Author Report for: Kaur JNo contact information in database for Kaur J
Anand PK, Kaur G, Saini V, Kaur J Ref: Biochimie, :, 2023 : PubMed ESTHER: Anand_2023_Biochimie__ PubMedSearch: Anand 2023 Biochimie PubMedID: 37156406 Gene_locus related to this paper: myctu-ppe63 Abstract Multigene PE/PPE family is exclusively present in mycobacterium species. Only few selected genes of this family have been characterized till date. Rv3539 was annotated as PPE63 with conserved PPE domain at N-terminal and PE-PPE at C-terminal. An alpha/beta hydrolase structural fold, characteristic of lipase/esterase, was present in the PE-PPE domain. To assign the biochemical function to Rv3539, the corresponding gene was cloned in pET-32a (+) as full-length, PPE, and PE-PPE domains individually, followed by expression in E. Coli (DE3). All three proteins demonstrated esterase activity. However, the enzyme activity in the N-terminal PPE domain was very low. The enzyme activity of Rv3539 and PE-PPE proteins was approximately same with the pNP-C4 as optimum substrate at 40 degreesC and pH 8.0. The loss of enzyme activity after mutating the predicted catalytic triad (Ser296Ala, Asp369Ala, and His395Ala) found only in the PE-PPE domain, confirmed the candidature of the bioinformatically predicted active site residue. The optimal activity and thermostability of the Rv3539 protein was altered by removing the PPE domain. CD-spectroscopy analysis confirmed the role of PPE domain to the thermostability of Rv3539 by maintaining the structural integrity at higher temperatures. The presence of the N-terminal PPE domain directed the Rv3539 protein to the cell membrane/wall and the extracellular compartment. The Rv3539 protein could generate humoral response in TB patients. Therefore, results demonstrated that Rv3539 demonstrated esterase activity. PE-PPE domain of Rv3539 is functionally automated, however N-terminus domain played a role in protein stabilization and its transportation. Both domains participated in immunomodulation. Title: Cell wall and immune modulation by Rv1800 (PPE28) helps M. smegmatis to evade intracellular killing Anand PK, Saini V, Kaur J, Kumar A Ref: Int J Biol Macromol, :125837, 2023 : PubMed ESTHER: Anand_2023_Int.J.Biol.Macromol__125837 PubMedSearch: Anand 2023 Int.J.Biol.Macromol 125837 PubMedID: 37455004 Gene_locus related to this paper: myctu-Rv1800 Abstract Rv1800 is predicted as PPE family protein found in pathogenic mycobacteria only. Under acidic stress, the rv1800 gene was expressed in M. tuberculosis H37Ra. In-silico study showed lipase/esterase activity in C-terminus PE-PPE domain having pentapeptide motif with catalytic Ser-Asp-His residue. Full-length Rv1800 and C-terminus PE-PPE domain proteins showed esterase activity with pNP-C4 at the optimum temperature of 40 degreesC and pH 8.0. However, the N-terminus PPE domain showed no esterase activity, but involved in thermostability of Rv1800 full-length protein. M. smegmatis expressing rv1800 (MS_Rv1800) showed altered colony morphology and a significant resistance to numerous environmental stresses, antibiotics and higher lipid content. In extracellular and membrane fraction, Rv1800 protein was detected, while C terminus PE-PPE was present in cytoplasm, suggesting the role of N-terminus PPE domain in transportation of protein. MS_Rv1800 infected macrophage showed higher intracellular survival and low production of ROS, NO and expression levels of iNOS and pro-inflammatory cytokines, while induced expression of the anti-inflammatory cytokines. The Rv1800, PPE and PE-PPE showed antibody-mediated immunity in MDR-TB and PTB patients. Overall, these results confirmed the esterase activity in the C-terminus and function of N-terminus in thermostabilization and transportation; predicting the role of Rv1800 in immune/lipid modulation to support intracellular mycobacterium survival. Title: Pathogenesis of Alzheimer's Disease and Diversity of 1,2,3-Triazole Scaffold in Drug Development: Design Strategies, Structural Insights, and Therapeutic Potential Singh A, Singh K, Kaur J, Kaur R, Sharma A, Kaur U, Chadha R, Bedi PMS Ref: ACS Chem Neurosci, :, 2023 : PubMed ESTHER: Singh_2023_ACS.Chem.Neurosci__ PubMedSearch: Singh 2023 ACS.Chem.Neurosci PubMedID: 37683129 Abstract Alzheimer's disease is a most prevalent form of dementia all around the globe and currently poses a significant challenge to the healthcare system. Currently available drugs only slow the progression of this disease rather than provide proper containment. Identification of multiple targets responsible for this disease in the last three decades established it as a multifactorial neurodegenerative disorder that needs novel multifunctional agents for its management and the possible reason for the failure of currently available single target clinical drugs. 1,2,3-Triazole is a miraculous nucleus in medicinal chemistry and the first choice for development of multifunctional hybrid molecules. Apart from that, it is an integral component of various drugs in clinical trials as well as in clinical practice. This review is focused on the pathogenesis of Alzheimer's disease and 1,2,3-triazole containing derivatives developed in recent decades as potential anti-Alzheimer's agents. The review will provide (A) precise insight of various established targets of Alzheimer's disease including cholinergic, amyloid, tau, monoamine oxidases, glutamate, calcium, and reactive oxygen species hypothesis and (B) design hypothesis, structure-activity relationships, and pharmacological outcomes of 1,2,3-triazole containing multifunctional anti-Alzheimer's agents. This review will provide a baseline for various research groups working on Alzheimer's drug development in designing potent, safer, and effective multifunctional anti-Alzheimer's candidates of the future. Title: Environment dependent expression of mycobacterium hormone sensitive lipases: expression pattern under ex-vivo and individual in-vitro stress conditions in M. tuberculosis H37Ra Arya S, Singh P, Kaur J, Kumar A Ref: Mol Biol Rep, :, 2022 : PubMed ESTHER: Arya_2022_Mol.Biol.Rep__ PubMedSearch: Arya 2022 Mol.Biol.Rep PubMedID: 35301657 Abstract BACKGROUND: Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kinds of lipids. In comparison to single human Hormone Sensitive Lipase (hHSL), that is induced under nutritional stress, twelve serine hydrolases are annotated as HSL in Mycobacterium tuberculosis (mHSL). Mycobacterium is exposed to multiple stresses inside the host. Therefore, the present study was carried out to investigate if mHSL are also expressed under stress condition and if there is any correlation between various stress conditions and expression pattern of mHSL. METHODS AND RESULTS: The expression pattern of mHSL under different environmental conditions (in-vitro and ex-vivo) were studied using qRT-PCR in M. tuberculosis H37Ra strain with 16 S rRNA as internal control. Out of 12, only two genes (lipU and lipY) were expressed at very low level in mid log phase culture under aerobic conditions, while 9 genes were expressed at stationary phase of growth. Ten mHSLs were expressed post-infection under ex-vivo conditions in time dependent manner. LipH and lipQ did not express at any time point under ex-vivo condition. The relative expression of most of the genes under individual stress was much higher than observed in ex-vivo conditions. The expression pattern of genes varied with change in stress condition. CONCLUSIONS: Different sets of mHSL genes were expressed under different individual stress conditions pointing towards the requirement of different mHSL to combat different stress conditions. Overall, most of the mHSLs have demonstrated stress dependent expression pointing towards their role in intracellular survival of mycobacteria. Title: Anti-inflammatory effects of ellagic acid and vanillic acid against quinolinic acid-induced rat model of Huntington's disease by targeting IKK-NF-B pathway Bains M, Kaur J, Akhtar A, Kuhad A, Sah SP Ref: European Journal of Pharmacology, :175316, 2022 : PubMed ESTHER: Bains_2022_Eur.J.Pharmacol__175316 PubMedSearch: Bains 2022 Eur.J.Pharmacol 175316 PubMedID: 36209926 Abstract Huntington disease (HD), an autosomal dominant neurodegenerative disorder characterized by involuntary choreatic movements with cognitive and behavioral disturbances. HD striatum has increased conversion of kynurenine to quinolinic acid (QA) which activates NMDA receptors leading to activation of microglia and increased levels of nuclear factor kappa B (NF-kappaB) leading to elevated transcription of inducible nitric oxide synthase (iNOS) and various cytokines causing neuronal death via neuroinflammation, oxidative stress, mitochondrial dysfunction and apoptosis. Therefore, inhibiting IKK-NF-kappaB pathway induced excitotoxicity, oxidative stress and neuroinflammation could be a potential intervention in slowing down the disease progression. QA injection intrastriatally (IS-QA) produce damage mimicking HD where neuroinflammation, oxidative stress and mitochondrial dysfunction play crucial role. Ellagic acid (EA) and vanillic acid (VA) are well reported to possess antioxidant and NF-kappaB inhibiting effect. Hence, in present study, rats administered IS-QA were treated with EA and VA for 21 days