腈水解酶克隆表达、固定化及分子改造的研究进展

随着基因工程技术的快速发展,通过对不同菌株腈水解酶基因的分析,将其克隆到表达菌株内,可以构建高效并且稳定的基因工程菌。对腈水解酶进行分子改造可以明显提高酶的活性、稳定性、底物耐受性和底物特异性等性能,为腈水解酶的工业化应用提供了可能。综述了腈水解酶的来源、结构、催化机制、克隆表达、固定化及分子改造等方面的研究进展。同时对腈水解酶的研究进行了展望,具有重要的指导意义。     

微生物环氧化合物水解酶在有机合成中的应用*

环氧化合物水解酶是一种在自然界广泛存在的水解酶,它能高对映体选择性、高区域选择性地将环氧化合物水解为相应的邻位二醇。近年来,国际上利用微生物环氧化合物水解酶催化的不对称水解反应获得了高光学纯度环氧化合物和邻二醇,可用于多种精细化学品和生物活性物质的合成,为该酶在合成工业上的应用开辟了一条新途径。这里综述了该酶的来源、催化机理、催化特性和应用研究情况。     

枯草杆菌脂肪水解酶LipA和LipB

源自枯草杆菌的分泌型脂肪水解酶LipA及LipB已经被克隆、表达并表征. 它们的分子结构特点、催化机理也已经被深入研究. 枯草杆菌脂肪水解酶因为具有较好的食品工业及化学工业等方面的应用潜力,已经吸引了越来越多的关注. 通过定向进化及高通量筛选的方法对酶分子进行改造,提高其热稳定性及立体选择性是近年的研究热点. 结合国外及本研究组的工作,本文对LipA和LipB的生化性质、结构特点以及采用基因工程突变的方法进行分子改造等方面的研究进展做一简要综述. 另外,对其中一些研究论文做了简要的评价,并提出对未来工作的展望.     

纤维素酶家族及其催化结构域分子改造的新进展

纤维素酶的分子改造是其催化性能改进及催化效率提升的重要手段。近年来,组学技术与结构测定技术的迅速发展,人们已建立了包括糖苷水解酶(Glycoside hydrolase,GH)在内的碳水化合物活性酶组分数据库。通过对同一蛋白家族进行序列比对、分子进化分析与祖先基因重构,以结构模建分析为指导的纤维素酶分子改造,可以明显缩小序列或结构的搜索空间,加快酶分子改造的速度,增大理性设计成功的概率;同时针对催化中心活性架构的分析可以进一步阐明纤维素酶的催化机理与酶分子持续性降解机制。文中主要对纤维素酶家族及其催化结构域的分子改造取得的最新进展作了综述。在后基因组时代基于蛋白质家族中的海量数据分析,以其保守结构信息为指导的理性设计,将会成为纤维素酶分子改造的重要方向,从而推动生物质转化工艺的快速发展。     

水稻水溶性环氧化合物水解酶的生物信息学分析

水溶性环氧化合物水解酶（Soluble Epoxide Hydrolase,SEH）是一组催化环氧化合物水解为相应邻位二醇的酶类,在哺乳动物、植物、昆虫和微生物体内广泛存在。通过BLAST对水稻基因组的蛋白质数据库进行搜索,获得10个水溶性环氧化物水解酶（Soluble Epoxide Hydrolase SEH）sEH蛋白的同源序列。经分析发现这些基因在水稻不同胁迫处理下各个部位都有所表达,而且不同成员之间的表达模式存在较大的差异。水稻sEH蛋白主要参与角质层形成,应激反应,以及病原防御等生理过程,特别在脱毒过程中扮演着重要的角色。对蛋白质多序列联配和三级结构预测结果表明：水溶性环氧化合物水解酶的核心结构域由3个催化残基Asp、His和Asp形成三位一体的催化活性构象。这类基因的表达受抗逆环境诱导,其功能与抗逆性有关,为基因工程抗逆育种提供了参考。     

