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Recombinant expression of Aspergillus niger GH10 endo‐xylanase in Pichia pastoris by constructing a double‐plasmid co‐expression system
Author(s) -
Long Lingfeng,
Zhang Yunbo,
Ren Hongyan,
Sun Haiyan,
Sun Fubao F,
Qin Wensheng
Publication year - 2020
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.6250
Subject(s) - pichia pastoris , xylanase , aspergillus niger , plasmid , expression vector , heterologous expression , recombinant dna , heterologous , biochemistry , biology , enzyme , chemistry , gene
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 ∼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 °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. © 2019 Society of Chemical Industry