Purification And Gene Sequence Identification Of Carbonyl Reductase From Candida Krusei SW 2026

Stereoseletive Oxidoreductase has been widely used as an important biocatalyst for asymmetric synthesis of chiral compounds, due to its particular advantages such as high enantioselectivity, moderate reaction conditions and environmental friendly. More than 200 microorganisms have been reported for the production of enantiopure alcohols by asymmetric reduction of carbonyl group. These chiral alcohols are key intermediates of high value-added chiral synthons for the production of pharmaceuticals, agrochemicals and foods. The optically pure chiral pharmaceuticals are becoming a new profit growth point and are critical for the commercialization of new durgs nowadays because of the intensive study on distinct pharmacological effects of optical isomers and the strict limitations on the application and approval of racemic drugs. Due to its unique advantages, biocatalytic synthesis is an important approach for the production of optically pure drugs and their intermediates. Optically active ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] is used as a key chiral building block in the synthesis of a series of angiotensin-converting enzyme inhibitors.In our previous study, (R)-HPBE was produced by Candida krusei SW 2026 cells from OPBE. To further study the carbonyl reductase responsible for the reaction, the purification, characterization, and gene sequence identification of the key carbonyl enzyme is carried out.An NADPH-dependent carbonyl reductase was purified from Candida krusei SW 2026, through ammonium sulfate fractionation (40-90%), HiPrep DEAE FF anion exchange chromatography, HiTrap Blue HP affinity chromatography and SuperdexTM 75 gel filtration from cell-free extract. The relative molecular mass of the enzyme was estimated to be around 46 kDa by SDS-polyacrylamide gel electrophoresis and 45.5 kDa by gel filtration.It was observed the purified enzyme exhibited maximum activity at pH 6.0 and 30℃, and retained over 80% of its activity over an acidic pH range of 4.5-7.0. The maximum reaction rate (Vmax) and apparent Michaelis-Menten constant (Km) for OPBE and NADPH were 18.7μmol/min·mg protein and 0.319 mmol/L,14.9μmol/min·mg protein and 0.306 mmol/L, respectively. The carbonyl reductase showed high substrate specificity towards ketone esters substrates, especially phenyl esters. It was observed that Mn2+,β-mercaptoethanol and dithiothreitol exhibited different degree positive effect on the activity of this carbonyl reductase while heavy metal ions, thiol group binding metal ions, inhibitory reagents, reducing reagents, organic solvents and detergents showed inhibitory effect. For the asymmetric reduction of OPBE catalyzed by the purified enzyme, (R)-HPBE was produced with nearly 100%e.e. and approximately 84.0% yield after 4 h.The LC-MS-MS analysis of the purified carbonyl reductase shows that the amino acid sequences of one peptide shares some identity with NADP-dependent isocitrate dehydrogenase, and bears approximate 2.4% coverage. The partial gene sequence was cloned, using genomic DNA of Candida krusei SW 2026 as the template and primers designed based on the LC-MS-MS results. The PCR sequence was ligated to pMD18-T simple vector and was sequenced. The PCR fragment consists of 677 bp, and encodes 225 amino acids from the second base pair. And the partial sequence was verified through Blast in NCBI.