| 2,3-Butanediol is a promising bulk chemical with extensive industry applications. Particularly, optically pure 2,3-butanediol with two vicinal stereogenic centers are valuable pharmaceutical intermediates as an excellent building block in asymmetric synthesis. In this study, it was found that Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation and its genome DNA harbored two genes encoding 2,3-butanediol dehydrogenase. Both genes were cloned and over-expressed in Escherichia coli BL21(DE3), and then the gene products were further characterized in terms of enzymatic properties and applications.1. During alcoholic fermentation, R. erythropolis WZ010 produced trace amounts of optically pure (2S,3S)-2,3-butanediol (<0.5mM) as product, while (2R,3R)-2,3-butanediol was also detectable when 2,3-butanedione was supplemented into the fermentation medium. The genes (rebdh-s and rebdh-m) encoding putative 2,3-butanediol dehydrogenase were ligated onto the vector pEASY-E1 and the recombinant vectors were functionally overexpressed in the E. coli BL21(DE3). After purification, the specific activity of recombinant ReBDH-S and ReBDH-M were 9.16 and 11.3 U/mg, respectively. Structure analysis of both enzymes revealed that ReBDH-S with a calculated subunit size of 26.9 kDa was a homodimer and belonged to the family of short-chain dehydrogenases/reductases, while ReBDH-M with a calculated subunit size of 37.2 kDa was a member of the family of medium-chain alcohol dehydrogenases/reductases and a monomer with two Zn2+ per subunit.2. Both ReBDH-S and ReBDH-M were fully characterized. In the oxidation reaction, the optimal values of temperature and pH for ReBDH-S were 25℃ and pH 9.5,whereas those in the reduction reaction were 30℃ and pH 7.0. The optimal substrates for oxidation and reduction were 2-pentanol and 2,3-butanedione, respectively. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6% of the initial activity after 4 h incubation with 30% (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. In comparison with ReBDH-S, ReBDH-M showed a poor tolerance towards organic solvent and metal ions. The optimal pH and temperature for alcohol oxidation were pH 10.0 and 45℃,while those for diacetyl reduction were pH 6.5 and 55℃. The optimal substrates for oxidation and reduction were (2R,3R)-2,3-butanediol and acetoin. Similar to ReBDH-S, the enzyme also displayed absolute stereospecificity in the reduction of diacetyl to (2R,3R)-2,3-butanediol via (R)-acetoin. Kinetic parameters of both enzymes showed lower Km values and higher catalytic efficiency for diacetyl and NADH in comparison to those for 2,3-butanediol and NAD+, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation.3.Two enzymatic approaches were developed for asymmetric synthesis of chiral alcohols,in which the coenzyme regeneration was highly efficient. In the first approach, the ReBDH-S catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversion and enantiomeric excess values. The total turnover number (TTN) of NADH was also as high as 210, indicating efficient coenzyme recycling. In the other appraoch, both ReBDH-S and ReBDH-M was used as a pair of stereoselectivity-complementary biocatalysts aiming for deracemization of 2-pentanol. The results indicated that (S)-2-pentanol was partially transferred to (R)-2-pentanol, which TTN value of NADH was 23 meanwhile the final yeild and e.e. value of (R)-2-pentanol were 76.3 and 62.5%, respectively. |
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