Gene_locus Report for: bacsp-lip

Lipase biocatalysts offer unique properties which are often impaired by low thermal and methanol stability. In this study, the rational design was employed to engineer a disulfide bond in the protein structure of Geobacillus zalihae T1 lipase in order to improve its stability. The selection of targeted disulfide bond sites was based on analysis of protein spatial configuration and change of Gibbs free energy. Two mutation points (S2C and A384C) were generated to rigidify the N-terminal and C-terminal regions of T1 lipase. The results showed the mutant 2DC lipase improved methanol stability from 35 to 40% (v/v) after 30 min of pre-incubation. Enhancement in thermostability for the mutant 2DC lipase at 70 degreesC and 75 degreesC showed higher half-life at 70 degreesC and 75 degreesC for 30 min and 52 min, respectively. The mutant 2DC lipase maintained the same optimum temperature (70 degreesC) as T1 lipase, while thermally induced unfolding showed the mutant maintained higher rigidity. The kcat/Km values demonstrated a relatively small difference between the T1 lipase (WT) and 2DC lipase (mutant). The kcat/Km (s(-1) mM(-1)) of the T1 and 2DC showed values of 13,043 +/- 224 and 13,047 +/- 312, respectively. X-ray diffraction of 2DC lipase crystal structure with a resolution of 2.04 A revealed that the introduced single disulfide bond did not lower initial structural interactions within the residues. Enhanced methanol and thermal stability are suggested to be strongly related to the newly disulfide bridge formation and the enhanced compactness and rigidity of the mutant structure. KEY POINTS: Protein engineering via rational design revealed relative improved enzymatic performance. The presence of disulfide bond impacts on the rigidity and structural function of proteins. X-ray crystallography reveals structural changes accompanying protein modification.

5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 A with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl-esters.

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 degC and 76.0 degC in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in beta-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.

The regioselectivity characteristic of lipases facilitate a wide range of novel molecule unit constructions and fat modifications. Lipases can be categorized as sn-1,3, sn-2, and random regiospecific. Geobacillus zalihae T1 lipase catalyzes the hydrolysis of the sn-1,3 acylglycerol chain. The T1 lipase structural analysis shows that the oxyanion hole F16 and its lid domain undergo structural rearrangement upon activation. Site-directed mutagenesis was performed by substituting the lid domain residues (F180G and F181S) and the oxyanion hole residue (F16W) in order to study their effects on the structural changes and regioselectivity. The novel lipase mutant 3M switches the regioselectivity from sn-1,3 to only sn-3. The mutant 3M shifts the optimum pH to 10, alters selectivity toward p-nitrophenyl ester selectivity to C14-C18, and maintains a similar catalytic efficiency of 518.4 x 10-6 (s-1/mM). The secondary structure of 3M lipase comprises 15.8% and 26.3% of the alpha-helix and beta-sheet, respectively, with a predicted melting temperature (Tm) value of 67.8 C. The in silico analysis was conducted to reveal the structural changes caused by the F180G/F181S/F16W mutations in blocking the binding of the sn-1 acylglycerol chain and orientating the substrate to bond to the sn-3 acylglycerol, which resulted in switching the T1 lipase regioselectivity.

Lipase biocatalysts offer unique properties which are often impaired by low thermal and methanol stability. In this study, the rational design was employed to engineer a disulfide bond in the protein structure of Geobacillus zalihae T1 lipase in order to improve its stability. The selection of targeted disulfide bond sites was based on analysis of protein spatial configuration and change of Gibbs free energy. Two mutation points (S2C and A384C) were generated to rigidify the N-terminal and C-terminal regions of T1 lipase. The results showed the mutant 2DC lipase improved methanol stability from 35 to 40% (v/v) after 30 min of pre-incubation. Enhancement in thermostability for the mutant 2DC lipase at 70 degreesC and 75 degreesC showed higher half-life at 70 degreesC and 75 degreesC for 30 min and 52 min, respectively. The mutant 2DC lipase maintained the same optimum temperature (70 degreesC) as T1 lipase, while thermally induced unfolding showed the mutant maintained higher rigidity. The kcat/Km values demonstrated a relatively small difference between the T1 lipase (WT) and 2DC lipase (mutant). The kcat/Km (s(-1) mM(-1)) of the T1 and 2DC showed values of 13,043 +/- 224 and 13,047 +/- 312, respectively. X-ray diffraction of 2DC lipase crystal structure with a resolution of 2.04 A revealed that the introduced single disulfide bond did not lower initial structural interactions within the residues. Enhanced methanol and thermal stability are suggested to be strongly related to the newly disulfide bridge formation and the enhanced compactness and rigidity of the mutant structure. KEY POINTS: Protein engineering via rational design revealed relative improved enzymatic performance. The presence of disulfide bond impacts on the rigidity and structural function of proteins. X-ray crystallography reveals structural changes accompanying protein modification.

