Exploring the Protein Stability Landscape: Bacillus subtilis Lipase A as a Model for Detergent Tolerance

A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal α/β‐hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts.     

Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection

Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis-Menten parameters. The 3D structures of the Sc and Rc inhibitor-lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.     

Towards quantitative computer-aided studies of enzymatic enantioselectivity: the case of Candida antarctica lipase A

The prospect for consistent computer-aided refinement of stereoselective enzymes is explored by simulating the hydrolysis of enantiomers of an α-substituted ester by wild-type and mutant Candida antarctica lipase A, using several strategies. In particular, we focused on the use of the empirical valence bond (EVB) method in a quantitative screening for enantioselectivity, and evaluate both k(cat) and k(cat)/K(M) of the R and S stereoisomers. We found that an extensive sampling is essential for obtaining converging results. This requirement points towards possible problems with approaches that use a limited conformational sampling. However, performing the proper sampling appears to give encouraging results and to offer a powerful tool for the computer-aided design of enantioselective enzymes. We also explore faster strategies for identifying mutations that will help in augmenting directed-evolution experiments, but these approaches require further refinement.     

Learning from directed evolution: Further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity

An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2-methyl-decanoic acid p-nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E = 1.1 for the wild-type to E = 51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular-dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).     

氯灭活水中枯草芽孢杆菌的效果

以枯草芽孢杆菌(ATCC6633)作为难灭活微生物的代表,研究了氯对水体中芽孢的灭活效果,考察了氯浓度、作用时间、反应体系pH值、温度以及芽孢初始浓度等因素的影响。结果表明,氯对芽孢的灭活过程可分为延滞期和灭活期;初始氯浓度在2.06~10.30 mg·L^-1,反应时间0~166 min,pH值6~9,温度1~30℃,初始芽孢浓度10²~10¹² cfu·ml^-1范围内,消毒剂浓度和反应时间共同影响着氯对芽孢的灭活效果,提高消毒剂投量或延长消毒反应时间,均可提高灭活率;酸性条件下氯灭活芽孢的能力强于碱性条件下;随着温度的上升,氯对芽孢的灭活能力增强;芽孢的初始浓度对氯灭活芽孢的效能影响不大。初始氯投量为8.30 mg·L^-1,pH=7,芽孢初始浓度10⁶ cfu·ml^-1,温度分别为5℃和25℃下,枯草芽孢杆菌对氯消毒剂的抗性强于炭疽芽孢杆菌。     

基于脯氨酸理论提高枯草芽孢杆菌脂肪酶A的热稳定性

以枯草芽孢杆菌脂肪酶A(LipA)为研究对象,根据从RCSB数据库中获取的晶体结构,采用分子动力学模拟和分子生物学实验相结合的方法进行脂肪酶热稳定性位点突变的理性设计。首先,利用分子动力学模拟获得晶体结构中柔性较高的Loop区域;进而,结合"脯氨酸理论",将位于该区域附近的Gly残基突变为Pro,分析引入Pro突变对LipA热稳定性的影响,筛选得到Gly52和Gly158两个突变位点;最后,通过定点突变操作对突变株LipAG52P和LipAG158P进行热稳定性实验验证。结果显示,突变株LipAG52P、LipAG158P的比活力分别是野生型LipA的5.6倍和2.7倍,Tm值分别提高了15℃和7℃,催化效率分别提高了85%和22%。     

Enrichment of glutaminase production by Bacillus subtilis RSP‐GLU in submerged cultivation based on neural network—genetic algorithm approach

BACKGROUND: Owing to the importance of glutaminase in biotech product production, its production with isolated Bacillus subtilis RSP‐GLU (MTCC 9727) was investigated. Fermentation factors play an important role in product enhancement. Hence, glutaminase production was optimized using an artificial neural network (ANN) coupled genetic algorithm (GA). RESULTS: A ‘6–12–1’ topology ANN was constructed to identify the nonlinear relationship between fermentation factors and enzyme yield. ANN‐predicted values were optimized for glutaminase production using a GA. The overall mean absolute predictive error (MAPE) and the mean square errors (MSE) were observed to be 0.00125 and 1.77 and 0.002 and 3.06 for training and testing, respectively. The goodness of neural network prediction (coefficient of R²) was found to be 0.996. The maximum interactive impact on glutaminase production was noted with rpm versus medium volume. The use of ANN–GA hybrid methodology resulted in a significant improvement (47%) in glutaminase yield. CONCLUSION: Five different optimum fermentation conditions out of 500 revealed maximum enzyme production. Glutaminase enzyme production in this Bacillus subtilis RSP‐GLU is strongly influenced by aeration of the fermentation. A hybrid ANN‐GA effectively identifies the different fermentation conditions for optimum production of enzyme in a given large set of conditions. Copyright © 2009 Society of Chemical Industry     

