黑曲霉h408阿魏酸酯酶基因的克隆及在毕赤酵母中的高效表达

Cloning of feruloyl esterase gene from Aspergillus niger h408 and high-efficient expression in Pichia pastoris

摘要:

摘要:【目的】实现在巴斯德毕赤酵母(Pichia pastoris)中高效表达黑曲霉(Aspergillus niger) h408阿魏酸酯酶A基因(AnfaeA)，并对重组酶特性进行表征。【方法】采用重叠延伸PCR扩增黑曲霉h408 的阿魏酸酯酶A基因。将AnfaeA基因和毕赤酵母表达载体pPIC9K连接，成功构建重组质粒pPIC9K-Anfae，经线性化后电转化P. pastoris GS115，透明圈法筛选活性高的转化子后进行诱导表达。利用紫外吸收法测定温度及pH对重组阿魏酸酯酶活性的影响。【结果】成功从A．niger h408中克隆得到阿魏酸酯酶A的cDNA基因(GenBank: KF911349)，并实现了其在P．pastoris GS115中的高效表达。该基因长度为783bp，含有1个开放阅读框架(ORF)，编码260个氨基酸，Blast分析显示该基因和GenBank中黑曲霉阿魏酸酯酶序列同源性为99%。翻译的氨基酸序列含有脂酶典型的活性盖子和催化三联体结构。从转化板上获得1株编号为pPIC9K-Anfae5的转化子阿魏酸酯酶活性最高，酶活达24.72 U/mL，比活力为40.84 U/mg，比黑曲霉出发菌株(22.1 mU/mL)提高了1100倍左右。重组阿魏酸酯酶的最适pH为5.0，且在pH 4.0－9.0稳定性较好;最适反应温度50℃，在40－60℃时较稳定。【结论】阿魏酸酯酶在毕赤酵母中的高效分泌表达为其在饲料工业和造纸工业等工业化应用提供了前提，也为后续改进酶学特性的定向进化奠定实验基础。

Abstract:

Abstract:［Objective］To achieve the high-efficiency expression of feruloyl estrase gene (AnfaeA) from Aspergillus niger h408 in Pichia pastoris and characterize the recombinant feruloyl esterase (FAE).［Methods］ Using gene splicing by overlap extension (SOE)，we cloned AnfaeA gene from A．niger h408 and subcloned into T vector for sequencing analysis．The expression vector pPIC9K-Anfae was constructed by the ligation of the Anfae A gene into the shuttle vector pPIC9K．The plasmid pPIC9K-Anfae was linearized and then electrotransformed into P．pastoris GS115． The recombinant strain with high level of FAE activity was obtained through plate screening． Effects of pH and temperature on recombinant FAE were determined by ultraviolet (UV) methods．［Results］We have successfully cloned and high-efficiently expressed the AnfaeA gene (GenBank: KF911349) from A．niger h408 in P．pastoris GS115．The sequencing result showed that the length of Anfae A was 783bp．The gene contained an Open Ｒeading Frame encoding 260 amino acids and was similar to feruloyl esterase A from A．niger by homology analysis． The deduced amino acids contained a typical active lid and catalytic triad of lipase．The SDS-PAGE result indicated that molecular weight of the recombinant FAE was about 30 kDa and the activity of the recombinant enzyme was 24. 72 U/mL．The specific activity of the recombinant FAE was 40.84 U/mg．Compared with A．niger h408，the recombinant enzyme activity increased about to 1100 times．The optimal temperature and pH for recombinant FAE was 50℃ and 5.0，respectively．Recombinant FAE showed nearly 80% of its maximal activity at 60℃ and was active in the pH range 4.0-9.0.［Conclusions］The high-efficient expression of AnfaeA gene in P．pastoris provided a prerequisite for achieving industrial application in feed and paper-making industry．In addition，the results established the experimental basis for further improvement of recombinant feruloyl esterase by directed evolution．