过量表达乳酸脱氢酶的重组干酪乳杆菌构建及发酵性能

doi:10.7506/spkx1002-6630-201309050

食品科学 ›› 2013, Vol. 34 ›› Issue (9): 245-249.doi: 10.7506/spkx1002-6630-201309050

过量表达乳酸脱氢酶的重组干酪乳杆菌构建及发酵性能

朱龙宝，葛飞，李婉珍，陶玉贵*

安徽工程大学生物与化学工程学院，安徽芜湖 241000

收稿日期:2012-05-15 修回日期:2013-03-15 出版日期:2013-05-15 发布日期:2013-05-07
通讯作者: 陶玉贵 E-mail:lbzhu2008@126.c0m
基金资助:
安徽省高校自然科学研究基金项目(KJ2010B014)

Over-expression of Lactate Dehydrogenase in L.casei and Fermentation Property of the Recombinant Strain

ZHU Long-bao，GE Fei，LI Wan-zhen，TAO Yu-gui*

School of Biochemical Engineering, Anhui Polytechnic University, Wuhu 241000, China

Received:2012-05-15 Revised:2013-03-15 Online:2013-05-15 Published:2013-05-07
Contact: TAO Yu-gui E-mail:lbzhu2008@126.c0m

摘要/Abstract

摘要：

乳酸脱氢酶(LDH)是干酪乳酸菌(L.casei)合成L-乳酸途径中的关键酶，通过构建过量表达乳酸脱氢酶的重组干酪乳杆菌，提高干酪乳杆菌发酵产乳酸的能力。根据干酪乳杆菌乳酸脱氢酶的基因序列设计引物，PCR扩增获得乳酸脱氢酶基因(ldh)，将其连接到乳酸杆菌穿梭质粒pMG36e，构建重组质粒pMG-ldh，电转入L.casei，利用红霉素MRS平板筛选阳性克隆，获得表达乳酸脱氢酶的重组菌L.casei pMG-ldh，SDS-PAGE和酶活分析结果表明成功表达了一个约37kD的乳酸脱氢酶蛋白，重组酶活力最高达95U/mL，较原始菌23U/mL提高了近3.1倍。为进一步提高其产乳酸能力，添加4mmol/L激活剂FBP和8mg/L烟酰胺腺嘌呤二核苷酸(NADH)前体物烟酸，结果表明：重组菌L.caseipMG-ldh乳酸积累128.2g/L，生产强度达到3.04g/(L•h)，较原始菌株L.casei分别提高了35%和53%，发酵周期缩短6h。

关键词: 乳酸脱氢酶, 重组干酪乳酸菌, 高效表达, 发酵特性

Abstract:

Lactate dehydrogenase (LDH) is a key enzyme for lactic acid synthesis in L. casei. A LDH over- expressing
recombinant L.casei was constructed in this study to enhance the production of lactic acid. The lactate dehydrogenase gene ldh
of L. casei was first cloned into vector pMG36e. The recombinant vector was then transferred into L. casei. SDS-PAGE analysis
showed that LDH was successfully over expressed in the recombinant strains, which was about 37 kD. Enzyme activity analysis
showed that LDH activity of the recombinant strain increased by 3.1 times when compared to that of the wild type strain.
4 mmol/L activator FBP and 8 mg/L nicotinic acid were then added to fermentation medium to further improve lactic acid production
and analysis showed that accumulation of lactic acid was 128.2 g/L, and the lactic acid productivity was 3.04 g/(L•h), which were
increased by 35% and 53%, respectively, when compared to control. Addition of FBP and nicotinic acid significantly increased
the consumption rate of glucose and shortened fermentation time by 6 h.

Key words: LDH, recombinant L.casei, over-expression, fermentation characteristics

中图分类号:

朱龙宝，葛飞，李婉珍，陶玉贵*. 过量表达乳酸脱氢酶的重组干酪乳杆菌构建及发酵性能[J]. 食品科学, 2013, 34(9): 245-249.

ZHU Long-bao，GE Fei，LI Wan-zhen，TAO Yu-gui*. Over-expression of Lactate Dehydrogenase in L.casei and Fermentation Property of the Recombinant Strain[J]. FOOD SCIENCE, 2013, 34(9): 245-249.

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过量表达乳酸脱氢酶的重组干酪乳杆菌构建及发酵性能

Over-expression of Lactate Dehydrogenase in L.casei and Fermentation Property of the Recombinant Strain

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