食品科学

• 生物工程 • 上一篇    下一篇

1株古细菌海藻糖合成酶系在枯草芽孢杆菌中的诱导表达

王珊瑛,李由然,顾正华,张 梁,丁重阳,石贵阳*   

  1. 1.江南大学 粮食发酵工艺与技术国家工程实验室,江苏 无锡 214122;
    2.江南大学生物工程学院,江苏 无锡 214122
  • 出版日期:2016-11-15 发布日期:2016-11-18

Heterologous Expression of Enzymes for Trehalose Synthesis from Sulfolobus acidocaldarius in Bacillus subtilis

WANG Shanying, LI Youran, GU Zhenghua, ZHANG Liang, DING Zhongyang, SHI Guiyang*   

  1. 1. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China;
    2. School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2016-11-15 Published:2016-11-18

摘要:

来源于古细菌嗜酸硫化叶菌( S u l f o l o b u s a c i d o c a l d a r i u s ) 的麦芽寡糖基海藻糖合成酶(maltooligosylterhalose synthase,MTSase)和麦芽寡糖基海藻糖水解酶(maltooligosyltrehalose trehalohydrolase,MTHase)联合作用,可以利用淀粉为底物生成海藻糖。本研究构建了6 种枯草芽孢杆菌-大肠杆菌穿梭表达载体,将密码子优化后的MTSase和MTHase编码基因在木糖异构酶基因的启动子及其阻遏蛋白介导下实现了在枯草芽孢杆菌中的功能表达,木糖启动子分别来源于枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillusmegaterium)、地衣芽孢杆菌(Bacillus licheniformis)。在以5 g/L甘油为碳源,24 g/L蛋白胨为氮源时,菌体培养8 h后加入终质量浓度为6 g/L的木糖,37 ℃诱导16 h后重组MTSase、MTHase产生联合作用,海藻糖转化率达到33.57%。实现了MTSase、MTHase在枯草芽孢杆菌中的功能表达。

关键词: 穿梭质粒, 嗜酸硫化叶菌, 枯草芽孢杆菌, 诱导表达

Abstract:

Trehalose has great potential applications in both the medical and food industries due to its unique properties.
Enzymatic transformation is superior to extraction from yeast cells for the industrial production of trehalose. Starch can
be transformed into trehalose via maltooligosylterhalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase
(MTHase) from Sulfolobus acidocaldarius. In this experiment, we constructed six recombinant expression plasmids
harboring the codon-optimized MTSase and MTHase encoding genes from Sulfolobus acidocaldarius, which could be
expressed under the control of the xylose operons from Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis.
The recombinant plasmids were transformed into Bacillus subtilis, respectively. The effects of different culture conditions
on the production of trehalose was investigated and the optimal culture conditions were determined as follows: glycerol
concentration as the best carbon source, 5 g/L; peptone concentration as the best nitrogen source, 24 g/L; xylose
concentration, 6 g/L; incubation duration, 8 h; and induction temperature, 37 ℃; induction time, 16 h. Experiments carried
out under these conditions gave a conversion rate of trehalose of 33.57%. This study has successfully achieved the functional
expression of MTSase and MTHase in Bacillus subtilis.

Key words: shuttle vector, Sulfolobus acidocaldarius, Bacillus subtilis, inducible expression

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