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Enantioselective oxidation of 2‐hydroxy carboxylic acids by glycolate oxidase and catalase coexpressed in methylotrophic Pichia pastoris
Author(s) -
Das Shuvendu,
Glenn IV James H.,
Subramanian Mani
Publication year - 2009
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.363
Subject(s) - pichia pastoris , chemistry , catalase , hydrogen peroxide , oxidase test , lactic acid , stereochemistry , biochemistry , enzyme , biology , recombinant dna , genetics , bacteria , gene
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