生物催化立体选择性氧化还原中存在问题及其发展策略

以立体选择性氧化还原酶或其全细胞催化的不对称氧化还原反应已经成为转化光学活性手性醇及其他手性化合物的有效手段。然而,生物催化氧化还原反应体系存在着催化活性与专一性、反应体系与催化稳定性等生物催化剂所固有的局限性问题,而且,生物氧化还原反应必需辅酶及其再生问题也是限制该转化途径产业化应用的一个重要因素。围绕上述生物催化立体选择性氧化还原中存在的关键问题,现代分子生物技术及反应工程的不断突破和发展为改善生物催化立体选择性氧化还原在催化剂本身和反应工程方面的局限性提供了有效的发展策略,为其进一步大规模产业应用提供了发展基础。     

Bacillaene酮还原酶结构域的异源表达及底物特异性分析

Bacillaene生物合成过程中,聚酮合酶第一个延伸模块的酮还原酶结构域(Bac KR1)既催化α酮基的还原,也催化β酮基的还原,具有天然的底物宽泛性。为进一步研究该结构域的底物特异性,在大肠杆菌中对其进行了异源表达。体外酶学分析表明Bac KR1可以催化聚酮类底物(±)-2-甲基-3-氧代戊酸-乙酰半胱胺硫酯外消旋体的立体选择性还原,仅生成4种非对映异构体中的一种,此外Bac KR1还可以催化环己酮和对氯苯乙酮等非聚酮类底物的还原,暗示了聚酮合酶中酮还原酶结构域作为生物催化剂的潜力。     

生物催化中酰胺酶立体选择性的影响因素

立体选择性酰胺酶是一种重要的手性合成工具酶,在制备手性羧酸及其衍生物方面具有广阔的应用前景,日益受到重视。在酰胺酶的应用中,其立体选择性影响巨大。从底物、反应温度、pH、添加共溶剂和微生物来源5个方面综述了其对酰胺酶立体选择性的影响,对提高酰胺酶的立体选择性,扩大其在制备光学活性化合物领域的应用具有重要的意义。     

Baeyer-Villiger单加氧酶非保守Hinge影响酶的催化活性和立体选择性

Baeyer-Villiger单加氧酶是一种重要的生物催化剂,可用于合成一系列有价值的酯和内酯化合物。通过序列比对和晶体结构分析推测连接NADPH结构域和FAD结构域的一段非保守Hinge可能在酶对底物识别和催化氧化过程中扮演着重要角色。在以环己酮单加氧酶为模型的研究中发现,对该Hinge结构进行同源序列替换得到的突变体几乎完全丧失了催化活性,证明了其整体水平的重要性。丙氨酸扫描突变揭示其中一些位点对酶的功能有显著影响:K153位点的改变使酶的活性下降,立体选择性却更优化;L143位点的改变对酶的活性影响较小,却降低了立体选择性;L144位点的改变则同时大幅度削弱酶的活性和立体选择性。将同样的方法运用在苯丙酮单加氧酶中,我们得到了相似的结论,证明这些位点的重要功能在Baeyer-Villiger单加氧酶家族中有一定的普遍性。这一研究增进了对Baeyer-Villiger单加氧酶的结构与功能关系的认识,有助于底物结合口袋的精确描述和Baeyer-Villiger单加氧酶催化图景的进一步细化,对未来相关的理性设计和定向改造研究提供了借鉴。     

来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶的手性识别过程研究

立体选择性是2-卤代酸脱卤酶最重要的性质之一,但目前其手性识别过程尚不明确,对其进行研究和解析具有重要意义。以来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶dehDIV-R为模型,研究了R-2-卤代酸脱卤酶的手性识别过程。首先通过测定反应产物的构型,确定dehDIV-R催化底物为SN2反应。通过Discovery Studio 3.0对dehDIV-R进行同源模建及底物分子对接,由对接结果和序列比对确定dehDIV-R立体选择性的关键位点Asn236,预测dehDIV-R的立体选择性与反应时底物到达反应位置的空间位阻密切相关。对dehDIV-R进行虚拟突变,将Asn236位点突变成具有不同空间位阻的残基Ala和Ser,并分别与底物分子进行分子对接,预测突变酶的立体选择性。根据预测结果,对Asn236氨基酸残基进行定点突变,发现在Asn236突变为Ala后的A1酶显示出对RS底物的活力;在Asn236突变为Ser后的S1酶显示出与原始酶相反的立体选择性,实现了立体选择性的反转。与模型的预测结果相符,证明了模型的合理性。     