5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 A with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl-esters.

Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The k(m) and k(cat) values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 degC and 76.0 degC in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in beta-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.

Thermostable T1 lipase from Geobacillus zalihae has been crystallized using counter-diffusion method under space and Earth conditions. The comparison of the three-dimensional structures from both crystallized proteins show differences in the formation of hydrogen bond and ion interactions. Hydrogen bond and ion interaction are important in the stabilization of protein structure towards extreme temperature and organic solvents. In this study, the differences of hydrogen bond interactions at position Asp43, Thr118, Glu250, and Asn304 and ion interaction at position Glu226 was chosen to imitate space-grown crystal structure, and the impact of these combined interactions in T1 lipase-mutated structure was studied. Using space-grown T1 lipase structure as a reference, subsequent simultaneous mutation D43E, T118N, E226D, E250L, and N304E was performed on recombinant wild-type T1 lipase (wt-HT1) to generate a quintuple mutant term as 5M mutant lipase. This mutant lipase shared similar characteristics to its wild-type in terms of optimal pH and temperature. The stability of mutant 5M lipase improved significantly in acidic and alkaline pH as compared to wt-HT1. 5M lipase was highly stable in organic solvents such as dimethyl sulfoxide (DMSO), methanol, and n-hexane compared to wt-HT1. Both wild-type and mutant lipases were found highly activated in calcium as compared to other metal ions due to the presence of calcium-binding site for thermostability. The presence of calcium prolonged the half-life of mutant 5M and wt-HT1, and at the same time increased their melting temperature (Tm). The melting temperature of 5M and wt-HT1 lipases increased at 8.4 and 12.1 degrees C, respectively, in the presence of calcium as compared to those without. Calcium enhanced the stability of mutant 5M in 25% (v/v) DMSO, n-hexane, and n-heptane. The lipase activity of wt-HT1 also increased in 25% (v/v) ethanol, methanol, acetonitrile, n-hexane, and n-heptane in the presence of calcium. The current study showed that the accumulation of amino acid substitutions D43E, T118N, E226D, E250L, and N304E produced highly stable T1 mutant when hydrolyzing oil in selected organic solvents such as DMSO, n-hexane, and n-heptane. It is also believed that calcium ion plays important role in regulating lipase thermostability.

Thermo-alkalophilic bacterium, Geobacillus thermoleovorans secrets many enzymes including a 43kDa extracellular lipase. Significant thermostability, organic solvent stability and wide substrate preferences for hydrolysis drew our attention to solve its structure by crystallography. The structure was solved by molecular replacement method and refined up to 2.14A resolution. Structure of the lipase showed an alpha-beta fold with 19 alpha-helices and 10 beta-sheets. The active site remains covered by a lid. One calcium and one zinc atom was found in the crystal. The structure showed a major difference (rmsd 5.6A) from its closest homolog in the amino acid region 191 to 203. Thermal unfolding of the lipase showed that the lipase is highly stable with Tm of 76 degrees C. (13)C NMR spectra of products upon triglyceride hydrolysate revealed that the lipase hydrolyses at both sn-1 and sn-2 positions with equal efficiency.

The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 degrees C in Tris-HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 degrees C temperature in Tris-HCl pH 8, while for L2 lipase it was at 70 degrees C in Glycine-NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m) value was at 85.96 degrees C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 degrees C within 16 h preincubation.

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93-97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7-12) and temperature (5-85 degrees C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6-12, thermoactivity (5-85 degrees C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.