to explore their neuroprotective effects. Behavioral studies, biochemical estimations for oxidative stress and acetylcholinesterase assay were performed. Mitochondrial function was determined by estimating mitochondrial enzyme complexes; inflammatory markers like TNF-alpha, IL-6, NF-kappaB by ELISA and apoptosis by caspase-3 levels. Brain damage was determined by histopathology which revealed their neuroprotective effects. Various doses of EA and VA produced improved motor and cognitive functions, oxidative stress and neuroinflammation were also reduced and mitochondrial functioning was improved. In a nutshell, these results signify improved motor and cognitive functions by EA and VA in QA model of HD, along with declined oxidative stress, mitochondrial dysfunction and neuroinflammation. Title: Recovery of hydroxytyrosol from olive mill wastewater using the promiscuous hydrolase/acyltransferase PestE Terholsen H, Kaur J, Kaloudis N, Staudt A, Muller H, Pavlidis IV, Bornscheuer U Ref: Chembiochem, 23:e202200254, 2022 : PubMed ESTHER: Terholsen_2022_Chembiochem_23_e202200254 PubMedSearch: Terholsen 2022 Chembiochem 23 e202200254 PubMedID: 35579388 Gene_locus related to this paper: pyrca-PCEST Abstract Olive mill wastewater (OMWW) is produced annually during olive oil extraction and contains most of the health-promoting 3-hydroxytyrosol of the olive fruit. To facilitate its recovery, enzymatic transesterification of hydroxytyrosol (HT) was directly performed in an aqueous system in the presence of ethyl acetate, yielding a 3-hydroxytyrosol acetate rich extract. For this, the promiscuous acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was engineered by rational design. The best mutant for the acetylation of hydroxytyrosol PestE_I208A_L209F_N288A was immobilized on EziG2 beads, resulting in hydroxytyrosol conversions between 82 and 89% in one hour, for at least ten reaction cycles in a buffered hydroxytyrosol solution. Due to inhibition by other phenols in OMWW the conversions of hydroxytyrosol from this source were between 51 and 62%. In a preparative scale reaction, 13.8 mg (57%) of 3-hydroxytyrosol acetate was extracted from 60 mL OMWW. Title: Protocol for in-vitro purification and refolding of hexachlorocyclohexane degrading enzyme haloalkane dehalogenase LinB from inclusion bodies Kaur J, Singh A, Panda AK, Lal R Ref: Enzyme Microb Technol, 146:109760, 2021 : PubMed ESTHER: Kaur_2021_Enzyme.Microb.Technol_146_109760 PubMedSearch: Kaur 2021 Enzyme.Microb.Technol 146 109760 PubMedID: 33812559 Gene_locus related to this paper: sphpi-linb Abstract LinB is an important haloalkane dehalogenase involved in the degradation pathway of different isomers of hexachlorocyclohexane (HCH), mainly in catalyzing degradation of the notorious beta-HCH. The HCH isomers are known to have neurotoxic, carcinogenic and estrogenic effects. Enzymatic bioremediation for decontamination of beta- as well as other HCH isomers can prove to be a potential remediation strategy. For any bioremediation technology that is to be developed, apart from having high turnover number, the candidate enzyme must also be available in sufficient amounts. In this direction, the LinB variants reported in database were tested in laboratory studies. The variant LinBSSO4-3 however could not be obtained in soluble fraction by using standard procedures. The protein LinBSSO4-3 was cloned in pDEST17 vector and codon optimized for better expression in Escherichia coli BL21AI using a strong T7 promoter. However, the over-expression of this protein in ectopic host E. coli, led to aggregation of the protein in form of inclusion bodies, which are insoluble aggregates of misfolded or partially folded proteins. SEM analysis of the inclusion bodies showed them as aggregated spherical particles. The inclusion bodies were isolated using high speed sonication and homogenization. This was followed by solubilization in the strong denaturing agent urea. Refolding into its native state was done by using pulsatile refolding. This was done by slowly decreasing the denaturant concentration in the presence of sucrose. The turnover number of the refolded protein was then determined for different isomers of HCH. The protein was found to have a turnover number of -43 molecules min(-1) on beta-HCH and -13 molecules min(-1) on delta-HCH. Additionally, a mutation I253 M in the active site of the enzyme was found to drastically decrease the enzyme activity on beta-HCH. Taking into consideration the wide range of substrates of haloalkane dehalogenases, such a protocol for inclusion body refolding will contribute to the field of bioremediation technology development for organochlorines, specifically HCH. Such a protocol for refolding of haloalkane dehalogenases from inclusion bodies has not been developed or reported before. Title: The lipolytic activity of LipJ, a stress-induced enzyme, is regulated by its C-terminal adenylate cyclase domain Kumari B, Kaur J, Maan P, Kumar A Ref: Future Microbiol, :, 2021 : PubMed ESTHER: Kumari_2021_Future.Microbiol__ PubMedSearch: Kumari 2021 Future.Microbiol PubMedID: 33960821 Abstract Aim: The confirmation of lipolytic activity and role of Rv1900c in the mycobacterium physiology Methods: rv1900c/N-terminus domain (rv1900NT) were cloned in pET28a/Escherichia coli, purified by affinity chromatography and characterized. Results: A zone of clearance on tributyrin-agar and activity with pNP-decanoate confirmed the lipolytic activity of Rv1900c. The Rv1900NT demonstrated higher enzyme specific activity, V(max) and k(cat), but Rv1900c was more thermostable. The lipolytic activity of Rv1900c decreased in presence of ATP. Mycobacterium smegmatis expressing rv1900c/rv1900NT altered colony morphology, growth, cell surface properties and survival under stress conditions. The effect was more prominent with Rv1900NT as compared with Rv1900c. Conclusion: The study confirmed the lipolytic activity of Rv1900c and suggested its regulation by the adenylate cyclase domain and role in intracellular survival of bacteria. Title: 1H-1,2,3-triazole grafted tacrine-chalcone conjugates as potential cholinesterase inhibitors with the evaluation of their behavioral tests and oxidative stress in mice brain cells Rani A, Singh A, Kaur J, Singh G, Bhatti R, Gumede N, Kisten P, Singh P, Sumanjit, Kumar V Ref: Bioorg Chem, 114:105053, 2021 : PubMed ESTHER: Rani_2021_Bioorg.Chem_114_105053 PubMedSearch: Rani 2021 Bioorg.Chem 114 105053 PubMedID: 34120027 Abstract The present paper explicates the synthesis of 1H-1,2,3-triazole tethered tacrine-chalcone conjugates and evaluation of their AChE and BuChE inhibitory activity. In-vitroAChE inhibition assay revealed three compounds, 9h, 9i, and 11f, being more potent than the standard drug tacrine and further evaluated against butyrylcholinesterase. The present study was extended to investigate the anti-amnestic effect of promising compoundson scopolamine-induced behavioral and neurochemical changes in mice. Inclined plane model and Elevated plus-maze model were performed to assess general limb motor activity and anxiety-like behavior, respectively, in mice pre-treated with scopolamine. Oxidative stress parameters reduced glutathione contents (GSH) and lipid peroxidation products (TBARS) in the brain homogenates as estimated using ex-vivo studies. Furthermore, molecular docking studies were performed for the potent compounds to decipher the mechanism of observed activities. Title: Rational Design for Enhanced Acyltransferase Activity in Water Catalyzed by the Pyrobaculum calidifontis VA1 Esterase Staudt A, Terholsen H, Kaur J, Muller H, Godehard SP, Itabaiana I, Jr., Leal ICR, Bornscheuer UT Ref: Microorganisms, 9:1790, 2021 : PubMed ESTHER: Staudt_2021_Microorganisms_9_1780 PubMedSearch: Staudt 2021 Microorganisms 9 1780 PubMedID: 34442869 Gene_locus related to this paper: pyrca-PCEST Abstract Biocatalytic transesterification is commonly carried out employing lipases in anhydrous organic solvents since hydrolases usually prefer hydrolysis over acyl transfer in bulk water. However, some promiscuous acyltransferases can catalyze acylation in an aqueous solution. In this study, a rational design was performed to enhance the acyltransferase selectivity and substrate scope of the Pyrobaculum calidifontis VA1 esterase (PestE). PestE wild type and variants were applied for the acylation of monoterpene alcohols. The mutant PestE_I208A is selective for (-)-menthyl acetate (E-Value = 55). Highly active acyltransferases were designed, allowing for complete conversion of (-)-citronellol to citronellyl acetate. Additionally, carvacrol was acetylated but with lower conversions. To the best of our knowledge, this is the first example of the biocatalytic acylation of a phenolic alcohol in bulk water. In addition, a high citronellol conversion of 92% was achieved with the more environmentally friendly and inexpensive acyl donor ethyl acetate using PestE_N288F as a catalyst. PestE_N288F exhibits good acyl transfer activity in an aqueous medium and low hydrolysis activity at the same time. Thus, our study demonstrates an alternative synthetic strategy for acylation of compounds without organic solvents. Title: Enzyme-based optical biosensors for organophosphate class of pesticide detection Kaur