profen drug family. Title: Systematic comparison of HEA and HEMA as initiators in enzymatic ring-opening polymerizations Xiao Y, Takwa M, Hult K, Koning CE, Heise A, Martinelle M Ref: Macromol Biosci, 9:713, 2009 : PubMed ESTHER: Xiao_2009_Macromol.Biosci_9_713 PubMedSearch: Xiao 2009 Macromol.Biosci 9 713 PubMedID: 19242919 Gene_locus related to this paper: canar-LipB Abstract Two initiators containing a cleavable ester bond were compared in the lipase-catalyzed ROP of CL and PDL. The results show that transesterification reactions are present at high rates throughout the enzymatic ROP and start at low conversion. HEA and HEMA displayed similar reaction efficiencies as initiators (acyl acceptors) in the enzymatic ROP. However, transacylation reactions on the HEA-initiated polyesters were found to be 15 times faster. While in both cases the amount of HEA- and HEMA-initiated polymers could be maximized by short reaction times, a well-defined (meth)acrylation by this approach was not possible. Our results show that transesterification reactions have to be considered when performing an enzyme-catalyzed ROP. Title: Candida antarctica lipase B chemically immobilized on epoxy-activated micro- and nanobeads: catalysts for polyester synthesis Chen B, Hu J, Miller EM, Xie W, Cai M, Gross RA Ref: Biomacromolecules, 9:463, 2008 : PubMed ESTHER: Chen_2008_Biomacromolecules_9_463 PubMedSearch: Chen 2008 Biomacromolecules 9 463 PubMedID: 18197630 Gene_locus related to this paper: canar-LipB Abstract Candida antarctica Lipase B (CALB) was covalently immobilized onto epoxy-activated macroporous poly(methyl methacrylate) Amberzyme beads (235 microm particle size, 220 A pore size) and nanoparticles (nanoPSG, diameter 68 nm) with a poly(glycidyl methacrylate) outer region. Amberzyme beads allowed CALB loading up to 0.16 g of enzyme per gram of support. IR microspectroscopy generated images of Amberzyme-CALB beads showed CALB is localized within a 50 microm thick loading front. IR microspectroscopy images, recorded prior to and after treatment of Amberzyme-CALB with DMSO/aqueous Triton X-100, are similar, confirming that CALB is largely chemically linked to Amberzyme. The activity of CALB immobilized on Amberzyme, Lewatit (i.e., Novozym 435 catalyst), and nanoPSG was assessed for lactone ring-opening and step-condensation polymerizations. For example, the percent conversion of -caprolactone using the same amount of enzyme catalyzed by Amberzym-CALB, Novozym 435, and nanoPSG-CALB for 20 min was 7.0, 16, and 65%, respectively. Differences in CALB reactivity were discussed based on resin physical parameters and availability of active sites determined by active site titrations. Regardless of the matrix used and chemical versus physical immobilization, -CL ring-opening polymerizations occur by a chain growth mechanism without chain termination. To test Amberzyme-CALB stability, the catalyst was reused over three reaction cycles for -CL ring-opening polymerization (70 degrees C, 70 min reactions) and glycerol/1,8-octanediol/adipic acid polycondensation reactions (90 degrees C, 64 h). Amberzyme-CALB was found to have far better stability for reuse relative to Novozym 435 for the polycondensation reaction. Title: Understanding promiscuous amidase activity of an esterase from Bacillus subtilis Kourist R, Bartsch S, Fransson L, Hult K, Bornscheuer UT Ref: Chembiochem, 9:67, 2008 : PubMed ESTHER: Kourist_2008_Chembiochem_9_67 PubMedSearch: Kourist 2008 Chembiochem 9 67 PubMedID: 18022973 Gene_locus related to this paper: bacsu-pnbae, canar-LipB Title: A genetic selection system for evolving enantioselectivity of enzymes Reetz MT, Hobenreich H, Soni P, Fernandez L Ref: Chem Commun (Camb), :5502, 2008 : PubMed ESTHER: Reetz_2008_Chem.Commun.(Camb)__5502 PubMedSearch: Reetz 2008 Chem.Commun.(Camb) 5502 PubMedID: 18997932 Gene_locus related to this paper: canar-LipB Abstract As an alternative to screening in the directed evolution of enantioselective enzymes, a selection system has been implemented for a lipase-catalyzed hydrolytic kinetic resolution of a chiral ester. Title: Lipase catalyzed HEMA initiated ring-opening polymerization: in situ formation of mixed polyester methacrylates by transesterification Takwa M, Xiao Y, Simpson N, Malmstrom E, Hult K, Koning CE, Heise A, Martinelle M Ref: Biomacromolecules, 9:704, 2008 : PubMed ESTHER: Takwa_2008_Biomacromolecules_9_704 PubMedSearch: Takwa 2008 Biomacromolecules 9 704 PubMedID: 18198845 Gene_locus related to this paper: canar-LipB