摘要
L-天冬氨酸α-脱羧酶(L-aspartateα-decarboxylase,Pan D)能选择性脱去L-天冬氨酸的α-羧基生成β-丙氨酸,具有重要的工业应用价值。以特基拉芽孢杆菌(Bacillus tequilensis)Pan D37的基因组为模板,扩增Pan D表达基因panD,构建表达质粒p ET32a(+)-panD,转入Escherichia coli BL21(DE3)成功实现异源表达。利用摇床和发酵罐优化培养条件实现高密度发酵,确定最佳培养条件为:37℃培养8 h,加入终浓度0.5 mmol/L的异丙基硫代-β-D-半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)降温至26℃诱导,采用葡萄糖为碳源并控制初始质量浓度为5 g/L,发酵过程中采用pH-stat方式补料。在此基础上利用5 L发酵罐进行发酵产酶,酶活力最高可达1 109.8 U/m L,OD_(600 nm)达到106.3。全细胞催化100 g/L L-天冬氨酸,反应10 h物质的量转化率为99.2%。构建的重组菌经发酵优化后具有较高的细胞浓度和Pan D活力,为生物法β-丙氨酸的工业化生产与应用提供理论支持。
L-Aspartate α-decarboxylase is an important industrial enzyme which can stereo-selectively transform L-aspartate acid into β-alanine.In the present study,we cloned and expressed the L-aspartate α-decarboxylase gene（panD） from Bacillus tequilensis to construct an L-aspartate α-decarboxylase-producing Escherichia coli strain and optimized the culture conditions for high cell density fermentation.Using the genome of B.tequilensis PanD37 as template,the panD gene was amplified,and the recombinant plasmid p ET32 a（＋）-panD was constructed and transformed into E.coli BL21（DE3） for expression.For high cell density growth and efficient L-aspartate α-decarboxylase expression,the optimum fermentation conditions in shake flasks were determined as follows： a pH-stat fed-batch culture was performed using glucose as the carbon source at an initial concentration of 5 g/L; after 8 h of culture at 37 ℃,isopropyl-β-D-thiogalactopyranoside（IPTG） was added to a final concentration of 0.5 mmol/L as an inducer and the temperature was reduced to 26 ℃.Maximum L-aspartate α-decarboxylase activity of 1 109.8 U/m L and OD（600 nm） value of 106.3 were obtained when the fermentation was carried out in a 5 L fermentor.Whole-cell catalysis of 100 g/L L-aspartate gave a molar conversion rate of 99.2% after 10 h of reaction.This work can provides a promising basis for further application of L-aspartate α-decarboxylase in industrial β-alanine production.
作者
范雪萍
冯志彬
房美芳
张娟
陈国忠
李丽娜
FAN Xueping;FENG Zhibin;FANG Meifang;ZHANG Juan;CHEN Guozhong;LI Lina(School of Life Sciences, Ludong University, Yantai 264025, China;School of Agriculture, Ludong University, Yantai 264025, China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2018年第2期144-150,共7页
Food Science
基金
山东省农业重大应用技术创新项目(鲁财指2014-38)
作者简介
范雪萍（1995-）,女,学士,研究方向为生物工程.E-mail：xuepingFan95@163.com;通信作者简介：冯志彬（1977-）,男,讲师,硕士,研究方向为微生物发酵与酶催化.E-mail：zhibinfeng@126.com
