交联醇脱氢酶聚集体的制备及其在(R)- 4-氯-3-羟基丁酸乙酯合成中的应用

沙凤，顾金海，许琳，严明

(南京工业大学 生物与制药工程学院，江苏 南京 211800)

摘要：基于基因组序列数据库挖掘新酶的技术，从白色念珠菌Candida albicans基因组中克隆了一条新型醇脱氢酶(CADH)基因，并在大肠杆菌Escherichia coli Rosetta(DE3)中表达。为克服游离酶稳定性差、不能重复使用的缺点，探索并优化了交联醇脱氢酶聚集体(CLEAs-CA)的制备条件。结果表明：重组CADH对底物四氯乙酰乙酸乙酯(COBE)的比活力为1.8 U/mg，产物(R)-4-氯-3-羟基丁酸乙酯((R)-CHBE)的对映体过量值大于99%。CLEAs-CA沉淀剂选择为60%饱和度的(NH4)2SO4，交联剂为10 mmol/L戊二醛。在固定化操作前，加入50 mmol/L异丙醇和0.1 mmol/L NAD`+对CADH催化活性位点、辅酶结合位点进行保护，CLEAs-CA的活力回收率提高了48.3%。将CLEAs-CA用于不对称合成(R)-CHBE，经过19次的重复使用，CLEAs-CA的活性仍保留有50%。

关键词：(R)-4-氯-3-羟基丁酸乙酯；醇脱氢酶；交联醇脱氢酶聚体；固定化

doi:10.3969/j.issn.1672-3678.2015.06.005

收稿日期：2015-04-28

基金项目：国家重点基础研究发展计划(973计划)(2011CBA00804)；国家高技术研究发展计划(863计划)(2012AA022101)；江苏高校优势学科建设工程

作者简介：沙凤(1991—)，女，江苏靖江人，硕士研究生，研究方向：手性生物催化；严明(联系人)，副教授，E-mail:yanming@njtech.edu.cn

中图分类号：TQ225.24

文献标志码：A

文章编号：1672-3678(2015)06-0024-06

Abstract：A novel alcohol dehydrogenase (CADH) from Candida albicans was discovered by genome data mining for ketoreductases. CADH was cloned and expressed in Escherichia coli Rosetta(DE3). Free enzymes usually have poor stability and are difficult to recover and reuse. To overcome such drawbacks, CADH was immobilized as cross-linked enzyme aggregates (CLEAs-CA) and the optimum conditions of the immobilization process were investigated. The recombinant product (CADH) exhibited specific activity of 1.8 U/mg toward ethyl 4-chloro-3-oxobutanoate (COBE),and the enantiomeric excess purity of (R)-CHBE was over 99%. Ammonium sulfate (60%) and 10 mmol/L glutaraldehyde were chosen as the optimum precipitant and cross-linker for the preparation of CLEAs. Moreover,addition of substrates (50 mmol/L isopropanol and 0.1 mmol/L NAD`+) in the immobilization process enhanced the activity recovery by 48.3% as compared to the CLEAs prepared without substrates. CLEAs-CA could be reused and still remained about 50% of its initial activity after 19 cycles.

Keywords：ethyl(R)-4-chloro-3-hydroxybutanoate; alcohol dehydrogenase; cross-linked enzyme aggregates (CLEAs); immobilization

Preparation of alcohol dehydrogenase cross- linked enzyme aggregates and its application to asymmetric synthesis of (R)-4- chloro-3- hydroxybutanoate

SHAFeng,GUJinhai,XULin,YANMing

(CollegeofBiotechnologyandPharmaceuticalEngineering,NanjingTechUniversity,Nanjing211800,China)

(R)-4-氯-3-羟基丁酸乙酯((R)-CHBE)是合成L-肉碱(L-carnitine)的前体物质，也是(-)-大内酰亚胺A((-)-macrolactinA)和(R)-γ-氨基-β-羟基丁酸(GABOB)等手性化合物合成的重要砌块[1-3]。与传统的化学法相比，利用氧化还原酶不对称还原前手性羰基化合物制备手性醇在催化效率、立体选择性等方面具有显著优势。目前，已有数种氧化还原酶被报道能用于不对称合成(R)-CHBE[5-11]，例如来源于醛酮还原酶超家族的ARI、CmAR，来源于短链脱氢酶超家族的Gox2036，然而这些酶大都以昂贵的辅酶NADPH作为氢供体。……