中温α-淀粉酶在地衣芽孢杆菌中的异源表达

摘要

To increase the fermentation temperature of α-amylase is of great significance for decreasing cooling water consumption and cost of production. In this study,the gene amyL encoding the thermostableα-amylase of B. licheniformis CBBD302 was deleted by homologous recombination. The resulting mutant strain B. licheniformis D402 was then used as a host cell to overexpress BaA. Afterwards,the recombinant plasmid pHY-WZX-BaA harboring the gene encoding B. amyloliquefaciens α-amylase (BaA)was constructed and transformed to D402 strain. The recombinant D402/pHY-WZX-BaA strain was obtained and also used for fermentation experiments. Under shake-flask conditions,the optimal fermen-tation temperature of the recombinant D402/pHY-WZX-BaA strain was 42 ℃. By comparison,the original strain B. amyloliquefaciens M23 exhibited optimal performance at 34 ℃. Furthermore,301 U/mL was the highest amount of produced enzyme obtained under the shake-flask condition and 531 U/mL was the highest amount of produced enzyme obtained after 78 h of fermentation in a 30 L fermentor. The recombinant enzyme showed an optimal performance at 60 ℃and pH 6. 5. This enzyme could also be inactivated completely by incubating at 90 ℃ for 20 min. Similar to the original products,the recombinant enzyme could remain stable at starch gelatinization temperature and be inactivated conveniently.%提高中温α-淀粉酶生产菌株的发酵温度，对减少冷却水消耗降低生产成本有重要意义。本文利用基因删除技术删除了地衣芽孢杆菌CBBD302菌株α-淀粉酶的编码基因（amyL）获得突变株D402。将表达解淀粉芽孢杆菌中温α-淀粉酶基因BaA的重组质粒pHY-WZX-BaA转化D402，获得表达中温α-淀粉酶的重组地衣芽孢杆菌D402／pHY-WZX-BaA。摇瓶发酵实验显示，重组菌最适发酵温度为42℃，比原生产菌株提高8℃，最高产酶水平达到301 U／mL。30 L发酵罐发酵试验，78 h达到最高酶活531 U／mL。重组酶的最适作用温度为60℃，最适作用pH 6．5，在90℃保温20 min可以完全失活，保持了中温α-淀粉酶既能在淀粉糊化温度下保持稳定又便于灭酶的优良性能。