Cholinesterases act as bio scavengers to clear organophosphorus (OP) compounds and prodrugs. The butyrylcholinesterase (BChE) gene has been found in several types of teleost fish but this gene has yet to be identified in cyprinid fish. Indeed, BChE homologs have not been found in the zebrafish (Danio rerio) genomic database. Here, we demonstrate that BChE activity is present in zebrafish, in line with other groups' findings. Using in-gel native-PAGE enzymatic activity staining and LC-MS/MS technique, an atypical BChE-like protein was identified in zebrafish. The si:ch211-93f2.1 gene was cloned, and His-tagged recombinant protein was expressed using the Pichia yeast system. The purified protein (molecular weight ~ 180 kDa) showed BChE activity, and degraded acetylcholinesterase (ACh) at a higher rate than BCh. However, phylogram analysis shows that this novel cholinesterase shared an evolutionary origin with carboxylic esterase rather than BChE. The zebrafish BChE-like protein shares structural characteristics with cholinesterase and carboxylesterase. The 2-arachidonoylglycerol (2-AG), nicosulfuron, and triacetin exhibited a higher binding affinity to the zebrafish BChE-like protein than BCh and ACh. With the identification of BChE-like protein in zebrafish, this study could shed light on the origin of BChE and may contribute towards the development of a BChE knockout zebrafish model for sensitive drug or toxin screening.
The spike protein of SARS-CoV-2 and the host ACE2 receptor plays a vital role in the entry to the cell. Among which the hotspot residue 501 is continuously subjected to positive selection pressure and induces unusual virulence. Keeping in view the importance of the hot spot residue 501, we predicted the potentially emerging structural variants of 501 residue. We analyzed the binding pattern of wild type and mutants (Spike RBD) to the ACE2 receptor by deciphering variations in the amino acids' interaction networks by graph kernels along with evolutionary, network metrics, and energetic information. Our analysis revealed that N501I, N501T, and N501V increase the binding affinity and alter the intra and inter-residue bonding networks. The N501T has shown strong positive selection and fitness in other animals. Docking results and repeated simulations (three times) confirmed the structural stability and tighter binding of these three variants, correlated with the previous results following the global stability trend. Consequently, we reported three variants N501I, N501T, and N501V could worsen the situation further if they emerged. The relations between the viral fitness and binding affinity is a complicated game thus the emergence of high affinity mutations in the SARS-CoV-2 RBD brings up the question of whether or not positive selection favours these mutations or not?
Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.
Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.
DEPTOR is an inhibitor of the mTOR kinase which controls cell growth. DEPTOR consists of two DEP domains and a PDZ domain connected by an unstructured linker, and its stability is tightly regulated through post-translational modifications of its linker region that contains the 286SSGYFS291 degron. Based on the mTORC1 complex, our modelling suggests a possible spatial arrangement of DEPTOR which is characterised to form a dimer. Our model shows that the two PDZ domains of a DEPTOR dimer bind separately to the dimeric mTOR's FAT domains ~130 Å apart, while each of the two extended linkers is sufficiently long to span from the FAT domain to the kinase domain of mTOR and beyond to join a shared dimer of the DEP domains. This places the linker's S299 closest to the kinase's catalytic site, indicating that phosphorylation would start with it and successively upstream towards DEPTOR's degron. The CK1α kinase is reportedly responsible for the phosphorylation of the degron, and our docking analysis further reveals that CK1α contains sites to bind DEPTOR's pS286, pS287 and pT295, which may act as priming phosphates for the phosphorylation of the degron's S291. DEPTOR's linker can also be ubiquitylated by the UbcH5A-SCFβ-TrCP complex without its PDZ dissociating from mTOR according to the modelling. As the catalytic cleft of mTOR's kinase is restricted, interactions between the kinase's unstructured segment surrounding the cleft and DEPTOR's linker, which may involve S293 and S299, may be critical to controlling DEPTOR's access to the catalytic cleft and hence its phosphorylation by mTOR in a manner dependent on mTOR's activation.
Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD) of heat shock 70 kDa protein (PDB: 1HJO) with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD) simulation. Human DNA binding domain of p53 motif (SCMGGMNR) retrieved from UniProt (UniProtKB: P04637) was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were -0.44 Kcal/mol and -9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.
