肌酐酶在大肠杆菌中外源表达及发酵优化  

Exogenous expression and fermentation optimization of creatininase in Escherichia coli

作　　者：周丽亚[1] 张悦 刘瑞琪 陈佳立 刘运亭 姜艳军[1] 马丽[1] ZHOU Liya;ZHANG Yue;LIU Ruiqi;CHEN Jiali;LIU Yunting;JIANG Yanjun;MA Li(School of Chemical Engineering and Technology,Hebei University of Technology,Tianjin 300000)

机构地区：[1]河北工业大学化工学院,天津300000

出　　处：《食品与发酵工业》2023年第23期33-40,共8页Food and Fermentation Industries

基　　金：河北省省级科技计划项目(21372805D,21372804D,20372802D);国家自然科学基金项目(21878068,21576068);河北省自然科学基金项目(B2020202036)。

摘　　要：肌酐酶(creatininase,CA)是一种应用于体外检测试剂的重要工具酶,目前主要由微生物法生产,存在产量低和活性差的问题,限制了其实际应用。该研究通过全基因合成和异源表达构建了重组大肠杆菌BL21(DE3)/pET28a-CA,获得了高性能CA。通过单因素试验和响应面设计分析确定最优培养基组成,并系统地优化了发酵条件,从而提高了CA表达量。培养基优化实验结果表明,最优培养基各组分为:葡萄糖10.33 g/L、酵母浸粉21.80 g/L、磷酸盐137.63 mmol/L和胰蛋白胨15.60 g/L。经过5 L发酵罐发酵工艺优化,极大地提高了CA的活力,在接种量4%、诱导温度25℃、诱导时间18 h、异丙基-β-D-硫代半乳糖苷浓度0.2 mmol/L、溶氧值30%条件下,其活力达到470.24 U/mL,比酶活力为367.38 U/mg。该研究为CA的生产和工业化应用提供了理论基础和技术支持。Creatininase,an important tool enzyme for in vitro detection reagents,is currently produced mainly by microbiological methods,which suffer from low yield and poor activity,limiting its practical application.Herein,recombinant Escherichia coli BL21(DE3)/pET28a-CA was constructed by whole gene synthesis and heterologous expression,obtaining high-performance creatininase.The optimal medium composition was determined by single-factor test and response surface design analysis,and the fermentation conditions were systematically optimized,resulting in improved creatininase expression.The medium optimization results showed that the optimal medium consisted of glucose 10.33 g/L,yeast extract 21.80 g/L,phosphate 137.63 mmol/L,tryptone 15.60 g/L.After optimization of the fermentation process in 5 L fermenters,the enzyme activity and specific enzyme activity of creatinine was greatly improved and reached 470.24 U/mL and 367.38 U/mg,respectively,under the conditions of 4%inoculum,25℃induction temperature,18 h induction time,0.2 mmol/L IPTG and 30%dissolved oxygen value.This study provides a theoretical basis and technical support for the production and industrial application of creatininase.

关 键 词：肌酐酶 外源表达 发酵优化 大肠杆菌 响应面法 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]