J, Singh PK Ref: Phys Chem Chem Phys, 22:15105, 2020 : PubMed ESTHER: Kaur_2020_Phys.Chem.Chem.Phys_22_15105 PubMedSearch: Kaur 2020 Phys.Chem.Chem.Phys 22 15105 PubMedID: 32613964 Abstract Organophosphorus pesticides, widely used as insecticides for crop protection, are classified as an extremely toxic class of chemical compounds by the World Health Organization (WHO). Organophosphorus pesticides show marked specificity for the enzyme acetylcholinesterase, and can cause irreversible harm to the nervous system. The excessive use of organophosphorus pesticides, specifically in developing countries, made WHO to impose a ban on certain organophosphorus pesticides being sold to the developing nations. WHO and the Food and Agriculture Organization of United Nations, in joint meetings on pesticide residues, have stressed the need to develop easy, rapid, and sensitive methods to detect the presence of organophosphorus pesticides in food and water. In this regard, a number of review articles have been published explaining a myriad of detection methods for organophosphorus pesticides. Among these, optical detection methods, using biological molecules as the recognition element, provide a number of advantages, such as, high sensitivity and selectivity, simple operation, fast response and cheap instrumentation, and can really prove to be an effective alternative to the time-consuming traditional methods in the current scenario. Therefore, in this review article, we have discussed the recent advances in the field of biosensors employing the optical detection of organophosphorus pesticides. Specifically, various biosensors developed, using enzymes acetylcholinesterase and organophosphate hydrolase, have been categorized on the basis of the material used for their fabrication. A systematic discussion of the working principles, analytical parameters, and advantages and disadvantages of the various biosensors, has been attempted, along with a future perspective, on the challenges and possible improvements in the field of optical detection of organophosphorus pesticides. Title: Molecular dynamics assisted mechanistic insight of Val430-Ala mutation of Rv1592c protein in isoniazid resistant Mycobacterium tuberculosis Kumar A, Anand PK, Chandel S, Shrivatava A, Kaur J Ref: Curr Comput Aided Drug Des, :, 2020 : PubMed ESTHER: Kumar_2020_Curr.Comput.Aided.Drug.Des__ PubMedSearch: Kumar 2020 Curr.Comput.Aided.Drug.Des PubMedID: 31939732 Gene_locus related to this paper: myctu-MT1628 Abstract BACKGROUND: Multi drug-resistant tuberculosis is a major health threat to humans. Whole genome sequencing of several isoniazid (INH) resistant strains of M. tuberculosis revealed mutations in several genes. Rv1592c was demonstrated as lipolytic enzyme and its expression was up-regulated during isoniazid (INH) treatment. The valine at position 430 of Rv1592c was mutated to alanine frequently in the INH resistant strain of M. tuberculosis. METHODS: In this report, an array of computational approaches was used to understand the role of Val430-Ala mutation in Rv1592c in INH resistance. The impact of mutations on structural stability and degree of INH modification was demonstrated using the molecular dynamics method. The mutation in the Rv1592c gene at V430 position was created by the PCR primer walking method. Mutant and wild type gene was cloned into E. coli-mycobacteria shuttle vector (pVV-16) and expressed in Mycobacterium smegmatis system. The isoniazid susceptibility assay was performed by agar plate culture spot and CFUs count assay. RESULTS: This study demonstrated that the Val430 in Rv1592c makes the part of flap covering the substrate binding cavity. Mutation at Val430-Ala in Rv1592c caused the displacement of the flap region, resulting in uncovering a cavity, which allows accessibility of substrate to the active site cleft. The Val430-Ala mutation in Rv1592c created its structure energetically more stable. RMSD, RMSF and Rg simulation of mutant maintained overall stability throughout the simulation period while the native protein displayed comparatively more fluctuations. Moreover, docking studies showed that INH was bound into the active pocket of the mutant with considerable binding energy (-6.3 kcal/mol). In order to observe constant binding for INH, complexes were simulated for 50 ns. It was observed that after simulation, INH remained bound in the pocket with an increased molecular bonding network with the neighbor amino acid residues. In vitro studies clearly suggested that M. smegmatis expressing mutant has a better survival rate in isoniazid treatment as compared to wild type. CONCLUSION: Overall, this study at the outset suggested that the mutation observed in drug resistant strain provides stability to the Rv1592c protein and increased affinity towards the INH due to flap displacement, leading to the possibility for its modification. In vitro results supported our in silico findings. Title: Engineering lipases for temperature adaptation: Structure function correlation Kumar R, Goomber S, Kaur J Ref: Biochimica & Biophysica Acta Proteins Proteom, 1867:140261, 2019 : PubMed ESTHER: Kumar_2019_Biochim.Biophys.Acta.Proteins.Proteom_1867_140261 PubMedSearch: Kumar 2019 Biochim.Biophys.Acta.Proteins.Proteom 1867 140261 PubMedID: 31401312 Abstract Bacillus lipases are industrially attractive enzymes due to their broad substrate specificity and optimum alkaline pH. However, narrow temperature range of action and low thermostability restrain their optimal use and thus, necessitate attention. Several laboratories are engaged in protein engineering of Bacillus lipases to generate variants with improved attributes for decades using techniques such as directed evolution or rational design. This review summarizes the effect of mutations on the conformational changes through in silico modeling and their manifestation with respect to various biochemical parameters. Various studies have been put together to develop a perspective on the molecular basis of biocatalysis of lipases holding industrial importance. Title: Rv2223c, an acid inducible carboxyl-esterase of Mycobacterium tuberculosis enhanced the growth and survival of Mycobacterium smegmatis Maan P, Kaur J Ref: Future Microbiol, 14:1397, 2019 : PubMed ESTHER: Maan_2019_Future.Microbiol_14_1397 PubMedSearch: Maan 2019 Future.Microbiol 14 1397 PubMedID: 31797682 Gene_locus related to this paper: myctu-ym23 Abstract Aim: To elucidate the role of Rv2223c in Mycobacterium tuberculosis. Methods: Purified recombinant Rv2223c protein was characterized. Expression of rv2223c in the presence of different stress environment and subcellular localization were performed in M. tuberculosis H37Ra and Mycobacterium smegmatis (MS_2223c). Effect of its overexpression on growth rate, infection and intracellular survival in THP-1/PBMC cells were studied. Results: rRv2223c demonstrated esterase activity with preference for pNP-octanoate and hydrolyzed trioctanoate to di- and mono-octanoate. Expression of rv2223c was upregulated in acidic and nutritive stress conditions. rRv2223c was identified in extracellular and cell wall fractions. MS_2223c exhibited enhanced growth, survival during in vitro stress, infection and intracellular survival. Conclusions: Rv2223c is a secretary, carboxyl-esterase, with enhanced expression under acidic and nutritive stress condition and might help in intracellular survival of bacteria. Title: Molecular characterization and immunogenic function of ML1899 (LipG) of Mycobacterium leprae Rastogi R, Kaur G, Maan P, Bhatnagar A, Narang T, Dogra S, Kaur J Ref: J Med Microbiol, 68:1629, 2019 : PubMed ESTHER: Rastogi_2019_J.Med.Microbiol_68_1629 PubMedSearch: Rastogi 2019 J.Med.Microbiol 68 1629 PubMedID: 31553301 Gene_locus related to this paper: mycle-lipG Abstract Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of alpha/beta hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 degrees C with 5.56 microM(-1) min(-1) catalytic efficiency. The enzyme exhibited stability up to 60 degrees C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 microM, 40 micromoles min(-1) ml(-1) and 27 U mg(-) (1), respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-alpha, IFN-gamma, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration. Title: mbtJ: an iron stress-induced acetyl hydrolase/esterase of Mycobacterium tuberculosis helps bacteria to survive during iron stress Chownk M, Kaur J, Singh K Ref: Future Microbiol, 13:547, 2018 : PubMed ESTHER: Chownk_2018_Future.Microbiol_13_547 PubMedSearch: Chownk 2018 Future.Microbiol 13 547 PubMedID: 29519132 Gene_locus related to this paper: myctu-Rv2385 Abstract AIM: mbtJ from Mycobacterium tuberculosis H37Rv is a member of mbt A-J operon required for mycobactin biogenesis. MATERIALS & METHODS: The esterase/acetyl-hydrolase activity of mbtJ was determined by pNP-esters/native-PAGE and expression under iron stress by quantitative-PCR. Effect of gene on growth/survival of Mycobacterium was studied using antisense. Its effect on morphology, growth/infection was studied in Mycobacterium smegmatis. RESULTS: It showed acetyl hydrolase/esterase activity at pH 8.0 and 50 degrees C with pNP-butyrate. Its expression was upregulated under iron stress. The antisense inhibited the survival of bacterium during