Deposition of amyloid protein, particularly Aβ1-42 , is a major contributor to the onset of Alzheimer's disease (AD). However, almost no deposition of Aβ in the peripheral tissues could be found. Human serum albumin (HSA), the most abundant protein in the blood, has been reported to inhibit amyloid formation through binding Aβ, which is believed to play an important role in the peripheral clearance of Aβ. We identified the Aβ binding site on HSA and developed HSA mutants with high binding capacities for Aβ using a phage display method. HSA fragment 187-385 (Domain II) was found to exhibit the highest binding capacity for Aβ compared with the other two HSA fragments. To elucidate the sequence that forms the binding site for Aβ on Domain II, a random screening of Domain II display phage biopanning was constructed. A number of mutants with higher Aβ binding capacities than the wild type were identified. These mutants exhibited stronger scavenging abilities than the wild type, as revealed via in vitro equilibrium dialysis of Aβ experiments. These findings provide useful basic data for developing a safer alternative therapy than Aβ vaccines and for application in plasma exchange as well as extracorporeal dialysis.
This study investigated combination nanocarrier and microwave system for α-tocopherol and γ-tocotrienol delivery against dermatitis, without skin thinning effect of steroids. The vitamin E was formulated into water-rich/water-poor nanoemulsions, and had their droplet size, zeta potential, morphology, therapeutic content, encapsulation efficiency and release, in vitro skin therapeutics/nanoemulsion penetration, retention and permeation profiles, and in vivo pharmacodynamics characteristics examined, with skin pre-treated by precision microwave when applicable. The nanoemulsions had droplet sizes <150 nm and negative zeta potential values. The skin pre-treatment by microwave (1 mW/3985 MHz) promoted therapeutics accumulation in epidermis through enhancing nanoemulsion penetration into skin. The combination nano- and microwave technologies fluidized skin lipid and protein domains with epidermal microstructures being fluidized to a greater extent than dermis, allowing a relatively high epidermal-to-dermal nanoemulsion distribution. Microwave of lower or higher than 3985 MHz brought about lower skin therapeutics/nanoemulsion accumulation due to insufficient lipid/protein domain fluidization or microwave-skin interaction limiting at skin surfaces only. Using water-rich nanoemulsion with higher therapeutic release and skin pre-treatment with 3985 MHz microwave, dermatitis was alleviated in vivo without skin thinning of standard steroid. The use of combination microwave and nanotechnology promotes vitamin delivery and translates to positive dermatitis treatment outcome that warrants future investigation.
[Formula: see text]-Helical transmembrane proteins are the most important drug targets in rational drug development. However, solving the experimental structures of these proteins remains difficult, therefore computational methods to accurately and efficiently predict the structures are in great demand. We present an improved structure prediction method TMDIM based on Park et al. (Proteins 57:577-585, 2004) for predicting bitopic transmembrane protein dimers. Three major algorithmic improvements are introduction of the packing type classification, the multiple-condition decoy filtering, and the cluster-based candidate selection. In a test of predicting nine known bitopic dimers, approximately 78% of our predictions achieved a successful fit (RMSD <2.0 Å) and 78% of the cases are better predicted than the two other methods compared. Our method provides an alternative for modeling TM bitopic dimers of unknown structures for further computational studies. TMDIM is freely available on the web at https://cbbio.cis.umac.mo/TMDIM . Website is implemented in PHP, MySQL and Apache, with all major browsers supported.
