摘要
内切纤维素酶Cel5A缺乏是限制纤维素酶制剂高效酶解天然纤维素的关键因素。本文尝试构建高效表达里氏木霉Cel5A的毕赤酵母重组菌株以弥补目前Cel5A的天然分泌不足,通过基因密码子偏好性优化里氏木霉Cel5A基因和构建表达载体p PIC9K-eg2,并将其电转入毕赤酵母GS115以构建重组子,利用浓度梯度平板和摇瓶发酵筛选获得一株高产毕赤酵母Pichia pastoris菌株GS115-EGⅡ。重组酶的酶学性质分析显示,该酶分子量50 k Da、最适p H(p H 4.5)略有降低及最适反应温度为60℃,专一性地作用于非结晶纤维素,与天然里氏木霉Cel5A并无明显区别。通过摇瓶发酵的初步优化,该菌摇瓶培养条件:培养温度28℃、起始p H 5.0、接种量2%、每24 h添加甲醇1.5%(V/V)、每24 h添加山梨醇4 g/L及吐温80添加4 g/L,发酵192 h重组酶酶活达到24.0 U/m L。进一步上罐(5 L)发酵180 h,该重组酶Cel5A酶活高达270.9 U/m L,蛋白含量达到4.16 g/L。重组毕赤酵母P.pastoris GS115-EGⅡ是一株适合于外源表达Cel5A的工程菌,该重组酶可替代天然分泌Cel5A适用于当前酶基生物炼制模式下木质纤维素基质高效水解中。
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 Cel5 A in Pichia pastoris by constructing a recombinant strain.With the gene optimization based on codon bias,and the construction of expression vector p PIC9K-eg2,the optimized gene was electro-transformed into P.pastoris GS115 to form transformants.Then,a high Cel5 A 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 Cel5 A reacted optimally at p H 4.5 and 60 ℃,with 50 k Da of molecular weight,preferentially degrading amorphous cellulose.Recombinant Cel5 A was not significantly different from the native T.reesei Cel5 A.Moreover,a shake-flask fermentation of the recombinant strain was optimized as below:incubation temperature 28 ℃,initial p H 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/m L of the Cel5 A after 192 h fermentation.When incubated in a 5 L fermentation,Cel5 A enzyme activity reached 270.9 U/m L 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 Cel5 A.And the recombinant Cel5 A can be used as an alternative to the native T.reesei Cel5 A in development of a commercially relevant enzyme based biorefinery process.
作者
白仁惠
张云博
王春迪
张斐洋
张喆
孙付保
张震宇
Renhui Bai Yunbo Zhang Chundi Wang Feiyang Zhang Zhe Zhang Fubao Sun Zhenyu Zhang(Laboratory of Industrial Biotechnology of Department of Education,Jiangnan University,Jiangsu Key Laboratory of Biomass-based Green Fuels and Chemicals,Nanjing Forestry University,State Key Laboratory of Motor Vehicle Biofuel Technology,Henan Tianguan Group Co.,Lt)
出处
《生物工程学报》
CAS
CSCD
北大核心
2016年第10期1381-1394,共14页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.21176106)
车用生物燃料技术国家重点实验室开放基金(No.KFKT2013010)
中国博士后科学基金(No.2015M571666)
江苏省生物质绿色燃料与化学品重点实验室课题(No.JSBGFC14006)资助~~