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。     

Improved enantioselectivity of a lipase by rational protein engineering

A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity. 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutations gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity. The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.     

A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A

In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects beta7 and alphaE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.     

多元电解质对脂肪酶有机相拆分炔戊醇的激活

脂肪酶是一种应用节广泛的重要生物催化剂,提高脂肪酶在非天然环境中的催化性能逐步成为了一个研究热点。研究表明,某些电解质的加入可显著提高提脂肪酶在有机溶剂体系中的酶活和选择性。然而有关研究主要考察单一电解质的作用,且涉及的电解质种类较少,更为系统的研究未见报道。本文以洋葱假单胞菌脂肪酶（Pseudomonas cepacia lipase,即PcL）催化手性菊酯农药前体4-甲基庚-4-烯基-1-炔基-3-醇（炔戊醇）在甲苯中选择性转酯化为模型,系统考察了卤化物、硝酸盐、硫酸盐、磷酸盐等8类35种电解质单独以及多种电解质协同对PcL催化性能的影响。结果表明,NaF-Na2HPO4/NaH2PO4的二元电解质体系具有远高于任意单一电解质组分的性能,该体系使PcL活力提高23倍。进一步研究表明,这一电解质体系对PcL催化不同对映体转酯化的活力提高程度不同,从而将其对映体选择率（E值）由11提高到21。     

Computational Study of the Lipase‐Mediated Desymmetrisation of 2‐Substituted‐Propane‐1,3‐Diamines

The enantioselectivity displayed by the lipase from Pseudomonas cepacia towards a wide range of prochiral 2‐substituted‐propane‐1,3‐diamines was studied by means of molecular dynamics simulations (MDS). In all cases the enzyme allows the recovery of the corresponding amino carbamates of R configuration. However, the enantioselectivity is only synthetically useful if no ortho substituent is present and the aromatic ring is directly bonded to the 2‐carbon of the 1,3‐diamine core. Analysis of the MDS trajectories revealed that the homologation of 2‐aryl substituents by means of a methylene group lowers enantioselectivity by alleviating the conformational tension of the slow‐reacting orientations due to unfavourable intramolecular contacts between the ortho carbons of the aryl group and the nucleophilic nitrogen, as well as between the chiral carbon and the oxyanion. Additionally, the relative solvent accessible surfaces of the atoms of the aryl ring nicely correlate with the effect of the location of the substituent on enantioselectivity.     

Simple conformation space search protocols for the evaluation of enantioselectivity of lipases

Two computational protocols have been evaluated regarding their ability to reproduce the enthalpic part of lipase enantioselectivity by forcefield potential energy differences (deltaV#R-S). Though the shortcomings of the approach are numerous, good qualitative results have been obtained here and elsewhere. The anticipated improvement of quantitative results by use of a second protocol, which did not impose any atom movement restrictions on the total system, was realized only in part. Seemingly, results depended not only on the design of the computational procedure but also on the enzyme-substrate combination modelled. With Candida antarctica lipase B, results diverged significantly more from an estimated deltadeltaH#R-S than with Rhizomucor miehei lipase and cutinase.      

分子模拟指导化学修饰对有机相中脂肪酶的性能强化

脂肪酶是一种应用广泛的重要生物催化剂,它对手性底物具有良好的立体选择性,而且能适应包括疏水有机溶剂在内的多种非天然反应介质。提高脂肪酶在非天然环境中的立体选择性已经成为关于脂肪酶的研究热点。然而已有研究在提高选择性的同时,往往伴随催化活力的降低。本文提出一种基于计算机模拟的理性改造方法,能够在不降低脂肪酶活力的情况下大幅提高其在有机介质中的立体选择性。以洋葱假单胞菌脂肪酶(Pseudomonas cepacia lipase,PcL)在正己烷中催化手性仲醇的转酯化反应为模型,借助分子动力学模拟,预测 N-乙酰咪唑(NAI)修饰 PcL的效果。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。进一步通过实验验证,发现经 NAI 修饰酪氨酸残基(Tyr)后 PcL催化拆分手性仲醇的对映体选择性最高从 E = 12.6 提高到 48.1,同时催化活力也有所提高。     

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).     

