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Characterization of R Aldo-keto Redutase from Lodderomyces Elongisporus and Catalytic Synthesis of (R)-CHBE with Coenzyme Regeneration System

Author: YeZuoZuo
Tutor: WangQiuYan; YinXiaoPu
School: Hangzhou Normal University
Course: Genetics
Keywords: aldo-keto reductase biotransformation ethyl4-chloro-3-oxobutano-ate (COBE) (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) coenzyme regeneration site-directed mutation
CLC: Q814
Type: Master's thesis
Year: 2013
Downloads: 34
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Abstract


Aldo-keto reductases can be used as biological catalyst for asymmetric reduction reaction with highly regional, chemical and stereo selectivity. In this study, a novel aldo-keto reductase was cloned and expressed in E.coli. The recombinant enzyme can be used to synthesize of (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) with high optical purity, which is one of the important chiral intermediates.The new gene of (R)-aldo-keto reductase cloned from the genome of Lodderomyces elongisporus CGMCC2.1589has been overexpressed in E.coli. The research on enzymatic properties of LEKRED has been systematically performmed. As NADPH dependent aldo-keto reductase, LEKRED can be used to catalyze asymmetric reduction of ethyl4-chloro-3-oxobutanoate (COBE) to (R)-CHBE. The specific activity of the enzyme is up to0.1784μmol/min/mg. The Michaelis-Menten constant (Km) and the maximum rate (Vmax) are up to37.01mM and0.04537μmol/min, respectively. The optimal temperature for reaction is35℃and the optimal pH for reaction is6.0. LEKRED has certain catalytic activity to aldehydes such as benzaldehyde, butyraldehyde, valeraldehyde, glutaraldehyde and caprylaldehyde or other ketones such as2,3-butanedione and2,3-pentanedione.The biotransformation for production of CHBE has been performed. For the lower cost in the large-scale industrial production, the aldo-keto reductase and glucose dehydrogenase were coupled in the catalytic reaction. The recombinant strain was designed to coexpress the two enzymes, which constructed the coenzyme regeneration cycle. Aldo-keto reductase LEKRED and glucose dehydrogenase GDH2genes were cloned into the plasmid pETDuet-1, and then the recombinant plasmid was transformed into expression host E.coli BL21(DE3). With22.2mM COBE and97.5mM glucose as substrates and the recombinant E.coli cell as catalyst, the capability of biotransformation was6.03mmol/L/h. The result showed that the enantiomeric excess and conversion yield of (R)-CHBE was99%and95%, respectively. Further with51.8mM COBE and97.5mM glucose as substrates, two strains for expression of LEKRED and GDH2as catalysts, the capability of biotransformation was56.51mmol/L/h. The result showed that the enantiomeric excess and conversion yield of (R)-CHBE was99%and100%, respectively. In two patterns of coenzyme regeneration cycle, the coversion yield of the chiral compound was improved significantly with higher substrate concentration and shorter period, lack of expensive coenzyme NADPH. The site-directed mutation for the key amino acids of aldo-keto reductase was performed to optimize the catalytic properties. After simulating3D structure of aldo-keto reductase LEKRED and analyzing amino acid sequences, the amion acid sites W28, L207and S209was choosen as the targets. Primers of site-directed mutation were designed according to codon preferences of E. coli. With the mutants W28A and S209G as catalysts, biotransformations were carried out. The results showed conversion yields of (R)-CHBE increased by12.7%and27.1%respectively, and the enantiomeric excess was99%consistent with LEKRED. With other mutations as catalysts, the results of the biotransformation showed that either conversion yields or enantiomeric excess declined.To sum up, a novel aldo-keto reductase LEKRED was cloned and overexpressed in E. coli. LEKRED can be added to the reductase library as a promising catalyst for multi-purpose bio-reduction systems.

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