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oai:ebyu-rdlopen.rawdatalibrary.net:ebyu/106002026-10-18T21:13:12Zopenairedrivercom_ebyu_10000col_ebyu_10001

Co-Expression of Chaperones for Improvement of Recombinant Geobacillus stearothermophilus Lipase Production in Pichia pastorisErden-Karaoglan, Fidanchaperonesfolding-assisting proteinslipasePDIPichia pastorisBacterial ProteinsGeobacillus stearothermophilusKomagataella pastorisMolecular ChaperonesRecombinant ProteinsSaccharomycetalesClone cellsCloningKinetic parametersSubstratesSulfur compoundsYeastchaperonedimethyl sulfoxiderecombinant enzymetriacylglycerol lipasebacterial proteinrecombinant proteinChaperonCo-expressionExpression systemFolding-assisting proteinFoldingsLipase productionSulphoxidePichia pastoris is among the most popular expression systems for recombinant protein production. The ability to secrete high titers of recombinant proteins is one of its primary advantages. Co-expression of folding-assisting factors is considered one of the strategies to improve protein production with this expression system. In this study, the effect of PDI, KAR2, HAC1, and ERO1 co-expression on Geobacillus stearothermophilus lipase production in P. pastoris was investigated. The chaperones were co-expressed under the regulation of the GAP promoter in clones with different lipase production levels (LPS#2 and LPS#8). Results showed that lipase-PDI co-expression clones have the highest activity. The effect of the other chaperones had varying effects for LPS#2 and LPS#8 clones. LPS#2-PDI and LPS#8-PDI were further analyzed to determine the effect of dimethyl sulphoxide (DMSO). Different concentrations (0.5%, 1%, and 2%) of DMSO were tested. The highest activity was obtained with approximately 1.5-fold activity in the LPS#8-PDI clone with 0.5% and 1% DMSO concentration levels. Comparison of the fermentation kinetic parameters revealed that the activity level of 56.54 U/mL provided with LPS#8 increased to 73.84 U/mL in the LPS#8-PDI clone with PDI co-expression, and when PDI co-expression was combined with DMSO conditions (0.5%), it reached 114.46 U/mL. It was observed that the productivity (U/g wet cell/g substrate/h) of the LPS#8 clone was increased 1.27-fold with LPS#8-PDI and 2.05-fold with LPS#8-PDI-DMSO conditions. In future studies, it is possible to achieve higher protein production levels by optimization at the fermenter scale. © 2025 International Union of Biochemistry and Molecular Biology, Inc.John Wiley and Sons Inc2025Articlehttps://rdlopen.rawdatalibrary.net/r/ebyu/10600https://doi.org/10.1002/bab.700170885-4513enBiotechnology and Applied Biochemistryinfo:eu-repo/semantics/openAccessinfo:eu-repo/semantics/articlehttps://ebyu-rdlopen.rawdatalibrary.net/tr/items/ebyu/10600