Utilization of Catalytic Properties of the Encapsulated Lipase with Calix[4]arene-Adorned Sporopollenin

The dihydrazide calix[4]arene was immobilized onto sporopollenin in order to encapsulate Candida rugosa lipase (CRL) via sol-gel entrapment. The kinetic resolution of the new encapsulated lipase was investigated for enantioselective hydrolysis of racemic naproxen methyl ester and 2-phenoxypropionic acid methyl ester. The results demonstrated that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calix[4]arene-immobilized sporopollenin. The encapsulated lipase showed an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E = 350 and 295, respectively, compared to the free enzyme.     

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.     

脂肪酶催化拆分外消旋6-羟基-8-氯辛酸乙酯

研究了脂肪酶拆分外消旋6-羟基-8-氯辛酸乙酯。从10种脂肪酶中筛选出了脂肪酶Lipase PS-D,该酶能够有效拆分外消旋6-羟基-8-氯辛酸乙酯,并对反应条件进行了优化,确定了该酶的最适反应条件:温度30℃,pH=7.0,摇床转速170 r/min,确定了有效表面活性剂为乳化剂OP,在此优化条件下反应1 h,得到(R)-6-羟基-8-氯辛酸乙酯的对映体过量值ee为93.1%,底物摩尔转化率为54.3%,总收率为91.4%。该研究为(R)-硫辛酸的制备提供了一条可行途径。     

Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2‐substituted racemic acid derivatives, which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild‐type BCL could be improved by chemical engineering of the substrate's molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aimed at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for enantioselectivity towards (R,S)‐2‐chloro ethyl 2‐bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable tenfold enhanced or reversed enantioselectivity. Analysis of substrate docking and access trajectories in the active site was then performed. From this analysis, the construction of 13 double mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E value of 178 and a 15‐fold enhanced specific activity compared to the parental enzyme; thus, this study demonstrates the efficiency of the semirational engineering strategy.     

Optical resolution of 2-alkanol by lipase-catalyzed acetylation with vinyl acetate in packed-bed reactor with recycling system

The lipase-catalyzed acetylation of 2-alkanol with vinyl acetate was studied using Burkholderia cepacia lipase (BCL), three alcohol and three organic solvents in a packed-bet reactor with a recycling system (flow method). The optical resolution data were found in agreement with those of the batch method in which BCL was suspended in the substrate solution. Repeated reaction results clearly indicated BCL in the packed-bed to be quite stable and to be usable for at least 50 reaction runs or to remain effective for as long as two months in the water-insoluble solvents such as hexane and 1,2-dichloroethane. In the reaction using a water-soluble solvent such as acetonitrile, the catalytic power of BCL showed only a 1% decrease of conversion per run or solvent recycling possibly owing to compression of BCL in the bed although enantioselectivity was independent of the number of reaction repetitions. The present method showed thus be applicable to kinetic resolution by enzyme-catalyzed acylation in hydrophobic organic solvents with no waste of enzyme.     

Biocatalytic kinetic resolution of rac‐1‐phenylethanol and rac‐2‐pentanol in hexane medium: ACYL donor and water content effects

The kinetic resolutions of rac‐1‐phenylethanol and rac‐2‐pentanol by transesterification with vinyl esters catalysed by a commercial immobilised Candida antarctica lipase B were successfully carried out in hexane medium. This enzyme showed very high enantioselectivity for both substrates. The influence of the water content of the medium on the synthetic activity, selectivity and enantioselectivity of the enzyme was analysed, with the optimal amount of water about 100 ppm. Our results also showed that the activity per gram enzymatic derivate of CaLB was slightly higher with butyl butyrate as acyl donor.     

Influence of the Procedure to Immobilize Lipase on SBA-15 for Biodiesel Production from Palm Kernel Oil

Catalysis Letters - Microbial lipase from Burkholderia cepacia was immobilized by covalent bond and physical adsorption on SBA-15 mesoporous support and its catalytic efficiency was measured in the...     

