Abstract
A gene encoding A-mannanase from Aspergillus niger GIM3.452 was amplified and inserted into a pPIC9K vector. The resulting recombinant plasmid, pPIC9K-MAN, was transformed into Pichia pastoris GS115. One strain (GSKM-1) having the highest A-mannanase activity of 26.6 U/mL was obtained. In order to increase the secretion of A-mannanase in P. pastoris, we constructed a double recombinant yeast and made it coexpress protein disulfide isomerase. One strain (GSKZ\alphaM2) with the highest A-mannanase activity of 40 U/mL was then obtained and used to optimize expression conditions. When the GSKZ\alphaM2 strain was induced under the optimized conditions (methanol concentration 1.5%, induction time 7 days), A-mannanase activity reached 222.8 U/mL. SDS-PAGE and deglycosylation assays demonstrated that the recombinant A. niger A-mannanase, a glycosylated protein with an apparent molecular weight 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 pH range of 2.4-8.0 and at a temperature of 60 °C or below. Our results suggested that coexpression chaperones could improve the yield of A-mannanase.
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