Abstract 2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the enzymatic ring-opening polymerization (ROP) of omega-pentadecalactone (PDL) and epsilon-caprolactone (CL). The lipase B from Candida antarctica was found to catalyze the cleavage of the ester bond in the HEMA end group of the formed polyesters, resulting in two major transesterification processes, methacrylate transfer and polyester transfer. This resulted in a number of different polyester methacrylate structures, such as polymers without, with one, and with two methacrylate end groups. Furthermore, the 1,2-ethanediol moiety (from HEMA) was found in the polyester products as an integral part of HEMA, as an end group (with one hydroxyl group) and incorporated within the polyester (polyester chains acylated on both hydroxyl groups). After 72 h, as a result of the methacrylate transfer, 79% (48%) of the initial amount of the methacrylate moiety (from HEMA) was situated (acylated) on the end hydroxyl group of the PPDL (PCL) polyester. In order to prepare materials for polymer networks, fully dimethacrylated polymers were synthesized in a one-pot procedure by combining HEMA-initiated ROP with end-capping using vinyl methacrylate. The novel PPDL dimethacrylate (>95% incorporated methacrylate end groups) is currently in use for polymer network formation. Our results show that initiators with cleavable ester groups are of limited use to obtain well-defined monomethacrylated macromonomers due to the enzyme-based transesterification processes. On the other hand, when combined with end-capping, well-defined dimethacrylated polymers (PPDL, PCL) were prepared. Title: Biocatalytic ammonolysis of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester: preparation of an intermediate to the dipeptidyl peptidase IV inhibitor Saxagliptin Gill I, Patel R Ref: Bioorganic & Medicinal Chemistry Lett, 16:705, 2006 : PubMed ESTHER: Gill_2006_Bioorg.Med.Chem.Lett_16_705 PubMedSearch: Gill 2006 Bioorg.Med.Chem.Lett 16 705 PubMedID: 16257208 Gene_locus related to this paper: canar-LipB Inhibitor(s) related to this paper: Saxagliptin Abstract An efficient biocatalytic method has been developed for the conversion of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester (1) into the corresponding amide (5S)-5-aminocarbonyl-4,5-dihydro-1H-pyrrole-1-carboxylic acid, 1-(1,1-dimethylethyl)ester (2), which is a critical intermediate in the synthesis of the dipeptidyl peptidase IV (DPP4) inhibitor Saxagliptin (3). Candida antartica lipase B mediates ammonolysis of the ester with ammonium carbamate as ammonia donor to yield up to 71% of the amide. The inclusion of Ascarite and calcium chloride as adsorbents for carbon dioxide and ethanol byproducts, respectively, increases the yield to 98%, thereby offering an efficient and practical alternative to chemical routes which yield 57-64%. Title: Lipase-mediated transformation of vegetable oils into biodiesel using propan-2-ol as acyl acceptor Modi MK, Reddy JR, Rao BV, Prasad RB Ref: Biotechnol Lett, 28:637, 2006 : PubMed ESTHER: Modi_2006_Biotechnol.Lett_28_637 PubMedSearch: Modi 2006 Biotechnol.Lett 28 637 PubMedID: 16642301 Gene_locus related to this paper: canar-LipB Abstract Propan-2-ol was used as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel. The optimum conditions for transesterification of crude jatropha (Jatropha curcas), karanj (Pongamia pinnata) and sunflower (Helianthus annuus) oils were 10% Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, alcohol to oil molar ratio of 4:1 at 50 degrees C for 8 h. The maximum conversions achieved using propan-2-ol were 92.8, 91.7 and 93.4% from crude jatropha, karanj and sunflower oils, respectively. Reusability of the lipase was maintained over 12 repeated cycles with propan-2-ol while it reached to zero by 7(th) cycle when methanol was used as an acyl acceptor, under standard reaction conditions. Title: Cocrystallization of random copolymers of omega-pentadecalactone and epsilon-caprolactone synthesized by lipase catalysis Ceccorulli G, Scandola M, Kumar A, Kalra B, Gross RA Ref: Biomacromolecules, 6:902, 2005 : PubMed ESTHER: Ceccorulli_2005_Biomacromolecules_6_902 PubMedSearch: Ceccorulli 2005 Biomacromolecules 6 902 PubMedID: 15762658 Gene_locus related to this paper: canar-LipB Abstract Random copolymers were prepared by Candida antarctica lipase B (Novozyme-435) catalyzed copolymerization of omega-pentadecalactone (PDL) with epsilon-caprolactone (CL). Over the whole composition range PDL-CL