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1.
Introduction of well-packed residues to the interior of a protein structure could be considered as a stabilization strategy since the reduction of buried cavities might stabilize protein structure. In this study, the less-packed residues with no water-contact were selected as target sites for increasing residual packing. When Lipase A from Bacillus subtilis (179 amino acids) was used as a model system, 43 less-packed residues were initially considered by analyzing their residual packing value and residual exposure ratio. Among the 43 residues, small amino acids such as GLY and ALA were chosen as target sites. Packing increases of ALA to VAL and GLY to ALA were estimated, by molecular modeling, to give 0.5368~0.7433?kcal mol?1 stabilization. Mutants of Lipase A such as A38V, A75V, G80A, A105V A146V, and G172A were obtained via protein engineering. Thermostability assays revealed that A38V, G80A and G172V were the most stable mutants. This procedure for selecting the target residues for improved thermostability of Lipase A could be applied for improving the thermostability of other proteins and enzymes. 相似文献
2.
Introduction of well-packed residues to the interior of a protein structure could be considered as a stabilization strategy since the reduction of buried cavities might stabilize protein structure. In this study, the less-packed residues with no water-contact were selected as target sites for increasing residual packing. When Lipase A from Bacillus subtilis (179 amino acids) was used as a model system, 43 less-packed residues were initially considered by analyzing their residual packing value and residual exposure ratio. Among the 43 residues, small amino acids such as GLY and ALA were chosen as target sites. Packing increases of ALA to VAL and GLY to ALA were estimated, by molecular modeling, to give 0.5368∼0.7433 kcal mol-1 stabilization. Mutants of Lipase A such as A38V, A75V, G80A, A105V A146V, and G172A were obtained via protein engineering. Thermostability assays revealed that A38V, G80A and G172V were the most stable mutants. This procedure for selecting the target residues for improved thermostability of Lipase A could be applied for improving the thermostability of other proteins and enzymes. 相似文献
3.
Prakash Chandra Rathi Alexander Fulton Karl-Erich Jaeger Holger Gohlke 《PLoS computational biology》2016,12(3)
Protein thermostability is a crucial factor for biotechnological enzyme applications. Protein engineering studies aimed at improving thermostability have successfully applied both directed evolution and rational design. However, for rational approaches, the major challenge remains the prediction of mutation sites and optimal amino acid substitutions. Recently, we showed that such mutation sites can be identified as structural weak spots by rigidity theory-based thermal unfolding simulations of proteins. Here, we describe and validate a unique, ensemble-based, yet highly efficient strategy to predict optimal amino acid substitutions at structural weak spots for improving a protein’s thermostability. For this, we exploit the fact that in the majority of cases an increased structural rigidity of the folded state has been found as the cause for thermostability. When applied prospectively to lipase A from Bacillus subtilis, we achieved both a high success rate (25% over all experimentally tested mutations, which raises to 60% if small-to-large residue mutations and mutations in the active site are excluded) in predicting significantly thermostabilized lipase variants and a remarkably large increase in those variants’ thermostability (up to 6.6°C) based on single amino acid mutations. When considering negative controls in addition and evaluating the performance of our approach as a binary classifier, the accuracy is 63% and increases to 83% if small-to-large residue mutations and mutations in the active site are excluded. The gain in precision (predictive value for increased thermostability) over random classification is 1.6-fold (2.4-fold). Furthermore, an increase in thermostability predicted by our approach significantly points to increased experimental thermostability (p < 0.05). These results suggest that our strategy is a valuable complement to existing methods for rational protein design aimed at improving thermostability. 相似文献
4.
Habibi AE Khajeh K Naderi-Manesh H Ranjbar B Nemat-Gorgani M 《Journal of biotechnology》2006,123(4):434-442
Chemical cross-linking of a mesophilic alpha-amylase from Bacillus amyloliquefaciens (BAA) was carried out. Intra-molecular cross-links between lysine residues upon treatment of the enzyme with ethylene glycol bis(succinic acid N-hydroxy succinimide ester) resulted in enhancement of thermostability as indicated by irreversible thermoinactivation experiments. Enhancement of thermostability coincided with a dramatic protection against aggregation, combined with a decrease in surface hydrophobicity. Deamidation, another important mechanism of irreversible thermoinactivation, was also diminished upon modification. While no significant changes in the kinetic parameters are evident, rigidification of the protein structure is suggested by circular dichroism (CD) and fluorescence studies. 相似文献
5.
