KMID : 1094720160210060695
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Biotechnology and Bioprocess Engineering 2016 Volume.21 No. 6 p.695 ~ p.703
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Improvement of 1,3-propanediol oxidoreductase (DhaT) stability against 3-hydroxypropionaldehyde by substitution of cysteine residues
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Li Zhengbin
Ro Soo-Moon Sekar Balaji Sundara Seol Eun-Hee Lama Suman Lee Sun-Gu Wang Guangyi Park Sung-Hoon
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Abstract
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1,3-propanediol oxidoreductase (DhaT), which catalyzes the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) with the oxidation of NADH to NAD+, is a key enzyme in the production of 1,3-PD from glycerol. DhaT is known to be severely inactivated by its physiological substrate, 3-HPA, due to the reaction of 3-HPA with the thiol group of the cysteine residues. In this study, using site-directed mutagenesis, four cysteine residues in Klebsiella pneumoniae J2B DhaT were substituted to alanine, the amino acid commonly found in cysteine¡¯s positions in other DhaT, individually and in combination. Among the total of 15 mutants developed, a double mutant (C28A_C107A) and a triple mutant (C28A_C93A_C107A) exhibited approximately 50 and 16% higher activity than the wild-type counterpart, respectively, after 1 h incubation with 10 mM 3-HPA. According to detailed kinetic studies, the double mutant had slightly better kinetic properties (V max , K cat , and K m for both 3-HPA and NADH) than wild-type DhaT. This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal, albeit to a limited extent.
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KEYWORD
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1,3-propanediol oxidoreductase, stability, 3-hydroxypropionaldehyde, cysteine, site-directed mutagenesis
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