基于化学修饰法探讨脂肪酶对手性伯醇的识别机理

洋葱假单胞菌脂肪酶（PcL）催化多种手性伯醇酯的不对称水解反应显示其对结构中含有额外的非酯键氧原子的手性伯醇酯的对映体选择性较高,而对不含额外氧原子的选择性差。分别用多种氨基酸残基特异性的修饰剂修饰 PcL,结果发现经 N-乙酰咪唑（NAI）修饰酪氨酸残基（Tyr）后 PcL 对含额外非酯键氧原子的手性伯醇酯对映体选择性显著降低,而对不含额外氧的伯醇酯选择性几乎不受影响。蛋白质谱证实产生主要影响的是位于 PcL 活性口袋内的 Tyr29 残基,手性伯醇酯能否与 Tyr29 形成额外氢键决定了 PcL对其选择性。分子动力学模拟表明,Tyr29被修饰的 PcL 不能与底物形成氢键,因此选择性明显下降。这一发现成功揭示了脂肪酶对手性伯醇对映体选择性的分子识别机理。     

Manipulating the expression rate and enantioselectivity of an epoxide hydrolase by using directed evolution

We describe here a strategy to improve the expression efficiency and enantioselectivity of Aspergillus niger epoxide hydrolase (ANEH) by directed evolution. Based on a blue‐colony screening system using the LacZα (β‐galactosidase α peptide) complementation solubility reporter, several ANEH variants out of 15 000 transformants from a random‐mutagenesis library were identified that show improved recombinant expression in E. coli. Among them, Pro221Ser was subsequently used as a template for iterative saturation mutagenesis (ISM) at sites around the ANEH binding pocket. Following four rounds of ISM, a highly enantioselective mutant was identified that catalyzes the hydrolytic kinetic resolution of racemic glycidyl phenyl ether with a selectivity factor of E=160 in favor of the (S)‐diol compared to WT ANEH characterized by E=4.6. Expression of this mutant is 50 times higher than that of WT ANEH. It also serves as an excellent stereoselective catalyst in the hydrolytic kinetic resolution and desymmetrization of several other structurally diverse epoxides.     

枯草芽孢杆菌BSD-2菌株喷雾干燥工艺优化

为研制生物农药新剂型,利用喷雾干燥法制备浓缩枯草芽孢杆菌BSD-2菌粉。以集粉率和抑菌活性为指标,确定最佳喷雾干燥工艺条件。在发酵液中加入15%β-环糊精作为填料,最佳入口温度为170℃,料液温度为30℃,热风量为20 m3/h,入料流量为15 m L/min。此条件下喷雾集粉率达到25.4%,效价保留率为66%,BSD-2菌体存活率为75%。     

绿色荧光蛋白基因在枯草芽孢杆菌中的表达

目的探讨异源基因在枯草芽孢杆菌中表达的可行性。方法以大肠杆菌和枯草芽孢杆菌的穿梭质粒为基本骨架,在大肠杆菌中完成含有绿色荧光蛋白(GFP)基因的重组表达质粒的构建,通过酶切鉴定筛选阳性克隆,采用电转化的方法,将重组质粒转入枯草芽孢杆菌进行表达,通过检测GFP的存在,评价枯草芽孢杆菌对异源基因的表达。结果重组表达质粒pGJP-GFP酶切鉴定结果与预期片段大小相符。绿色荧光蛋白可在枯草芽孢杆菌中表达,且表达蛋白具有较好的生物学活性。结论利用枯草芽孢杆菌的自身启动子,可以实现外源基因在枯草芽孢杆菌中的表达。     

