[논문]Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris

Qiao, Hanzhen; Zhang, Wenfei; Guan, Wutai; Chen, Fang; Zhang, Shihai; Deng, Zixiao

doi:10.1016/j.pep.2017.05.005

Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris

Protein expression and purification, v.138, 2017년, pp.34 - 45

Qiao, Hanzhen (South China Agricultural University, College of Animal Science, South China Agricultural University, Wushan Avenue, Tianhe District, Guangzhou, 510642, China) , Zhang, Wenfei (South China Agricultural University, College of Animal Science, South China Agricultural University, Wushan Avenue, Tianhe District, Guangzhou, 510642, China) , Guan, Wutai (South China Agricultural University, College of Animal Science, South China Agricultural University, Wushan Avenue, Tianhe District, Guangzhou, 510642, China) , Chen, Fang (South China Agricultural University, College of Animal Science, South China Agricultural University, Wushan Avenue, Tianhe District, Guangzhou, 510642, China) , Zhang, Shihai (South China Agricultural University, College of Animal Science, South China Agricultural University, Wushan Avenue, Tianhe District, Guangzhou, 510642, China) , Deng, Zixiao (South China Agricultural University, College of Animal Science, South China Agricultural University, Wushan Avenue, Tianhe District, Guangzhou, 510642, China)

Abstract ▼ AI-Helper

Abstract Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C, respectively. The enzyme was active over a broad pH range (7.0–9.0) and temperature range (20 °C–45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg2+, Ca2+, Mn2+ and Cu2+ and inhibited by Fe2+, Fe3+, Zn2+ and Co2+. This lipase may prove useful to the detergent industry and in organic synthesis reactions. Highlights Codon-optimized GGl I is overexpressed in P. pastoris with activity of 150 U/mL. The enzyme was active over a broad pH range and temperature range. Exhibiting high activities towards medium and long chain fatty acid methyl esters. Presumed glycosylation of the recombinant GGl I was confirmed. Graphical abstract [DISPLAY OMISSION]

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Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris

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