高选择性脂肪酶的筛选及其拆分布洛芬

为了实现脂肪酶在有机相中对外消旋布洛芬的高效拆分,筛选得到了一株具有高立体选择性的脂肪酶产生菌-扩展青霉TS414(Penicillium expansum TS414). 实验探讨了水分、温度、有机溶剂、酶浓度、醇结构和醇浓度对酶促拆分反应的影响,确立了酯化反应的最佳反应体系：布洛芬6.46 mmol/L,脂肪酶53.3 mg/mL,正丙醇40 mmol/L,异辛烷15 mL,水0.5%(j),60℃条件下反应50 h. 在此条件下,拆分反应的转化率和对映体选择率分别为42%和429.63. 结果表明,Penicillium expansum TS414脂肪酶是一种较为理想的用于外消旋布洛芬拆分的工具酶,在布洛芬手性拆分方面具有广阔的应用前景.     

A water molecule in the stereospecificity pocket of Candida antarctica lipase B enhances enantioselectivity towards pentan-2-ol

The effect of water activity on enzyme-catalyzed enantioselective transesterification was studied by using a solid/gas reactor. The experimental results were compared with predictions from molecular modelling. The system studied was the esterification of pentan-2-ol with methylpropanoate as acyl donor and lipase B from Candida antarctica as catalyst. The data showed a pronounced water-activity effect on both reaction rate and enantioselectivity. The enantioselectivity increased from 100, at water activity close to zero, to a maximum of 320, at a water activity of 0.2. Molecular modelling revealed how a water molecule could bind in the active site and obstruct the binding of the slowly reacting enantiomer. Measurements of enantioselectivity at different water-activity values and temperatures showed that the water molecule had a high affinity for the stereospecificity pocket of the active site with a binding energy of 9 kJ mol-1, and that it lost all its degrees of rotation, corresponding to an entropic energy of 37 J mol-1 K-1.     

脂肪酶不对称立体选择性能改善的研究进展

脂肪酶已广泛用于制备光学纯手性化合物的不对称反应中，但大多数酶催化拆分外消旋化合物的立体选择性不很理想. 目前，改善脂肪酶立体选择性的研究主要从改造脂肪酶酶蛋白结构、优化体系的反应条件、改善反应过程以及对映选择性抑制等方面进行. 通过微波照射也能在一定程度上改善脂肪酶催化反应的立体选择性. 本文主要介绍了几种改善脂肪酶催化不对称反应的立体选择性的方法.     

Influence of delta-functional groups on the enantiorecognition of secondary alcohols by Candida antarctica lipase B

The selectivity of acetylation of delta-functionalized secondary alcohols catalyzed by Candida antarctica lipase B has been examined by molecular dynamics. The results from the simulation show that a delta-alcohol functionality forms a hydrogen bond with the carbonyl group of Thr 40. This interaction stabilizes the tetrahedral intermediate and thus leads to selective acetylation of the R enantiomer. A stabilizing interaction of the delta-(R)-acetoxy group with the peptide NH of alanine 282 was also observed. No stabilizing interaction could be found for the delta-keto functionality, and it is proposed that this is the reason for the experimentally observed decrease in enantioselectivity. From these results, it was hypothesized that the enantioselectivity could be restored by mutating the alanine in position 281 for serine. The mutation was made experimentally, and the results show that the E value increased from 9 to 120.     

Modulation of Immobilized Lipase Enantioselectivity via Chemical Amination

The aspartic and glutamic carboxylic groups of the surface of three different immobilized lipases (those from Candida antarctica (form B) (CAL‐B), Thermomyces lanuginose (TLL) and Pseudomonas fluorescens (PFL) have been modified with ethylenediamine (after activation of the carboxylic function with carbodiimide). The exchange of groups with a negative charge for positively charged groups permitted us to greatly alter the activity, specificity and stereoselectivity of the lipase. Thus, in some cases, the activity of the lipases increased after the chemical modification while in other cases the activity was strongly reduced. Moreover, the effect of the experimental conditions on the activity was also strongly altered. Remarkably, the enantioselectivity of the enzymes in the hydrolysis of different mandelic acid derivatives was strongly modulated. For example, amination of the CNBr‐CAL‐B preparation greatly increased the enantioselectivity of the enzyme in the hydrolysis of (±)‐2‐hydroxyphenylacetic acid methyl ester, from an E value of 2 without modification up to E>100, affording (R)‐mandelic acid in high purity (ee>99 % at 50 % conversion) at pH 7 and 4 °C. Thus, the chemical modification of lipases seems to be a very powerful tool to improve their performance as enantioselective biocatalysts.