笔者所在实验室前期筛选到1株产脂肪酶粘质沙雷氏菌,克隆其脂肪酶基因,构建重组枯草芽胞杆菌Bacillus subtilis 168/pMA5-lipA,成功实现了来源于粘质沙雷氏菌的脂肪酶基因在枯草芽胞杆菌中的表达。基于以上工作基础上,对B.subtilis 168/pMA5-lipA进行了摇瓶水平上的产酶发酵优化。首先通过单因素和正交试验确定了有利于产脂肪酶的最佳培养基成分,并对发酵条件进行了优化。结果表明:优化后的培养基组分为蔗糖35 g/L,玉米浆27.5 g/L,(NH4)2SO41.25 g/L,CaCl24 g/L,pH 7.0。在最优发酵培养基的条件下,37℃、160 r/min摇床培养33 h,每毫升发酵液中重组菌脂肪酶酶活可达98.6 U,是优化前的3倍。 相似文献
6.
Ma J Zhang Z Wang B Kong X Wang Y Cao S Feng Y 《Protein expression and purification》2006,45(1):22-29
A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, was cloned into the plasmid pBSR2 and transformed into B. subtilis A.S.1.1655. Thus, an overexpression strain, BSL2, was obtained. The yield of lipase is about 8.6 mg protein/g of wet weight of cell mass and 100-fold higher than that in B. subtilis strain IFFI10210. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Characterizations of the purified enzyme revealed a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, maximum activity at 43 degrees C and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km and Vm were found to be 0.37 mM and 303 micromol mg-1 min-1, respectively. The substrate specificity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+, and Mg2+ can activate the lipase, whereas Fe2+, Cu2+, and Co2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7 mM and inhibited at concentrations over 10 mM. 相似文献
7.
Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor 总被引:3,自引:0,他引:3
Dröge MJ Rüggeberg CJ van der Sloot AM Schimmel J Dijkstra DS Verhaert RM Reetz MT Quax WJ 《Journal of biotechnology》2003,101(1):19-28
Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates. 相似文献
8.
Stabilization of the phosphorylated form of Bacillus subtilis DegU caused by degU9 mutation 总被引:1,自引:0,他引:1
Abstract A Bacillus subtilis response regulator, DegU9, carrying an amino acid alteration caused by the degU9 (Hy) mutation was partially purified, and phosphorylation and dephosphorylation of the protein was studied. The extent of phosphorylation was not as high as the level attained with wild-type DegU, but the DegU9-phosphate once formed was more stable than the wild-type DegU-phosphate. An in vivo study with a degU9 mutant showed that degS was necessary for the overproduction of exoproteases. These results suggest that phosphorylation is necessary for the mutant DegU9 to exert its effect and that the higher stability of phosphorylated DegU9 is responsible for the overproulation phenotype. 相似文献
9.
Temperature-sensitive sporulation caused by a mutation in the Bacillus subtilis secY gene.
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H Yoshikawa S M Jeong A Hirata F Kawamura R H Doi H Takahashi 《Journal of bacteriology》1993,175(11):3656-3660
A thermosensitive sporulation mutant of Bacillus subtilis containing a mutation in the secY gene was isolated and characterized. No asymmetric septum specific to the sporulation was detected by electron microscopy at the nonpermissive temperature, indicating that the block occurred at a very early stage of sporulation. Furthermore, competence development in the mutant cell was affected even at the sporulation-proficient temperature. It is assumed that the SecY protein of B. subtilis has multiple roles both in the regulation of spore formation and in stationary-phase-associated phenomena. 相似文献
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12.
Mormeneo M Andrés I Bofill C Díaz P Zueco J 《Applied microbiology and biotechnology》2008,80(3):437-445
Both the secretion and the cell surface display of Bacillus subtilis lipase A (Lip A) in Saccharomyces cerevisiae was investigated using different domains of the cell wall protein Pir4 as translational fusion partners. LipA gene minus its leader peptide was fused inframe in two places of PIR4 to achieve cell wall targeting, or substituting most of the PIR4 sequence, after the signal peptide and the Kex2 processed subunit I of Pir4 to achieve secretion to the growth medium. Expression of the recombinant fusion proteins was investigated in a standard and a glycosylation-deficient strain of S. cerevisiae, grown in selective or rich medium. Fusion proteins intended to be retained at the cell wall were secreted to the growth medium, most likely as result of the degradation of the Pir4 moiety containing the cell wall retention domain, giving low levels of lipase activity. However, the fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 400 IU of lipase activity per milliliter of cell supernatant. This is, to our knowledge, the first report of the efficient production, as a secreted protein, of lipase A of B. subtilis in baker's yeast. 相似文献
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14.
