tailieunhanh - Enhancing the expression of Aspergillus niger β-mannanase in Pichia pastoris by coexpression of protein disulfide isomerase
A gene encoding β-mannanase from Aspergillus niger 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 β-mannanase activity of U/mL was obtained. | Turkish Journal of Biology Turk J Biol (2015) 39: 312-319 © TÜBİTAK doi: Research Article Enhancing the expression of Aspergillus niger β-mannanase in Pichia pastoris by coexpression of protein disulfide isomerase 1, 1 1, 1 1, 1 2 1 Xiaoling CHEN *, Bo ZHOU *, Meng XU , Zhiqing HUANG **, Gang JIA , Jiayun QIAO , Guangmang LIU Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, Sichuan, P. R. China 2 Tianjin Institute of Animal Science and Veterinary Medicine, Tianjin, P. R. China Received: Accepted: Published Online: Printed: Abstract: A gene encoding β-mannanase from Aspergillus niger 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 β-mannanase activity of U/mL was obtained. In order to increase the secretion of β-mannanase in P. pastoris, we constructed a double recombinant yeast and made it coexpress protein disulfide isomerase. One strain (GSKZαM2) with the highest β-mannanase activity of 40 U/mL was then obtained and used to optimize expression conditions. When the GSKZαM2 strain was induced under the optimized conditions (methanol concentration , induction time 7 days), β-mannanase activity reached U/mL. SDS-PAGE and deglycosylation assays demonstrated that the recombinant A. niger β-mannanase, a glycosylated protein with an apparent molecular weight of 45 kDa, was secreted into the culture medium. It displayed maximum activity at pH and 60 °C, and it was stable in a pH range of – and at a temperature of 60 °C or below. Our results suggested that coexpression chaperones could improve the yield of β-mannanase. Key words: Aspergillus niger, β-mannanase, Pichia pastoris, .
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