产β-苯丙氨酸变位酶基因工程菌的构建及其发酵条件优化

Construction of recombinant E coli.of producing β-phenylalanine aminomutase and optimization of fermentation conditions

构建β-苯丙氨酸变位酶基因表达重组质粒pET-28apam,并转化大肠杆菌BL21,获得β-苯丙氨酸变位酶基因重组菌。考察培养基初始pH、摇床转速、种龄、发酵时间以及装液量5个因素对β-苯丙氨酸变位酶基因工程菌产酶活性的影响,确定较佳发酵条件为:培养基初始pH 7.0、摇床转速200r/min、发酵时间24h、装液量50mL/500mL、种龄10h。采用Box-Behnken试验设计,选择了种龄、初始pH、发酵时间3个对β-苯丙氨酸变位酶活力影响较大的因素进行响应面优化。优化结果为:在种龄12.8h、初始pH 6.7、发酵时间25.2h条件下,PAM活力达到11.15 U/mL,比优化前酶活4.87U/mL提高了128.9%。

The genetic engineering strain with recombinant plasmid pET-28a-pam was expressed in E.coli BL21.The fermentation conditions of initial pH, shaking speed, inoculation time and fermentation time were optimized with the PAM activity by using single factor.The results showed that the optimal fermentation conditions were initial pH 7.0,200r/min,the tenth hour broth and liquid volume 50 mL/500 mL,fermented for 24 h.Furthermore,the three factors were optimized by Box-Behnken design,and the results showed that inoculation time,initial pH and fermentation time are main affecting factors and their optimal levels are 12.8h,6.7and25.2h.Under the optimal conditions,th activity of PAM reached11.15U/mL,improved by 128.9% compared with that under initial culture conditions.