Acharya P Rajakumara E Sankaranarayanan R Rao NM 《Journal of molecular biology》2004,341(5):1271-1281
Variation in gene sequences generated by directed evolution approaches often does not assure a minimalist design for obtaining a desired property in proteins. While screening for enhanced thermostability, structural information was utilized in selecting mutations that are generated by error-prone PCR. By this approach we have increased the half-life of denaturation by 300-fold compared to the wild-type Bacillus subtilis lipase through three point mutations generated by only two cycles of error-prone PCR. At lower temperatures the activity parameters of the thermostable mutants are unaltered. High-resolution crystal structures of the mutants show subtle changes, which include stacking of tyrosine residues, peptide plane flipping and a better anchoring of the terminus, that challenge rational design and explain the structural basis for enhanced thermostability. The approach may offer an efficient and minimalist solution for the enhancement of a desired property of a protein. 相似文献
15.
J A Lepesant A Billault J Kejzlarová-Lepesant M Pascal F Kunst R Dedonder 《Biochimie》1974,56(11-12):1465-1470
16.
Structural basis for the remarkable stability of Bacillus subtilis lipase (Lip A) at low pH 总被引:1,自引:0,他引:1
Rajakumara E Acharya P Ahmad S Sankaranaryanan R Rao NM 《Biochimica et biophysica acta》2008,1784(2):302-311
Understanding the structural basis of altered properties of proteins due to changes in temperature or pH provides useful insights in designing proteins with improved stability. Here we report the basis for the pH-dependent thermostability of the Bacillus subtilis lipase (Lip A) using spectroscopic and X-ray crystallographic studies. At pH values above 7, lipase denatures and aggregates when heated at temperatures above 45 degrees C. However, at pH below 6 lipase denatures upon heating but the activity and its native structure is completely recovered upon cooling. In order to obtain the structural basis of this unusual stability of lipase, we determined high-resolution crystal structures of the lipase in two different crystal forms at pH 4.5 and 5. These structures show linear oligomerization of lipase using only two types of dimeric associations and these inter-molecular interactions are completely absent in several crystal forms of wild-type and mutant proteins obtained at basic pH. In accordance with the crystallographic studies, spectroscopic investigations reveal an invariant secondary structure in the pH range of 4-10. Quaternary organization of lipase at low pH resulted in changes in the tryptophan environment and binding of 1-anilino-8-naphthalene sulfate (ANS) at low pH. Low pH stability of the lipase is not observed in the presence of sodium chloride (>0.2 M) indicating the importance of ionic interactions at low pH. Inter- and intra-molecular ionic interactions that occur at pH below 6.0 are proposed to trap the molecule in a conformation that allows its complete refolding upon cooling. 相似文献
17.
Dissection of the functional and structural domains of phosphorelay histidine kinase A of Bacillus subtilis
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Wang L Fabret C Kanamaru K Stephenson K Dartois V Perego M Hoch JA 《Journal of bacteriology》2001,183(9):2795-2802
The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains. 相似文献
18.
The macrolide toxin brefeldin A is a determinant of Alternaria leaf blight disease in safflower, which causes severe economic losses worldwide. Soilborne bacteria, classified as Bacillus subtilis spp., were isolated and shown to readily metabolize brefeldin A in laboratory culture to one major product. This product was identified by high resolution 2D 1H NMR and FAB mass spectroscopies as the acid resulting from hydrolysis of the macrolide ring in brefeldin A . In contrast to brefeldin A, the acid completely lacked phytotoxic activity in the standard leaf bioassay. Detoxification of brefeldin A by the lactonase activity from Bacillus subtilis may be exploited in the future to introduce resistance to Alternaria leaf blight in safflower. 相似文献
19.
Khan MI Ito K Kim H Ashida H Ishikawa T Shibata H Sawa Y 《Bioscience, biotechnology, and biochemistry》2005,69(10):1861-1870
The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in Escherichia coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined to have a hexameric structure (M(r) 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate, requiring NADH and NAD+ as cofactors respectively. The enzyme showed low thermostability with T(m) = 41 degrees C due to dissociation of the hexamer. To improve the thermostability of this enzyme, we performed error-prone PCR, introducing random mutagenesis on cloned GluDH. Two single mutant enzymes, Q144R and E27F, were isolated from the final mutant library. Their T(m) values were 61 degrees C and 49 degrees C respectively. Furthermore, Q144R had a remarkably high k(cat) value (435 s(-1)) for amination reaction at 37 degrees C, 1.3 times higher than that of the wild-type. Thus, Q144R can be used as a template gene to modify the substrate specificity of Bs-GluDH for industrial use. 相似文献
20.
Annaluru N Watanabe S Pack SP Saleh AA Kodaki T Makino K 《Journal of biotechnology》2007,129(4):717-722
Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 degrees C higher thermal transition temperature (T(CD)) and 20.8 degrees C higher half denaturation temperature (T(1/2)) compared with wild-type. 相似文献