猪源枯草芽孢杆菌喷雾干燥工艺的优化

目的优化猪源枯草芽孢杆菌喷雾干燥工艺,提高菌体喷雾干燥后的存活率。方法应用SAS V 8.0统计分析软件中的二次正交旋转组合设计方案进行试验设计,分析各因素对Y值的效应关系;应用优化的喷雾干燥工艺进行3次重复喷雾干燥试验,验证预测结果的准确性;并检测芽孢率对菌体喷雾干燥存活率的影响。结果优化的喷雾干燥工艺中各条件的最佳参数为:出口温度90℃,入口温度150℃,保护剂浓度20%,入料速度1 200 ml/h;应用优化的喷雾干燥工艺进行3次重复喷雾干燥试验,菌体平均存活率为75.7%,与预测值(77%)基本相符;芽孢率对菌体喷雾干燥存活率影响较大。结论优化了猪源枯草芽孢杆菌的喷雾干燥工艺,为其工业化生产奠定了基础。     

枯草芽孢杆菌对黑土中可培养微生物的生态效应

研究了微生物农药枯草芽孢杆菌对东北黑土中可培养微生物的生态影响.其动态变化表明:低质量分数枯草芽孢杆菌对细菌总数没有明显影响,较高质量分数枯草芽孢杆菌町促进细菌总数的显著增加,其中质量分数在3200 mg/kg时,枯草芽孢杆菌对细菌的刺激强度最高,为对照的11倍.枯草芽孢杆菌对土壤中的放线菌也有刺激作用,刺激强度最高时,放线菌数量可增至对照的8.3倍左右.枯草芽孢杆菌对真菌的敏感性较低,只有质量分数高达3200 mg/kg时,才对真菌产生明显刺激作用,最高刺激强度为同时期对照的29倍.     

Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block

A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).     

枯草芽孢杆菌对土壤呼吸作用和脲酶活性的影响

通过模拟实验研究了微生物农药枯草芽孢杆菌对黑土的呼吸作用和脲酶活性的生态毒理效应.结果表明:枯草芽孢杆菌各质量分数处理均表现为对土壤呼吸作用的刺激效应,并且土壤巾枯草芽孢杆菌质量分数越大,对土壤呼吸强度的刺激作用越大,其中最高质量分数(3200 mg/kg干土)处理在第42天时达到最大刺激强度,刺激率为69.1%.与对照相比,除第1天外,所有处理(32～3200 mg/kg干土)对土壤脲酶均表现出刺激效应,其中最高质量分数(3200 mg/kg干土)处理在第28天脲酶活性上升到最高,刺激率达到101.1%.     

氨基甲酸乙酯水解酶在枯草芽孢杆菌中的表达及发酵优化

将来源于赖氨酸芽孢杆菌SC02的氨基甲酸乙酯水解酶(UH)基因在枯草芽孢杆菌Bacillus subtilis WB600中进行克隆和表达,在枯草芽孢杆菌中实现了UH活性表达,在摇瓶水平通过单因素考察和响应面分析实验对氨基甲酸乙酯水解酶发酵进行优化. 结表明,酶活最高可达到14.20 U/mL,产酶最佳培养基成分为：淀粉10 g/L、磷酸氢二钾9 g/L、麦芽浸膏25 g/L、硫酸镁1 g/L、胰蛋白胨55 g/L,最适发酵温度为37℃,最佳接种量4%. 在3 L发酵罐中采用最优发酵条件,酶活在16 h达到18.03 U/mL.     

A structure-controlled investigation of lipase enantioselectivity by a path-planning approach

A novel approach based on efficient path-planning algorithms was applied to investigate the influence of substrate access on Burkholderia cepacia lipase enantioselectivity. The system studied was the transesterification of 2-substituted racemic acid derivatives catalysed by B. cepacia lipase. In silico data provided by this approach showed a fair qualitative agreement with experimental results, and hence the potential of this computational method for fast screening of racemates. In addition, a collision detector algorithm used during the pathway searches enabled the rapid identification of amino acid residues hindering the displacement of substrates along the deep, narrow active-site pocket of B. cepacia lipase and thus provided valuable information to guide the molecular engineering of lipase enantioselectivity.