合成生物学, 2020, 1(5): 528-539 DOI: 10.12211/2096-8280.2020-004

特约评述

微生物环氧水解酶催化机制及应用研究进展

曹菲,1,2, 李永泉1,2, 毛旭明1,2

1.浙江大学药物生物技术研究所，浙江 杭州 310058

2.浙江省微生物生化与代谢工程重点实验室，浙江 杭州 310058

Recent development in catalytic mechanisms and applications of microbial epoxide hydrolases

CAO Fei,1,2, LI Yongquan1,2, MAO Xuming1,2

1.Institute of Pharmaceutical Biotechnology，Zhejiang University，Hangzhou 310058，Zhejiang，China

2.Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering，Hangzhou 310058，Zhejiang，China

收稿日期: 2020-02-27   修回日期: 2020-08-07  

基金资助: 国家重点研发计划.  2019YFA0905400

Received: 2020-02-27   Revised: 2020-08-07  

作者简介 About authors

作者简介:曹菲（1995—），男，博士研究生，研究方向为微生物药物生物合成、微生物天然产物生物合成的酶学机制和化学机制研究、基于合成生物学的微生物药物开发。E-mail：feecao@zju.edu.cn

李永泉（1962—），男，博士，浙江大学求是特聘教授。研究方向为微生物合成生物学、微生物次级代谢调控和微生物制药。E-mail：lyq@zju.edu.cn

毛旭明（1978—），男，博士，教授，研究方向为基于合成生物学的微生物药物开发、微生物药物生物合成的调控机制研究、基于多组学的新活性和新结构微生物天然产物挖掘、微生物天然产物生物合成的酶学机制和化学机制研究。E-mail：xmmao@zju.edu.cn

摘要

微生物来源的环氧水解酶（epoxide hydrolases，EHs，EC 3.3.2.3）能不对称水解外消旋环氧化物生成手性环氧化物和邻二醇，催化效率高且区域、立体选择性强，有利于合成高纯度的手性化合物。因此，微生物EHs成为了手性药物合成的一种非常重要的生物催化剂，也是一种强有力的生物合成元件。近10年来，随着基因组学、分子生物学、化学生物学、结构生物学等技术的快速发展，研究者又从多种微生物体内发现了多种具有潜在应用价值的EHs。与此同时，研究者也对微生物来源的EHs的酶学特性、生物大分子结构与催化机理进行了深入的研究。本文介绍了几种目前研究比较透彻的EHs催化机制，并综述了近10年来最新发现的微生物来源EHs，这些EHs具备潜在的应用价值。此外，本文总结了EHs的应用进展，重点介绍了EHs在合成生物学领域的应用。利用酶的定向进化等技术提高EHs催化性能和串联多个合成元件高效合成手性产物，是当前主要的研究趋势。

关键词： 微生物 ; 环氧水解酶 ; 催化机制 ; 合成生物学 ; 手性药物

Abstract

Microbial epoxide hydrolases (EHs, EC 3.3.2.3) catalyze the enantioselective ring opening of racemic epoxides to the chiral epoxides and vicinal diols. EHs have high catalytic efficiency and chemical-, region-, and stereo-selectivity without cofactors, and are conducive to the synthesis of high-purity chiral compounds by biotransformation. Since they were discovered, EHs have become very important catalysts and powerful biosynthetic elements for the green synthesis of chiral drugs in industry. In the last 10 years, with the rapid development of genomics, molecular biology, chemical biology and structural biology, researchers have found a variety of EHs from various microorganisms with potential applications. At the same time, researchers have also paid a lot of attention to the enzymatic properties, protein structure and catalytic mechanism of microbial EHs. This review introduces the currently well-understood catalytic mechanism of microbial EHs, such as ArEH from Agrobacterium radiobacter and limonene 1,2-EH from Rhodococcus erythropolis. Moreover, it summarizes 30+ microbial EHs with potential values, which were newly discovered in the last decade based on genome mining of microorganisms, along with the cognate biosynthetic pathways for natural product production. At least 15 crystal structures of new microbial EHs have been elucidated, helping people further understand the catalytic mechanisms at atomic levels and laying the foundation of their evolutions and applications. In addition, the applications of EHs in the field of pharmaceuticals and synthetic biology are also highlighted. Nowadays, researchers are mainly focusing on the improvement of catalytic activity, specificity and efficiency, as well as enantio-purity and yield of the products from microbial EHs with two main strategies: random or rational mutations of enzymes and strains, and artificial design of enzyme cascades within chassis cells. In the future, the synthesis of chiral drugs catalyzed by EHs will commence on the evolution of enzymatic activity, and assembly of multi-enzyme instead of single-enzyme in a cell factory for efficient biocatalysis.

Keywords： microorganisms ; epoxide hydrolases ; catalytic mechanism ; synthetic biology ; chiral drugs

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曹菲, 李永泉, 毛旭明. 微生物环氧水解酶催化机制及应用研究进展. 合成生物学[J], 2020, 1(5): 528-539 DOI:10.12211/2096-8280.2020-004

CAO Fei, LI Yongquan, MAO Xuming. Recent development in catalytic mechanisms and applications of microbial epoxide hydrolases. Synthetic Biology Journal[J], 2020, 1(5): 528-539 DOI:10.12211/2096-8280.2020-004

环氧水解酶（epoxide hydrolases，EHs）是一类非常重要的生物催化剂，能选择性水解外消旋环氧化物生成具有高度手性选择性的环氧化物和邻二醇［1-3］，在制药工程、生物工程、化学工程等领域有广泛的应用。微生物来源的EHs大多属于α/β折叠酶［4］，相比于动植物来源的EHs，它的优点是容易大量制备、催化效率高且立体选择性强［5-6］，同时催化反应不需要辅酶的参与［6-7］。因此，微生物来源的EHs在生物催化领域被视为一类非常有价值的生物催化剂［8-10］。

