以热硫梭菌Clostridium thermosulfurogenes基因组为模板,PCR扩增出β-淀粉酶基因;将该基因克隆到pET-22b(+)载体上,转入大肠杆菌BL21-SI,采用NaCl形成的高渗透压诱导表达。研究了不同诱导条件对重组菌产β-淀粉酶的影响,结果以菌体密度达到OD600为0.6,诱导剂NaCl浓度为0.6 mol/L,诱导温度为35℃最佳。重组菌以LBON为培养基分批发酵时,由于营养限制,菌体密度OD600仅为4.60,β-淀粉酶活力为118U/mL。采用pH-Stat分批补料发酵时,在菌体密度仅是分批发酵的2.35倍的条件下,得到6.12倍的产酶量,酶活力达722U/mL。重组β-淀粉酶70℃水解可溶性淀粉3h的麦芽糖转化率为62.2%,高于大麦β-淀粉酶的转化率。

A β-amylase gene from Clostridium thermosulfurogenes was cloned and heterologously expressed in Escherichia coli BL 21-SI.The influences of cell density at induction,temperature,and NaCl concentration were studied.The optimum condition of induction was at cell density of OD600,=0.6,temperature of 35 ℃,and NaCl concentration of 0.6M.The expression of recombinant β-amylase reached 118U/ mL in a 20L batch culture.In a 20L pH-Stat fed-batch culture,the cell density and enzyme activity were 2.35 times and 6.12 times of those obtained in batch culture.The soluble starch was converted to maltose by recombinant β-amylase and the conversion ratio was 62.2% after 3h reaction at 70℃.