copolymers are highly crystalline (melting enthalpy by differential scanning calorimetry, above 100 J/g; crystallinity degree by wide-angle X-ray scattering, WAXS, 60-70%). The copolymers melt at temperatures that linearly decrease with composition from that of poly(omega-pentadecalactone) (PPDL; 97 degrees C) to that of poly(epsilon-caprolactone) (PCL; 59 degrees C). The WAXS profiles of PCL and PPDL homopolymers are very similar, except for the presence in PPDL of the (001) reflection at 2theta = 4.58 degrees that corresponds to a 19.3 angstroms periodicity in the chain direction. In PDL-CL copolymers the intensity of this reflection decreases with increasing content of CL units and vanishes at 50 mol % CL, as a result of randomization of the ester group alignment and loss of chain periodicity. PDL-CL copolymers crystallize in a lattice that gradually changes from that of one homopolymer to that of the other, owing to comonomer isomorphous substitution. Cocrystallization of comonomer units is also shown by a random PDL-CL copolymer obtained in a polymerization/transesterification reaction catalyzed by C. antarctica lipase B (Novozyme-435) starting from preformed PCL and PDL monomer. Title: Understanding structure-stability relationships of Candida antartica lipase B in ionic liquids De Diego T, Lozano P, Gmouh S, Vaultier M, Iborra JL Ref: Biomacromolecules, 6:1457, 2005 : PubMed ESTHER: De Diego_2005_Biomacromolecules_6_1457 PubMedSearch: De Diego 2005 Biomacromolecules 6 1457 PubMedID: 15877365 Gene_locus related to this paper: canar-LipB Abstract Two different water-immiscible ionic liquids (ILs), 1-ethyl-3-methylimidizolium bis(trifluoromethylsulfonyl)imide and butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, were used for butyl butyrate synthesis from vinyl butyrate catalyzed by Candida antarctica lipase B (CALB) at 2% (v/v) water content and 50 degrees C. Both the synthetic activity and stability of the enzyme in these ILs were enhanced as compared to those in hexane. Circular dichroism and intrinsic fluorescence spectroscopic techniques have been used over a period of 4 days to determine structural changes in the enzyme associated with differences in its stability for each assayed medium. CALB showed a loss in residual activity higher than 75% after 4 days of incubation in both water and hexane media at 50 degrees C, being related to great changes in both alpha-helix and beta-strand secondary structures. The stabilization of CALB, which was observed in the two ILs studied, was associated with both the maintenance of the 50% of initial alpha-helix content and the enhancement of beta-strands. Furthermore, intrinsic fluorescence studies clearly showed how a classical enzyme unfolding was occurring with time in both water and hexane media. However, the structural changes associated with the incubation of the enzyme in both ILs might be attributed to a compact and active enzyme conformation, resulting in an enhancement of the stability in these nonaqueous environments. Title: An inverse substrate orientation for the regioselective acylation of 3',5'-diaminonucleosides catalyzed by Candida antarctica lipase B? Lavandera I, Fernandez S, Magdalena J, Ferrero M, Kazlauskas RJ, Gotor V Ref: Chembiochem, 6:1381, 2005 : PubMed ESTHER: Lavandera_2005_Chembiochem_6_1381 PubMedSearch: Lavandera 2005 Chembiochem 6 1381 PubMedID: 15977272 Gene_locus related to this paper: canar-LipB Abstract Candida antarctica lipase B (CAL-B) catalyzes the regioselective acylation of natural thymidine with oxime esters and also the regioselective acylation of an analogue, 3',5'-diamino-3',5'-dideoxythymidine with nonactivated esters. In both cases, acylation favors the less hindered 5'-position over the 3'-position by upto 80-fold. Computer modeling of phosphonate transition-state analogues for the acylation of thymidine suggests that CAL-B favors acylation of the 5'-position because this orientation allows the thymine ring to bind in a hydrophobic pocket and forms stronger key hydrogen bonds than acylation of the 3'-position. On the other hand, computer modeling of phosphonamidate analogues of the transition states for acylation of either the 3'- or 5'-amino groups in 3',5'-diamino-3',5'-dideoxythymidine shows similar orientations and hydrogen bonds and, thus, does not explain the high regioselectivity. However, computer modeling of inverse structures, in which the acyl chain binds in the nucleophile pocket and vice versa, does rationalize the observed