作为人造极端酶的交联酶晶体

作为人造极端酶的交联酶晶体林影卢荣德郭勇（华南理工大学生物工程系,广州５１０６４０关键词交联酶晶体极端酶酶作为生物催化剂,催化效率高,立体选择性和底物专一性强,是普通化学催化剂所不可比拟的。然而,在酶的应用过程中还有很多令人不满意之处,如在环境中常常...     

w-转氨酶分子改造研究进展

ω-转氨酶能催化羰基化合物发生不对称还原胺化反应,在制备手性胺类化合物方面具有较好的应用前景。由于底物结合区域特殊的空间结构,野生型ω-转氨酶在合成大位阻手性胺方面的应用受到了限制。此外,在立体选择性和稳定性方面这一类酶也存在一些不足,目前满足工业应用需求的ω-转氨酶仍较为有限。文中首先介绍了ω-转氨酶的结构特征和催化机制,并探讨S型和R型酶在结构特征方面的主要差异。然后对ω-转氨酶的分子改造研究进行了综述,重点阐述了基于结构特征和催化机制进行的分子改造研究,包括底物特异性改造、立体选择性改造和稳定性改造三方面。最后,对ω-转氨酶分子改造研究进展进行总结和展望。     

ω-转氨酶不对称合成手性胺及非天然氨基酸的研究进展

ω-转氨酶不对称合成手性胺及非天然氨基酸是目前生物加工过程的研究热点之一。ω-转氨酶具有优良的立体选择性及区域选择性,利用其进行生物催化生产手性胺,已被应用于医药、农药和化工等领域。本文中,笔者综述了ω-转氨酶的基本结构特性,并以转氨酶法制备西他列汀关键中间体等为例,同时阐述了该酶的高通量筛选方法及分子改造方面的研究进展,并对级联反应提高手性胺产量的策略作了进一步讨论。最后,本文简要总结了ω-转氨酶在不对称合成非天然氨基酸中的具体应用。     

ω-转氨酶分子改造研究进展

ω-转氨酶能催化羰基化合物发生不对称还原胺化反应,在制备手性胺类化合物方面具有较好的应用前景。由于底物结合区域特殊的空间结构,野生型ω-转氨酶在合成大位阻手性胺方面的应用受到了限制。此外,在立体选择性和稳定性方面这一类酶也存在一些不足,目前满足工业应用需求的ω-转氨酶仍较为有限。文中首先介绍了ω-转氨酶的结构特征和催化机制,并探讨S型和R型酶在结构特征方面的主要差异。然后对ω-转氨酶的分子改造研究进行了综述,重点阐述了基于结构特征和催化机制进行的分子改造研究,包括底物特异性改造、立体选择性改造和稳定性改造三方面。最后,对ω-转氨酶分子改造研究进展进行总结和展望。     

一种来源于Burkholderia phytofirmans PsJN的ω-转氨酶的表达纯化及性质分析

ω-转氨酶(ω-transaminase)可以通过手性拆分和不对称合成的催化反应来获得光学纯的手性胺类化合物和非天然氨基酸,在医药中间体合成中是一种重要的生物催化剂。采用基因挖掘技术,在基因组数据库中获得一个来自伯克氏菌Burkholderia phytofirmans Ps JN的ω-转氨酶基因(hbp),将该基因在大肠杆菌BL21(DE3)中克隆、表达,利用镍柱亲和层析将该酶(HBP)进行纯化并研究了其酶学性质和底物谱。结果表明,以β-苯丙氨酸(β-Phe)为氨基供体、丙酮酸为氨基受体,HBP具有较高的活力(33.80 U/mg)和立体选择性;其最适温度为40℃左右,最适p H在8.0–8.5之间。研究过程中,建立了一种简便快捷的紫外吸收法来检测β-Phe的脱氨反应,证明了该反应的热力学平衡性质。底物谱研究表明HBP可以以β-Phe及其衍生物为氨基供体。结果表明HBP能够有效地手性拆分rac-β-Phe及其衍生物,转化率在50%左右,ee99%。     

