摘要羟基腈裂解酶作为用途广泛的一类水解酶，不仅在植物体内起着抵御微生物侵袭的作 用，更被广泛运用于手性氰醇的合成。从自然界中获取羟基腈裂解酶工艺复杂，且无法满足 工业生产。为了大量的获得羟基腈裂解酶，克隆拟南芥(Arabidopsis thaliana),以 pET-28a(+) 为载体，构建重组质粒 pET-28a(+)-HNL-At，将其在大肠杆菌 BL21(DE3)中进行诱导表达。 本实验通过单因素法考察诱导温度、诱导浓度、诱导时机和诱导时间对产酶条件的影响。优 化诱导表达的最佳条件：加入 IPTG 浓度为 0。504mmol·L-1、诱导温度为 30℃、诱导时间 6h, 诱导时机 OD600 值为 0。9。在此条件下，获得最大的酶活力 29。3U·mL-1。表达工艺优化后， 粗酶活提升 114。8%，酶学性质表征正确。本研究也为羟基腈裂解酶的工业化应用提供了理 论依据。88683

Hydroxynitrile lyases, as a widely used type of hydrolase, not only plays an important role in plant system against microbial attack , more widely used in synthesis of cyanohydrin。 But it is difficult to meet the requirement of industrial production by the process of obtaining Hydroxynitrile lyases from nature plants。 In order to gain a large number of Hydroxyl- acrylic lyase, we can construct pET-28a(+)-HNL-At in E。 coli   BL21    (DE3)   from   Arabidopsis   thalianaby   pET-28a(+)    as   the     vector。

And we explored the effects of starting time of induction, inducer concentration, induction duration and induction temperature on HNL

production by single  factor  tests。  The  results  show  that  the  recombinant expression vector is successfully constructed, and best optimization condition is: the

tendency for 0。504mmol·L-1  IPTG amount, culture temperature 30 ℃, time of 6 h,源Q于D优G尔X论V文Y网wwW.yOueRw.com 原文+QQ75201`8766

induction time OD600 value of 0。9。 The maximum activity is 29。3U·mL-1。 After the optimization of the expression process, the crude enzyme activity was improved by 114。8%, and the characterization of the enzyme was correct。 And this study has provided a theoretical basis for industrial application of Hydroxynitrile lyases。

关键词：羟基腈裂解酶；大肠杆菌；基因重组；功能表达

Keyword: Hydroxynitrile lyase; Escherichia coli; Genetic recombination; Functional expression

 目录

1 前言 5

2 实验材料与方法 7

2。1 实验材料 7

2。1。1 菌种和质粒 7

2。1。2 主要实验仪器 7

2。1。3  主要药品与试剂 9

2。2 实验方法 10

2。2。1 培养基与试剂配置