Cloning And Expression Of Dera Gene To Prepare The Chiral Amine Of Atorvastatin Calcium

Atorvastatin calcium can reduce total cholesterol and triglycerides while it belongs to3-hydroxy-3-methyl-glutaryl-coenzyme A （HMG-CoA） reductase inhibitors. Currently, there are two synthesismethods atorvastatin calcium statin chiral intermediates, one is the chemical synthesis, i.e., the racemicmixture of the chiral separation, the other one is biocatalytic synthesis. However, the chemical synthesismethod requires using hazardous or highly toxic substances and expensive compounds, and synthetic stepsare tedious, low utilization rate of raw materials, product impurities, pollutants serious. At present, moreand more people attention biosynthesis by2-deoxy-D-ribose-5-phosphate aldolase （DeoxyribosePhosphate Aldolase, DERA）.Bacillus subtilis UD-20was a endophyte that isolated by our laboratory, which has DERA activity.After cloning the DERA gene by PCR from Bacillus subtilis UD-20genomic DNA, sequence analysisshowed that the open reading frame （ORF） is672bp, which encodes223amino acids, pI of amino acids（isoelectric point） is5.04, a theoretical molecular weight of the protein is about23.23KDa. DERAenzyme amino acid sequence of the amino-line analysis software ClustalW registered in GenBank othersources for multiple comparison, the results showed that the amino acids and Bacillus subtilis subsp.Subtilis str. SC-8（WP₀03227127.1）, Bacillus subtilis subsp. spizizenii str. W23（YP₀03868275.1） andBacillus subtilis subsp. subtilis str.168（CAA57662.1） similarities were more than98%. The Bacillussubtilis UD-20DERA amino acid sequence and template ID （PDB:3ngj.1.A） was submitted to theSWISS-MODEL homology modeling. By observing the model relates to the discovery of the enzymeactive site Cys47, Asp89, Lys152and Lys181completely conserved in other sources DERA enzyme, TIM（α/β）8barrel with a classic structure, belonging TIM-phosphate-superfamily Deoc binding family, whereinthe Schiff base formed in the active β6Lys side chains on the residues, the phosphate-binding site located atthe end of β7.DERA gene was cloned into restriction sites between BamH I and Hind III, and connected with theexpression vector pET32a to build the expression plasmid pET-DERA, and transformed into E. coliexpression system BL-21DE3（pLysS）. This enzyme was highly expressed after the inducer （IPTG）induced. The result showed that the enzyme activity reached0.32U/g. Expression products was analyzed by SDS-PAGE, protein expression pattern displays that the induced pET32a have a specific band about43.4KDa.Affection from the experimental temperature, IPTG concentration and induction time of three singlefactor to be tested, and based on the results to design response surface experiment. Univariate tests showedthat the best combination: fermentation temperature26℃, IPTG0.1mM, induction10h, and in thisfermentation conditions, the enzyme activity is2.4U/g, which is7.5fold of the initial activity. Afterexperimental design and response surface regression analysis, the impact on the production of enzymeinduction time was not significant, the final optimum induction temperature of26.5℃, the optimal IPTGconcentration was0.11mmol/L. Under fermentation conditions, the enzyme activity of up to2.6U/g, is8.2fold of the initial enzyme activity. Theoretical response surface optimization results analysis andregression equations derived from highly fit.Characterization studies have shown that, DERA enzyme optimum reaction temperature and pH were40℃and8.0; enzyme in the range of35-45℃stable enzyme activity after incubated for30min50℃,DERA enzyme activity can still be retained25.71%, but was kept at65℃-70℃basically no activityafter30min; under conditions of pH8.0DERA’s most stable enzyme activity, pH within the range of6.0-10.0incubated for15min25℃activity can keep46.86%or more, within the range of pH7.0-9.0were incubated15min enzyme retains more than53.13%, indicating that the enzyme at pH7.0-9.0environment better stability. Na+, K+, Ca2+, Mg2+and Ba2+promote DERA enzyme action, in which Ca2+activation of the strongest, the enzyme activity increased by approximately29.8%; Mn2+, Co2+and EDTAthe enzyme inhibition, the relative enzyme activity was89.1%,68.6%and87.2%; through Cu2+, Fe2+andZn2+relative activity of enzyme treated DERA retained only44.2%,51.9%and37.5%.Catalyst chloroacetaldehyde and acetaldehyde using whole-cell catalysis, and analyze with GC-MSand GC-FID. Through analysis the main product with the Gas-mass and fitting the structural formula.Results show that the main product for paraldehyde, but the degree of fitting is only83%, the results needsto validate with nuclear magnetic resonance （NMR）.