iron stress. Expression of mbtJ changed colony morphology and enhanced the growth/infection in M. smegmatis. CONCLUSION: mbtJ, an acetyl-hydrolase/esterase, enhanced the survival of M. tuberculosis under iron stress, affected the growth/infection efficiency in M. smegmatis, suggesting its pivotal role in the intracellular survival of bacterium. Title: Characterization of ML0314c of Mycobacterium leprae and deciphering its role in the immune response in leprosy patients Kaur G, Sharma A, Narang T, Dogra S, Kaur J Ref: Gene, 643:26, 2018 : PubMed ESTHER: Kaur_2018_Gene_643_26 PubMedSearch: Kaur 2018 Gene 643 26 PubMedID: 29208413 Gene_locus related to this paper: mycle-ML0314 Abstract Mycobacterium leprae has a reduced genome size due to the reductive evolution over a long period of time. Lipid metabolism plays an important role in the life cycle and pathogenesis of this bacterium. In comparison to 26 lip genes (Lip A-Z) of M. tuberculosis, M. leprae retained only three orthologs indicating their importance in its life cycle. ML0314c (LipU) is one of them. It is conserved throughout the mycobacterium species. Bioinformatics analysis showed the presence of an alpha/beta hydrolase fold and 'GXSXG' characteristic of the esterases/lipases. The gene was expressed in E. coli and purified to homogeneity. It showed preference towards short chain esters with pNP-acetate as the preferred substrate. The enzyme showed optimal activity at 45 degrees C and pH8.0. ML0314c protein was stable between temperatures ranging from 20 to 60 degrees C and pH5.0-8.0, i.e., relatively acidic and neutral conditions. The active site residues predicted bioinformatically were confirmed to be Ser168, Glu267, and His297 by site directed mutagenesis. E-serine, DEPC and Tetrahydrolipstatin (THL) completely inhibited the activity of ML0314c. The protein was localized in cell wall and extracellular medium. Several antigenic epitopes were predicted in ML0314c. Protein elicited strong humoral immune response in leprosy patients, whereas, a reduced immune response was observed in the relapsed cases. No humoral response was observed in treatment completed patients. Overexpression of ml0314c in the surrogate host M. smegmatis showed marked difference in the colony morphology and growth rate. In conclusion, ML0314c is a secretary carboxyl esterase that could modulate the immune response in leprosy patients. Title: Rv1288, a Two Domain, Cell Wall Anchored, Nutrient Stress Inducible Carboxyl-Esterase of Mycobacterium tuberculosis, Modulates Cell Wall Lipid Maan P, Kumar A, Kaur J Ref: Front Cell Infect Microbiol, 8:421, 2018 : PubMed ESTHER: Maan_2018_Front.Cell.Infect.Microbiol_8_421 PubMedSearch: Maan 2018 Front.Cell.Infect.Microbiol 8 421 PubMedID: 30560095 Gene_locus related to this paper: myctu-yc88 Abstract Rv1288, a conserved hypothetical protein of M. tuberculosis (M.tb), was recently characterized as two-domain esterase enzyme by in silico study. In the present study, Rv1288 and its domains (Est and Lyt) were cloned individually from M.tb into E. coli for expression and purification. The purified rRv1288 and rEst proteins exhibited lipolytic activity with medium chain length esters as optimum substrates, while Lyt domain did not show enzymatic activity. However, presence of Lyt domain resulted in enhanced rate of protein aggregation at higher temperature. Both rRv1288 and rEst followed the similar patterns of substrate specificity, temperature and pH activity. Site directed mutagenesis confirmed the Ser-294, Asp-391 and His-425 as catalytic site residues. Rv1288 was found to be present in cell wall fraction of M.tb H37Ra. Peptidoglycan binding activity of Rv1288 and its domains demonstrated that the Lyt domain is essential for anchoring protein to the cell wall. Expression of rv1288 was up regulated in M.tb under nutrient starved condition. Over expression of rv1288 in surrogate host M. smegmatis led to change in colony morphology, enhanced pellicle and aggregate formation that might be linked with the changed lipid composition of bacterial cell wall. Cell wall of M. smegmatis expressing rv1288 had higher amount of lipids, with a significant increase in trehalose dimycolate content. Rv1288 also leads to increase in drug resistance of M. smegmatis. Rv1288 also enhanced the intracellular survival of M. smegmatis in Raw264.7 cell line. Overall, this study suggested that Rv1288, a cell wall localized carboxyl hydrolase with mycolyl-transferase activity, modulated the cell wall lipids to favor the survival of bacteria under stress condition. Title: mesT, a unique epoxide hydrolase, is essential for optimal growth of Mycobacterium tuberculosis in the presence of styrene oxide Chownk M, Sharma A, Singh K, Kaur J Ref: Future Microbiol, 12:527, 2017 : PubMed ESTHER: Chownk_2017_Future.Microbiol_12_527 PubMedSearch: Chownk 2017 Future.Microbiol 12 527 PubMedID: 28492351 Gene_locus related to this paper: myctu-LIPS Abstract AIM: mesT of Mycobacterium tuberculosis, a hypothetical/putative epoxide hydrolase, is predicted to convert toxic epoxides to the more water-soluble and less toxic diols. Detailed characterization of the protein was carried out. Title: Characterization of an extracellular protein, Rv1076 from M. tuberculosis with a potential role in humoral response Kaur G, Saini V, Kumari B, Kaur J Ref: Int J Biol Macromol, 101:621, 2017 : PubMed ESTHER: Kaur_2017_Int.J.Biol.Macromol_101_621 PubMedSearch: Kaur 2017 Int.J.Biol.Macromol 101 621 PubMedID: 28327423 Gene_locus related to this paper: myctu-Rv1076 Abstract Many mycobacterial proteins involved in lipid metabolism are reported to be essential for survival and pathogenesis of M. tuberculosis. Rv1076 of M. tuberculosis has been annotated as a putative esterase/lipase based on the consensus sequence 'GXSXG'. It is conserved in all the mycobacterial species. Therefore, in the present study we have characterized Rv1076 gene product in detail. The gene rv1076 was expressed in E. coli and purified from inclusion bodies with approx. 40% yield. The protein showed high specific activity with pNP- butyrate as preferred substrate. The enzyme was stable upto 50 degrees C and in pH range of 6-8 i.e. under acidic conditions. Ser-140, Glu-239 and His-269 were confirmed as active site residues using site directed mutagenesis. The specific activity, Km and Vmax of enzyme was determined to be 177Umg-1 protein, 334muM and 262mumolml-1min-1, respectively. Western blot analysis established Rv1076 to be an extracellular protein. Several putative immunodominant epitopes were predicted in Rv1076. Rv1076 elicited strong humoral response in both extrapulmonary and relapsed cases of TB patients. Therefore, we conclude that Rv1076 is a novel secretory esterase of M. tuberculosis which could be a potential immunodominant antigen of M. tuberculosis. Title: Enantiomeric separation of pharmaceutically important drug intermediates using a Metagenomic lipase and optimization of its large scale production Kumar R, Banoth L, Banerjee UC, Kaur J Ref: Int J Biol Macromol, 95:995, 2017 : PubMed ESTHER: Kumar_2017_Int.J.Biol.Macromol_95_995 PubMedSearch: Kumar 2017 Int.J.Biol.Macromol 95 995 PubMedID: 27984150 Abstract In the present study, efficient enzymatic methods were developed using a recombinant metagenomic lipase (LipR1) for the synthesis of corresponding esters by the transesterification of five different pharmaceutically important secondary alcohols. The recombinant lipase (specific activity=87m6U/mg) showed maximum conversion in presence of ionic liquid with Naphthyl-ethanol (eeP=99%), Indanol and Methyl-4 pyridine methanol (eeS of 98% and 99%) respectively in 1h. Vinyl acetate was found as suitable acyl donor in transesterification reactions. It was interesting to observe that maximum eeP of 85% was observed in just 15min with 1-indanol. As this enzyme demonstrated pharmaceutical applications, attempts were made to scale up the enzyme production on a pilot scale in a 5litre bioreactor. Different physical parameters affecting enzyme production and biomass concentration such as agitation rate, aeration rate and inoculum concentration were evaluated. Maximum lipase activity of 8463U/ml was obtained at 7h of cultivation at 1 lpm, 300rpm and 1.5% inoculum. Title: Rv0774c, an iron stress inducible, extracellular esterase is involved in immune-suppression associated with altered cytokine and TLR2 expression Kumar A, Singh SM, Singh R, Kaur J Ref: Int J Med Microbiol, 307:126, 2017 : PubMed ESTHER: Kumar_2017_Int.J.Med.Microbiol_307_126 PubMedSearch: Kumar 2017 Int.J.Med.Microbiol 307 126 PubMedID: 28161108 Gene_locus related to this paper: myctu-RV0774C Abstract Tuberculosis, one of the leading cause of death from infectious diseases, is caused by Mycobacterium tuberculosis. The genome of M. tuberculosis has been sequenced and nearly 40% of the whole genome sequence was categorized as hypothetical. Rv0774c was annotated as membrane exported hypothetical protein in TB database. In silico analysis revealed that Rv0774c is a paralog of PE-PGRS multi gene family with 100 aa N-terminal domain similar to PE domain of PE-PGRS proteins. Its C-terminal domain is quite different from PGRS domain, having characteristic lipase signature GXSXG & HG and catalytic residues predicted