普鲁兰酶基因在解淀粉芽孢杆菌BF7658菌株中的分泌表达

根据文献报道的核苷酸序列合成Bacillus deramificans普鲁兰酶成熟肽编码基因BdP。将BdP基因插入芽孢杆菌分泌表达载体pUC980信号肽编码区下游，获得重组质粒pUC980-BdP，重组质粒转化中温α-淀粉酶生产菌解淀粉芽孢杆菌BF7658菌株。摇瓶发酵实验表明，重组转化子发酵液有明显普鲁兰酶酶活，约48h酶活达到最高水平，为2．8ASPU／mL。酶学性质分析表明，重组酶最适作用温度约为60℃，最适反应pH为5．0，60℃保温3h仍保存50％的活性。重组酶性质适合淀粉糖化工艺的要求。

Secreted expression of the pullulanase in Bacillus amyloliquefaciens BF7658

According to the sequence of puUulanase gene from Bacillus deramificans （NCBI accession number： BD140672）, the gene encoding mature peptide of pullulanase was synthesized and designated BdP. BdP was inserted into the downstream of the DNA fragment encoding signal peptide of pUC980, a secreting expression vector of Bacillus. The constructed plasmid was designated pUC980-/3dP. Subsequently, pUC980-BdP was introduced into Bacillus arnyloliquefaciens BF7658, an industrial mesophilic amylase producer. Active pullulanase was expressed by recombinant t3. aznyloliquefaciens. Recombinant pullulanase was secreted into medium, the optimal time of pullulanase production was 48 h and the enzymatic activity was 2.8 U/mL. Its optimum pH was 5.0 and its optimum temperature was 60 ℃. More than 50 % of its initial enzymatic activity was still detectable after incubated at 60 ℃ for 3 h. The enzymatic property showed that the recombinant pullulanase could be used in saccharification process of starch .