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首页> 外文期刊>Biotechnology Progress >Enantioselective Oxidation of 2-Hydroxy Carboxylic Acids by Glycolate Oxidase and Catalase Coexpressed in Methylotrophic Pichia pastoris
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Enantioselective Oxidation of 2-Hydroxy Carboxylic Acids by Glycolate Oxidase and Catalase Coexpressed in Methylotrophic Pichia pastoris

机译：在甲基营养巴斯德毕赤酵母中共表达的乙醇酸氧化酶和过氧化氢酶对2-羟基羧酸的对映选择性氧化

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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-hydroxybuta-noic 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-Phenyl-lactic 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.
机译：乙醇酸氧化酶（GO;（S）-2-羟基酸氧化酶，EC 1.1.3.15）是一种黄素单核苷酸（FMN）依赖性酶，可催化2-羟基羧酸氧化为相应的2-酮酸。过氧化氢酶已被用作分解反应中产生的过氧化氢的助催化剂，从而限制了基于过氧化物的副反应和GO失活。菠菜中的GO和先前在毕赤酵母菌株NRRL Y-21001中共表达的啤酒酵母中的过氧化氢酶T被透化并用于氧化3-苯基乳酸，3-吲哚乳酸，3-氯乳酸，2-羟基丁酸，和2-羟基癸酸显示出对（S）-对映异构体的高度选择性，而（R）-异构体完好无损。相对于乳酸到丙酮酸的氧化，氧化速率为1.3-120.0％。最好的底物是3-氯乳酸（110％）和2-羟基丁酸（120％）。用500 mL规模的（R）-，（S）-和（RS）-3-苯基乳酸，（RS）-乳酸和（RS）-2-羟基丁酸进行氧化以表征产物和反应的化学计量。所有（RS）-和（S）-2-羟基酸在1-9小时内产生的2-酮酸的产率接近理论收率。 （R）-3-苯基-乳酸在9小时内未被氧化。使用双重重组毕赤酵母全细胞进行该氧化不需要添加外源FMN和过氧化氢酶。由于GO对（S）-对映异构体是绝对特异的，因此它可用于将外消旋2-羟基酸拆分为（R）-2-羟基酸，以及用于生产2-酮酸。这是关于GO对多种2-羟基酸的选择性的首次报道。

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