XIAOLING CHEN, BO ZHOU, MENG XU, ZHIQING HUANG, GANG JIA, JIAYUN QIAO, GUANGMANG LIU

95142

Enhancing the expression of Aspergillus niger ß-mannanase in Pichia pastoris by coexpression of protein disulfide isomerase

A gene encoding ß-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 ß-mannanase activity of 26.6 U/mL was obtained. In order to increase the secretion of ß-mannanase in P. pastoris, we constructed a double recombinant yeast and made it coexpress protein disulfide isomerase. One strain (GSKZ\alphaM2) with the highest ß-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), ß-mannanase activity reached 222.8 U/mL. SDS-PAGE and deglycosylation assays demonstrated that the recombinant A. niger ß-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 ß-mannanase.
Anahtar Kelimeler:

Aspergillus niger, ß-mannanase, Pichia pastoris, protein disulfide isomerase, expression condition optimization

Enhancing the expression of Aspergillus niger ß-mannanase in Pichia pastoris by coexpression of protein disulfide isomerase

A gene encoding ß-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 ß-mannanase activity of 26.6 U/mL was obtained. In order to increase the secretion of ß-mannanase in P. pastoris, we constructed a double recombinant yeast and made it coexpress protein disulfide isomerase. One strain (GSKZ\alphaM2) with the highest ß-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), ß-mannanase activity reached 222.8 U/mL. SDS-PAGE and deglycosylation assays demonstrated that the recombinant A. niger ß-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 ß-mannanase.
Keywords:

Aspergillus niger, ß-mannanase, Pichia pastoris, protein disulfide isomerase, expression condition optimization,

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Turkish Journal of Biology-Cover
  • ISSN: 1300-0152
  • Yayın Aralığı: 6
  • Yayıncı: TÜBİTAK
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