九香虫保幼激素环氧水解酶基因克隆、生物信息学及表达分析

[目的]保幼激素环氧水解酶(Juvenile hormone epoxide hydrolase,JHEH)是昆虫体内保幼激素(Juvenile hormone,JH)的主要降解酶.本文旨在分析九香虫Aspongopus chinensis Dallas保幼激素环氧水解酶基因序列(AcJHEH)信息,探索其在九香虫生长发育过程中的作用.[方法]以九香虫成虫的cDNA为模板,采用RT-PCR技术克隆获得AcJHEH基因序列,并利用生物信息学软件对其编码蛋白的理化特性、结构特征和系统进化进行分析;采用qRT-PCR技术检测并分析九香虫不同龄期和不同组织中AcJHEH的相对表达量.[结果]成功获得了AcJHEH的完整开放阅读框ORF序列,全长均为1 363 bp,共编码453个氨基酸,预测分子量为51.74 ku,等电点(pI)为7.66,分子蛋白式C2414H3683N581O653S14,含有N-端跨膜基序XWG、催化三联体、保守基序HGWP和2个酪氨酸,属于环氧水解酶家族.系统进化树分析表明,AcJHEH与茶翅蝽Halyomorpha halys的JHEH聚为一支,再与同为半翅目的臭虫Cimex lectulariu和绿盲蝽Apolygus lucorum的JHEH聚为一类.荧光定量PCR结果显示,AcJHEH在九香虫不同发育阶段和各组织中均有表达,且脂肪中的表达量极显著高于其它组织(P＜0.001);滞育九香虫成虫表达量最高,其次为4-5龄若虫都极显著高于其它发育阶段(P＜0.001).[结论]九香虫JHEH属于环氧水解酶家族,确定为保幼激素环氧水解酶.依据qPCR结果推测AcJHEH通过改变九香虫体内保幼激素浓度来影响九香虫4-5龄若虫蜕皮发育、成虫生殖系统发育和滞育等.     

细菌编码β-1,3-1,4-葡聚糖酶催化活性优化方法

β-1,3-1,4-葡聚糖酶是一类专一降解β-葡聚糖的内切水解酶。高效β-葡聚糖酶在啤酒酿造工业上具有十分重要的应用价值。目前,研究较多的β-1,3-1,4-葡聚糖酶主要来源于细菌。文中概述了细菌编码β-1,3-1,4-葡聚糖酶的分子生物学性质,并且从蛋白分子改造、表达调控和发酵条件优化三方面阐述了其催化活性提高的方法和成果。     

可溶性环氧化物水解酶在疾病中的作用

花生四烯酸经过细胞色素P450(cytochrome P450,CYP)表氧化酶途径生成环氧二十碳三烯酸(epoxy eicosatrienoic acid,EETs),具有扩张血管、降低血压、抗炎等多种生物学功能。在哺乳动物系统中的可溶性环氧化物水解酶（soluble epoxide hydrolase,sEH)具有α/β水解酶折叠结构,对环氧脂肪酸具有高度的选择性。sEH能够快速水解EETs,增加患心血管疾病的风险。目前,研究发现sEH抑制剂具有抑制sEH活性、提高EETs的含量的重要功能。 在多种疾病动物模型中应用sEH抑制剂或sEH基因敲除,证实sEH在心肌肥厚、糖尿病、高血压和肾病等疾病中发挥重要的生理作用。因此,sEH已被作为疾病治疗的新靶点而进行研究。本文就sEH的分布、作用机制以及sEH与疾病的关系等方面进行了讨论。     

热稳定型α-半乳糖苷酶的应用及基因工程研究进展

α-半乳糖苷酶是一种水解酶,可以将食品、饲料中的不良寡糖等抗营养因子水解,改变其营养成分并被动物吸收利用。该酶广泛存在于植物、动物及微生物中,在饲料、食品等工业以及现代医学中都有应用。目前,开发热稳定的α-半乳糖苷酶并利用基因工程方法进行分子改造和外源表达已成为研究热点。本文对近年来热稳定的α-半乳糖苷酶的应用和基因工程研究进展进行综述。     

PET水解酶传统与智能分子设计研究进展

塑料由于其耐久性和耐降解性造成的环境污染日趋严重,而塑料废弃物的处理回收方法存在着缺陷。聚对苯二甲酸乙二醇酯（polyethylene terephthalate,PET）是应用最广泛的塑料类型之一,但在自然条件下很难被降解。近年来,虽然多种具有PET降解活性的酶被发现,但这些酶的催化活性和热稳定性难以支撑实际工业所需,因此提高PET水解酶的降解能力已成为研究热点而备受关注。脂肪酶、角质酶、IsPETase和IsMHETase是目前研究最为广泛的PET水解酶,就这几种酶的结构、活性特征进行了总结,重点阐述了传统蛋白质工程和人工智能分子设计在增强PET水解酶应用性能方面的研究进展。期望塑料降解酶可以进一步发展优化,为循环塑料经济做出有价值的贡献。     