regioselectivity. The inverse structures fit the 5'-, but not the 3'-intermediate thymine ring, into the hydrophobic pocket, and form a weak new hydrogen bond between the O-2 carbonyl atom of the thymine and the nucleophile amine only for the 5'-intermediate. A water molecule might transfer a proton from the ammonium group to the active-site histidine. As a test of this inverse orientation, we compared the acylation of thymidine and 3',5'-diamino-3',5'-dideoxythymidine with butyryl acyl donors and with isosteric methoxyacetyl acyl donors. Both acyl donors reacted at equal rates with thymidine, but the methoxyacetyl acyl donor reacted four times faster than the butyryl acyl donor with 3',5'-diamino-3',5'-dideoxythymidine. This faster rate is consistent with an inverse orientation for 3',5'-diamino-3',5'-dideoxythymidine, in which the ether oxygen atom of the methoxyacetyl group can form a similar hydrogen bond to the nucleophilic amine. This combination of modeling and experiments suggests that such lipase-catalyzed reactions of apparently close substrate analogues like alcohols and amines might follow different pathways. Title: Creating space for large secondary alcohols by rational redesign of Candida antarctica lipase B Magnusson AO, Rotticci-Mulder JC, Santagostino A, Hult K Ref: Chembiochem, 6:1051, 2005 : PubMed ESTHER: Magnusson_2005_Chembiochem_6_1051 PubMedSearch: Magnusson 2005 Chembiochem 6 1051 PubMedID: 15883973 Gene_locus related to this paper: canar-LipB Abstract The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser-His-Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast-reacting enantiomer of secondary alcohols places its medium-sized substituent in the stereospecificity pocket and its large substituent towards the active-site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild-type lipase increased 270 times towards heptan-4-ol and 5500 times towards nonan-5-ol; this resulted in the high specificity constants 1100 and 830 s(-1) M(-1), respectively. The substrate selectivity changed over 400,000 times for nonan-5-ol over propan-2-ol with both Trp104Ala and the Trp104Gln mutations. Title: Improving the catalytic activity of Candida antarctica lipase B by circular permutation Qian Z, Lutz S Ref: Journal of the American Chemical Society, 127:13466, 2005 : PubMed ESTHER: Qian_2005_J.Am.Chem.Soc_127_13466 PubMedSearch: Qian 2005 J.Am.Chem.Soc 127 13466 PubMedID: 16190688 Gene_locus related to this paper: canar-LipB Abstract Lipases (EC 3.1.1.3) play an important role in asymmetric biocatalysis. Tailoring these enzymes to novel, unnatural substrates is one of the primary challenges of protein engineering. We have used circular permutation, the intramolecular relocation of a protein's N- and C-termini, to explore the effects of altered active site accessibility and protein backbone flexibility on the catalytic performance of lipase B from Candida antarctica (CALB). Our combinatorial approach identified 63 unique functional protein permutants of CALB, and kinetic analysis of selected candidates indicated that a majority of enzyme variants either retained or surpassed wild-type CALB activity on a series of standard substrates. Beyond the potential benefits of these tailor-made lipases as new catalysts for unnatural substrates, our study validates circular permutation as a promising general method for lipase engineering. Title: Synthesis of flavor and fragrance esters using Candida antarctica lipase Larios A, Garcia HS, Oliart RM, Valerio-Alfaro G Ref: Applied Microbiology & Biotechnology, 65:373, 2004 : PubMed ESTHER: Larios_2004_Appl.Microbiol.Biotechnol_65_373 PubMedSearch: Larios 2004 Appl.Microbiol.Biotechnol 65 373 PubMedID: 15248036 Gene_locus related to this paper: canar-LipB Abstract Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral alpha-substituted or beta-substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids. Title: Synthesis of novel D-glucuronic acid fatty esters using Candida antarctica lipase in tert-butanol Moreau B, Lognay GC, Blecker C, Brohee JC, Chery F, Rollin P, Paquot M, Marlier M Ref: Biotechnol Lett, 26:419, 2004 : PubMed ESTHER: Moreau_2004_Biotechnol.Lett_26_419 PubMedSearch: Moreau 2004 Biotechnol.Lett 26 419 PubMedID: 15104141 Gene_locus related to this paper: canar-LipB Abstract Glucuronic acid n-alkyl esters, a novel class of promising biosurfactants and their corresponding glucose esters with the same side-chain length, were synthesized by direct esterification in a non-aqueous phase (tert-butanol) using an immobilized lipase. Title: Kinetic resolution of rac-2-pentanol catalyzed by Candida antarctica lipase B in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide Noel M, Lozano P, Vaultier M, Iborra JL Ref: Biotechnol Lett, 26:301, 2004 : PubMed ESTHER: Noel_2004_Biotechnol.Lett_26_301 PubMedSearch: Noel 2004 Biotechnol.Lett 26 301 PubMedID: 15055765 Gene_locus related to this paper: canar-LipB Abstract The ionic liquid, l-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide ([Bmim] [NTf2]), was used as a reaction medium for the kinetic resolution of rac-2-pentanol catalyzed by free Candida antarctica lipase B, using vinyl propionate at 2% (v/v) water content. The synthetic activity of lipase in [Bmim] [NTf2] was up 2.5-times greater than in hexane, and showed high enantioselectivity (ee > 99.99%). The optimal temperature and pH were 60 degrees C and 7, respectively. A decrease in water activity (aw) produced a decay in synthetic activity, and an exponential increase in selectivity. Title: Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure Passicos E, Santarelli X, Coulon D Ref: Biotechnol Lett, 26:1073, 2004 : PubMed ESTHER: Passicos_2004_Biotechnol.Lett_26_1073 PubMedSearch: Passicos 2004 Biotechnol.Lett 26 1073 PubMedID: 15218382 Gene_locus related to this paper: canar-LipB Abstract A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated. Title: Stability improvement of immobilized Candida antarctica lipase B in an organic medium under microwave radiation Rejasse B, Lamare S, Legoy MD, Besson T Ref: Org Biomol Chem, 2:1086, 2004 : PubMed ESTHER: Rejasse_2004_Org.Biomol.Chem_2_1086 PubMedSearch: Rejasse 2004 Org.Biomol.Chem 2 1086 PubMedID: 15034633 Gene_locus related to this paper: canar-LipB Abstract The influence of microwave heating on the stability of immobilized Candida antarctica lipase B was studied at 100 degrees in an organic medium. The microwave radiation was carried out before enzymatic reaction (storage conditions) or during the enzymatic catalysis (use conditions). In both cases, enzymatic stability was higher under microwave heating than under conventional thermal heating, in strictly identical operating conditions. Furthermore, the gain of enzymatic stability under microwave heating appears to be higher in a more polar solvent, which interacts strongly with the microwave field. Our results suggest that microwave radiation has an effect, not related to temperature, on the process of enzymatic inactivation. Title: Lipase catalysed resolution of the Lotrafiban intermediate 2,3,4,5-tetrahydro-4-methyl-3-oxo-1 H-1,4-benzodiazepine-2-acetic acid methyl ester in ionic liquids: comparison to the industrial t-butanol process Roberts NJ, Seago A, Carey JS, Freer R, Preston C, Lye GJ Ref: Green Chem, 6:475 , 2004 : PubMed ESTHER: Roberts_2004_Green.Chem_6_475 PubMedSearch: Roberts 2004 Green.Chem 6 475 Gene_locus related to this paper: canar-LipB Inhibitor(s) related to this paper: W22 Abstract The Candida antarctica lipase B (Novozyme 435) catalysed resolution of2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid methyl ester (SB-235349), a key Lotrafiban intermediate, has been investigated in six ionic liquids including [BMIM][PF6] and [BMIM][N(SO2CF3)2]. The initial rate and final yield of the reaction have subsequently been determined in [BMIM][PF6] as a function of initial substrateconcentration (5-40 g L-1), temperature (25-100 oC) and initial water content (3-15% H2O w/w). In each case the results have been compared to those obtained for the optimised industrial process operated in t-butanol (88% v/v). Simply replacing the organic solvent with an ionic liquid under otherwise identical reaction conditions reduced the rate of conversion. However, exploiting the increased solubility of the substrate in ionic liquids and the ability to operate at higher temperatures increased the overall rate of reaction by a factor of four while maintaining the same overall yield of 47%. In each case the ee of the product was 99%. Further experiments demonstrated the ability to re-use the enzyme over 10 reaction cycles and suggested that solute mass transfer in ionic liquids might be an issue for reactions carried out at larger scale. Overall the results suggest that ionic liquids can be very favourable reaction media for industrial bioconversion processes, which also overcome many of the safety and environmental concerns of conventional organic solvents. Title: Regeneration of immobilized Candida antarctica lipase