Lipase plays an important role in industrial and biotechnological applications. Lipases have been subject to modification at the N and C terminals, allowing better understanding of lipase stability and the discovery of novel properties. A thermotolerant lipase has been isolated from Antarctic Pseudomonas sp. The purified Antarctic AMS3 lipase (native) was found to be stable across a broad range of temperatures and pH levels. The lipase has a partial Glutathione-S-transferase type C (GST-C) domain at the N-terminal not found in other lipases. To understand the influence of N-terminal GST-C domain on the biochemical and structural features of the native lipase, the deletion of the GST-C domain was carried out. The truncated protein was successfully expressed in E. coli BL21(DE3). The molecular weight of truncated AMS3 lipase was approximately ~45 kDa. The number of truncated AMS3 lipase purification folds was higher than native lipase. Various mono and divalent metal ions increased the activity of the AMS3 lipase. The truncated AMS3 lipase demonstrated a similarly broad temperature range, with the pH profile exhibiting higher activity under alkaline conditions. The purified lipase showed a substrate preference for a long carbon chain substrate. In addition, the enzyme activity in organic solvents was enhanced, especially for toluene, Dimethylsulfoxide (DMSO), chloroform and xylene. Molecular simulation revealed that the truncated lipase had increased structural compactness and rigidity as compared to native lipase. Removal of the N terminal GST-C generally improved the lipase biochemical characteristics. This enzyme may be utilized for industrial purposes.
The inability of existing vaccines to cope with the mutation rate has highlighted the need for effective preventative strategies for COVID-19. Through the secretion of immunoglobulin A, mucosal delivery of vaccines can effectively stimulate mucosal immunity for better protection against SARS-CoV-2 infection. In this study, various immunoinformatic tools were used to design a multi-epitope oral vaccine against SARS-CoV-2 based on its receptor-binding domain (RBD) and heptad repeat (HR) domains. T and B lymphocyte epitopes were initially predicted from the RBD and HR domains of SARS-CoV-2, and potential antigenic, immunogenic, non-allergenic, and non-toxic epitopes were identified. Epitopes that are highly conserved and have no significant similarity to human proteome were selected. The epitopes were joined with appropriate linkers, and an adjuvant was added to enhance the vaccine efficacy. The vaccine 3D structure constructs were docked with toll-like receptor 4 (TLR-4) and TLR1-TLR2, and the binding affinity was calculated. The designed multi-epitope vaccine construct (MEVC) consisted of 33 antigenic T and B lymphocyte epitopes. The results of molecular dockings and free binding energies confirmed that the MEVC effectively binds to TLR molecules, and the complexes were stable. The results suggested that the designed MEVC is a potentially safe and effective oral vaccine against SARS-CoV-2. This in silico study presents a novel approach for creating an oral multi-epitope vaccine against the rapidly evolving SARS-CoV-2 variants. These findings offer valuable insights for developing an effective strategy to combat COVID-19. Further preclinical and clinical studies are required to confirm the efficacy of the MEVC vaccine.
Carbapenem resistance in Gram-negative pathogens has become a global concern for health workers worldwide. In one of our earlier studies, a Klebsiella pneumoniae-carbapenemase-2 producing strain was induced with meropenem to explore differentially expressed proteins under induced and uninduced conditions. There is, LysM domain BON family protein, was found over 12-fold expressed under the induced state. A hypothesis was proposed that LysM domain protein might have an affinity towards carbapenem antibiotics making them unavailable to bind with their target. Hence, we initiated a study to understand the binding mode of carbapenem with LysM domain protein. MICs of imipenem and meropenem against LysM clone were increased by several folds as compared to NP-6 clinical strain as well as DH5 α (PET-28a KPC-2) clone. This study further revealed a strong binding of both antibiotics to LysM domain protein. Molecular simulation studies of LysM domain protein with meropenem and imipenem for 80 ns has also showed stable structure. We concluded that overexpressed LysM domain under induced condition interacted with carbapenems, leading to enhanced resistance as proved by high MIC values. Hence, the study proved the proposed hypothesis that the LysM domain plays a significant role in the putative mechanism of antibiotics resistance.