Sequence and structure of epoxide hydrolases: a systematic analysis

Epoxide hydrolases (EC 3.3.2.3) are ubiquitous enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. More than 100 epoxide hydrolases (EH) have been identified or predicted, and 3 structures are available. Although they catalyze the same chemical reaction, sequence similarity is low. To identify conserved regions, all EHs were aligned. Phylogenetic analysis identified 12 homologous families, which were grouped into 2 major superfamilies: the microsomal EH superfamily, which includes the homologous families of Mammalian, Insect, Fungal, and Bacterial EHs, and the cytosolic EH superfamily, which includes Mammalian, Plant, and Bacterial EHs. Bacterial EHs show a high sequence diversity. Based on structure comparison of three known structures from Agrobacterium radiobacter AD1 (cytosolic EH), Aspergillus niger (microsomal EH), Mus musculus (cytosolic EH), and multisequence alignment and phylogenetic analysis of 95 EHs, the modular architecture of this enzyme family was analyzed. Although core and cap domain are highly conserved, the structural differences between the EHs are restricted to only two loops: the NC-loop connecting the core and the cap and the cap-loop, which is inserted into the cap domain. EHs were assigned to either of three clusters based on loop length. By using this classification, core and cap region of all EHs, NC-loops and cap-loops of 78% and 89% of all EHs, respectively, could be modeled. Representative models are available from the Lipase Engineering Database, http://www.led.uni-stuttgart.de.     

An Overview on the Enhancement of Enantioselectivity and Stability of Microbial Epoxide Hydrolases

Epoxide hydrolases (EHs; 3.3.2.x) catalyze the enantioselective ring opening of racemic epoxides to the corresponding enantiopure vicinal diols and remaining equivalent unreacted epoxides. These epoxides and diols are used for the synthesis of chiral drug intermediates. With an upsurge in the methods for identification of novel microbial EHs, a lot of EHs have been discovered and utilized for kinetic resolution of racemic epoxides. However, there is still a constraint on the account of limited EHs being successfully applied on the preparative scale for industrial biotransformations. This limitation has to be overcome before application of identified functional EHs on large scale. Many strategies such as optimizing reaction media, immobilizing EHs and laboratory-scale directed evolution of EHs have been adopted for enhancing the industrial potential of EHs. In this review, these approaches have been highlighted which can serve as a pathway for the enrichment of already identified EHs for their application on an industrial scale in future studies.     

Molecular engineering of epoxide hydrolase and its application to asymmetric and enantioconvergent hydrolysis

Safety and regulatory issues favor increasing use of enantiopure compounds in pharmaceuticals. Enantiopure epoxides and diols are valuable intermediates in organic synthesis for the production of optically active pharmaceuticals. Enantiopure epoxide can be prepared using epoxide hydrolase (EH)-catalyzed asymmetric hydrolysis of its racemate. Enantioconvergent hydrolysis of racemic epoxides by EHs possessing complementary enantioselectivity and regioselectivity can lead to the formation of enantiopure vicinal diols with high yield. EHs are cofactor-independent and easy-to-use catalysts. EHs will attract much attention as commercial biocatalysts for the preparation of enantiopure epoxides and diols. In this paper, recent progress in molecular engineering of EHs is reviewed. Some examples and prospects of asymmetric and enantioconvergent hydrolysis reactions are discussed as supplements to molecular engineering to improve EH performance.     

One-pot biotransformation of racemic styrene oxide into (R)-1,2-phenylethandiol by two recombinant microbial epoxide hydrolases

An enantioconvergent biotransformation of racemic styrene oxide by using two recombinant microbial epoxide hydrolases (EHs) in one pot has been investigated to prepare enantiopure vicinal diols. The recombinant whole cell possessing EH gene from Aspergillus niger LK or Rhodotorula glutinis exhibited a complementary enantioselectivity and regioselectivity, compared to the recombinant cell containing Caulobacter crescentus EH gene. When two recombinant microbial EHs were used in combination, 1.3 g of enantiopure (R)-1,2-phenylethandiol with more than 90% enantiopurity and 95% overall yield was obtained from 1.2 g of racemic styrene oxide in a preparative-scale batch enantioconvergent biotransformation.     