for transesterification Chen JW, Wu WT Ref: J Biosci Bioeng, 95:466, 2003 : PubMed ESTHER: Chen_2003_J.Biosci.Bioeng_95_466 PubMedSearch: Chen 2003 J.Biosci.Bioeng 95 466 PubMedID: 16233441 Gene_locus related to this paper: canar-LipB Abstract Immobilized lipase from Candida antarctica was employed to convert triglycerides to biodiesel using alcohol. Immobilized lipase is frequently deactivated by lower alcohols with deactivation being caused by the immiscibility between triglycerides and methanol or ethanol. When the lower alcohol is adsorbed to the immobilized enzyme, the entry of triglycerides is blocked, which causes the reaction to stop. An alcohol with three or more carbon atoms, preferably 2-butanol or tert-butanol, can regenerate the deactivated immobilized enzyme. The present work established that the activity of immobilized lipase could be significantly increased when such alcohols were used for an immersion pretreatment of the enzyme. The activity of the commercially available immobilized enzyme, Novozyme 435, increased about tenfold in comparison to the enzyme not subjected to any pretreatment. Following complete deactivation of the enzyme by methanol, washing with 2-butanol and tert-butanol successfully regenerated the enzyme and restored it to about 56% and 75% of its original activity level, respectively. Title: Fluorophoric assay for the high-throughput determination of amidase activity Henke E, Bornscheuer UT Ref: Analytical Chemistry, 75:255, 2003 : PubMed ESTHER: Henke_2003_Anal.Chem_75_255 PubMedSearch: Henke 2003 Anal.Chem 75 255 PubMedID: 12553759 Gene_locus related to this paper: canar-LipB Abstract An assay has been developed for the high-throughput identification of amidase activity. Amines released from the enzyme-catalyzed hydrolysis of corresponding amides were detected by the formation of a fluorescent dye by coupling with 4-nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-CI). Using this format, 22 lipases and esterases were tested for their ability to hydrolyze aromatic substituted N-acylamines in a microtiter plate format. Identified active enzymes were further characterized toward a broad range of compounds to determine the influence of substrate structure on activity. For recombinantly produced esterases, it could be shown that the assay works with high reproducibility and sensitivity, even in the presence of amino acids and proteins present in culture media and cell debris. Title: Water activity effects on geranyl acetate synthesis catalyzed by novozym in supercritical ethane and in supercritical carbon dioxide Peres C, Gomes da Silva MD, Barreiros S Ref: Journal of Agricultural and Food Chemistry, 51:1884, 2003 : PubMed ESTHER: Peres_2003_J.Agric.Food.Chem_51_1884 PubMedSearch: Peres 2003 J.Agric.Food.Chem 51 1884 PubMedID: 12643646 Gene_locus related to this paper: canar-LipB Abstract The esterification reaction of geraniol with acetic acid catalyzed by Novozym was studied in supercritical ethane (sc-ethane) and in supercritical carbon dioxide (sc-CO(2)). Water activity (a(W)) had a very strong effect on enzyme activity, with reaction rates increasing up to a(W) = 0.25 and then decreasing for higher a(W). Salt hydrate pairs could not prevent changes in a(W) during the course of reaction but were able to control a(W) to some extent and had a beneficial effect on both initial rates of esterification and conversion in sc-ethane. The enzyme was more active in sc-ethane than in sc-CO(2), confirming the deleterious effect of the latter already observed with some enzymes. Temperatures between 40 and 60 degrees C did not have a strong effect on initial rates of esterification, although reaction progress declined considerably in that temperature range. For the mixture of 50 mM acetic acid plus 200 mM geraniol, 100% conversion was achieved at a reaction time of 10 h at 40 degrees C, 100 bar, an a(W) of incubation of 0.25, and a Novozym concentration of 0.55 mg cm(-)(3) in sc-ethane. Conversion was below 50% in sc-CO(2) at otherwise identical conditions. With an equimolar mixture of the two substrates (100 mM), 98% conversion was reached at 10 h of reaction in sc-ethane (73% conversion in sc-CO(2)). Title: Regiospecific analysis by ethanolysis of oil with immobilized Candida antarctica lipase Shimada Y, Ogawa J, Watanabe Y, Nagao T, Kawashima A, Kobayashi T, Shimizu S Ref: Lipids, 38:1281, 2003 : PubMed ESTHER: Shimada_2003_Lipids_38_1281 PubMedSearch: Shimada 2003 Lipids 38 1281 PubMedID: 14870932 Gene_locus related