Lactobacillus plantarum苯丙酮酸还原酶的异源表达及其在苯乳酸制备中的应用

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  • 1 (江南大学食品学院,江苏无锡 214122) 2 (江南大学无锡医学院,江苏无锡 214122)
袁风娇 (1991?),女,山东菏泽人,硕士研究生,主要从事生物催化研究。E-mail: yuanfj2015@163.com

网络出版日期: 2017-11-27

基金资助

国家自然科学基金项目 (21676117) 江苏省普通高校研究生科研创新计划项目 (SJLX16-0472)

Heterologous Expression of phenylpyruvate reductase from Lactobacillus plantarum and Its Application in the Preparation of Phenyllactic Acid

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  • (1School of Food Science and Technology, Jiangnan University, Wuxi 214122, China) (2Wuxi Medical School, Jiangnan University, Wuxi 214122, China)

Online published: 2017-11-27

摘要

构建产苯丙酮酸还原酶的基因工程菌E. coli/ppr,优化其目的蛋白表达条件,并利用全细胞催化制备光学纯的D-苯乳酸。以Lactobacillus plantarum的基因组DNA为模板,经PCR扩增出一种编码苯丙酮酸还原酶的基因 (lpppr),并将其在大肠杆菌BL21中进行表达。以苯丙酮酸为底物,通过单因素和正交试验优化诱导表达条件,然后对E. coli/ppr全细胞制备苯乳酸的工艺进行研究。结果表明:E. coli/ppr在IPTG终浓度为0.5 mmol/L,20℃诱导8 h时具有最高的苯丙酮酸还原酶活性。由其催化制备苯乳酸的最佳条件为:反应温度40℃,反应初始pH 6.5,葡萄糖浓度20 mmol/L。在上述条件下增加苯丙酮酸的浓度至25 mmol/L,5 h后苯乳酸的最终产率达到98.4%,产物D-苯乳酸光学纯度ee>99.9%。

本文引用格式

袁风娇, 李雪晴, 李剑芳, 等 . Lactobacillus plantarum苯丙酮酸还原酶的异源表达及其在苯乳酸制备中的应用[J]. 食品与发酵工业, 2017 , 43(11) : 16 -21 . DOI: 10.13995/j.cnki.11-1802/ts.015007

Abstract

In orderto construct a genetic engineering phenylpyruvate reductase strain (E. coli/ppr), the expression conditions and bioconversion medium were optimized to increase the production of PLA with whole-cell transformation. A phenylpyruvate reductase-encoding gene, lpppr, was obtained by PCR from the genomic DNA of Lactobacillus plantarum and heterologously expressed in E. coli BL21 (DE3). Taking phenylpyruvic acid as the substrate, single factor and orthogonal experiments was used to optimized the induced expression and whole-cell catalysis conditions. Results showed that after inductionwith 0.5 mmol/L IPTG at 20 ℃ for 8 h, E. coli/ppr displayed the highest LpPPR activity. When phenylpyruvic acid was added up to 25 mmol/L, the product enantiomeric excess percent was over 99.9% and the final yield of PLA could reach 98.38% at 5 h under the obtained optimal catalytic conditionsas follows: temperature 40 ℃, initial pH 6.5 and 20 mmol/L glucose.
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