麦芽糖诱导软化芽孢杆菌α-环糊精葡萄糖基转移酶在枯草杆菌中的表达

通过PCR扩增软化芽孢杆菌α-环糊精葡萄糖基转移酶基因,将基因片段克隆到大肠杆菌-枯草杆菌穿梭载体pGJ103中,转化枯草杆菌WB600得基因工程菌进行外源表达。在1.5%的麦芽糖初始发酵培养基上摇瓶培养,48 h后重组枯草杆菌产酶活性为6.1U/ml。通过单因素分析和响应面分析对重组枯草杆菌产CGT酶摇瓶发酵条件进行优化。分析得到培养基关键组分麦芽糖,玉米淀粉和酵母粉三者最佳浓度分别为:15.5g/L,13g/L和20g/L。在此条件下,摇瓶培养36h后α-CGT酶活性为17.6U/ml,5L罐分批发酵30h后酶活达到20U/ml (水解活性为1.4×10⁴ IU/ml)。

Expression of Paenibacillus macerans α-cyclodextrin Glycosyltraferase in Bacillus subtilis by Maltose Induction

The α-cgt gene was obtained from Paenibacillus macerans by PCR, and then it was cloned into Escherichia coli-Bacillus subtilis shuttle vector pGJ103 and transformed into B. subtilis WB600. After cultivation for 48 h with shake flask in 1.5% maltose initial medium, the α-CGTase activity of recombinant B. subtilis was 6.1U/ml. In addition, the experiment optimized the culture conditions of the recombinant B. subtilis strain in shaking flask by means of a single factor synthesis and Box-Behnken design (BBD). The analysis predicted the concentrations of maltose, corn starch and yeast extract were at 15 g/L, 13 g/L, and 20 g/L, respectively. In this condition, the experimental result of 17.6 U/ml could be obtained when the cells were cultured for 36 h in shaking flasks. The CGTase activity reached 20 U/ml at 30 h of culture in a 5 L bioreactor (hydrolysis activity was 1.4×10⁴ IU/ml).