- Turkish Journal of Biology
- Vol: 39 Issue: 2
- Enhancing the expression of Aspergillus niger Ã-mannanase in Pichia pastoris by coexpression of pro...
Enhancing the expression of Aspergillus niger Ã-mannanase in Pichia pastoris by coexpression of protein disulfide isomerase
Authors : Xiaoling Chen, Bo Zhou, Meng Xu, Zhiqing Huang, Gang Jia, Jiayun Qiao, Guangmang Liu
Pages : 312-319
Doi:10.3906/biy-1408-33
View : 8 | Download : 3
Publication Date : 9999-12-31
Article Type : Makaleler
Abstract :A gene encoding Ã-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 Ã-mannanase activity of 26.6 U/mL was obtained. In order to increase the secretion of Ã-mannanase in P. pastoris, we constructed a double recombinant yeast and made it coexpress protein disulfide isomerase. One strain (GSKZ\alphaM2) with the highest Ã-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), Ã-mannanase activity reached 222.8 U/mL. SDS-PAGE and deglycosylation assays demonstrated that the recombinant A. niger Ã-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 Ã-mannanase.Keywords : Aspergillus niger, Ã-mannanase, Pichia pastoris, protein disulfide isomerase, expression condition optimization