for lipolytic activity. Therefore, DNA coding for Rv0774c (303 aa), its N-terminal (1-100 aa) and C- terminal domain (100-303 aa) were separately cloned from M. tuberculosis and were over expressed in E. coli. Rv0774c gene and its C-terminal lipolytic domain preferably hydrolyzed short chain esters. Though no enzyme activity was observed in N-terminus PE like domain, it was demonstrated to enhance the thermostability of full length Rv0774c. Tetrahydrolipstatin inhibited the enzyme activity and predicted catalytic residues (Ser-185, Asp-255 and His-281) were confirmed by site directed mutagenesis. Rv0774c was secreted out in culture media by M. tuberculosis and was up-regulated in iron limiting conditions. Treatment of THP-1 cells with rRv0774c resulted in a decline in the LPS induced production of NO and expression of iNOS. rRv0774c treated THP-1 cells also showed an enhanced expression of IL-10 and TLR2. On contrary, it suppressed the LPS induced production of IL-12, chemokines MCP-1 and IL-8. Rv0774c inhibited the LPS induced phosphorylation of p38. These observations suggested that Rv0774c could modulate the pro-inflammatory immune response to support intracellular survival of the mycobacterium. Title: The immunosuppressive effects of a novel recombinant LipQ (Rv2485c) protein of Mycobacterium tuberculosis on human macrophage cell lines Kumar A, Manisha, Sangha GK, Shrivastava A, Kaur J Ref: Microb Pathog, 107:361, 2017 : PubMed ESTHER: Kumar_2017_Microb.Pathog_107_361 PubMedSearch: Kumar 2017 Microb.Pathog 107 361 PubMedID: 28412202 Gene_locus related to this paper: myctu-Rv2485c Abstract Mycobacterium tuberculosis (MTB), an intracellular pathogen, still represents a major global health challenge. A number of mycobacterial macromolecules have been shown to target biological processes within host macrophages; however, the exact mechanism for the majority of these host pathogen interactions is still poorly understood. Moreover, the lipid metabolic pathway is one of the most important physiologic pathways that plays a vital role in the survival and infection of Mycobacterium tuberculosis. In present study, we investigated the effect of rLipQ from Mycobacterium tuberculosis H37Rv on macrophage functions in vitro.Our results demonstrate that rLipQ significantly lowers the expression level of pro-inflammatory cytokines (TNF-alpha& IFN-gamma) and augments the level of anti inflammatory cytokines such as IL-4 & IL-10as compared to LPS stimulated macrophages. An up-regulation of anti-inflammatory and down-regulation of pro-inflammatory cytokines levels in rLipQ pretreated macrophages implies immuno-modulatory functions in TB patients. Interestingly, rLipQ also inhibited the expression of iNOS, TLR-2 and transcription factor NF-kB in LPS stimulated macrophages whereas the expression of TLR-4 remains unchanged. The inhibition in the expression of these signaling molecules has been correlated to the inhibition of NO production in macrophages. Taken together, these studies demonstrate that rLipQ is a novel lipase that is highly immunogenic and may play an important role in the virulence and pathogenesis of M. tuberculosis infection, by altering the balance of cytokines, which might help to assess prognosis and contribute to a better understanding against host-pathogen interactions. Title: In-Silico Characterization of a Hypothetical Protein, Rv1288 of Mycobacterium tuberculosis Containing an Esterase Signature and an Uncommon LytE Domain Kumar A, Maan P, Singh G, Kaur J Ref: Curr Comput Aided Drug Des, 13:101, 2017 : PubMed ESTHER: Kumar_2017_Curr.Comput.Aided.Drug.Des_13_101 PubMedSearch: Kumar 2017 Curr.Comput.Aided.Drug.Des 13 101 PubMedID: 27890013 Gene_locus related to this paper: myctu-yc88 Abstract BACKGROUND: Death toll due to tuberculosis is still rising day by day. Whole genome sequence of Mycobacterium tuberculosis has provided a platform to conduct research in order to identify the probable drug target. OBJECTIVES: Out of 4000 gene products of M. tuberculosis, approximately 40% of proteins are annotated as hypothetical. Identifying and characterizing these proteins could provide a new prescriptive for developing new TB drugs. Rv1288, a protein of M. tuberculosis H37Rv has been annotated as a hypothetical protein in database. Attempt has been made to assign a meaningful role to rv1288 gene product in M. tuberculosis life cycle. METHODS: A homology 3D structure of both domains was separately generated and assigned as Rv1288LytE and Rv1288est. Molecular simulation of Rv1288est was carried out for proper structure analysis. To further confirm the predictive role of Rv1288 in mycobacterium life cycle, molecular docking was performed. N-acetyl glucosamine, a major constituent of cell wall was docked with LytE domain, whereas, esterase domain was docked with lipolytic substrate, pNP-ester derivatives and inhibitors THL/PMSF. RESULTS: In-silico analysis revealed that Rv1288 is a two domain protein, an N-terminal LytE domain containing three consecutive LysM motifs and a C-terminal esterase domain of esterase D family. LytE domain has the property to bind N-acetyl glucosamine moieties of peptidoglycan, a major component of cell wall. Detailed in-silico sequence analysis revealed that this LytE domain may help in positioning the esterase domain to the cell wall of mycobacterium. Esterase domain comprised a tetrapeptide motif HGGG, a pentapeptide sequence motif GxSxG and conserved amino acid residues Ser-141, Asp-238 and His-272 which constitute a catalytic triad characteristic of other hormone sensitive lipases/ esterases. Docking studies suggested that THL and PMSF could be the potent inhibitors for Rv1288 protein. CONCLUSION: In the present investigation, we bioinformatically confirmed that Rv1288 is most likely a LytE domain containing lipolytic enzyme showing similarity to hormone sensitive lipases/esterases. Title: Standardized extract of Lactuca sativa Linn. and its fractions abrogates scopolamine-induced amnesia in mice: A possible cholinergic and antioxidant mechanism Malik J, Kaur J, Choudhary S Ref: Nutr Neurosci, :1, 2017 : PubMed ESTHER: Malik_2017_Nutr.Neurosci__1 PubMedSearch: Malik 2017 Nutr.Neurosci 1 PubMedID: 28245707 Abstract OBJECTIVES: The present study was designed to evaluate the efficacy of Lactuca sativa (LS) Linn. (Asteraceae) against scopolamine- induced amnesia and to validate its traditional claim as memory enhancer. Title: Thirty-degree shift in optimum temperature of a thermophilic lipase by a single-point mutation: effect of serine to threonine mutation on structural flexibility Sharma M, Kumar R, Singh R, Kaur J Ref: Molecular & Cellular Biochemistry, 430:21, 2017 : PubMed ESTHER: Sharma_2017_Mol.Cell.Biochem_430_21 PubMedSearch: Sharma 2017 Mol.Cell.Biochem 430 21 PubMedID: 28190170 Abstract In order to understand the molecular basis of cold adaptation, we have used directed evolution to transform a thermophilic lipase LipR1 into its psychrophilic counterpart. A single round of random mutagenesis followed by screening for improved variants yielded a mutant with single-point mutation LipR1M1 (S130T), with optimum activity at 20 degrees C. Its activity at 50 degrees C is only 20% as compared to wild type (100%). It showed catalytic rate constant (k cat) 3 times higher and a catalytic efficiency (k cat/K m) 4 times that of wild type. Circular dichroism and fluorescence studies also supported our observation of mutant structural flexibility. Structure analysis using homology models showed that Threonine 130 is exposed to solvent and has lost H-bond interaction with neighboring amino acid, thereby increasing flexibility of this lipase structure. Title: Disruption of N terminus long range non covalent interactions shifted temp.opt 25 degrees C to cold: Evolution of point mutant Bacillus lipase by error prone PCR Goomber S, Kumar A, Kaur J Ref: Gene, 576:237, 2016 : PubMed ESTHER: Goomber_2016_Gene_576_237 PubMedSearch: Goomber 2016 Gene 576 237 PubMedID: 26456196 Abstract Cold adapted enzymes have applications in detergent, textile, food, bioremediation and biotechnology processes. Bacillus lipases are 'generally recognized as safe' (GRAS) and hence are industrially attractive. Bacillus lipase of 1.4 subfamily are of lowest molecular weight and are reversibly unfolded due to absence of disulphide bonds. Therefore these are largely used to study energetic of protein stability that represents unfolding of native protein to fully unfolded state. In present study, metagenomically isolated Bacillus LipJ was laboratory evolved for cold adaptation by error Prone PCR. Library of variants were screened for high relative activity at low temperature of 10 degrees C compared to native protein LipJ. Point mutant sequenced as Phe19-->Leu was determined to be active at cold and was selected for extensive biochemical, biophysical characterization. Variant F19L showed its maximum activity at 10 degrees C where parent protein LipJ had 20% relative activity. Psychrophilic nature of F19L was established with about 50% relative active at 5 degrees C where native protein was frozen to act. Variant F19L showed no activity at temperature 40 degrees C and above, establishing its thermolabile nature. Thermostability studies determined mutant to be unstable