近10年来，随着基因组学、分子生物学等领域的快速发展，又有许多微生物EHs通过基因组挖掘被发现［11-28］。同时越来越多需要EHs参与的复杂天然产物合成途径被解析［29-41］。还有部分病原微生物中也发现了新的EHs，这些EHs参与微生物生长代谢，可以作为药物靶点［42-49］。这些新发现的微生物EHs在手性化合物合成中具有潜在的应用价值。与此同时，研究者也对EHs的酶学特性、蛋白质结构与催化机理进行了深入的研究。

21世纪以来，合成生物学领域发展十分迅速，EHs作为一类重要的工程酶同时也是一类强有力的合成元件。许多研究者改变了手性药物中间体的合成思路，从简单的单酶催化到将合成元件（含EHs）流水线式组装入微生物细胞工厂，从头合成或从中间体通过一系列反应最终得到产物［50-51］。运用合成生物学理念，改变EHs应用方法，有利于降低底物成本、提高酶利用率，从而进一步降低手性药物中间体生产成本。本文作者介绍了微生物来源的EHs催化机制，总结了近10年来发现的新型微生物来源的EHs，还介绍了目前EHs在生物催化和合成生物学领域应用的进展。

1 微生物来源的环氧水解酶

1.1 酶学特性

微生物来源的EHs能不对称水解外消旋环氧化物生成有价值的手性化合物，具有优良的酶学特性，已经成为手性药物中间体合成领域重要的生物催化剂。EHs的催化效率高［5］，底物区域与立体选择性高［6］，反应不需辅酶或辅基参与，同时产物的手性控制强［6-7］。来源于放射形土壤农杆菌（Agrobacterium radiobacter AD1）的EH（ArEH）研究得比较透彻。1997年，Rink等［52］报道A. radiobacter AD1能在以环氧氯丙烷为唯一碳源的培养基上生长，原因是体内的ArEH能将环氧氯丙烷水解为3-氯-1，2-丙二醇，然后通过卤代醇脱卤酶将其转化为缩水甘油，缩水甘油进一步被中心代谢转化为甘油。研究者通过克隆ArEH基因，在Escherichia coli BL21（DE3）中大量表达与纯化，测定了ArEH的酶学特性。研究发现：①ArEH底物选择性高，底物谱分析表明环氧氯丙烷和环氧溴丙烷是最好的底物；②ArEH催化速率高，ArEH与环氧氯丙烷反应的Km远小于50 mmol/L，vmax约为 38 U/mg蛋白，催化常数kcat值约为21 s-1。

1.2 EHs催化机理

大部分微生物EHs属于α/β折叠水解酶家族，因其含有的核心结构域和帽子结构域两个功能性的结构单元都由α螺旋和β折叠组成［53］，活性位点位于核心和帽子结构域之间的主要疏水环境中。ArEH是具代表性的、研究比较透彻的一种α/β折叠型EH。以ArEH为代表的EHs活性位点由催化三联体残基构成，包括活性结构域中的亲核残基Asp、His和羧酸残基Asp/Glu；帽子结构域中Tyr为反应提供质子。现在比较公认的EHs催化的反应历程是通过两步法进行的。例如，ArEH的催化反应机理为：①Asp107亲核攻击环氧化物上的碳原子将环打开，形成共价酯中间体，Tyr152提供质子形成醇；②Asp246辅助His275活化水分子，使酯中间体水解形成邻二醇，并使产物与酶分离［54-55］。

也有一些EHs与上述ArEH有不同的结构与催化机制，例如红平红球菌Rhodococcus erythropolis DCL14来源的柠檬烯1，2-环氧水解酶（limonene-1，2-epoxide hydrolase，LEH）。LEH类EHs与α/β折叠型EHs区别是：LEH分子量较小，不具备α/β折叠结构，由α螺旋和β折叠弯曲形成疏水口袋。LEH活性位点由Arg99、Asp101和Asp132构成，其催化反应机理为：①Arg99分别与Asp131、Asp246的羧基形成氢键，稳定电荷，Tyr53、Asn55和Asp132与水分子形成氢键，将水分子定位于有利于亲核攻击底物的氧环；②Asp101为环氧化物的氧提供质子形成醇，Asp132吸引水分子中的质子，辅助亲核攻击环氧化物环碳原子直接形成二醇，而不形成酯中间体［56-57］。不同菌种来源的EHs基本都与上述两类EHs具有相似性，目前尚未有文章报道有新类型的微生物EHs。

1.3 微生物来源的新型EHs

近10年来，随着基因组学和分子生物学技术的发展，不断地有新微生物EHs被发现（表1）。2010—2019年，至少在30余种微生物中发现了新的能催化不同底物的EHs，并且至少有15个微生物EHs已经完成蛋白质结构解析。表1中总结了近10年报道的新发现的EHs或具备EH活性的菌株，罗列了EHs能催化的底物以及生成的产物。通过总结EHs来源菌株发现，挖掘新型微生物EHs的主要方法有：①筛选能催化生成手性环氧化物或二醇的菌株，并进一步鉴定相应的EHs（表1编号1～19）；②表征微生物天然产物的合成途径中的后修饰酶（表1编号20～34）；③分析鉴定能引起人或动物疾病的病原微生物体内的药物靶标。此外，这些EHs催化的底物既包含结构简单的环氧化物，也包含结构复杂的天然产物，说明微生物EHs具有宽泛的底物谱。这些新发现的EHs在手性药物中间体、复杂天然产物合成中具备很好的应用开发前景。