Epoxide hydrolases: their roles and interactions with lipid metabolism

The epoxide hydrolases (EHs) are enzymes present in all living organisms, which transform epoxide containing lipids by the addition of water. In plants and animals, many of these lipid substrates have potent biologically activities, such as host defenses, control of development, regulation of inflammation and blood pressure. Thus the EHs have important and diverse biological roles with profound effects on the physiological state of the host organisms. Currently, seven distinct epoxide hydrolase sub-types are recognized in higher organisms. These include the plant soluble EHs, the mammalian soluble epoxide hydrolase, the hepoxilin hydrolase, leukotriene A4 hydrolase, the microsomal epoxide hydrolase, and the insect juvenile hormone epoxide hydrolase. While our understanding of these enzymes has progressed at different rates, here we discuss the current state of knowledge for each of these enzymes, along with a distillation of our current understanding of their endogenous roles. By reviewing the entire enzyme class together, both commonalities and discrepancies in our understanding are highlighted and important directions for future research pertaining to these enzymes are indicated.     

Microbiological transformations 50: selection of epoxide hydrolases for enzymatic resolution of 2-, 3- or 4-pyridyloxirane

A study aimed to select efficient epoxide hydrolases (EHs) allowing to achieve the enzymatic resolution of 2-, 3- and 4-pyridyloxirane (1–3) has been achieved, using 2-pyridyloxirane 1 as test substrate. Five thus selected EH-sources that showed interesting enantioselectivity were looked at in more detail for the conversion of 1–3.     

Epoxide hydrolase-mediated enantioconvergent bioconversions to prepare chiral epoxides and alcohols

A number of epoxide hydrolase (EH)-mediated bioconversions have been developed to prepare single enantiomeric product from racemic substrates with a yield greater than 50%. Enantioconvergent hydrolysis using single or two EHs possessing complementary enantio- and regio-selectivity, EH-based chemoenzymatic reactions, and EH-triggered cascade-reactions have been developed for the preparation of chiral epoxides, epoxyalcohols, tetrahydrofuran derivatives and vicinal diols. All these bioconversions are based on stereochemical flexibilities of various EHs and can be used in total synthesis of biologically active compounds without the formation of unwanted enantiomers.     

Cloning and characterization of a microsomal epoxide hydrolase from Heliothis virescens

Epoxide hydrolases (EHs) are α/β-hydrolase fold superfamily enzymes that convert epoxides to 1,2-trans diols. In insects EHs play critical roles in the metabolism of toxic compounds and allelochemicals found in the diet and for the regulation of endogenous juvenile hormones (JHs). In this study we obtained a full-length cDNA, hvmeh1, from the generalist feeder Heliothis virescens that encoded a highly active EH, Hv-mEH1. Of the 10 different EH substrates that were tested, Hv-mEH1 showed the highest specific activity (1180 nmol min^?1 mg^?1) for a 1,2-disubstituted epoxide-containing fluorescent substrate. This specific activity was more than 25- and 3900-fold higher than that for the general EH substrates cis-stilbene oxide and trans-stilbene oxide, respectively. Although phylogenetic analysis placed Hv-mEH1 in a clade with some lepidopteran JH metabolizing EHs (JHEHs), JH III was a relatively poor substrate for Hv-mEH1. Hv-mEH1 showed a unique substrate selectivity profile for the substrates tested in comparison to those of MsJHEH, a well-characterized JHEH from Manduca sexta, and hmEH, a human microsomal EH. Hv-mEH1 also showed unique enzyme inhibition profiles to JH-like urea, JH-like secondary amide, JH-like primary amide, and non-JH-like primary amide compounds in comparison to MsJHEH and hmEH. Although Hv-mEH1 is capable of metabolizing JH III, our findings suggest that this enzymatic activity does not play a significant role in the metabolism of JH in the caterpillar. The ability of Hv-mEH1 to rapidly hydrolyze 1,2-disubstituted epoxides suggests that it may play roles in the metabolism of fatty acid epoxides such as those that are commonly found in the diet of Heliothis.     

Substrate and inhibitor selectivity,and biological activity of an epoxide hydrolase from Trichoderma reesei

Molecular Biology Reports - Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the...