介导两种半纤维素酶分泌表达的信号肽比较  

Comparison of Signal Peptides for Two Hemicellulase Secretory Expression

作　　者：杨青[1] 汪斌[1] 王亚伟[1] 张华山[2] 熊海容[1] 张莉[1] 

机构地区：[1]中南民族大学生命科学学院,武汉430074 [2]湖北工业大学发酵工程省部共建教育部重点实验室,武汉430068

出　　处：《中国生物工程杂志》2017年第8期15-22,共8页China Biotechnology

基　　金：中央高校基本科研业务费专项资金资助项目(CZW15026;CZW15012)

摘　　要：木聚糖酶和甘露聚糖酶是两种主要的半纤维素酶,广泛应用于诸多领域。选择了3种毕赤酵母内源信号肽Scw11、Dse4和Exg1,以α-factor为参照,分别在毕赤酵母X33中用于表达实验室前期获得的耐热木聚糖酶DSB和耐热甘露聚糖酶Man A,选择适合DSB和Man A表达的信号肽以提高胞外酶活水平。结果表明不同信号肽引导的两种半纤维素酶的酶活水平相差较大:对DSB(分子量为23k Da),α-factor介导的表达效率明显优于其它3种信号肽;但对Man A(分子量为30k Da),Dse4和α-factor介导的表达效率相当且明显优于Scw11和Exg1。因此在X33中表达DSB时应选用α-factor,而表达Man A时应选用Dse4或α-factor。此外胞内酶活的结果显示α-factor介导的DSB和Man A重组菌胞内滞留酶活明显高于其它信号肽,而分子量为30k Da的Man A的滞留酶活又明显高于分子量为23k Da的DSB,因此在表达Man A蛋白时,选用其它的信号肽,如Dse4,会减少外源蛋白在胞内的滞留从而在一定程度上提高外源蛋白的分泌量。因此为毕赤酵母表达系统鉴定更多可用的信号肽并筛选到针对目的蛋白的最优信号肽奠定了一定的基础。Xylanase and mannanase are two hemicellulases,which are widely used in many fields. To improve the expression of thermostable xylanase DSB and thermostable mannanase Man A in Pichia pastoris,three endogenous signal peptides of Pichia pastoris（ Scw11,Dse4 and Exg1） were chosed. Their capability to mediate the secretion of DSB and Man A with that of the Saccharomyces cerevisiae α-factor were compared. In shake-flask cultivation,three endogenous signal peptides and α-factor efficiently mediated the secretion of DSB and Man A,but the secretion efficiency has obvious difference. As for DSB,the expression efficiency of α-factor was much higher than three endogenous signal peptides. But as for Man A,the expression efficiency of Dse4 was equal to α-factor and much higher than Scw11 and Exg1. Therefore,α-factoris the most efficient signal peptide for DSB expression and Dse4 or α-factor are the most efficient signal peptide for Man A expression in Pichia pastoris X33.Moreover,the intracellular activities of DSB and Man A by α-factor are higher than Scw11,Dse4 and Exg1,and the intracellular activity of Man A was higher than DSB（ the molecular weight of Man A was larger than DSB）.Thus,when Man A were expressed in Pichia pastoris,different signal peptide,such as Dse4,could be used for improving the secretion efficiency. A basis for identifying more available signal peptides and screening for the optimal signal peptide for the target protein was provided.

关 键 词：信号肽 巴斯德毕赤酵母 木聚糖酶(DSB) 甘露聚糖酶(ManA) 

分 类 号：Q819[生物学—生物工程]