Dataset Information

High-level expression of lipase from Galactomyces geotrichum mafic-0601 by codon optimization in Pichia pastoris and its application in hydrolysis of various oils.

ABSTRACT: A Galactomyces geotrichum strain with lipolytic activity was isolated and identified by the analysis of internal transcribed spacer (ITS) sequence of 18 s rDNA. Full-length lipase gene of this stain is composed of 1692 base pairs (bp) without intron, which encodes a 563-amino-acid protein. A catalytic triad (Ser217-Glu354-His463) was found by constructing the three-dimensional structure of the lipase. In shake flasks, the lipase (LIP) catalytic activity in the supernatant of the recombinant Pichia pastoris increased 48.7% by codon optimization. LIP purified by anion exchange column showed a single protein band on 12% SDS-PAGE. The molecular weight (MW) of LIP was approximately 62 kDa. The specific activity of purified LIP reached 1257.9 U/mg. The optimum temperature and pH of LIP catalysis were 45 °C and pH 8.2, respectively. LIP was stable over the pH range of 4.2-11.2. LIP maintained its activity constantly at 40 °C and 50 °C for 120 min. Zn2+ inhibited LIP activity; Ba2+, Mn2+, Ca2+ and EDTA increased the enzyme activity. Referring the amount of hydrolyzed olive oil by LIP as 100%, various oils including lard, peanut oil, rapeseed oil, sunflower oil, soybean oil and linseed oil were efficiently hydrolyzed by 17.24?±?1.34%, 40.34?±?2.56%, 105.86?±?2.78%, 115.51?±?2.32%, 116.21?±?2.15%, 120.69?±?1.98%, respectively. The characteristics allow LIP as a potential biocatalyst in various fields of industry.

SUBMITTER: Wang J

PROVIDER: S-EPMC6726727 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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High-level expression of lipase from Galactomyces geotrichum mafic-0601 by codon optimization in Pichia pastoris and its application in hydrolysis of various oils.

Wang Jian J Liu Yajing Y Guo Xiaojing X Dong Bing B Cao Yunhe Y

3 Biotech 20190904 10

A Galactomyces geotrichum strain with lipolytic activity was isolated and identified by the analysis of internal transcribed spacer (ITS) sequence of 18 s rDNA. Full-length lipase gene of this stain is composed of 1692 base pairs (bp) without intron, which encodes a 563-amino-acid protein. A catalytic triad (Ser217-Glu354-His463) was found by constructing the three-dimensional structure of the lipase. In shake flasks, the lipase (LIP) catalytic activity in ...[more]

PMID: 31501755

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Project description:Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P. pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), Km (0.041 mM), and Vmax (2.008 µmol x mg protein(-1) x min(-1)) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg x L(-1) vs 36 mg x L(-1)) and total enzyme activity increased about 5 folds (1900 IU x L(-1) vs 367 IU x L(-1)) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.

Dataset Information

High-level expression of lipase from Galactomyces geotrichum mafic-0601 by codon optimization in Pichia pastoris and its application in hydrolysis of various oils.

Publications

High-level expression of lipase from <i>Galactomyces geotrichum</i> mafic-0601 by codon optimization in <i>Pichia pastoris</i> and its application in hydrolysis of various oils.

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