The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD) of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol) and water mixture systems to a concentration of 50%. On the basis of the MD results, the structural deviations of the backbone atoms elucidated the dynamic effects of water/organic solvent mixtures on the equilibrium state of the protein simulations in decreasing solvent polarity. The results show that the solvent mixture gives rise to deviations in enzyme structure from the native one simulated in water. The drop in the flexibility in H2O, MtOH, EtOH and PrOH simulation mixtures shows that greater motions of residues were influenced in BtOH and PtOH simulation mixtures. Comparing the root mean square fluctuations value with the accessible solvent area (SASA) for every residue showed an almost correspondingly high SASA value of residues to high flexibility and low SASA value to low flexibility. The study further revealed that the organic solvents influenced the formation of more hydrogen bonds in MtOH, EtOH and PrOH and thus, it is assumed that increased intraprotein hydrogen bonding is ultimately correlated to the stability of the protein. However, the solvent accessibility analysis showed that in all solvent systems, hydrophobic residues were exposed and polar residues tended to be buried away from the solvent. Distance variation of the tetrahedral intermediate packing of the active pocket was not conserved in organic solvent systems, which could lead to weaknesses in the catalytic H-bond network and most likely a drop in catalytic activity. The conformational variation of the lid domain caused by the solvent molecules influenced its gradual opening. Formation of additional hydrogen bonds and hydrophobic interactions indicates that the contribution of the cooperative network of interactions could retain the stability of the protein in some solvent systems. Time-correlated atomic motions were used to characterize the correlations between the motions of the atoms from atomic coordinates. The resulting cross-correlation map revealed that the organic solvent mixtures performed functional, concerted, correlated motions in regions of residues of the lid domain to other residues. These observations suggest that varying lengths of polar organic solvents play a significant role in introducing dynamic conformational diversity in proteins in a decreasing order of polarity.

GD-95-10 and GD-95-20 lipases are modified GD-95 lipase variants, which lack 10 and 20 C-terminal amino acids, respectively. Previous analysis showed that GD-95-10 lipase has higher activity than GD-95 lipase, while GD-95-20 lipase almost completely loses its activity. Analysis in silico suggested three conservative amino acids at region between 369 and 378 amino acids which can be relevant to the activity of GD-95-10 lipase. These amino acids have direct contacts with residues involved in substrate binding, stabilization of the serine loop or form oxyanion hole. In this work, the role of Asp371, Phe375, and Tyr376 on activity, functionality, and structure of GD-95-10 lipase was analyzed by Ala scanning mutagenesis. We showed that even a single mutation can impact the main structure and activity of Geobacillus lipases. Our experiments provide new knowledge about lipases from thermophilic Geobacillus bacteria and are important for protein engineering and synthetic biology. These enzymes and their engineering can be basis for future biocatalysts applied in production of biofuel or other industrial esters.

Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50 degrees C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.

GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80 % lipolytic activity at 5 degrees C. It has maximum activity at 55 degrees C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an alpha helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.

Three-dimensional structure of thermostable lipase is much sought after nowadays as it is important for industrial application mainly found in the food, detergent, and pharmaceutical sectors. Crystallization utilizing the counter diffusion method in space was performed with the aim to obtain high resolution diffracting crystals with better internal order to improve the accuracy of the structure. Thermostable T1 lipase enzyme has been crystallized in laboratory on earth and also under microgravity condition aboard Progress spacecraft to the ISS in collaboration with JAXA (Japanese Aerospace Exploration Agency). This study is conducted with the aims of improving crystal packing and structure resolution. The diffraction data set for ground grown crystal was collected to 1.3 A resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.40 A, b = 80.95 A, and c = 99.81 A, whereas the diffraction data set for space grown crystal was collected to 1.1 A resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.31 A, b = 80.85 A, and c = 99.81 A. The major difference between the two crystal growth systems is the lack of convection and sedimentation in microgravity environment resulted in the growth of much higher quality crystals of T1 lipase.

BACKGROUND: T1 lipase has received considerable attention due to its thermostability. Fatty acid specificity of T1 lipase (crude and purified) was investigated, and its potential in the synthesis of acylglycerols was also evaluated.
RESULTS: Fatty acid specificity of T1 lipase (crude and purified) was investigated in the esterification of fatty acids (C6:0 to C18:3), suggesting that crude and purified T1 lipase had the lowest preference for C18:0 [specificity constant (1/alpha) = 0.08] followed by C18:1 (1/alpha = 0.12) and showed the highest preference for C8:0 (1/alpha = 1). A structural model was constructed to briefly explore interactions between the lipase and its substrate. Furthermore, crude T1 lipase-catalysed synthesis of diacylglycerols (DAGs) and monoacylglycerols (MAGs) by esterification of glycerol with C18:1 was studied for evaluating its potential in acylglycerols synthesis. The optimal conditions were glycerol/oleic acid molar ratio 5:1, the lipase concentration 9.7 U g(-1) of substrates, water content 50 g kg(-1) of substrates and temperature 50 degrees C, which yielded 42.25% DAGs, 26.34% MAGs and 9.18% triacylglycerols at 2 h. CONCLUSION: DAGs and MAGs were synthesised in good yields although C18:1 (a much poorer substrate) was used. Our work demonstrates that T1 lipase, which was discovered to show 1,3-regio-selectivity, is a promising biocatalyst for lipids modification.