to this paper: canar-LipB Abstract A mixture of oil/ethanol (1:3, w/w) was shaken at 30 degrees C with 4% immobilized Candida antarctica lipase by weight of the reaction mixture. The reaction regiospecifically converted FA at the 1- and 3-positions to FA ethyl esters, and the lipase acted on C14-C24 FA to a similar degree. The content of 2-MAG reached a maximum after 4 h; the content was 28-29 mol% based on the total amount of FA in the reaction mixture at 59-69% ethanolysis. Only 2-MAG were present in the reaction mixture during the first 4 h, and 1(3)-MAG were detected after 7 h. After removal of ethanol from the 4-h reaction mixture by evaporation, 2-MAG were fractionated by silica gel column chromatography. The contents of FA in the 2-MAG obtained by ethanolysis of several oils coincided well with FA compositions at the 2-position, which was analyzed by Grignard degradation. It was shown that ethanolysis of oil with C. antarctica lipase can be applied to analysis of FA composition at the 2-position in TAG. Title: Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution Zhang N, Suen WC, Windsor W, Xiao L, Madison V, Zaks A Ref: Protein Engineering, 16:599, 2003 : PubMed ESTHER: Zhang_2003_Protein.Eng_16_599 PubMedSearch: Zhang 2003 Protein.Eng 16 599 PubMedID: 12968077 Gene_locus related to this paper: canar-LipB Abstract To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20-fold increase in half-life at 70 degrees C compared with the wild-type CALB (WT-CALB). The increase in half-life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error-prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error-prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyl octanoate was also found to be superior to that of WT-CALB. Title: Ethyl esterification of docosahexaenoic acid in an organic solvent-free system with immobilized Candida antarctica lipase Shimada Y, Watanabe Y, Sugihara A, Baba T, Ooguri T, Moriyama S, Terai T, Tominaga Y Ref: J Biosci Bioeng, 92:19, 2001 : PubMed ESTHER: Shimada_2001_J.Biosci.Bioeng_92_19 PubMedSearch: Shimada 2001 J.Biosci.Bioeng 92 19 PubMedID: 16233051 Gene_locus related to this paper: canar-LipB Abstract Ethyl docosahexaenoate (EtDHA) is regarded as a potentially useful pharmaceutical substance on account of its beneficial physiological activities. We attempted the ethyl esterification of docosahexaenoic acid (DHA) in an organic solvent-free system using Candida antarctica lipase, which acts strongly on DHA and ethanol. Esterification of 88% was attained by shaking a mixture of DHA/ethanol (1:1, mol/mol) and 2 wt% immobilized C. antarctica lipase at 30 degrees C for 24 h. However, even in the presence of an excess amount of ethanol, the extent of esterification could not be raised above 90%. To attain a higher level of esterification, a two-step reaction was found to be effective. The first step was performed in a mixture of DHA/ethanol (1:1, mol/mol), and the reaction mixture was then dehydrated. In the second step, the resulting mixture was shaken at 30 degrees C for 24 h with 5 molar equivalents of ethanol against the remaining DHA using 2 wt% immobilized lipase. By means of this two-step procedure, 96% esterification was attained. Repetition of the first and second reactions showed that the immobilized lipase was reusable for at least 50 cycles. In addition, DHA remaining in the second-step reaction mixture was removed by a conventional alkali refining process, giving purified EtDHA with a high yield. Title: Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil Samukawa T, Kaieda M, Matsumoto T, Ban K, Kondo A, Shimada Y, Noda H, Fukuda H Ref: J Biosci Bioeng, 90:180, 2000 : PubMed ESTHER: Samukawa_2000_J.Biosci.Bioeng_90_180 PubMedSearch: Samukawa 2000 J.Biosci.Bioeng 90 180 PubMedID: 16232839 Gene_locus related to this paper: canar-LipB Abstract The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated. Methanolysis progressed much faster when Novozym 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. The initial reaction rate of methanolysis catalyzed by both the non-treated and preincubated enzyme decreased significantly with increasing water content. The initial reaction rate increased with increasing methanol content, showed a maximum, and thereafter decreased when the methanol content was increased further. The variation of the initial reaction rate with the methanol content was therefore analyzed using a Michaelis-Menten-type equation with substrate inhibition. Based on this