The Expression Of Candida Parapsilosis ATCC 7330 Carbonyl Reductase CpCR And The Effect On Enzyme Stability Of Its Molecular Modification

Carbonyl reductase belongs to the class of oxidoreductases,which are widely found in bacteria,fungi,yeasts and animals and plants.With coenzyme NAD(P)⁺or NAD(P)H as electron acceptors and donors,they can specifically catalyze the mutual conversion between ketones(aldehydes)and alcohols.They are used to synthesize important value hydroxyl compounds.In recent years,becauseof the wide substrate spectrum and excellent catalytic propertiesthe,the whole cell of Candida parapsilosis or its carbonyl reductase has been widely used in the development of flavors,functional foods and drugs.Our team obtained a strain of Candida parapsilosis ATCC 7330 in the previous study.it can catalyze the asymmetric reduction of a variety of carbonyl compounds to produce the corresponding alcohols.Because of its excellent substrate specificity and stereoselectivity,carbonyl reductase has huge industrial application prospects.In this study,by constructing recombinant E.coli BL21(DE3)/p ACYCDuet-1-cpcr,the carbonyl reductase CpCR gene(cpcr)from Candida parapsilosis ATCC 7330 was expressed.The recombinant enzyme CpCR was separated and purified by Ni-Agarose affinity chromatography to obtain pure enzyme.Using benzaldehyde as a substrate system,the enzymatic properties of recombinant CpCR under different temperature,p H,metal ions and other conditions were studied.In addition,Discovery Studio 2017 was used to conduct homology modeling and molecular docking of the carbonyl reductase CpCR,to screen out several key amino acids in the flexible region,and rationally design the mutant of the enzyme.Through site-directed mutagenesis,the effects of these sites on enzyme activity and enzyme stability under different temperature,shear force,coenzyme concentration,and single-phase/biphasic conditions were investigated.The corresponding results are as follows:(1)Successfully cloned the carbonyl reductase gene cpcr from Candida parapsilosis ATCC 7330,and realized heterologous expression in E.coli BL21(DE3).The results showed that the full length of the carbonyl reductase gene cpcr was 1 107 bp,containing 368 amino acids with molecular weight around 41 kD and specific enzyme activity of CpCR 20 U/mg.(2)The recombinant CpCR was stable in the temperature range of 4-35? with the relative enzyme activity above 80%.With the increase of temperature,the enzyme activity decreased rapidly,and the T₅₀value was 37?.The suitable p H range was 6.2-7.5,and it has the optimal stability under neutral conditions.The Cu²⁺had a strong inhibition on the recombinant CpCR,and the relative enzyme activity decreased by 30% at a concentration of 1mmol/L,and by 50% at a concentration of 5mmol/L.The affinity of the enzyme to benzaldehyde and n-butyraldehyde was stronger than that to 4-chlor-3-keto-butyrate-ethyl ester(COBE).The catalytic efficiency for benzaldehyde or n-butyraldehyde was about 20 times that for COBE.The recombinant CpCR was an NADPH-dependent carbonyl reductase.(3)By using virtual amino acids for saturation mutation scanning of the soft region amino acid residues of the enzyme and coenzyme NADPH,the mutation energy and mutation sites are calculated and screened.and the amino acid mutation points that affect the stability of the combination of carbonyl reductase CpCR and NADPH are determined.They are Ala98,Ser258,Ser216,Ser307,Gly262.Finaly,we constructed mutant libraries A98N,S307N,G262N,S216N and S258N,and prepared mutant enzymes.(4)The five kinds of mutants about A98N?S307N?G262N?S216N?S258N were constructed and investigated on the changeable characterization of their stabilities tolerant to organic solvents,heat,the shearing force,oxygen,and the NADPH concentration based on carbonyl reductase CpCR from Candida parapsilosis ATCC 7330 through protein rational design and site-directed mutagenesis technology.The results showed that by compared to the enzymatic activity of wt CpCR in potassium dihydrogen phosphate/dibasic potassium phosphate(PB)buffer,the enzymatic activity of the A98N increased by 10%,but the mutant S307N had no enzymatic activity and the activity of other mutants slightly decreased.A98N and G262N in the PB-methyl tert-butyl ether biphasic media increased 1.7 and 1.4-fold higher interfacial stability than that of the wt CpCR,respectively.In the remaining four mutants,their thermal resistance properties are slightly reduced,with T₅₀values ranging from 31-36?.Under the conditions of 100 rpm and 200 rpm,the shear resistance of the four mutants was enhanced.When the speed was 300 rpm,the stability of the G262N was 1.3 times higher than that of wt CpCR.The G262N mutant enzyme has better stability under aerobic conditions,which the t_1/2value was 11.87h and was 1.4 times higher than the wild-type enzyme.When the NADPH concentration was in the range of 0-0.4 mmol/L,the G262N was much more stable.