The murine double minute 2 (MDM2) protein is a major negative regulator of the tumour suppressor protein p53. Under normal conditions, MDM2 constantly binds to p53 transactivation domain and/or ubiquinates p53 via its role as E3 ubiquitin ligase to promote p53 degradation as well as nuclear export to maintain p53 levels in cells. Meanwhile, amplification of MDM2 and appearance of MDM2 spliced variants occur in many tumours and normal tissues making it a prognostic indicator for human cancers. The mutation or deletion of p53 protein in half of human cancers inactivates its tumour suppressor activity. However, cancers with wild type p53 have its function effectively inhibited through direct interaction with MDM2 oncoprotein. Here, we described the construction of a MDM2 spliced variant (rMDM215kDa) consisting of SWIB/MDM2 domain and its central region for antibody generation. Biopanning with a human naïve scFv library generated four scFv clones specific to rMDM215kDa. Additionally, the selected scFv clones were able to bind to the recombinant full length MDM2 (rMDM2-FL). Computational prediction showed that the selected scFv clones potentially bind to exon 7-8 of MDM2 while leaving the MDM2/SWIB domain free for p53 interaction. The developed antibodies exhibit good specificity can be further investigated for downstream biomedical and research applications.
CATH (https://www.cathdb.info) identifies domains in protein structures from wwPDB and classifies these into evolutionary superfamilies, thereby providing structural and functional annotations. There are two levels: CATH-B, a daily snapshot of the latest domain structures and superfamily assignments, and CATH+, with additional derived data, such as predicted sequence domains, and functionally coherent sequence subsets (Functional Families or FunFams). The latest CATH+ release, version 4.3, significantly increases coverage of structural and sequence data, with an addition of 65,351 fully-classified domains structures (+15%), providing 500 238 structural domains, and 151 million predicted sequence domains (+59%) assigned to 5481 superfamilies. The FunFam generation pipeline has been re-engineered to cope with the increased influx of data. Three times more sequences are captured in FunFams, with a concomitant increase in functional purity, information content and structural coverage. FunFam expansion increases the structural annotations provided for experimental GO terms (+59%). We also present CATH-FunVar web-pages displaying variations in protein sequences and their proximity to known or predicted functional sites. We present two case studies (1) putative cancer drivers and (2) SARS-CoV-2 proteins. Finally, we have improved links to and from CATH including SCOP, InterPro, Aquaria and 2DProt.
Understanding Macrobrachium rosenbergii ovarian maturation control at the genome level is an important aspect for increasing larvae production. In this study, an ovarian maturation related gene, M. rosenbergii vWD domain and three Kazal-type domains of a gene (MrvWD-Kazal) have been studied. The MrvWD-Kazal gene was isolated using a rapid amplification of cDNA end (RACE) method and the relative abundances of MrvWD-Kazal mRNA in the ovary, hepatopancreas, stomach, intestine and gill were determined by using the quantitative PCR technique. The MrvWD-Kazal gene is composed of 2194 bp with an open reading frame (ORF) of 1998 bp encoding 665 amino acids and has great similarity to the M. nipponense vWD-Kazal gene (91%). The qPCR analyses indicated the relative abundance of MrvWD-Kazal mRNA transcript varied among different stages of ovarian function (P < 0.05), but there were no differences abundance in hepatopancreas, stomach, intestine and gill (P> 0.05). In the ovary, relative abundance of MrvWD-Kazal mRNA transcript gradually increased with ovarian maturation from Stages 1 (Spent; 1.00-fold), to 2 (Proliferative; 3.47-fold) to 3 (Premature; 6.18-fold) and decreased at Stage 4 (Mature; 1.31-fold). Differential relative abundances of MrvWD-Kazal mRNA transcript in the ovary indicate the MrvWD-Kazal protein may have an important function in ovarian maturation of M. rosenbergii. The results of this study also indicate the MrvWD-Kazal is not involved in regulation of the reproductive related function of the hepatopancreas, digestive system (stomach and intestine) and respiratory system (gill).
