Bai Renhui, Zhang Yunbo, Wang Chundi, Zhang Feiyang, Zhang Zhe, Sun Fubao, Zhang Zhenyu
Laboratory of Industrial Biotechnology of Department of Education, Jiangnan University, Wuxi 214122, Jiangsu, China.
Jiangsu Key Laboratory of Biomass-based Green Fuels and Chemicals, Nanjing Forestry University, Nanjing 210037, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2016 Oct 25;32(10):1381-1394. doi: 10.13345/j.cjb.160017.
Deficient activity of endo-1,4-beta-glucanase II (Cel5A) secreted by Trichoderma reesei is one of the challenges involved in effective cellulase saccharification of cellulosic substrates. Therefore, we expressed Cel5A in Pichia pastoris by constructing a recombinant strain. With the gene optimization based on codon bias, and the construction of expression vector pPIC9K-eg2, the optimized gene was electro-transformed into P. pastoris GS115 to form transformants. Then, a high Cel5A activity producing recombinant, namely P. pastoris GS115-EG Ⅱ, was selected on G-418 resistant plates, followed by shake-flask cultivation. Enzyme characterization showed that the recombinant Cel5A reacted optimally at pH 4.5 and 60 ℃, with 50 kDa of molecular weight, preferentially degrading amorphous cellulose. Recombinant Cel5A was not significantly different from the native T. reesei Cel5A. Moreover, a shake-flask fermentation of the recombinant strain was optimized as below: incubation temperature 28 ℃, initial pH 5.0, inoculum volume 2%, methanol addition (per 24 h) 1.5% (V/V), sorbitol addition (per 24 h) 4 g/L and Tween 80 4 g/L. Under above optimized condition, the recombinant produced 24.0 U/mL of the Cel5A after 192 h fermentation. When incubated in a 5 L fermentation, Cel5A enzyme activity reached 270.9 U/mL at 180 h, with 4.16 g/L of the total protein. The study indicates that the recombinant strain P. pastoris GS115-EG Ⅱ is extremely suitable for heterologous expression of T. reesei cellulase Cel5A. And the recombinant Cel5A can be used as an alternative to the native T. reesei Cel5A in development of a commercially relevant enzyme based biorefinery process.
里氏木霉分泌的内切-1,4-β-葡聚糖酶II（Cel5A）活性不足是纤维素底物有效纤维素酶糖化过程中面临的挑战之一。因此，我们通过构建重组菌株在毕赤酵母中表达Cel5A。基于密码子偏好性进行基因优化，并构建表达载体pPIC9K-eg2，将优化后的基因电转化至毕赤酵母GS115中以形成转化子。然后，在G-418抗性平板上筛选出高Cel5A活性产生重组体，即毕赤酵母GS115-EGⅡ，随后进行摇瓶培养。酶学特性表明，重组Cel5A在pH 4.5和60℃时反应最佳，分子量为50 kDa，优先降解无定形纤维素。重组Cel5A与天然里氏木霉Cel5A无显著差异。此外，对重组菌株的摇瓶发酵进行了如下优化：培养温度28℃，初始pH 5.0，接种量2%，甲醇添加量（每24小时）1.5%（V/V），山梨醇添加量（每24小时）4 g/L，吐温80 4 g/L。在上述优化条件下，重组体在发酵192小时后产生24.0 U/mL的Cel5A。在5 L发酵中培养时，180小时时Cel5A酶活性达到270.9 U/mL，总蛋白为4.16 g/L。该研究表明，重组菌株毕赤酵母GS115-EGⅡ非常适合里氏木霉纤维素酶Cel5A的异源表达。并且重组Cel5A可在基于商业相关酶构建生物精炼工艺中替代天然里氏木霉Cel5A。
