Abstract
A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. The fragment was then amplified by polymerase chain reaction and inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using SalI and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0–8.0 and 41–53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca2+ and Mg2+ had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant (K m) of 1.89 mg/mL and a maximum reaction rate (V max) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris, and the achieved PVADH yield is the highest ever reported.
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Acknowledgments
This project was financially supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (111-2-06), the National High Technology Research and Development Program of China (863 Program, 2011AA100905), the Priority Academic Program Development of Jiangsu Higher Education Institution, the PhD Research Fund of Jiangnan University, and the 973 Program (2012CB720802 and 2012CB720806).
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Jianghua Li equally contributed with the first author.
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Jia, D., Li, J., Liu, L. et al. High-level expression, purification, and enzymatic characterization of truncated poly(vinyl alcohol) dehydrogenase in methylotrophic yeast Pichia pastoris . Appl Microbiol Biotechnol 97, 1113–1120 (2013). https://doi.org/10.1007/s00253-012-3986-3
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DOI: https://doi.org/10.1007/s00253-012-3986-3