表1   2010—2019年微生物中新发现的EHs

Tab. 1  Newly discovered microbial EHs in 2010—2019

编号环氧水 解酶①菌株底物产物参考文献
14R9LRhodococcus erythropolis DCL14[11]
24NZZBacillus megaterium[12]
35JPURhodococcus erythropolis[13]
46IX4Aspergillus usamii E001[14]
5未报道Dentipellis sp. KUC8613[15]
6vEH-AmAgromyces mediolanus[16]
7未报道Aspergillus tubingensis TF1[17]
8未报道Aspergillus niger ZJUTZQ208[18]
9未报道Rhodobacterales bacterium HTCC2654[19]
10McEHCaulobacter crescentus[20]
11SpoFSalinospora tropica CNB-440[21]
12SgrFStreptomyces griseus IFO
13BMEHB. megateriumQMB 1551[22]
14CMEH

Cupriavidus

metallidurans-CH34

[23]
15AmEHAgromyces mediolanus ZJB120203[24]
16TpEH1Tsukamurella paurometabola[25]
17未报道Galactomyces geotrichum ZJUTZQ200[26]
18AbEH

Aspergillus

brasiliensis CCT 1435

[27]
19EphDMycobacterium tuberculosis[28]
203RGAStreptomyces lasaliensis[29]
215CXO

Streptomyces albus

ATCC 21838

[30]
226FXDPseudomonas fluorescens[31]
23AurDCalcarisporium arbuscula[32]
24CtvDAspergillus terreus var. aureus[33]
25NcsF2Streptomyces carzinostaticus ATCC 15944[34]
26PenJPenicillium sp.[35]
27AsqBAspergillus nidulans
28MonBⅠ/ⅡStreptomyces cinnamonesis[36]
29SalBⅠ/Ⅱ/ⅢStreptomyces albus DSM 41398[37]
30AuaⅠ

Stigmatella aurantiaca

Sg a15

[38]
31NsnG

Nocardiopsis sp.

CMB-M0232

[39]

32

33

Alp1U

Lom6

Streptomyces ambofaciens

Salinispora pacifica

[40]
34TsrIStreptomyces laurentii[41]

①环氧水解酶的名称来源于PDB ID （Protein Data Bank：http：//www.rcsb.org/） 或者参考文献中的蛋白名称。

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2 微生物EHs的应用

2.1 基于生物转化的手性药物中间体合成

手性环氧化物及邻二醇制备主要有化学合成法［58-59］和生物催化法［60-61］。化学合成手性环氧化物目前存在着反应条件苛刻、选择性差、重金属催化剂昂贵、环境不友好等问题［62］。而微生物来源的EHs制备成本低、酶催化效率高且不需辅酶与辅基参与反应［7］。更为重要的是，EHs对对映体选择性很高，能不对称水解外消旋环氧化物制备光学纯的环氧化物和邻二醇，生成多种有重要价值的手性药物中间体［62-63］，例如（R）-环氧氯丙烷［64-66］、手性苯基乙二醇［67］、（S）-芳基缩水甘油醚等［68-69］。

许多研究者通过对反应条件的摸索与优化［70］，结合随机突变［71-72］、迭代饱和突变［73-74］、定点突变等［75-77］酶的定向进化技术，对不同来源的微生物EHs加以优化改造提升催化活性、对映选择性以及产物产率等性质。Saini等［70］从Streptomyces griseus NBRC 13350基因组中发现了一种新的EH，该EH能将消旋苯基缩水甘油醚（PGE）动力学分解为（R）-PGE。通过催化条件摸索与优化，当反应中细胞与底物比20：1（质量比）、pH 7.5、20 ℃、体系含10%二甲基甲酰胺（体积分数）时，（R）-PGE产率为34%，e.e.达到99%。

按照随机突变策略，Jin等［78］使用60Co γ射线和紫外线照射诱变EH生产菌株Aspergillus niger ZJB-09103，筛选到了阳性突变菌株的EH，其活性达到33.7 U/g，是野生型菌株EH活性的2.7倍。突变株催化生成（S）-环氧氯丙烷所需的反应时间缩短了2.5倍，e.e.达到99%，收率约为17.6%。运用酶的定向进化技术，Zou等［73］通过迭代饱和突变ArEH，发现T247K/I108L/D131S突变的ArEH与野生型酶相比，对环氧氯丙烷的催化效率提高了4.5倍，对环氧氯丙烷的对映体选择性提高了2.1倍，50℃条件下热稳定性提高了8倍。使用突变的ArEH对外消旋环氧氯丙烷进行动力学拆分，（R）-环氧氯丙烷收率为40.2%，且e.e.大于99.9%，生产率达到1410 g/（L·d）。

2.2 基于合成生物学理念的新型手性化合物合成方法

微生物来源的EHs不仅在手性药物中间体有重要的应用，随着近年来合成生物学领域的快速发展，EHs作为一个重要的合成元件，未来在微生物底盘细胞中重构化合物合成途径［51，79-80］、基因簇异源表达、天然产物组合生物合成［81-82］等方面展现出非常广阔的应用潜力。目前，以微生物EHs为合成元件组装化合物的生物合成途径，已经在以大肠杆菌为代表的原核底盘细胞［80］和以酵母菌为代表的真核底盘细胞中有了较好的应用［51］。