A thermoalkalophilic lipase (LIPSBS) from the newly isolated Geobacillus strain SBS-4S which hydrolyzes a wide range of fatty acids has been characterized. In the present study, the crystallization of purified LIPSBS using the sitting-drop vapour-diffusion method and its X-ray diffraction studies are described. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 55.13, b = 71.75, c = 126.26A. The structure was determined at 1.6A resolution by the molecular-replacement method using the lipase from G. stearothermophilus L1 as a model.

The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100 degrees C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises alpha6 and alpha7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.

A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 degrees C and showed stability over a temperature range of 40-60 degrees C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.

The crystallization of proteins makes it possible to determine their structure by X-ray crystallography, and is therefore important for the analysis of protein structure-function relationships. L2 lipase was crystallized by using the J-tube counter diffusion method. A crystallization consisting of 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl was found to be the best condition to produce crystals with good shape and size (0.5 x 0.1 x 0.2 mm). The protein concentration used for the crystallization was 3 mg/mL. L2 lipase crystal has two crystal forms, Shape 1 and Shape 2. Shape 2 L2 lipase crystal was diffracted at 1.5 A and the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 72.0, b = 81.8, c = 83.4 A, alpha = beta = gamma = 90 degrees . There is one molecule per asymmetric unit and the solvent content of the crystals is 56.9%, with a Matthew's coefficient of 2.85 A Da(-1). The 3D structure of L2 lipase revealed topological organization of alpha/beta-hydrolase fold consisting of 11 beta-strands and 13 alpha-helices. Ser-113, His-358 and Asp-317 were assigned as catalytic triad residues. One Ca(2+) and one Zn(2+) were found in the L2 lipase molecule.

Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 degrees C, 70.59 degrees C and 68.54 degrees C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 A using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 A, b = 81.16 A and c = 100.14 A. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.

The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65 degrees C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50 degrees C for more than 150 min.

An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 A resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09x0.19x0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=117.41, b=80.85, c=99.44 A, beta=96.40 degrees .

The use of lipase in hydrophilic solvent is usually hampered by inactivation. The solvent stability of a recombinant solvent stable lipase isolated from thermostable Bacillus sp. strain 42 (Lip 42), in DMSO and methanol were studied at different solvent-water compositions. The enzymatic activities were retained in up to 45% v/v solvent compositions. The near-UV CD spectra indicated that tertiary structures were perturbed at 60% v/v and above. Far-UV CD in methanol indicated the secondary structure in Lip 42 was retained throughout all solvent compositions. Fluorescence studies indicated formations of molten globules in solvent compositions of 60% v/v and above. The enzyme was able to retain its secondary structures in the presence of methanol; however, there was a general reduction in beta-sheet and an increase in alpha-helix contents. The H-bonding arrangements triggered in methanol and DMSO, respectively, caused different forms of tertiary structure perturbations on Lip 42, despite both showing partial denaturation with molten globule formations.

Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 A resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 A. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 57%.

A 1.2 kb lipase gene (AY 78735) from solvent stable and thermostableBacillus sp. strain 42 was overexpressed in a heterologous system that allowed for an extensive characterization of its solvent stability and thermostability. An overexpression was achieved using pET51b vector withEscherichia coli host strain BL21(DE3)pLysS, in which optimum expression was at 22-24 h incubation at 37C, with lipase activity reached at 80.0 U mL-1 (specific activity 160.0 U mg-1), after induction by 0.5 mM IPTG. This expression was 11.5 fold higher and superseded the pQE-30UA/M15 (pREP4) host-vector system, which only achieved at 17.0 U mL1 (34.0 U mg-1). The fusion lipase contains N-terminal Strep-tag II affinity tag that in one step of purification, the lipase was purified to homogeneity using Strep-tag II agarose column. The lipase was purified at 1.3 fold and 70% recovery with the elution fraction gave a band of 43 kDa in SDS-PAGE. The purified fusion lipase was most active at 70C and pH 8.0, and was stable in a broad pH range of 7-10. It showed hydrolysis preference towards olive, sunflower and corn oils. Based on solvent stability studies in 30 min pre-incubation in 25% v/v solvents with a shaking rate at 150 strokes per min, the purified Lip 42 showed a different residual activity profiles depending on solvents and temperatures. Lip 42 was found be stable in polar organic solvents such as DMSO, DMF, acetone, methanol, heptanol and octanol, which could make it as a potential biocatalyst for the use in industrial biodiesel production.

