内切葡聚糖酶与木聚糖酶在毕赤酵母中的共分泌表达

Co-Expression of Endoglucanase and Xylanase in Pichia pastoris

DOI：10.3969/j.issn.1673-1689.2015.03.005

中文关键词: 内切葡聚糖酶 木聚糖酶 毕赤酵母 共分泌表达

英文关键词: endoglucanase,xylanase,Pichia pastoris,co-expression

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中文摘要:

为实现内切葡聚糖酶和木聚糖酶在毕赤酵母中的共分泌表达,进而降低酶制剂的生产成本,构建含木聚糖酶基因的重组表达载体pPICZαA-Aoxyn11A,经Sac I线性化后,电转化至含内切葡聚糖酶基因Aucel12A的重组毕赤酵母GSC7中,获得双重重组毕赤酵母GSCX8。经甲醇诱导表达后,GSCX8发酵上清液中内切葡聚糖酶和木聚糖酶的活性分别为47.77 IU/mL和192.71 IU/mL,为单独表达菌株GSC7和GSX5的85%和80%。酶学性质分析显示,内切葡聚糖酶的最适pH为4.0,在pH 3.0~8.5稳定;最适温度为50 ℃,在60 ℃以下稳定。木聚糖酶的最适pH为5.5,在pH 3.0~10.0稳定;最适温度为55 ℃,在50 ℃以下稳定。GSCX8遗传稳定性测试结果表明,内切葡聚糖酶和木聚糖酶在毕赤酵母中实现了稳定的共表达。

英文摘要:

Both endoglucanase and xylanase are additives in feed for mono-gastric animal. Co-expression of endoglucanase and xylanase in P. pastoris is a useful way to reduce the production cost in feedstuff industry. An endoglucanase-encoding gene(Aucel12A) from Aspergillus usamii was expressed in P. pastoris,and the engineering strain with the highest endoglucanase activity was labeled GSC7. Then,a recombinant vector,pPICZαA-Aoxyn11A,was constructed and linearized by Sac I,followed by transforming it into GSC7. Ultimately,the endoglucanase and xylanase genes were successfully co-expressed in P. pastoris. One strain harbored genes Aoxyn11A and Aucel12A,with the highest xylanase and endoglucanase activities,was labeled GSCX8. The enzymatic properties of the recombinant AuCel12A and AoXyn11A,expressed in GSCX8,were characterized. Meanwhile,to analyze the genetic stability of GSCX8,the xylanase and endoglucanase activities of different strains after passing 10 generations were also measured. After strain GSCX8 was induced for 96 h,the activities of recombinant AuCel12A and AoXyn11A in the cultured supernatant were 47.77 IU/mL and 192.71 IU/mL,respectively,which were about 85% and 80% of that of enzymes expressed by the single gene engineering strains GSCZ and GSX5. The recombinant AuCel12A displayed the optimal activity at pH 4.0 and 50 ℃,and was stable over a pH range of 3.0~8.5 and at 60 ℃ or below. Besides,the recombinant AoXyn11A displayed the optimal activity at pH 5.5 and 55 ℃,and was stable over a pH range of 3.0~10.0 and at 50 ℃ or below. Additionally,the activities of the two enzymes were well maintained after the GSCX8 passing 10 generations.

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