大肠杆菌蛋白表达量高，遗传操作简便。许多研究者以大肠杆菌为底盘，将EHs与其他催化元件进行串联，从而合成手性药物中间体。Yu等［80］设计了一套不对称催化串联反应，该反应路线为过氧化酶OleTJE将底物苯丙酸脱羧氧化为苯乙烯，单加氧酶P450-BM3将苯乙烯氧化成（R）-氧化苯乙烯，最后通过Aspergillus niger来源的水解酶ANEH将氧化苯乙烯开环水解生成R型苯乙二醇类产物。该串联反应可对至少9种底物实现较高的转化率和对映选择性。此外，Sun等［50］设计了一种新的三步一锅法级联系统。该系统采用环氧水解酶、甘油脱氢酶和ω-转氨酶，将外消旋苯乙烯氧化物不对称合成（R）-苯基甘氨醇。还构建了AlaDH/L-Ala的辅助因子自给系统用于再生昂贵的辅助因子NAD+和去除副产物丙酮酸，并用阳离子树脂吸附去除产物使反应的热力学平衡朝产物生成的方向发展。最终，由外消旋苯乙烯氧化物成功制备光学纯的（R）-苯基甘氨醇，产率高达81.9%，e.e.达99％。Liu等［83］开发了从（S）-环氧化物一步合成光学纯的1，2-氨基醇的方法。该研究设计了一种非天然生物催化级联反应，通过偶联环氧水解酶SpEH、酒精脱氢酶MnADH和ω-转氨酶PAKω-TA，将级联反应路线构建在大肠杆菌BL21（SGMP）体内。另外，研究者通过过表达谷氨酸脱氢酶GluDH使得细胞可以同时释放辅助因子和氨基供体，建立了胞内辅因子和供体自足系统。该工程菌SGMP催化的产物e.e.大于99%，从（S）-环氧苯乙烷到1，2-氨基醇的转化率为99.6%；另外4种（S）-环氧化物转换为1，2-氨基醇的转化率在65%～96.4%。

除了上述以大肠杆菌为底盘设计串联反应外，Yuan等［51］首次尝试在真核生物酿酒酵母体内建立以苯乙烯为中间体生产苯乙二醇的人工生物合成途径。该研究使用苯丙氨酸氨裂合酶PAL和阿魏酸脱羧酶FDC将苯丙氨酸脱氨基/脱羧生成苯乙烯；对苯乙烯进行（S）-选择性环氧化，得到（S）-苯乙烯；再用苯乙烯单加氧酶SMO催化氧化生成（S）-环氧乙烷；最后，用鞘氨醇单胞菌HXN-200或马铃薯的环氧水解酶SpEH/StEH对（S）-环氧乙烷进行区域选择性水解，生成（S）-苯基乙二醇或（R）-苯基乙二醇。该研究证明，包含立体特异性环氧水解的级联反应可以在真核宿主酿酒酵母中功能性表达。小型摇瓶研究表明，经过工程改造的酵母细胞工厂在培养96 h后，可生产约100～120 mg/L的（S）-苯基乙二醇或（R）-苯基乙二醇。

3 展望

21世纪以来，基因组学的飞速发展使人们获取了大量微生物基因组信息，而其中蕴藏着大量具有环氧水解功能的催化酶基因。经过20年左右的努力，随着生物信息学、分子遗传学等领域的发展，已有数个EHs被发掘并成熟应用于不对称催化领域。当前，利用微生物EHs生物催化合成手性药物中间体已经成为了一种主流的工业生产方法。从庞大的微生物基因组中挖掘新型EHs的工作始终在进行中，天然产物生物合成研究构筑了EHs应用于合成生物学的坚实基础。值得注意的是，近年来又有许多新颖的微生物EHs从天然产物生物合成途径中被鉴定出来。在生物合成中，EHs往往作为一种重要的后修饰酶，介导环氧底物的开环反应，开环后的产物还可以被其他后修饰酶进一步催化，从而得到结构复杂的天然产物。因此，EHs为天然产物结构多样性提供了丰富前体。此外，EHs介导的反应常常具有高度的区域、立体选择性，这相比于化学合成具有很大的优势，十分具有成为生物催化剂或合成生物学元件的潜力。因此，在未来从微生物天然产物生物合成途径中挖掘EHs仍然是一项非常具有开创性的工作，这帮助人们不断充实EHs的底物谱、扩充备选的EHs生物催化及合成生物学元件库。

EHs结构生物学方面的研究帮助研究者更加清晰地了解了EHs的催化机制，尤其是其独特的手性选择性，这为未来酶的定向改造与产业化应用提供了坚实的理论基础。目前，许多微生物EHs生物大分子结构已经被解析，几个代表性的EHs催化机理已经研究得比较清楚，例如ArEH、LEH等。然而，新颖的微生物EHs数量仍在不断增加，其中有不少EHs具备特殊的结构与功能，识别独特而复杂的底物。作为一项基础性研究工作，利用结构生物学手段解析新型微生物EHs结构仍需要研究者们不断地投入大量的工作。为了将EHs进行产业化应用，必须通过不断提高EHs催化效率、稳定性和对映体产物纯度等酶学特性来降低生产成本。研究者基于酶的定向进化策略，通过反应体系优化、随机突变、定点突变、迭代饱和突变等技术，得到了众多具有优良活性的工程酶。这项工作是EHs走上产业化之路必不可少的环节。

21世纪新兴的合成生物学研究给传统的基于生物催化生产手性药物中间体的生产方式带来了冲击。其理念是将不同来源的生物合成相关的代谢途径模块化、元件化，并在底盘细胞上组装；人工设计目的产物的生物合成途径，提高代谢途径的效率，从而实现手性药物中间体的高效生物合成和规模化生产。基于合成生物学理念，已有研究将EHs作为合成元件与上游和下游催化元件串联组装入细胞工厂（大肠杆菌、酵母等），组成一条相对完整的代谢通路，用于高效合成手性药物中间体。未来，催化元件性能的不断提升、底盘细胞内供给系统的不断优化，将会进一步提升产物生成效率，手性药物中间体生产成本会逐步降低。这可能会使合成生物学生产方法逐渐取代单酶催化法，成为手性药物中间体生产的主流方法。


参考文献

CHEN Yijun, CHEN Chen, WU Xuri.

Dicarbonyl reduction by single enzyme for the preparation of chiral diols

[J]. Chemical Society Reviews, 2012, 41(5): 1742-1753.

[本文引用: 1]

STEINREIBER A, FABER K.

Microbial epoxide hydrolases for preparative biotransformations

[J]. Current Opinion in Biotechnology, 2001, 12(6): 552-558.

ZOCHER F, ENZELBERGER M M, BORNSCHEUER U T, et al.