above 20 degrees C and three fold decrease in its half life at 30 degrees C compared to native protein. Far UV-CD and intrinsic fluorescence study demonstrated unstable tertiary structure of point variant F19L leading to its unfolding at low temperature of 20 degrees C. Cold adaptation of mutant F19L is accompanied with increased specific activity. Mutant was catalytically more efficient with 1.3 fold increase in kcat. Homologue structure modelling predicted disruption of intersecondary hydrophobic core formed by aromatic ring of Phe19 with non polar residues placed at beta3, beta4, beta5, beta6, alphaF. Increased local flexibility of variant F19L explains molecular basis of its psychrophilic nature. Title: Point Mutation Ile137-Met Near Surface Conferred Psychrophilic Behaviour and Improved Catalytic Efficiency to Bacillus Lipase of 1.4 Subfamily Goomber S, Kumar A, Singh R, Kaur J Ref: Appl Biochem Biotechnol, 178:753, 2016 : PubMed ESTHER: Goomber_2016_Appl.Biochem.Biotechnol_178_753 PubMedSearch: Goomber 2016 Appl.Biochem.Biotechnol 178 753 PubMedID: 26520838 Abstract Bacillus lipolytic enzymes of subfamily 1.4 are industrially attractive because of its alkaline optimum pH and broad substrate specificity. The activity and stability of these enzymes for a limited temperature range (30-50 degrees C) need attention for its industrial application. In the present study, Bacillus subtilis LipJ was rationally designed for low-temperature adaptation. Small amino acids with lower volume and without side chain branches have high occurrence among psychrophilic proteins. Met residue is reported to be preferred for cold adaptation as it is thermolabile in nature and undergoes oxidation at high temperature. Therefore, the Ile137 residue, three residues downstream the catalytic residue Asp133, was substituted by Met. Biochemical study demonstrated that variant Ile137Met was optimally active at 20 degrees C whereas parent enzyme was most active at 37 degrees C. The variant retained 70-80 % relative activity at 10 degrees C where parent enzyme demonstrated low activity. Ile137Met was observed to be unstable at and above 30 degrees C. Kinetic study demonstrated increased K m and k cat values for variant referring improved catalytic efficiency but poor substrate affinity. Homolog modelling predicted lowered number of weak interactions by substituted Met137 as molecular basis of increased flexibility of variant. Hence, increased structure flexibility might be responsible for poor substrate affinity but increased molecular motion for higher catalysis at cold. Title: Point mutation Gln121-Arg increased temperature optima of Bacillus lipase (1.4 subfamily) by fifteen degrees Goomber S, Kumar R, Singh R, Mishra N, Kaur J Ref: Int J Biol Macromol, 88:507, 2016 : PubMed ESTHER: Goomber_2016_Int.J.Biol.Macromol_88_507 PubMedSearch: Goomber 2016 Int.J.Biol.Macromol 88 507 PubMedID: 27083848 Abstract Small molecular weight Bacillus lipases are industrially attractive because of its alkaline optimum pH, broad substrate specificity and production in high yield by overexpression both in Escherichia coli and Bacillus subtilis. Its major limitation of being mesophilic in nature is constantly targeted by laboratory evolution studies. Herein metagenomically isolated Bacillus LipJ was randomly evolved by error prone PCR and library of variants were screened for enhanced thermostability. Point mutant Gln121Arg was extensively characterized and it showed dramatic shift of Temp. opt to 50 degrees C compared to 37 degrees C for parent enzyme. Thermostability studies at 45 degrees C and 50 degrees C determined six fold increase in half life for point variant Gln121Arg compared to LipJ. Circular dichroism (CD) and tryptophan fluorescence study established enhanced thermostability of Gln121Arg. Specific activity of point variant Gln121Arg was comparable to wild type with increased substrate affinity (Km reduced). Reduced kcat for variant Gln121Arg infer that kinetic and catalytic efficiency of mutant was compromised. Structural implications by homolog modelling predicted Gln121 to be placed within longest loop of the structure at surface. Localization of loop due to additional polar interactions by Arg121 to protein core defines molecular basis of enhanced thermostability of random point variant Gln121Arg. Title: Characterization of LipN (Rv2970c) of Mycobacterium Tuberculosis H37Rv and its Probable Role in Xenobiotic Degradation Jadeja D, Dogra N, Arya S, Singh G, Kaur J Ref: Journal of Cellular Biochemistry, 117:390, 2016 : PubMed ESTHER: Jadeja_2016_J.Cell.Biochem_117_390 PubMedSearch: Jadeja 2016 J.Cell.Biochem 117 390 PubMedID: 26212120 Gene_locus related to this paper: myctu-Rv2970c Abstract LipN (Rv2970c) belongs to the Lip family of M. tuberculosis H37Rv and is homologous to the human Hormone Sensitive Lipase. The enzyme demonstrated preference for short carbon chain substrates with optimal activity at 45 degrees C/pH 8.0 and stability between pH 6.0-9.0. The specific activity of the enzyme was 217 U/mg protein with pNP-butyrate as substrate. It hydrolyzed tributyrin to di- and monobutyrin. The active-site residues of the enzyme were confirmed to be Ser216, Asp316, and His346. Tetrahydrolipstatin, RHC-80267 and N-bromosuccinimide inhibited LipN enzyme activity completely. Interestingly, Trp145, a non active-site residue, demonstrated functional role to retain enzyme activity. The enzyme was localized in cytosolic fraction of M. tuberculosis H37Rv. The enzyme was able to synthesize ester of butyric acid, methyl butyrate, in presence of methanol. LipN was able to hydrolyze 4-hydroxyphenylacetate to hydroquinone. The gene was not expressed in in-vitro growth conditions while the expression of rv2970c gene was observed post 6h of macrophage infection by M. tuberculosis H37Ra. Under individual in-vitro stress conditions, the gene was expressed during acidic stress condition only. These findings suggested that LipN is a cytosolic, acid inducible carboxylesterase with no positional specificity in demonstrating activity with short carbon chain substrates. It requires Trp145, a non active site residue, for it's enzyme activity. J. Cell. Biochem. 117: 390-401, 2016. (c) 2015 Wiley Periodicals, Inc. Title: Neurokinin-1 receptor inhibition reverses ischaemic brain injury and dementia in bilateral common carotid artery occluded rats: possible mechanisms Kaur J, Sharma S, Sandhu M Ref: Inflammopharmacology, 24:133, 2016 : PubMed ESTHER: Kaur_2016_Inflammopharmacology_24_133 PubMedSearch: Kaur 2016 Inflammopharmacology 24 133 PubMedID: 27380492 Abstract BACKGROUNDS: Increase in SP release as a function of hypoxia of the rat carotid body is a tissue response to ischemia that leads to neurogenic inflammation and cognitive deficits. Substance P-mediated inflammation is reported to attenuate the neuroprotective PPAR-gamma. This study was undertaken to investigate the effect of aprepitant, a substance P-NK1 receptor antagonist in bilateral carotid artery occlusion (BCCAO)-induced ischaemic brain injury and vascular dementia. Title: Accelerated Stability Studies on Dried Extracts of Centella asiatica Through Chemical, HPLC, HPTLC, and Biological Activity Analyses Kaur I, Suthar N, Kaur J, Bansal Y, Bansal G Ref: J Evid Based Complementary Altern Med, 21:NP127, 2016 : PubMed ESTHER: Kaur_2016_J.Evid.Based.Complementary.Altern.Med_21_NP127 PubMedSearch: Kaur 2016 J.Evid.Based.Complementary.Altern.Med 21 NP127 PubMedID: 27486096 Abstract Regulatory guidelines recommend systematic stability studies on a herbal product to establish its shelf life. In the present study, commercial extracts (Types I and II) and freshly prepared extract (Type III) of Centella asiatica were subjected to accelerated stability testing for 6 months. Control and stability samples were evaluated for organoleptics, pH, moisture, total phenolic content (TPC), asiatic acid, kaempherol, and high-performance thin layer chromatography fingerprints, and for antioxidant and acetylcholinesterase inhibitory activities. Markers and TPC and both the activities of each extract decreased in stability samples with respect to control. These losses were maximum in Type I extract and minimum in Type III extract. Higher stability of Type III extract than others might be attributed to the additional phytoconstituents and/or preservatives in it. Pearson correlation analysis of the results suggested that TPC, asiatic acid, and kaempferol can be taken as chemical markers to assess chemical and therapeutic shelf lives of herbal products containing Centella asiatica. Title: Functional characterization of hypothetical proteins of Mycobacterium tuberculosis with possible esterase/lipase signature: A Cumulative in silico and in vitro approach Kumar A, Sharma A, Kaur G, Makkar P, Kaur J Ref: J Biomol Struct Dyn, 35:1226, 2016 : PubMed ESTHER: Kumar_2016_J.Biomol.Struct.Dyn_35_1226 PubMedSearch: Kumar 2016 J.Biomol.Struct.Dyn 35 1226 PubMedID: 27050490 Gene_locus related to this paper: myctu-MT1628, myctu-RV0421C, myctu-RV0519C, myctu-RV0774C, myctu-Rv1191, myctu-Rv3591c Abstract The functional aspect of several mycobacterium proteins annotated as hypothetical are yet to be discovered. In the present investigation, in silico approaches were used to predict the biological