Synthesis of Enantiopure ( S)-2-Hydroxyphenylbutanoic Acid Using Novel Hydroxy Acid Dehydrogenase from Enterobacter sp. BK2K

Abstract

Enterobacter sp. BK2K, screened from soil samples, can enantioselectively reduce 2-oxo-4-phenylbutanoic acid into (S)-2-hydroxy-4-phenylbutanoic acid. alpha-Hydroxy acid dehydrogenase (HADH) (specific activity 62.6 U/mg) was purified from the crude extract of Enterobacter sp. BK2K, and its gene was cloned and functionally expressed in E. coli BL21. The optimal pH and temperature for the HADH activity were 6.5 and 30 degrees C, respectively. The purified enzyme catalyzes the reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (S)-2-hydoxy carboxylic acids using NADH as cofactor. For example, the Km and kcat/Km for 2-oxo-4-phenylbutaonoic acid in the presence of 2 mM NADH were 6.8 mM and 350 M-1 min-1, respectively. For practical applications, a NADH recycle system employing the recombinant formate dehydrogenase from E. coli K12 was coupled with HADH in E. coli BL21. Using the recombinant HADH (110 U of 11 U/mg crude cell extract) and formate dehydrogenase (670 U of 67 U/mg crude cell extract) in 10 mL of 500 mM phosphate buffer (pH 6.5), 96 mM of (S)-phenyllactic acid (> 94% ee) and 95 mM of (S)-2-hydroxy-4-phenylbutanoic acid (> 94% ee) were produced in quantitative yields from 100 mM of phenylpyruvate and 2-oxo-4-phenylbutanoic acid.

Glycolate is a Novel Marker of Vitamin B2 Deficiency Involved in Gut Microbe Metabolism in Mice

“…The enzyme GO, also known as (L-2-) hydroxy-acid oxidase, which metabolizes GA to glyoxylate, is widely conserved in intestinal bacteria and mammals [33][34][35][36][37][38]. VB-diet feeding induced changes in the abundances of specific phylum, such as Firmicutes and Bacteroidetes; however, unlike the change in GA concentration, no consistent changes were observed between the 2-week and 4-week treatments (Supplemental Figure S5).…”

Assessing the stereoselectivity of carbonyl reductases toward the reduction of OPBE and docking analysis

“…BK2K produced ( S )‐2‐hydroxy phenylbutanoic acid ((S)‐HPBA) with >94% e.e. . Both ( S )‐HPBE and ( S )‐HPBA are important chiral building blocks in synthesizing of ACE inhibitors .…”

Highly stereoselective biosynthesis of (R)-α-hydroxy carboxylic acids through rationally re-designed mutation of d-lactate dehydrogenase

“…Many routes have been developed for the preparation of chiral α-hydroxy carboxylic acids, such as traditional chemical methods 8 9 , chemoenzymatic routes 5 7 10 , enzymatic kinetic resolution of racemic α-hydroxy carboxylic acids 4 11 12 13 14 , and asymmetric bioreduction of α-keto carboxylic acids 7 15 16 17 . Currently, asymmetric bioreduction of α-keto carboxylic acids by using dehydrogenases is the method of choice because of its excellent stereoselectivity, high theoretical yield up to 100%, and environment-friendly nature.…”

“…Currently, asymmetric bioreduction of α-keto carboxylic acids by using dehydrogenases is the method of choice because of its excellent stereoselectivity, high theoretical yield up to 100%, and environment-friendly nature. However, the used dehydrogenases exhibited low activities, especially toward substrates with large aliphatic or aromatic groups at C-3 16 17 18 . Therefore, there is still a requirement for new dehydrogenases that can reduce a broad spectrum of α-keto carboxylic acids with high efficiency.…”