Epoxide hydrolase activity of Streptomyces strains

[J]. Journal of Biotechnology, 2000, 77(2/3): 287-292.

[本文引用: 1]

MORISSEAU C, HAMMOCK B D.

Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles

[J]. Annual Review of Pharmacology and Toxicology, 2005, 45: 311-333.

[本文引用: 1]

MANOJ K, ARCHELAS A, BARATTI J, et al.

Microbiological transformations. Part 45: A green chemistry preparative scale synthesis of enantiopure building blocks of Eliprodil: elaboration of a high substrate concentration epoxide hydrolase-catalyzed hydrolytic kinetic resolution process

[J]. Tetrahedron, 2001, 57(4): 695-701.

[本文引用: 2]

ORRU R V, FABER K.

Stereoselectivities of microbial epoxide hydrolases

[J]. Current Opinion in Biotechnology, 1999, 3(1): 16-21.

[本文引用: 4]

NESTL B M, HAMMER S C, NEBEL B A, et al.

New generation of biocatalysts for organic synthesis

[J]. Angewandte Chemie International Edition, 2014, 53(12): 3070-3095.

[本文引用: 3]

潘海峰.

新颖水解酶CESH的催化机制及其高效催化工艺研究

[D]. 杭州: 浙江大学, 2011.

[本文引用: 1]

PAN Haifeng.

Study on the catalytic mechanism of a novel hydrolase CESH and its catalytic process with a high efficiency

[D]. Hangzhou: Zhejiang University, 2011.

[本文引用: 1]

壮晓健, 金火喜, 胡忠策, 等.

微生物环氧化物水解酶的研究进展

[J]. 生物技术, 2010, 20(1): 91-94.

ZHUANG Xiaojian, JIN Huoxi, HU Zhongce, et al.

Research progress of microbial epoxide hydrolase

[J]. Chinese Journal of Biotechnology, 2010, 20(1): 91-94.

CHOI Won Jae, CHOI Cha Yong.

Production of chiral epoxides: epoxide hydrolase-catalyzed enantioselective hydrolysis

[J]. Biotechnology and Bioprocess Engineering, 2005, 10(3): 167.

[本文引用: 1]

FLOOR R J, WIJMA H J, JEKEL P A, et al.

X-ray crystallographic validation of structure predictions used in computational design for protein stabilization

[J]. Proteins, 2015, 83(5): 940-951.

[本文引用: 2]

KONG Xudong, YUAN Shuguang, LI Lin, et al.

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

[J]. Proceedings of the National Academy of Sciences of The United States Of America, 2014, 111(44): 15717-15722.

[本文引用: 1]

LI Guangyue, ZHANG Hui, SUN Zhoutong, et al.

Multiparameter optimization in directed evolution: engineering thermostability, enantioselectivity, and activity of an epoxide hydrolase

[J]. ACS Catalysis, 2016, 6(6): 3679-3687.

[本文引用: 1]

HU Die, TANG Cunduo, YANG Biao, et al.

Expression of a novel epoxide hydrolase of Aspergillus usamii E001 in Escherichia coli and its performance in resolution of racemic styrene oxide

[J]. Journal of Industrial Microbiology & Biotechnology, 2015, 42(5): 671-680.

[本文引用: 1]

PARK Hongjae, MIN Byoungnam, JANG Yeongseon, et al.

Comprehensive genomic and transcriptomic analysis of polycyclic aromatic hydrocarbon degradation by a mycoremediation fungus, Dentipellis sp. KUC8613

[J]. Applied Microbiology and Biotechnology, 2019, 103(19): 8145-8155.

[本文引用: 1]

JIN Huoxi, LI Yan, ZHANG Qianwei, et al.

Enantioselective hydrolysis of styrene oxide and benzyl glycidyl ether by a variant of epoxide hydrolase from Agromyces mediolanus

[J]. Marine Drugs, 2019, 17(6): 367.

[本文引用: 1]

DUARAH A, GOSWAMI A, BORA T C, et al.

Enantioconvergent biohydrolysis of racemic styrene oxide to R-phenyl-1,2-ethanediol by a newly isolated filamentous fungus Aspergillus tubingensis TF1

[J]. Applied Biochemistry and Biotechnology, 2013, 170(8): 1965-1973.

[本文引用: 1]

CHEN Lin, SHEN ChenHonglei, WEI Chun, et al.

Bioresolution of (R)-glycidyl azide by Aspergillus niger ZJUTZQ208: a new and concise synthon for chiral vicinal amino alcohols

[J]. Applied Microbiology and Biotechnology, 2013, 97(6): 2609-2616.

[本文引用: 1]

Jung-Hee WOO, KANG Ji-Hyun, HWANG Young-Ok, et al.

Biocatalytic resolution of glycidyl phenyl ether using a novel epoxide hydrolase from a marine bacterium, Rhodobacterales bacterium HTCC2654

[J]. Journal of Bioscience and Bioengineering, 2010, 110(4): 509-509.

[本文引用: 1]

MIN Ji Young, Eun Yeol LEE.

Biosynthesis of (R)-1,2-phenylethanediol and (R)-4-chloro-1,2-phenylethanediol by using two recombinant cells expressing enantiocomplementary epoxide hydrolases

[J]. Journal of Industrial and Engineering Chemistry, 2012, 18(1): 160-164.

[本文引用: 1]

HORSMAN G P, LECHNER A, OHNISHI Y, et al.

Predictive model for epoxide hydrolase-generated stereochemistry in the biosynthesis of nine-membered enediyne antitumor antibiotics

[J]. Biochemistry, 2013, 52(31): 5217-5224.

[本文引用: 1]

ZHAO Jing, CHU Yanyan, LI Aitao, et al.

An unusual (R)-selective epoxide hydrolase with high activity for facile preparation of enantiopure glycidyl ethers

[J]. Advanced Synthesis & Catalysis, 2011, 353(9): 1510-1518.