Engineered enzymes for chemical production

In order to enable competitive manufacturing routes, most biocatalysts must be tailor-made for their processes. Enzymes from nature rarely have the combined properties necessary for industrial chemical production such as high activity and selectivity on non-natural substrates and toleration of high concentrations of organic media over the wide range of conditions (decreasing substrate, increasing product concentrations, solvents, etc.,) that will be present over the course of a manufacturing process. With the advances in protein engineering technologies, a variety of enzyme properties can be altered simultaneously, if the appropriate screening parameters are employed. Here we discuss the process of directed evolution for the generation of commercially viable biocatalysts for the production of fine chemicals, and how novel approaches have helped to overcome some of the challenges.     

微生物脂肪酶蛋白质工程*

微生物脂肪酶催化的化学反应具有严格的立体选择性、位点选择性等专一性,催化活性高而副反应少,催化反应不需要辅助因子等特点,因此广泛应用于工农业生产中的诸多领域。利用蛋白质工程技术,提高微生物脂肪酶的特异性、活性和稳定性,对提高微生物脂肪酶制剂产品的市场竞争能力,扩大微生物脂肪酶的应用领域,具有重要的意义。综述了蛋白质工程技术在微生物脂肪酶改性方面的应用现状、存在问题及前景。     

Probing and Engineering the Fatty Acyl Substrate Selectivity of Starter Condensation Domains of Nonribosomal Peptide Synthetases in Lipopeptide Biosynthesis

Lipopeptides are produced by nonribosomal peptide synthetases (NRPSs) and contain diverse fatty acyl moieties that are major determinants of antibiotic potency. The lipid chains are incorporated into peptidyl backbones via lipoinitiation, a process comprising free fatty acid activation and the subsequent starter condensation domain (C1)‐catalyzed conjugation of fatty acyl moieties onto the aminoacyl substrates. Thus, a thorough understanding of lipoinitiation biocatalysts would significantly expand their potential to produce novel antibiotics. Here, biochemical assays, in silico analysis, and mutagenesis studies are used to ultimately identify the specific amino acid residues that control the fatty acyl substrate selectivity of C1 in lipopeptide A54145. In silico docking study has identified four candidate amino acids, and subsequent in vitro assays confirmed their functional contribution to the channel that controls substrate selectivity. Two engineered variants with single point mutations in C1 are found to alter the substrate selectivity toward nonnatural fatty acyl substrates. The detailed mechanistic insights into the catalytic contribution of C1 obtained from the present study will facilitate future NPRS biocatalyst efforts     

A study of Raphanus sativus and its endophytes as carbonyl group bioreducing agents

The reported anti-Prelog selectivity in Raphanus sativus hairy roots prompted us to search for similar activity in other R. sativus systems, such as in vitro germinated sprouts, edible roots, and its associated endophytic microorganisms. Two test substrates were used in these studies: acetophenone and rac-ethyl 2-ethyl-3-oxobutanoate. Endophytes were isolated under substrate selective pressure and individual strains were evaluated for their ability to reduce both substrates. Among the isolates, two bacteria and a fungus having the potential to be used as biocatalysts were found: Bacillus megaterium which reduces acetophenone with excellent anti-Prelog selectivity, and Pseudomonas sp. and Penicillium chrysogenum with enantioselectivity and stereoselectivity for the reduction of rac-ethyl 2-ethyl-3-oxobutanoate, respectively.     

Improved biocatalysts by directed evolution and rational protein design

The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity. However, wild-type enzymes often need to be optimized to fulfill these requirements. Two rather contradictory tools can be used on a molecular level to create tailor-made biocatalysts: directed evolution and rational protein design.     

Bioconversions in aqueous two-phase systems.