function of some of the unknown Mtb proteins, which were further validated by wet lab experiments. After screening thousands of Mtb proteins, functionally unknown hypothetical proteins Rv0421c, Rv0519c, Rv0774c, Rv1191, Rv1592c and Rv3591c were chosen on the basis of their importance in Mtb life cycle. All these proteins posses the alpha/beta-hydrolase topological fold, characteristic of lipases/esterases, with serine, aspartate, and histidine as the putative members of the catalytic triad. The catalytic serine is located in pentapeptide motif "GXSXG" and oxyanion residue is in dipeptide motif HG. To further support our observation, molecular docking was performed with conventional synthetic lipolytic substrates (pNP-esterss) and specific lipase/esterase inhibitors (tetrahydrolipstatin, PMSF). Significant docking score and strong interaction of substrates/inhibitors with these proteins revealed that these could be possible lipases/esterases. To validate the in silico studies, these genes were cloned from Mtb genome and the proteins were over-expressed in pQE-30/Escherichia coli M15 system. The expressed proteins were purified to homogeneity and enzymatic activity was determined by using pNP esters as substrate. The enzyme activity of recombinant proteins was inhibited by tetrahydrolipstatin and PMSF pre-treatment. Outcome of the present investigation provided a basic platform to analyse and characterize unknown hypothetical proteins. Title: Intrinsically Unstructured Carboxy Terminus of Bacillus Lipase is Essential for its Function Khurana J, Manisha, Singh R, Kaur J Ref: Protein Pept Lett, 21:1265, 2014 : PubMed ESTHER: Khurana_2014_Protein.Pept.Lett_21_1265 PubMedSearch: Khurana 2014 Protein.Pept.Lett 21 1265 PubMedID: 25001212 Abstract We have identified intrinsically unstructured C-terminus of a Bacillus lipase. In an effort to understand the possible role of this C-terminus unstructured region, 10, 20 and 30 amino acids were serially deleted from C-terminal region of the lipase. The catalytic properties of wild type and resulted truncated enzymes were compared. Deletion of 10 amino acids from C-terminus region resulted in decrease in transcription of lipase, specific enzyme activity and extracellular secretion of lipase in comparison to wild type while no effect on lipase aggregation was observed. Negligible activity was observed upon deletion of 20 amino acids. The homology model of the protein demonstrated that the tertiary structure of the protein was held together by these C-terminus residues due to six critically placed hydrogen bonds. Therefore C terminus was essential for the tertiary structure and enzyme activity of lipase. Due to structural flexibility and plasticity originating from the lack of a definite-ordered 3D structure, such disordered regions might represent a major functional advantage for proteins. Title: Combinatorial reshaping of a lipase structure for thermostability: additive role of surface stabilizing single point mutations Kumar R, Singh R, Kaur J Ref: Biochemical & Biophysical Research Communications, 447:626, 2014 : PubMed ESTHER: Kumar_2014_Biochem.Biophys.Res.Commun_447_626 PubMedSearch: Kumar 2014 Biochem.Biophys.Res.Commun 447 626 PubMedID: 24751523 Abstract Thermostable lipases are of high priority for industrial applications. In the present study, targeted improvement of the thermostability of a lipase from metagenomic origin was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. A variant (LipR5) was generated after combination of two thermostabilizing mutations (R214C & N355K). Thermostability of the variant enzyme was analyzed by half-life measurement and circular dichroism (CD). To assess whether catalytic properties were affected by mutation, the optimal reaction conditions were determined. The protein LipR5, displayed optimum activity at 50 degrees C and pH 8.0. It showed two fold enhancement in thermostability (at 60 degrees C) as compared to LipR3 (R214C) and nearly 168 fold enhancement as compared to parent enzyme (LipR1). Circular dichroism and fluorescence study suggest that the protein structure had become more rigid and stable to denaturation. Study of 3D model suggested that Lys355 was involved in formation of a Hydrogen bond with OE1 of Glu284. Lys355 was also making salt bridge with OE2 of Glu284. Title: Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay Sharma P, Jindal S, Bala K, Kumari K, Niharika N, Kaur J, Pandey G, Pandey R, Russell RJ and Lal R <1 more author(s)> Sharma P, Jindal S, Bala K, Kumari K, Niharika N, Kaur J, Pandey G, Pandey R, Russell RJ, Oakeshott JG, Lal R (- 1) Ref: Biodegradation, 25:179, 2014 : PubMed ESTHER: Sharma_2014_Biodegradation_25_179 PubMedSearch: Sharma 2014 Biodegradation 25 179 PubMedID: 23740574 Abstract Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified. Title: Insights into controlling role of substitution mutation, E315G on thermostability of a lipase cloned from metagenome of hot spring soil Sharma PK, Kumar R, Garg P, Kaur J Ref: 3 Biotech, 4:189, 2014 : PubMed ESTHER: Sharma_2014_3.Biotech_4_189 PubMedSearch: Sharma 2014 3.Biotech 4 189 PubMedID: 28324449 Abstract Rational mutagenesis was performed (at the vicinity of the active site residues D317 and H358 of a mature polypeptide) to investigate the role of amino acids in the thermostability/activity of a lipase enzyme. The single variant enzyme created with E315G (lip M2) mutation near one of the active site residue (D317) found to be an important residue in controlling the thermal stability, the variant with E315G mutation demonstrated biochemical properties similar to that of native lipase. However, we found that this mutation strongly affected the activity and stability of the lip M1 mutant, reported in our previous study (Sharma et al. in Gene 491:264-271, 2012b). The dual mutant with E315G/N355K mutation in the Wt showed small increase in the protein thermostability compared to the native lipase, however, the thermostability of the mutant lip M1 was reduced several fold. Presumably, E315G (lip M2) mutation reverted the thermostability evolved by N355K (lip M1). The native and variant enzymes also displayed large variation in enzyme kinetics and their preference for pNP-esters (substrates). We further generated 3D models and studied the loop modelling of the WT and variants. Interestingly, loop region Leu314-Asn321 showed structural flexibility on introducing E315G mutation in the native lipase. On the other hand, lysine in mutant N355K exhibited side chain conformational changes in the loop Thr353-His358 which resulted in its H-bonding with Glu284. In addition, replacing glutamic acid by glycine at 315 position in lip M3 distorted the electrostatic interactions between Glu315 and Lys355 in the flexible loop region Leu314-Asn321. Title: Microbial degradation of an organophosphate pesticide, malathion Singh B, Kaur J, Singh K Ref: Crit Rev Microbiol, 40:146, 2014 : PubMed ESTHER: Singh_2014_Crit.Rev.Microbiol_40_146 PubMedSearch: Singh 2014 Crit.Rev.Microbiol 40 146 PubMedID: 23442144 Abstract Abstract Organophosphorus pesticide, malathion, is used in public health, residential, and agricultural settings worldwide to control the pest population. It is proven that exposure to malathion produce toxic effects in humans and other mammals. Due to high toxicity, studies are going on to design effective methods for removal of malathion and its associated compounds from the environment. Among various techniques available, degradation of malathion by microbes proves to be an effective and environment friendly method. Recently, research activities in this area have shown that a diverse range of microorganisms are capable of degrading malathion. Therefore, we aimed at providing an overview of research accomplishments on this subject and discussed the toxicity of malathion and its metabolites, various microorganisms involved in its biodegradation and effect of various environmental parameters on its degradation. Title: Draft Genome Sequence of Agrobacterium sp. Strain UHFBA-218, Isolated from Rhizosphere Soil of Crown Gall-Infected Cherry Rootstock Colt Dua A, Sangwan N, Kaur J, Saxena A, Kohli P, Gupta AK, Lal R Ref: Genome Announc, 1:, 2013 : PubMed ESTHER: Dua_2013_Genome.Announc_1_E00302 PubMedSearch: Dua 2013 Genome.Announc 1 E00302 PubMedID: 23723402 Gene_locus related to this paper: rhird-m8b858, rhird-m8b836, rhird-a0a067u386, rhird-m8b397 Abstract We report here the draft genome sequence of the alphaproteobacterium Agrobacterium sp. strain UHFBA-218, which was isolated from rhizosphere soil of crown gall-infected cherry rootstock Colt. The draft genome of strain UHFBA-218 consists of 112 contigs (5,425,303 bp) and 5,063 coding sequences with a G+C content of 59.8%. Title: Draft Genome Sequence of a Hexachlorocyclohexane-Degrading Bacterium, Sphingobium baderi Strain LL03T Kaur J, Verma H, Tripathi C, Khurana JP, Lal R Ref: Genome Announc, 1:, 2013 : PubMed ESTHER: Kaur_2013_Genome.Announc_1_e00751 PubMedSearch: Kaur 2013 Genome.Announc 1 e00751 PubMedID: 24051322 Gene_locus related to this paper: 9sphn-t0g9p6, 9sphn-t0g1r2 Abstract Sphingobium baderi strain LL03(T) was isolated from hexachlorocyclohexane (HCH)-contaminated soil from Spolana, Czech Republic. Strain LL03(T) is a mutant