Expression And Characterization Of Cyclodextrin Glycosyltransferase And Carbonyl Reductase

Biocatalysts （as both isolated enzyme and whole-cell systems） are increasingly being used to assist in synthetic routes to complex molecules of industrial interest because of the environmental pollution of traditional chemical industry.Cyclodextrin Glycosyltransferase/Glucanotransferase（CGTases EC 2.4.1.19） belongs to the a-amylase family and has the capability of converting starch into Cyclodextrin （CD）. The hollow cylinder radius of the β-CD is just right. So the β-CD has the same safety as the starch and the cyclodextrin. Because of its good functions, its development and application became to be the focuse.In this study,18 strains which can convert starch into β-CD has been screened from the soil.1 strain was selected for further study. According to physiological, biochemical and microbiological analysis the strain of V2-1 belongs to bacillus. The colonial morphology is as follows:the bacterial colony is yellow, opaque, irregular circular, smooth in surface. After 5 days cultured, the diameter of colony reaches 2 cm. Through the microscope, we observed that it was in form of short rod. It appears red after gram straining, so it belongs to Gram-negative bacteria.16S rDNA sequence was blasted with others of NCBI and there was 99% homology with Bacillus sp.The CGTase gene was cloned from wild strain V2-1 and inserted into PET28a. Then the constructed plasmid was translated into Escherichia coli BL21（DE 3） and CGTase was expressed successfully. CGTase was purified after two steps of purification.We draw the growth curve of V2-1 and the strain reaches stationary phase after 25 hours. According to physiological, biochemical and microbiological analysis the strain of V2-1 belongs to bacillus.The CGTase gene was cloned from wild strain V2-1. According information from NCBI, we designed degenerate primer and clone fragment between two primers. The sequence of cloned was blasted with other CGTase genes of NCBI. Then primers that clone purpose gene was designed according the result of blast. The CGTase gene was cloned successfully by PCR. CGTase gene is 2166 bp and encodes 722 aa including a signal peptide which plays a part in secretion of protein. CGTase was expressed successfully in E.coli BL21（DE3） at 25℃ and the induction time is 80 h. The acticity of fermentation broth reaches 1U/mL. The enzyme was purified through two steps:Q-Sepharose column and phenyl-Superose column.Optically active alcohols are key building blocks in the preparation of high-value fine chemicals. A straightforward approach to access these enantiomerically pure alcohols is the reduction of the prochiral ketones. Biocatalysis has emerged as a great addition to traditional chemical processes for production of some chiral alcohols. Carbonyl reductases catalyze prochiral ketone to afford chiral alcohols.Genome of Pichia pastoris GS115 has been sequenced. By genomic data mining we found a putative carbonyl reductase in Pichia pastoris GS115 chromosome 2 （locus_tag: PAS_chr2-1₀542）. It was amplified by PCR from the genomic DNA, and expressed in Escherichia coli BL21 （DE3）. The recombinant protein was purified to homogeneity.The gene of ppcr was 759bp with a his-tage in C terminal.The optimum temperature of PPCR was 37℃ and the optimum pH was 6.0 in sodium phosphate buffer. PPCR was stable below 45 stable below 45hate bufferasnd a putatifor ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s-1, respectively.The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, a-ketoesters, (3-ketoesters and aryl ketones to give the corresponding alcohols with>97% ee with only a few exceptions, showing its application potential in the synthesis of chiral alcohols.