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Recombinant expression of Aspergillus niger GH10 endo-xylanase in Pichia pastoris by constructing a double-plasmid co-expression system

文献类型： 外文期刊

作者： Long, Lingfeng 1 ; Zhang, Yunbo 1 ; Ren, Hongyan 2 ; Sun, Haiyan 3 ; Sun, Fubao F. 1 ; Qin, Wensheng 4 ;

作者机构： 1.Jiangnan Univ, Sch Biotechnol, Key Lab Carbohydrate Chem & Biotechnol, Minist Educ, Wuxi 214122, Jiangsu, Peoples R China

2.Jiangnan Univ, Sch Environm & Civil Engn, Jiangsu Key Lab Anaerob Biotechnol, Wuxi, Jiangsu, Peoples R China

3.Chinese Acad Trop Agr Sci, Inst Trop Bioscienceand Biotechnol, Haikou, Hainan, Peoples R China

4.Univ Lakehead, Dept Biol, Thunder Bay, ON, Canada

关键词： codon optimization; double-plasmid co-expression system; Pichia pastoris; recombinant expression; xylanase

期刊名称：JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY （ 影响因子：3.174； 五年影响因子：3.137 ）

ISSN： 0268-2575

年卷期： 2020 年 95 卷 3 期

页码：

收录情况： SCI

摘要： BACKGROUND Previous work has shown that GH10 endo-xylanase presented better synergistic cooperation with cellulase to achieve effective hydrolysis. However, most currently reported commercial xylanolytic enzymes from Aspergillus niger belong to GH11 and there have been few studies regarding GH10 xylanase. In addition, to increase overall expression of heterologous endo-xylanase in Pichia pastoris, a double-plasmid co-expression method, including the construction of recombinant P. pastoris using one expression vector followed by inserting another vector which targeted to different locus, was introduced. RESULTS A GH10 xylanase gene from A. niger BE-2 (XynC) was optimized and constitutively expressed in P. pastoris GS115. Compared with the conventional single-plasmid method, it appeared that the double-plasmid strategy could considerably increase the XynC yield by similar to 33%. To further improve enzyme production, cultivation conditions were optimized at shake-flask level and then scaled up to a 5-L bioreactor. Through high cell-density fermentation, the expression level of GH10 XynC reached 1650 U mL(-1). The XynC showed maximum activity at 55 degrees C and pH 5.0, and exhibited an excellent stability over a wide range of pH from 4.5 to 7.0. The kinetic parameters K-m and V-max values for beechwood xylan were 3.5 mg mL(-1) and 2327 U mg(-1), respectively. CONCLUSION The double-plasmid co-expression strategy introduced herein could greatly improve the XynC yield in P. pastoris. The GH10 xylanase from A. niger BE-2 shared similar reaction conditions with the existing enzymatic hydrolysis, which might be a good candidate to assist cellulases for industrial application. (c) 2019 Society of Chemical Industry

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