Bo ZHOU, Meng XU, Xiaoling CHEN, Zhiqing HUANG, Gang JIA, Jiayun QIAO, Guangmang LIU

Enhancing the expression of Aspergillus niger β-mannanase in Pichia pastoris by coexpression of protein disulfde isomerase

A gene encoding β-mannanase from Aspergillus niger GIM3.452 was amplifed and inserted into a pPIC9K vector. Teresulting recombinant plasmid, pPIC9K-MAN, was transformed into Pichia pastoris GS115. One strain (GSKM-1) having the highestβ-mannanase activity of 26.6 U/mL was obtained. In order to increase the secretion of β-mannanase in P. pastoris, we constructed adouble recombinant yeast and made it coexpress protein disulfde isomerase. One strain (GSKZαM2) with the highest β-mannanaseactivity of 40 U/mL was then obtained and used to optimize expression conditions. When the GSKZαM2 strain was induced under theoptimized conditions (methanol concentration 1.5%, induction time 7 days), β-mannanase activity reached 222.8 U/mL. SDS-PAGEand deglycosylation assays demonstrated that the recombinant A. niger β-mannanase, a glycosylated protein with an apparent molecularweight of 45 kDa, was secreted into the culture medium. It displayed maximum activity at pH 4.4 and 60 °C, and it was stable in a pHrange of 2.4 8.0 and at a temperature of 60 °C or below. Our results suggested that coexpression chaperones could improve the yield ofβ-mannanase.

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