Enantioselective oxidation of 2-hydroxy carboxylic acids by glycolate oxidase and catalase coexpressed in methylotrophic Pichia pastoris.
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.
Das
Shuvendu
S
Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, IA 52242, USA.
Glenn
James H
JH
Subramanian
Mani
M
D015086:2,6-Dichloroindophenol; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001548:Benzalkonium Compounds; D018533:Biomass; D002374:Catalase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002851:Chromatography, High Pressure Liquid; D005285:Fermentation; D006880:Hydroxy Acids / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010843:Pichia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019289:Pyruvic Acid / Q000378:metabolism; D013237:Stereoisomerism
D001548:Benzalkonium Compounds; D006880:Hydroxy Acids; D006885:Hydroxybutyrates; D019344:Lactic Acid; D019289:Pyruvic Acid; D015086:2,6-Dichloroindophenol; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase; D002374:Catalase; C031570:2-hydroxybutyric acid; D010100:Oxygen