Enhancing the expression of Aspergillus niger Ã-mannanase in Pichia pastoris by coexpression of protein disulfide isomerase
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.
Enhancing the expression of Aspergillus niger Ã-mannanase in Pichia pastoris by coexpression of protein disulfide isomerase
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.
___
- Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM (2002). Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13: 329–332.
- Cereghino JL, Cregg JM (2000). Heterologous expression system in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24: 45–66.
- Chen X, Qiao J, Yu H, Cao Y (2009). Overexpression of an optimized Aspergillus sulphureus β-mannanase gene in Pichia pastoris. Biologia 64: 235–238.
- Chen XL, Cao YH, Ding YH, Lu WQ, Li DF (2007). Cloning, functional expression and characterization of Aspergillus sulphureus β-mannanase in Pichia pastoris. J Biotechnol 128: 452–461.
- Chen XL, Huang ZQ, Zhou B, Wang H, Jia G, Qiao JY (2014). Expression and purification of porcine Akirin2 in Escherichia coli. Turk J Biol 38: 339–345.
- Gilbert HF (1998). Protein disulfide isomerase. Methods Enzymol 290: 26–50.
- Heidorne FO, Magalhães PO, Ferraz AL, Milagres AMF (2006). Characterization of hemicellulases and cellulases produced by Ceriporiopsis subvermispora grown on wood under biopulping conditions. Enzyme Microb Technol 38: 436–442.
- Hohenblum H (2004). Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85: 367–375.
- Inan M, Aryasomayajula D, Sinha J, Meagher MM (2006). Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase. Biotechnol Bioeng 93: 771–778.
- Morreira LR, Filho EX (2008). An overview of mannan structure and mannan-degrading enzyme systems. Appl Microbiol Biotechnol 79: 165–178.
- Ngiam C, Jeenes DJ, Punt PJ, Van Den Hondel CA, Archer DB (2000). Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. Appl Environ Microbiol 66: 775–782.
- Noiva R (1999). Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum. Semin Cell Dev Biol 10: 481–493.
- Powers SL, Robinson AS (2007). PDI improves secretion of redox- inactive beta-glucosidase. Biotechnol Prog 23: 364–369.
- Roth R, Moodley V, Zyl P (2009). Heterologous expression and optimized production of an Aspergillus aculeatus endo-1,4- β-mannanase in Yarrowia lipolytica. Mol Biotechnol 43: 112– 120.
- Sha C, Yu X, Lin N, Zhang M, Xu Y (2013). Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase. Enzyme Microb Tech 53: 438–443.
- Shen Q, Wu M, Wang H, Naranmandura H, Chen S (2012). The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris. Appl Microbiol Biotechnol 96: 763–772.
- Smith JD, Tang BC, Robinson AS (2004). Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast. Biotechnol Bioeng 85: 340–350.
- Sitia R, Braakman I (2003). Quality control in the endoplasmic reticulum protein factory. Nature 426: 891–894.
- Tang CD, Guo J, Li JF, Wei XH, Hu D, Gao SJ, Yin X, Wu MC (2014). Enhancing expression level of an acidophilic β-mannanase in Pichia pastoris by double vector system. Ann Microbiol 64: 561–569.
- Tsai CW, Duggan PF, Shimp RL Jr, Miller LH, Narum DL (2006). Overproduction of Pichia pastoris or Plasmodium falciparum protein disulfide isomerase affects expression, folding and O-linked glycosylation of a malaria vaccine candidate expressed in P. pastoris. J Biotechnol 121: 458–470.
- Wang H, Chen XL, Huang ZQ, Zhou B, Jia G, Liu GM, Zhao H (2014). Expression and purification of porcine PID1 gene in Escherichia coli. Turk J Biol 38: 523–527.
- Wu D, Yu XW, Wang TC, Wang R, Xu Y (2011). High yield Rhizopus chinenisis prolipase production in Pichia pastoris: impact of methanol concentration. Biotechnol Bioprocess Eng 16: 305–311.
- Wu MC, Tang CD, Li JF, Zhang HM, Guo J (2011). Bimutation breeding of Aspergillus niger strain for enhancing β-mannanase production by solid-state fermentation. Carbohydr Res 346: 2149–2155.
- Zhang J, Wu D, Chen J, Wu J (2011). Enhancing functional expression of β-glucosidase in Pichia pastoris by co-expression protein disulfide isomerase. Biotechnol Bioprocess Eng 16: 1196–1200.