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Enantioselective oxidation of 2-hydroxy carboxylic acids by glycolate oxidase and catalase coexpressed in methylotrophic Pichia pastoris

Authors

  • Shuvendu Das,

    1. Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, IA 52242
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  • James H. Glenn IV,

    1. Chemical and Biochemical Engineering, College of Engineering, The University of Iowa, Iowa City, IA 52242
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  • Mani Subramanian

    Corresponding author
    1. Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, IA 52242
    2. Chemical and Biochemical Engineering, College of Engineering, The University of Iowa, Iowa City, IA 52242
    • Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, IA 52242
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Abstract

Glycolate oxidase (GO; (S)-2-hydroxyacid oxidase, EC 1.1.3.15) is a flavin mononucleotide (FMN)-dependent enzyme, which catalyzes the oxidation of 2-hydroxy carboxylic acids to the corresponding 2-keto acids. Catalase has been used as cocatalyst to decompose hydrogen peroxide produced in the reaction, thus limiting peroxide-based side reactions and GO deactivation. GO from spinach and catalase T from Saccharomyces cerevisiae previously coexpressed in Pichia pastoris strain NRRL Y-21001, was permeabilized and used for the oxidation of 3-phenyllactic acid, 3-indolelactic acid, 3-chlorolactic acid, 2-hydroxybutanoic acid, and 2-hydroxydecanoic acid to demonstrate high degree of selectivity to the (S)-enantiomers, leaving (R)-isomers intact. The rates of oxidation ranged from 1.3 to 120.0%, relative to the oxidation of lactic acid to pyruvic acid. The best substrates were 3-chlorolactic acid (110%) and 2-hydroxybutanoic acid (120%). Oxidation was carried out with (R)-, (S)-, and (RS)-3-phenyllactic acid, (RS)-lactic acid, and (RS)-2-hydroxybutanoic acid in 500 mL scale to characterize the products and stoichiometry of the reaction. All (RS)- and (S)-2-hydroxy acids produced 2-keto acids at close to the theoretical yield in 1–9 h. (R)-3-Phenyllactic acid was not oxidized over a period of 9 h. Addition of exogenous FMN and catalase were not required for this oxidation using double recombinant Pichia pastoris whole cells. As GO is absolutely specific to (S)-enantiomers, it can be used for resolution of racemic 2-hydroxy acids to (R)-2-hydroxy acids as well as for production of 2-keto acids. This is the first report on the selectivity of a broad range of 2-hydroxy acids by GO. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010

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