[本文引用: 1]

KUMAR R, WANI S I, CHAUHAN N S, et al.

Cloning and characterization of an epoxide hydrolase from Cupriavidus metallidurans-CH34

[J]. Protein Expression and Purification, 2011, 79(1): 49-59.

[本文引用: 1]

XUE Feng, LIU Zhiqiang, ZOU Shuping, et al.

A novel enantioselective epoxide hydrolase from Agromyces mediolanus ZJB120203: cloning, characterization and application

[J]. Process Biochemistry, 2014, 49(3): 409-417.

[本文引用: 1]

WU Kai, WANG Hualei, SUN Huihui, et al.

Efficient kinetic resolution of phenyl glycidyl ether by a novel epoxide hydrolase from Tsukamurella paurometabola

[J]. Applied Microbiology and Biotechnology, 2015, 99(22): 9511-9521.

[本文引用: 1]

WEI Chun, LING Jinlong, SHEN Honglei, et al.

Bioresolution production of (2R, 3S)-ethyl-3-phenylglycidate for chemoenzymatic synthesis of the taxol C-13 side chain by Galactomyces geotrichum ZJUTZQ200, a new epoxide-hydrolase-producing strain

[J]. Molecules, 2014, 19(6): 8067-8079.

[本文引用: 1]

BELOTI L L, COSTA B Z, TOLEDO M A, et al.

A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization

[J]. Protein Expression and Purification, 2013, 91(2): 175-183.

[本文引用: 1]

MADACKI J, LAVAL F, GRZEGORZEWICZ A, et al.

Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria

[J]. The Journal of Biological Chemistry, 2018, 293(14): 5172-5184.

[本文引用: 2]

HOTTA K, CHEN Xi, PATON R S, et al.

Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis

[J]. Nature, 2012, 483(7389): 355-U154.

[本文引用: 2]

LUHAVAYA H, DIAS M V, WILLIAMS S R, et al.

Enzymology of pyran ring A formation in salinomycin biosynthesis

[J]. Angewandte Chemie (Weinheim an der Bergstrasse, Germany), 2015, 127(46): 13826-13829.

[本文引用: 1]

WANG Luoyi, PARNELL A, WILLIAMS C, et al.

A Rieske oxygenase/epoxide hydrolase-catalysed reaction cascade creates oxygen heterocycles in mupirocin biosynthesis

[J]. Nature Catalysis, 2018, 1(12): 968-976.

[本文引用: 1]

MAO Xuming, ZHAN Zhajun, GRAYSON M N, et al.

Efficient biosynthesis of fungal polyketides containing the dioxabicyclo-octane ring system

[J]. Journal of the American Chemical Society, 2015, 137(37): 11904-11907.

[本文引用: 1]

LIN Tzu-Shyang, CHIANG Yi-Ming, WANG C C.

Biosynthetic pathway of the reduced polyketide product citreoviridin in Aspergillus terreus var. aureus revealed by heterologous expression in Aspergillus nidulans

[J]. Organic Letters, 2016, 18(6): 1366-1369.

[本文引用: 1]

LIN Shuangjun, HORSMAN G P, SHEN Ben.

Characterization of the epoxide hydrolase NcsF2 from the neocarzinostatin biosynthetic gene cluster

[J]. Organic Letters, 2010, 12(17): 3816-3819.

[本文引用: 1]

ZOU Yi, GARCIA-BORRAS M, TANG Mancheng, et al.

Enzyme-catalyzed cationic epoxide rearrangements in quinolone alkaloid biosynthesis

[J]. Nature Chemical Biology, 2017, 13(3): 325-332.

[本文引用: 1]

SATO K, MINAMI A, OSE T, et al.

Remarkable synergistic effect between MonBI and MonBII on epoxide opening reaction in ionophore polyether monensin biosynthesis

[J]. Tetrahedron Letters, 2011, 52(41): 5277-5280.

[本文引用: 1]

YURKOVICH M E, TYRAKIS P A, HONG H, et al.

A late-stage intermediate in salinomycin biosynthesis is revealed by specific mutation in the biosynthetic gene cluster

[J]. ChemBioChem, 2012, 13(1): 66-71.

[本文引用: 1]

PISTORIUS D, LI Yanyan, SANDMANN A, et al.

Completing the puzzle of aurachin biosynthesis in Stigmatella aurantiaca Sg a15

[J]. Molecular BioSystems, 2011, 7(12): 3308-3315.

[本文引用: 1]

BIS D M, BAN Y H, JAMES E D, et al.

Characterization of the nocardiopsin biosynthetic gene cluster reveals similarities to and differences from the rapamycin and fk-506 pathways

[J]. ChemBioChem, 2015, 16(6): 990-997.

[本文引用: 1]

WANG Bin, GUO Fang, REN Jinwei, et al.

Identification of Alp1U and Lom6 as epoxy hydrolases and implications for kinamycin and lomaiviticin biosynthesis

[J]. Nature Communications, 2015, 6(1): 7674.

[本文引用: 1]

ZHENG Qingfei, WANG Shoufeng, DUAN Panpan, et al.

An α/β-hydrolase fold protein in the biosynthesis of thiostrepton exhibits a dual activity for endopeptidyl hydrolysis and epoxide ring opening/macrocyclization

[J]. Proceedings of the National Academy of Sciences of The United States Of America, 2016, 113(50): 14318-14323.

[本文引用: 2]

WANG F, TAN K, BIGELOW L, et al.

The crystal structure of an epoxide hydrolase from Streptomyces carzinostaticus subsp. neocarzinostaticus

[DB/OL]. 2014.

Protein Data Bank:

.

[本文引用: 1]

WANG F, TAN K, BIGELOW L, et al.

Ensemble refinement of an epoxide hydrolase from Streptomyces carzinostaticus subsp. neocarzinostaticus

[DB/OL]. 2014.