that is deficient in linB and linC (genes that encode hexachlorocyclohexane haloalkane dehalogenase and dehydrogenase, respectively). The draft genome sequence of LL03(T) (~4.85 Mb) consists of 92 contigs and 4,914 coding sequences, with a G+C content of 63.5%. Title: Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability Kumar R, Sharma M, Singh R, Kaur J Ref: Molecular & Cellular Biochemistry, 373:149, 2013 : PubMed ESTHER: Kumar_2013_Mol.Cell.Biochem_373_149 PubMedSearch: Kumar 2013 Mol.Cell.Biochem 373 149 PubMedID: 23104399 Abstract In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 degrees C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 degrees C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity. Title: Genome sequence of Sphingobium indicum B90A, a hexachlorocyclohexane-degrading bacterium Anand S, Sangwan N, Lata P, Kaur J, Dua A, Singh AK, Verma M, Khurana JP, Khurana P and Lal R <1 more author(s)> Anand S, Sangwan N, Lata P, Kaur J, Dua A, Singh AK, Verma M, Khurana JP, Khurana P, Mathur S, Lal R (- 1) Ref: Journal of Bacteriology, 194:4471, 2012 : PubMed ESTHER: Anand_2012_J.Bacteriol_194_4471 PubMedSearch: Anand 2012 J.Bacteriol 194 4471 PubMedID: 22843598 Gene_locus related to this paper: sphju-d4z363, sphpi-linb Abstract Sphingobium indicum B90A, an efficient degrader of hexachlorocyclohexane (HCH) isomers, was isolated in 1990 from sugarcane rhizosphere soil in Cuttack, India. Here we report the draft genome sequence of this bacterium, which has now become a model system for understanding the genetics, biochemistry, and physiology of HCH degradation. Title: Engineering of a metagenome derived lipase toward thermal tolerance: effect of asparagine to lysine mutation on the protein surface Sharma PK, Kumar R, Mohammad O, Singh R, Kaur J Ref: Gene, 491:264, 2012 : PubMed ESTHER: Sharma_2012_Gene_491_264 PubMedSearch: Sharma 2012 Gene 491 264 PubMedID: 22001407 Abstract A highly thermostable mutant lipase was generated and characterized. Mutant enzyme demonstrated 144 fold enhanced thermostability over the wild type enzyme at 60 degrees C. Interestingly, the overall catalytic efficiency (k(cat/)K(m)) of mutant was also enhanced (~20 folds). Circular dichroism spectroscopy, studied as function of temperature, demonstrated that the mutant lipase retained its secondary structure up to 70-80 degrees C, whereas wild type protein structure was completely distorted above 35 degrees C. Additionally, the intrinsic tryptophan fluorescence (a probe for the tertiary structure) also displayed difference in the conformation of two enzymes during temperature dependent unfolding. Furthermore, mutation N355K resulted in extensive H-bonding (Lys355 HZ1OE2 Glu284) with a distance 2.44 A. In contrast to this, Wt enzyme has not shown such H-bonding interaction. Title: Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature Sharma PK, Singh K, Singh R, Capalash N, Ali A, Mohammad O, Kaur J Ref: Mol Biol Rep, 39:2795, 2012 : PubMed ESTHER: Sharma_2012_Mol.Biol.Rep_39_2795 PubMedSearch: Sharma 2012 Mol.Biol.Rep 39 2795 PubMedID: 21678056 Abstract A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50 degrees C and pH 9.0. It showed thermal stability up to 40 degrees C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50 degrees C even after incubation for 75 min. However above 50 degrees C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60 degrees C. Interestingly the CD spectroscopic study carried out in the temperature range of 25-95 degrees C revealed distortion in solution structure above 35 degrees C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35 degrees C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K ( m ), V ( max ) and K ( cat ) of 0.73 +/- 0.18 muM, 239 +/- 16 mumol/ml/min and 569 s(-1) respectively. Enzyme activity was strongly inhibited by CuCl(2), HgCl(2) and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone. Title: Whole genome sequence of the rifamycin B-producing strain Amycolatopsis mediterranei S699 Verma M, Kaur J, Kumar M, Kumari K, Saxena A, Anand S, Nigam A, Ravi V, Raghuvanshi S and Lal R <3 more author(s)> Verma M, Kaur J, Kumar M, Kumari K, Saxena A, Anand S, Nigam A, Ravi V, Raghuvanshi S, Khurana P, Tyagi AK, Khurana JP, Lal R (- 3) Ref: Journal of Bacteriology, 193:5562, 2011 : PubMed ESTHER: Verma_2011_J.Bacteriol_193_5562 PubMedSearch: Verma 2011 J.Bacteriol 193 5562 PubMedID: 21914879 Gene_locus related to this paper: amymu-d8hka5, amymu-d8hpp2, amymu-d8hu68, amymu-d8hy73, amymu-d8i2j5, amymu-d8i8i8, amyms-g0fkj6, amyms-g0g7f0, amyms-g0fps5 Abstract Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences. Title: Lipid hydrolizing enzymes in virulence: Mycobacterium tuberculosis as a model system Singh G, Jadeja D, Kaur J Ref: Crit Rev Microbiol, 36:259, 2010 : PubMed ESTHER: Singh_2010_Crit.Rev.Microbiol_36_259 PubMedSearch: Singh 2010 Crit.Rev.Microbiol 36 259 PubMedID: 20500016 Abstract This review is focused on the virulent traits of lipolytic enzymes from bacteria with special emphasis on Mycobacterium tuberculosis. In vivo, triacylglycerols in the form of inclusion bodies are present in tubercle bacilli in the lungs. This pathogenic bacterium possesses a lipase gene (Lip) family, which is expressed and differentially regulated under a variety of in vitro conditions. Not much research work has been carried out on these lipolytic enzymes. A better understanding of lipolytic enzymes in mycobacteria would lead to develop new strategies for tuberculosis treatment. The present review highlights the recent work done in the field of mycobacterium lipolytic enzymes and their involvement in the virulence and pathogenicity. Title: A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: purification and characterization Nawani N, Khurana J, Kaur J Ref: Molecular & Cellular Biochemistry, 290:17, 2006 : PubMed ESTHER: Nawani_2006_Mol.Cell.Biochem_290_17 PubMedSearch: Nawani 2006 Mol.Cell.Biochem 290 17 PubMedID: 16924423 Abstract A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60-65 ( composite function)C and retained 100% activity after incubation at 60 ( composite function)C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ( composite function)C and 23 min incubation at 80 ( composite function)C. Catalytic function of lipase was activated by Mg(++) (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, betaME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K ( m ) and V (max) of 0.5 mM and 0.139 microM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap. Title: Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp Dosanjh NS, Kaur J Ref: Protein Expr Purif, 24:71, 2002 : PubMed ESTHER: Dosanjh_2002_Protein.Expr.Purif_24_71 PubMedSearch: Dosanjh 2002 Protein.Expr.Purif 24 71 PubMedID: 11812225 Abstract An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C. Title: Oxyaniliniums as acetylcholinesterase inhibitors for the reversal of neuromuscular block Grove SJ, Kaur J, Muir AW, Pow E, Tarver GJ, Zhang MQ Ref: Bioorganic & Medicinal Chemistry Lett, 12:193, 2002 : PubMed ESTHER: Grove_2002_Bioorg.Med.Chem.Lett_12_193 PubMedSearch: Grove 2002 Bioorg.Med.Chem.Lett 12 193 PubMedID: 11755352 Abstract A series of oxyanilinium-based AChE inhibitors have been synthesised and tested for the reversal of vecuronium-induced neuromuscular block Several compounds for example 2-hydroxy and 2-methoxy-N,N-dimethyl-N-allylanilinium bromide 3 and 6 showed comparable reversal potencies to edrophonium and clean in vivo cardiovascular profiles Title: Molecular modelling and QSAR of reversible acetylcholines-terase inhibitors Kaur J, Zhang MQ Ref: Curr Med Chem, 7:273, 2000 : PubMed ESTHER: Kaur_2000_Curr.Med.Chem_7_273 PubMedSearch: Kaur 2000 Curr.Med.Chem 7 273 PubMedID: 10637365 Inhibitor(s) related to this paper: Aricept~Donepezil~E2020 Abstract Acetylcholinesterase (AChE) inhibitors are an important class of medicinal agents useful for the treatment of Alzheimer s disease, glaucoma, myasthenia gravis and for the recovery of neuromuscular block in surgery. To rationalize the structural requirements of AChE inhibitors we attempt to derive a coherent AChE-inhibitor recognition pattern based on literature data of molecular modelling and quantitative structure-activity relationship (QSAR) analyses. These data are summarised from nearly all therapeutically important chemical classes of reversible AChE inhibitors, e.g., derivatives of physostigmine, tacrine, donepezil and huperzine A. Interactions observed from X-ray crystallography between these inhibitors and AChE have also been incorporated and compared with modelling and QSAR results. It is concluded that hydrophobicity and the presence of an ionizable nitrogen are the pre-requisites for the inhibitors to interact with AChE. However the mode of interaction i.e., the 3-dimensional (3D) positioning of the inhibitor in the active site of the enzyme varies among different chemical classes. It is also recognised that water molecules play crucial roles in defining these different 3D positioning. The information on AChE-inhibitor interactions provided should be useful for future discovery of new chemical classes of AChE inhibitors, especially from De Novo design and hybrid construction. | |||||||
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