嗜热芽胞杆菌α-环糊精葡萄糖基转移酶在枯草芽胞杆菌中的表达

陈龙军; 林陈强; 张慧; 贾宪波; 方宇; 陈济琛

doi:10.19303/j.issn.1008-0384.2019.05.014

嗜热芽胞杆菌α-环糊精葡萄糖基转移酶在枯草芽胞杆菌中的表达

Expression of α-Cyclodextrin Glycosyltransferase Gene of Gebacillius sp. CHB1 in Bacillus subtilis

摘要

摘要:
目的构建分泌表达α-环糊精葡萄糖基转移酶（α-CGTase）的重组枯草芽胞杆菌，实现α-CGTase的安全高效表达。
方法通过PCR法扩增嗜热地芽胞杆菌α-CGTase基因，运用EcoR Ⅰ/Xho Ⅰ分别对α-CGTase基因及pBES进行双酶切，然后将该基因片段插入到大肠杆菌-枯草芽胞杆菌穿梭载体pBES中，再电转化法转化枯草芽胞杆菌RIK1285；对重组枯草芽胞杆菌B.subtilis RIK1285/pBE-CGT发酵条件进行探索。
结果（1）利用发酵培养基分泌表达α-CGTase，重组枯草芽胞杆菌工程菌B.subtilis RIK1285/pBE-CGT发酵上清液产酶达到2.9 U·mL^-1。（2）TB为最适发酵培养基（配方：甘油0.5%，蛋白胨1.2%，酵母粉2.4%，K₂HPO₄ 1.64%，KH₂PO₄ 0.23%）；在初始pH 6.5，温度为37℃下，摇瓶发酵培养24 h后，α-环糊精酶的环化活性达到5.3 U·mL^-1，是野生菌株嗜热地芽胞杆菌（0.66 U·mL^-1）的8倍。
结论成功构建了枯草芽胞杆菌B.subtilis RIK1285/pBE-CGT工程菌，并确定其最适发酵培养基和培养条件。

Abstract:
Objective To construct a vector for expressing α-cyclodextrin glycosyltransferase (α-CGTase) gene in Bacillus subtilis.
Method The α-CGTase gene from Gebacillius sp. CHB1 was amplified by PCR. The EcoR Ⅰ-digested pBES and Xho Ⅰ-digested α-CGT gene were connected and transformed into B. subtilis RIK1285. Subsequently, fermentation of the recombinant B. subtilis RIK1285/pBE-CGT was optimized.
Result (1) The α-CGTase gene was expressed in a fermentation medium to show an enzymatic activity of 2.9 U·mL^-1 by B. subtilis RIK1285/pBE-CGT. (2) Medium TB with the formula of 0.5% glycerol, 1.2% peptone, 2.4% yeast extract, 1.64% K₂HPO₄, and 0.23% KH₂PO₄ was found to be optimal for the fermentation. After fermentation in TB at 37℃ for 24h, the α-CGTase activity reached 5.3 U·mL^-1, which was 8-fold of what the wild Gebacillius sp. CHB1 could generate.
Conclusion The engineered B. subtilis RIK1285/pBE-CGT was successfully obtained and the fermentation process optimized.

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