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration to the cerebral microvasculature via binding of DBLβ domains to intercellular adhesion molecule 1 (ICAM1) and is associated with severe cerebral malaria. In a cohort of 187 young children from Papua New Guinea (PNG), we examined baseline levels of antibody to the ICAM1-binding PfEMP1 domain, DBLβ3PF11_0521, in comparison to four control antigens, including NTS-DBLα and CIDR1 domains from another group A variant and a group B/C variant. Antibody levels for the group A antigens were strongly associated with age and exposure. Antibody responses to DBLβ3PF11_0521 were associated with a 37% reduced risk of high-density clinical malaria in the follow-up period (adjusted incidence risk ratio [aIRR] = 0.63 [95% confidence interval {CI}, 0.45 to 0.88; P = 0.007]) and a 25% reduction in risk of low-density clinical malaria (aIRR = 0.75 [95% CI, 0.55 to 1.01; P = 0.06]), while there was no such association for other variants. Children who experienced severe malaria also had significantly lower levels of antibody to DBLβ3PF11_0521 and the other group A domains than those that experienced nonsevere malaria. Furthermore, a subset of PNG DBLβ sequences had ICAM1-binding motifs, formed a distinct phylogenetic cluster, and were similar to sequences from other areas of endemicity. PfEMP1 variants associated with these DBLβ domains were enriched for DC4 and DC13 head structures implicated in endothelial protein C receptor (EPCR) binding and severe malaria, suggesting conservation of dual binding specificities. These results provide further support for the development of specific classes of PfEMP1 as vaccine candidates and as biomarkers for protective immunity against clinical P. falciparum malaria.
Chitinases play a vital role during the pathogenic invasion and immunosuppression in various organisms including invertebrates and vertebrates. In this study, we have investigated the participation of MrChit-3 (Macrobrachium rosenbergii Chitinase-3) during host-pathogenic interaction in freshwater prawn, M. rosenbergii. Quantitative real-time PCR analysis showed that the expression of MrChit-3 was up-regulated during bacterial, viral and laminarin challenge. Moreover, to understand the antimicrobial role of the GH18 domain, a putative membrane-targeting antimicrobial peptide (MrVG) was identified from the GH18 domain region of the protein and it was chemically synthesized. Physico-chemical features of the GH18 derived antimicrobial peptide (AMP) was assessed by various in silico tools and the antimicrobial property of the peptide was confirmed from in vitro studies. The membrane targeting mechanism of the peptide was determined by flow cytometry (FACS) and scanning electron microscope (SEM) analysis. Interestingly, the peptide was able to inhibit the growth of a chitinolytic fungal pathogen, Aspergillus niger, which was isolated from the shells of M. rosenbergii. The toxicity studies such as hemolysis activity on human blood erythrocytes and cell viability assay with primary kidney cells, HEK293 of MrVG revealed that the peptide was not involved in inducing any toxicity.
SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC50 of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.
Clostridial neurotoxins, including tetanus and botulinum neurotoxins, generally target vertebrates. We show here that this family of toxins has a much broader host spectrum, by identifying PMP1, a clostridial-like neurotoxin that selectively targets anopheline mosquitoes. Isolation of PMP1 from Paraclostridium bifermentans strains collected in anopheline endemic areas on two continents indicates it is widely distributed. The toxin likely evolved from an ancestral form that targets the nervous system of similar organisms, using a common mechanism that disrupts SNARE-mediated exocytosis. It cleaves the mosquito syntaxin and employs a unique receptor recognition strategy. Our research has an important impact on the study of the evolution of clostridial neurotoxins and provides the basis for the use of P. bifermentans strains and PMP1 as innovative, environmentally friendly approaches to reduce malaria through anopheline control.
The utilization of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages. However, an adaptation of enzyme towards organic solvent is unpredictable and not fully understood because of limited information on the organic solvent tolerant enzymes. To understand how the enzyme can adapt to the organic solvent environment, structural and computational approaches were employed. A recombinant elastase from Pseudomonas aeruginosa strain K was an organic solvent tolerant zinc metalloprotease was successfully crystallized and diffracted up to 1.39 Å. Crystal structure of elastase from strain K showed the typical, canonical alpha-beta hydrolase fold consisting of 10-helices (118 residues), 10- β-strands (38 residues) and 142 residues were formed other secondary structure such as loop and coil to whole structure. The elastase from Pseusomonas aeruginosa strain K possess His-140, His-144 and Glu-164 served as a ligand for zinc ion. The conserved catalytic triad was composed of Glu-141, Tyr-155 and His-223. Three-dimensional structure features such as calcium-binding and presence of disulphide-bridge contribute to the stabilizing the elastase structure. Molecular dynamic (MD) simulation of elastase revealed that, amino acid residues located at the surface area and disulphide bridge in Cys-30 to Cys-58 were responsible for enzyme stability in organic solvents.