equation, a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired methanol content was determined. When preincubated Novozym 435 was used, the ME content reached over 97% within 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25-0.4 h intervals. Title: Stepwise ethanolysis of tuna oil using immobilized Candida antarctica lipase Watanabe Y, Shimada Y, Sugihara A, Tominaga Y Ref: J Biosci Bioeng, 88:622, 1999 : PubMed ESTHER: Watanabe_1999_J.Biosci.Bioeng_88_622 PubMedSearch: Watanabe 1999 J.Biosci.Bioeng 88 622 PubMedID: 16232674 Gene_locus related to this paper: canar-LipB Abstract Ethanolysis of fish oil under mild conditions has been strongly desired for preparing the starting materials for the purification of ethyl docosahexaenoate. Thus, we attempted ethanolysis of tuna oil using immobilized Candida antarctica lipase. The immobilized lipase was inactivated in the presence of 2 3 molar equivalent of ethanol against the total fatty acids in tuna oil. To avoid such inactivation, the first step of ethanolysis was conducted at 40 degrees C in a mixture of tuna oil and 1 3 molar equivalent of ethanol using 4% immobilized lipase. After a 10-h reaction, ethanol was consumed and 33% of tuna oil was converted to its corresponding ethyl esters (E-FAs). The reactant is named Gly/E-FA33. The lipase was not inactivated in the presence of 2 3 molar equivalent of ethanol against the total fatty acids in Gly/E-FA33. These findings and the consideration of several factors affecting ethanolysis of tuna oil led to the development of the two- and three-step ethanolyses. The two-step reaction was performed as follows: the first step was carried out at 40 degrees C for 12 h in a mixture of tuna oil and 1 3 molar equivalent of ethanol with 4% immobilized lipase; the second step was performed for 36 h (total reaction period, 48 h) after adding 2 3 molar equivalent of ethanol. On the other hand, the three-step reaction was conducted as follows: the first step was conducted under the same conditions as those in the two-step ethanolysis; in the second and third steps, 1 3 molar equivalent of ethanol was added after 12 and 24 h, respectively; and in the third step, the mixture was shaken for 24 h (total, 48 h). Both types of ethanolyses achieved the conversion of 95% or more of tuna oil to its corresponding E-FAs. To investigate the lipase stability, the two- and three-step ethanolyses were repeated by transferring the enzyme to a fresh substrate mixture of the first step after finishing one cycle of reaction. The two- and three-step reactions maintained over 95% of the conversion for 70 d and over 100 d, respectively. Title: Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols Uppenberg J, Ohrner N, Norin M, Hult K, Kleywegt GJ, Patkar S, Waagen V, Anthonsen T, Jones TA Ref: Biochemistry, 34:16838, 1995 : PubMed ESTHER: Uppenberg_1995_Biochemistry_34_16838 PubMedSearch: Uppenberg 1995 Biochemistry 34 16838 PubMedID: 8527460 Gene_locus related to this paper: canar-LipB Inhibitor(s) related to this paper: N-Hexylphosphonate-Ethyl-Ester Abstract Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols. Title: Crystallization and preliminary X-ray studies of lipase B from Candida antarctica Uppenberg J, Patkar S, Bergfors T, Jones TA Ref: Journal of Molecular Biology, 235:790, 1994 : PubMed ESTHER: Uppenberg_1994_J.Mol.Biol_235_790 PubMedSearch: Uppenberg 1994 J.Mol.Biol 235 790 PubMedID: 8289302 Gene_locus related to this paper: canar-LipB Abstract Lipase B from Candida antarctica has been crystallized in five different crystal forms. The space groups and cell dimensions have been determined by X-ray diffraction methods. Four of the crystal forms have been judged to be of good quality for further X-ray studies. The best crystals diffract to 1.7 Angstrom. Title: The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica Uppenberg J, Hansen MT, Patkar S, Jones TA Ref: Structure, 2:293, 1994 : PubMed ESTHER: Uppenberg_1994_Structure_2_293 PubMedSearch: Uppenberg 1994 Structure 2 293 PubMedID: 8087556 Gene_locus related to this paper: canar-LipB Abstract BACKGROUND: Lipases constitute a family of enzymes that hydrolyze triglycerides. They occur in many organisms and display a wide variety of substrate specificities. In recent years, much progress has been made towards explaining the mechanism of these enzymes and their ability to hydrolyze their substrates at an oil-water interface. Other Papers | ||||||||||||||||||||||||||||||||
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