Purification And Characterization Of The Key Carbonyl Reductase From Microbacterium Sp.

Chiral alcohols are important intermediates to synthesize single enantiomeric purepharmaceutical. Asymmetric reduction of pro-chiral ketones to produce chiral alcohols catalyzedby whole cell or oxidoreductase has become a research hot field in asymmetric synthesis. Todiscover the mechanism for whole-cell catalysis and to find a potential appliable enzyme forpractical application, this work focus on isolating the key carbonyl reductase fromMicrobacterium sp., and investigating the enzymatic properties and its bioinformatics. Themain contents are as follows:At First, a purifying technology for isolating the key carbonyl reductase from theMicrobacterium sp. was developed for further study of enzymatic catalysis mechanism at proteinlevel. The cells were disintegrated with ultrasonic. Then the reductase was enriched and purifiedby (NH4)2SO4fractional precipitation, anion exchange chromatography and gel filtrationchromatography. The target enzyme was purified with a partially81fold,16.23U/mg specialactivity and29.62%yield. It is a dimer with an apparent molecular weight of61.9kDa.The enzymatic properties of the purified reductase were also evaluated. The carbonylreductase could catalyze the reduction of ketones with NADH as coenzyme. The optimaltemperature for enzymatic reaction was40°C and the optimal pH for enzymatic reduction was7.0. The enzyme was with good thermal stability and was sensitive of pH change. It is onlystable between pH6.0and8.0. Cu2+had strong inhibitory effect on enzyme activity, while Fe2+could remarkably improve the enzyme activity.Four carbonyl reductase sequences (carbonyl reductase from Candida parapsilosis, TrEMBLID: B2KJ46; carbonyl reductase from Candida magnoliae, TrEMBL ID: Q9C4B3; carbonylreductase from Pichia stipitis, TrEMBL ID: A3GF07; carbonyl reductase from Pichia stipitis,TrEMBL ID: A3GF05) were obtained from protein sequence database. Multiple sequencealignment, active sites and secondary structure prediction were also evaluated. The four targetsequences have good homology. Three of the target sequences contain a typical conserved regionPS00061and a tyrosine active site residue in short-chain dehydrogenases/reductases familysignature. The main secondary structure of the four target sequences are random coil-based,followed by the β-sheet and α-helix and no β-turn.The whole work provides the foundation for discovering the mechanism of the actioncatalyzed by enzyme. There is important realistic meaning and theoretical value through thecarbonyl reductase enztmatic and informatics properties research.