Arthrobacter ureafaciens CZ31丙氨酸脱氢酶可溶性表达及产酶条件优化

Expression and production optimization of alanine dehydrogenase from Arthrobacter ureafaciens CZ31

摘要:

[目的] 克隆Arthrobacter ureafaciens CZ31丙氨酸脱氢酶的编码基因（alanine dehydrogenase），转化至Escherichia coli Rosetta（DE3）中构建可溶性表达alanine dehydrogenase（ald）的工程菌CZR07并优化产酶条件。[方法] 提取A. ureafaciens CZ31菌株的全基因组DNA，设计引物扩增出ald基因，与pET-28a连接后导入E. coli Rosetta中表达并纯化重组蛋白，以单因素实验结果为依据，响应面法优化发酵条件。[结果] ald全长为1119 bp，编码含372个氨基酸残基的蛋白质，分子量约为40 kDa，酶活为2.65 U/mg。响应面分析温度、诱导时间及诱导剂浓度的影响强度为IPTG浓度 > 温度 > 温度×IPTG浓度 > 温度×诱导时间 > IPTG浓度×诱导时间 > 诱导时间。CZR07摇瓶发酵最佳条件为温度22 ℃、IPTG 0.7 mmol/L、诱导时间7 h，此条件下重组酶酶活达到15.23 U/mg，与响应面优化的预测值相似，较优化前提高5.75倍。[结论] 克隆并实现了CZ31中ald基因的可溶性表达，采用BBD法优化产酶的诱导条件，获得显著的优化效果，为其他工程菌株产酶条件优化提供借鉴。

关键词:Arthrobacter ureafaciens CZ31;丙氨酸脱氢酶;克隆表达;响应面优化

Abstract:

[Objective] Alanine dehydrogenase gene (ald) of Arthrobacter ureafaciens CZ31 was cloned and transformed into Escherichia coli Rosetta (DE3) to construct engineering bacteria CZR07 with soluble expression of ald, and the conditions of alanine dehydrogenase(AlaDH) production were optimized.[Methods] Genomic DNA of Arthrobacter ureafaciens CZ31 was extracted; A pair of specific primers was designed to obtain the ald gene, and then subcloned into expression plasmid, pET-28a-ald, and expressed in E. coli Rosetta. The recombinant protein, AlaDH, was purified by Ni²⁺ chromatography. Response surface methodology was used to optimize fermentation conditions.[Results] The length of ald gene was 1119 bp, encoding 372 amino acid residues, molecular weight of the target protein was 40 kDa according to SDS-PAGE analysis. Specific enzyme activity of the recombinant enzyme was 2.65 U/mg. The optimal induction conditions were:22℃, isopropyl-β-D-thiogalactoside 0.7 mmol/L, induction time 8 h. The specific activity of the enzyme was 15.23 U/mg under optimized conditions, about 5.75 times of the initial.[Conclusion] We optimized the induction conditions of the recombinant enzyme production through Box-Benhnken Design, and achieved the desired results, which provided a reference for the optimization of other genetic engineering bacteria.

Key words:Arthrobacter ureafaciens CZ31;alanine dehydrogenase;cloning and expression;response surface optimization