Bioconversions involving enzymes and/or microbial cells in aqueous two-phase systems are reviewed. The partitioning of biocatalysts, substrates, and products is discussed in relation to their size. The efficiency of retaining biocatalysts in aqueous two-phase systems is summarized in relation to other methods of recirculating. The influence of phase components on the activity and the stability of enzymatic biocatalysts is exemplified with penicillin acylase and the cellulolytic enzyme system, and the effect of phase components on biocatalytic living cells is exemplified with the production of alpha-amylase with Bacillus sp. Process design costs in bioconversions in aqueous two-phase systems are briefly summarized.     

Directed evolution of enzymes and biosynthetic pathways

Directed evolution is an important tool for overcoming the limitations of natural enzymes as biocatalysts. Recent advances have focused on applying directed evolution to a variety of enzymes, such as epoxide hydrolase, glyphosate N-acetyltransferase, xylanase and phosphotriesterase, in order to improve their activity, selectivity, stability and solubility. The focus has also shifted to manipulating biosynthetic pathways for the production of many naturally synthesized compounds, as well as the production of novel 'unnatural' compounds. A combined directed evolution and computational design approach is becoming increasingly important in exploring enzyme sequence-space and creating improved or novel enzymes. Fueled by recent breakthroughs in genomics and metagenomics, these developments should help expand the use of biocatalysts in industry.     

Microbial epoxide hydrolases for preparative biotransformations

Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolysed epoxide in nonracemic form, enantioconvergent processes are possible: these are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane. The data accumulated over recent years reveal a common picture of the substrate structure selectivity pattern of microbial epoxide hydrolases and indicate that substrates of various structural types can be selectively hydrolysed with enzymes from certain microbial sources.     

A systematic approach for yielding a potential pool of enzymes: practical case for chiral resolution of (R,S)-ketoprofen ethyl ester

A systematic approach for the selection of potential biocatalysts from a natural source was developed and then a practical application was addressed. The approach that involves systematically combined conventional screening methods and current tools comprises the following consecutive steps: strain enrichment for activity screening, identification of positive strains, choosing whole genome-sequenced strains as candidates, gathering information about responsible enzymes, bioinformatic analyses and gene mining, probing genetic molecules and then functional expression. The target compound (R,S)-ketoprofen ethyl ester was to be resolved into an enantiomer, and a potential esterase from Pseudomonas fluorescens KCTC 1767 was prepared by the proposed procedure. The enzyme had a high activity and also strict selectivity for the enantiomer (S)-ketoprofen and was suitable therefore as a biocatalyst for practical use. The result achieved by the combined approach could not easily be obtained using other approaches with typical procedures. Hence the approach proposed here should be of considerable use for the screening of potential enzymes, particularly for enzymes with desired activity to unnatural substrates, from conditionally expressed and/or repressed proteins that are distributed widely in natural pools under normal conditions.     

Predicting the impact of mutations on the specific activity of Bacillus thermocatenulatus lipase using a combined approach of docking and molecular dynamics

Lipases are important biocatalysts owing to their ability to catalyze diverse reactions with exceptional substrate specificities. A combined docking and molecular dynamics (MD) approach was applied to study the chain‐length selectivity of Bacillus thermocatenulatus lipase (BTL2) towards its natural substrates (triacylglycerols). A scoring function including electrostatic, van der Waals (vdW) and desolvation energies along with conformational entropy was developed to predict the impact of mutation. The native BTL2 and its 6 mutants (F17A, V175A, V175F, D176F, T178V and I320F) were experimentally analyzed to determine their specific activities towards tributyrin (C4) or tricaprylin (C8), which were used to test our approach. Our scoring methodology predicted the chain‐length selectivity of BTL2 with 85.7% (6/7) accuracy with a positive correlation between the calculated scores and the experimental activity values (r = 0.82, p = 0.0004). Additionally, the impact of mutation on activity was predicted with 75% (9/12) accuracy. The described study represents a fast and reliable approach to accurately predict the effect of mutations on the activity and selectivity of lipases and also of other enzymes. Copyright © 2016 John Wiley & Sons, Ltd.