Protein Data Bank:

.

TAN K, BIGELOW L, CLANCY S, et al.

The crystal structure of an epoxide hydrolase from Streptomyces carzinostaticus subsp. neocarzinostaticus

[DB/OL]. 2012.

Protein Data Bank:

.

SCHULZ E C, WILMANNS M, HENDERSON S, et al.

The crystal structure of Mycobacterial epoxide hydrolase A

[DB/OL]. 2016.

Protein Data Bank:

.

MOYNIE L, SCHNELL R, MCMAHON S A, et al.

The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery

[J]. Acta Crystallographica Section F, 2013, 69: 25-34.

DE OLIVEIRA G S, ADRIANI P P, RIBEIRO J A, et al.

The molecular structure of an epoxide hydrolase from Trichoderma reesei in complex with urea or amide-based inhibitors

[J]. International Journal of Biological Macromolecules, 2019, 129: 653-658.

WILSON C, DE OLIVEIRA G S, ADRIANI P P, et al.

Structure of a soluble epoxide hydrolase identified in Trichoderma reesei

[J]. Biochimica et Biophysica Acta - Proteins and Proteomics, 2017, 1865(8): 1039-1045.

BAHL C D, HVORECNY K L, BRIDGES A A, et al.

Signature motifs identify an acinetobacter Cif virulence factor with epoxide hydrolase activity

[J]. The Journal of Biological Chemistry, 2014, 289(11): 7460-7469.

[本文引用: 1]

SUN Zaibao, ZHANG Zhijun, LI Fulong, et al.

One pot asymmetric synthesis of (R)-phenylglycinol from racemic styrene oxide via cascade biocatalysis

[J]. ChemCatChem, 2019, 11(16): 3802-3807.

[本文引用: 2]

YUAN Jifeng, LUKITO B R, LI Zhi.

De novo biosynthesis of (S)- and (R)-phenylethanediol in yeast via artificial enzyme cascades

[J]. ACS Synthetic Biology, 2019, 8(8): 1801-1808.

[本文引用: 4]

RINK R, FENNEMA M, SMIDS M, et al.

Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

[J]. The Journal of Biological Chemistry, 1997, 272(23): 14650-14657.

[本文引用: 1]

孔旭东, 郁惠蕾, 周佳海, 等.

环氧水解酶的结构基础及新酶开发

[J]. 生物加工过程, 2013, 11(1): 77-86.

[本文引用: 1]

KONG Xudong, YU Huilei, ZHOU Jiahai, et al.

Structure basis of EHs and development of novel EHs as biocatalyst

[J]. Chinese Journal of Bioprocess Engineering, 2013, 11(1): 77-86.

[本文引用: 1]

ZOU Jinyu, HALLBERG B M, BERGFORS T, et al.

Structure of Aspergillus niger epoxide hydrolase at 1.8 Å resolution: implications for the structure and function of the mammalian microsomal class of epoxide hydrolases

[J]. Structure, 2000, 8(2): 111-122.

[本文引用: 1]

NARDINI M, RINK R, JANSSEN D B, et al.

Structure and mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

[J]. Journal of Molecular Catalysis B: Enzymatic, 2001, 11(4/5/6): 1035-1042.

[本文引用: 1]

HOPMANN K H, HALLBERG B M, HIMO F.

Catalytic mechanism of limonene epoxide hydrolase, a theoretical study

[J]. Journal of the American Chemical Society, 2005, 127(41): 14339-14347.

[本文引用: 1]

ARAND M, HALLBERG B M, ZOU Jinyu, et al.

Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site

[J]. The EMBO Journal, 2003, 22(11): 2583-2592.

[本文引用: 1]

李昆, 杨国强, 刘媛媛, 等.

手性双金属催化剂及其在不对称催化反应中的应用

[J]. 有机化学, 2013, 33(4): 749-759.

[本文引用: 1]

LI Kun, YANG Guoqiang, LIU Yuanyuan, et al.

Chiral polymetallic catalysts and their applications in asymmetric catalysis

[J]. Chinese Journal of Organic Chemistry, 2013, 33(4): 749-759.

[本文引用: 1]

曹靖.

大基团取代的末端手性环氧化物的制备及其聚合物的合成

[D]. 湘潭: 湘潭大学, 2007.

[本文引用: 1]

CAO Jing.

The synthesis and polymerization of chiral terminal epoxides with bulky group

[D]. Xiangtan: Xiangtan University, 2007.

[本文引用: 1]

张晓健, 郑裕国.

甘油原料转化生产手性环氧氯丙烷关键酶的开发

[J]. 生物产业技术, 2017(6): 65-72.

[本文引用: 1]

ZHANG Xiaojian, ZHENG Yuguo.

Research and development on key enzymes for biosynthesis of chiral epichlorohydrin using glycerol

[J]. Biotechnology&Business, 2017(6): 65-72.

[本文引用: 1]

赵剑伟.

转氨酶MVTA的克隆表达及其应用于级联催化合成手性β-氨基醇和邻二醇的研究

[D]. 太原: 太原理工大学, 2019.

[本文引用: 1]

ZHAO Jianwei.

Cloningand expression of ω-transaminase mvta applying in synthsis of chiral β-amino alcohols and vicinal diols via cascade catalysis

[D]. Taiyuan: Taiyuan University of Technology, 2019.

[本文引用: 1]

吴群, 邹树平, 郑裕国.

环氧化物水解酶在手性药物中间体合成中的应用

[J]. 精细与专用化学品, 2015, 23(6): 23-29.

[本文引用: 2]

WU Qun, ZOU Shuping, ZHENG Yuguo.

Applications of epoxide hydrolase in the synthesis of chiral drug intermediates

[J]. Fine and Special Chemicals, 2015, 23(6): 23-29.

[本文引用: 2]

吴群.

环氧化物水解酶分子改造及其催化拆分环氧氯丙烷的研究

[D]. 杭州: 浙江工业大学, 2015.