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae alpha-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 degrees C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca2+. L2 lipase showed a preference for medium to long chain triacylglycerols (C(10)-C(16)), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70 degrees C and pH 9, respectively. It was stable up to 65 degrees C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na(+), Ca(2+), Mn(2+), K(+) and Mg(2+ ), but inhibited by Cu(2+), Fe(3+) and Zn(2+). Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T(m) for T1 lipase was around 72.2 degrees C, as revealed by denatured protein analysis of CD spectra.

The gene encoding thermostable T1 lipase secreted by Geobacillus sp. strain T1 has been overexpressed in a prokaryotic system. Preliminary crystallization was conducted with crystal screen and crystal screen II through a sitting drop vapor diffusion method with 0.5 mg/mL purified T1 lipase at 16 C. Crystallization at 16 degC using formulation 21 of crystal screen II at 2.5 mg/mL yielded bigger and more defined crystals. Good crystals could easily be obtained as the temperature was increased further while retaining other conditions. In fact, crystallization of T1 lipase is still possible at 60 C. The ability to form crystals at 60 degC is a new discovery in lipase crystallization.

BACKGROUND: Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78 degrees C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5-99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.
RESULTS: Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1T was chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70 degrees C and was also stable up to 60 degrees C without significant loss of crude enzyme activity. Strain T1T was a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1T was shown to belong to the Bacillus rRNA group 5 related to Geobacillus thermoleovorans (DSM 5366T) and Geobacillus kaustophilus (DSM 7263T). Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1T to the genus Geobacillus. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1T from its validly published closest phylogenetic neighbors. Strain T1T therefore represents a novel species, for which the name Geobacillus zalihae sp. nov. is proposed, with the type strain T1T (=DSM 18318T; NBRC 101842T). CONCLUSION: Strain T1T was able to secrete extracellular thermostable lipase into culture medium. The strain T1T was identified as Geobacillus zalihae T1T as it differs from its type strains Geobacillus kaustophilus (DSM 7263T) and Geobacillus thermoleovorans (DSM 5366T) on some physiological studies, cellular fatty acids composition, RiboPrint analysis, length of lipase gene and protein profile.

A novel lipase-producing thermophilic strain TW1, assigned to Geobacillus sp. TW1 based on 16S rRNA sequence, was isolated from a hot spring in China. Based on this strain, a lipase gene encoding 417 amino acids was cloned. Subsequently, the lipase gene was expressed in Escherichia coli and purified as a fusion protein with glutathione S-transferase. The results showed that the recombinant lipase had an activity optimum at 40 degrees C and pH at 7.0-8.0. It was active up to 90 degrees C at pH 7.5, and stable over a wide pH ranging from 6.0 to 9.0. The recombinant lipase was stable in 1 mM enzyme inhibitors (EDTA, 2-ME, SDS, PMSF or DTT), as well as in 0.1% detergents (Tween 20, Chaps or Triton X-100). Its catalytic function was enhanced in the presence of Ca(2+), Mg(2+), Zn(2+), Fe(2+) or Fe(3+), but inhibited by Cu(2+), Mn(2+), and Li(+). By comparison with the crude lipase, the recombinant lipase had similar properties and was characteristic of thermostable enzymes. Our study presented a rapid overexpression and purification of the lipase gene from thermophile, aimed at improving the enzyme yield for industrial applications.

A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg(-1) which corresponds to 2927.15 Ug(-1) of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65 degrees C and pH 9, respectively. It was stable up to 65 degrees C at pH 7 and active over a wide pH range (pH 6-11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.

A thermophilic lipase of Bacillus thermoleovorans ID-1 was cloned and sequenced. The lipase gene codes 416 amino acid residues and contains the conserved pentapeptide Ala-X-Ser-X-Gly as other Bacillus lipase genes. The optimum temperature of the lipase is 75 degrees C, which is higher than other known Bacillus lipases. For expression in Escherichia coli, the lipase gene was subcloned in pET-22b(+) vector with a strong T7 promoter. Lipase activity was approximately 1.4-fold greater than under the native promoter.