[本文引用: 1]

WU Qun.

Molecular modification to epoxide hydrolase and its application in the resolution of epichlorohydrin

[D]. Hangzhou: Zhejiang University of Technology, 2015.

[本文引用: 1]

JIANG Chengjun, HONG Huabin.

A new practical synthesis of ethyl (R)-(-)-4-cyano-3-hydroxybutyrate from (S)-3-chloro-1,2-propanediol

[J]. Letters in Organic Chemistry, 2012, 9(7): 520-521.

[本文引用: 1]

LIANG Youxiang, JIAO Song, WANG Miaomiao, et al.

Overexpression of epoxide hydrolase in Rhodococcus ruber with high robustness for the synthesis of chiral epichlorohydrin

[J]. Process Biochemistry, 2019, 79: 49-56.

ZOU Shuping, WANG Zhicai, QIN Chao, et al.

Covalent immobilization of Agrobacterium radiobacter epoxide hydrolase on ethylenediamine functionalised epoxy supports for biocatalytical synthesis of (R)-epichlorohydrin

[J]. Biotechnology Letters, 2016, 38(9): 1579-1585.

[本文引用: 1]

JIA Xin, XU Yi, LI Zhi.

Regio-and stereoselective concurrent oxidations with whole cell biocatalyst: simple and green syntheses of enantiopure 1,2-diols via oxidative kinetic resolution

[J]. ACS Catalysis, 2011, 1(6): 591-596.

[本文引用: 1]

LI Keqiang, VIG S, UCKUN F M.

Stereocontrolled synthesis of the tetrahydrofuran unit of annonaceous acetogenins

[J]. Tetrahedron Letters, 1998, 39(15): 2063-2066.

[本文引用: 1]

HU Die, ZONG Xuncheng, XUE Feng, et al.

Manipulating regioselectivity of an epoxide hydrolase for single enzymatic synthesis of (R)-1,2-diols from racemic epoxides

[J]. Chemical Communications, 2020, 56(18): 2799-2802.

[本文引用: 1]

SAINI P, KUMAR N, WANI S I, et al.

Bioresolution of racemic phenyl glycidyl ether by a putative recombinant epoxide hydrolase from Streptomyces griseus NBRC 13350

[J]. World Journal of Microbiology and Biotechnology, 2017, 33(5): 82.

[本文引用: 2]

VAN LOO B, SPELBERG J H L, KINGMA J, et al.

Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling

[J]. Chemistry & Biology, 2004, 11(7): 981-990.

[本文引用: 1]

Myung Soo KO, LOVE J J.

Step-wise directed evolution of an epoxide hydrolase against progressively larger non-natural substrates

[J]. The FASEB Journal, 2017, 31(s1): 922.

1-922

.1.

[本文引用: 1]

ZOU Shuping, ZHENG Yuguo, WU Qun, et al.

Enhanced catalytic efficiency and enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter AD1 by iterative saturation mutagenesis for (R)-epichlorohydrin synthesis

[J]. Applied Microbiology and Biotechnology, 2018, 102(2): 733-742.

[本文引用: 2]

SUN Zhoutong, LONSDALE R, WU Lian, et al.

Structure-guided triple-code saturation mutagenesis: efficient tuning of the stereoselectivity of an epoxide hydrolase

[J]. ACS Catalysis, 2016, 6(3): 1590-1597.

[本文引用: 1]

ZHENG Huabao, REETZ M T.

Manipulating the stereoselectivity of limonene epoxide hydrolase by directed evolution based on iterative saturation mutagenesis

[J]. Journal of the American Chemical Society, 2010, 132(44): 15744-15751.

[本文引用: 1]

ZONG Xuncheng, LI Chuang, XU Yaohui, et al.

Substantially improving the enantioconvergence of Pv EH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution

[J]. Microbial Cell Factories, 2019, 18(1): 1-8.

LI Fulong, KONG Xudong, CHEN Qi, et al.

Regioselectivity engineering of epoxide hydrolase: near-perfect enantioconvergence through a single site mutation

[J]. ACS Catalysis, 2018, 8(9): 8314-8317.

[本文引用: 1]

JIN Huoxi, OUYANG Xiaokun, HU Zhongce.

Enhancement of epoxide hydrolase production by 60Co gamma and UV irradiation mutagenesis of Aspergillus niger ZJB-09103

[J]. Biotechnology and Applied Biochemistry, 2017, 64(3): 392-399.

[本文引用: 1]

LIANG Bo, HUANG Xuenian, TENG Yun, et al.

Enhanced single-step bioproduction of the simvastatin precursor monacolin J in an industrial strain of Aspergillus terreus by employing the evolved lovastatin hydrolase

[J]. Biotechnology Journal, 2018, 13(6): 1800094.

[本文引用: 1]

YU Da, WANG Jianbo, REETZ M T.

Exploiting designed oxidase-peroxygenase mutual benefit system for asymmetric cascade reactions

[J]. Journal of the American Chemical Society, 2019, 141(14): 5655-5658.

[本文引用: 3]

NIU Guoqing, ZHENG Jiazhen, TAN Huarong.

Biosynthesis and combinatorial biosynthesis of antifungal nucleoside antibiotics

[J]. Science China Life Sciences, 2017, 60(9): 939-947.

[本文引用: 1]

MOSS N A, SEILER G, LEÃO T F, et al.

Nature's combinatorial biosynthesis produces Vatiamides A-F

[J]. Angewandte Chemie International Edition, 2019, 58(27): 9027-9031.

[本文引用: 1]

LIU Song, ZHANG Xian, LIU Fei, et al.

Designing of a cofactor self-sufficient whole-cell biocatalyst system for production of 1,2-amino alcohols from epoxides

[J]. ACS Synthetic Biology, 2019, 8(4): 734-743.

[本文引用: 1]