Molecular Engineering, Product Specificity And Enzymatic Properties Of Cyclodextrin Glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) is a multifunctional enzyme which can not only catalyze intramolecular transglycosylation, namely cyclization reaction, but also participate in the coupling reaction, hydrolysis reaction and disproportionation reactions. The significant industrial application of CGTase is to catalyse starch or related matrix to cyclodextrins by cyclization reaction, the most common product are α-、 β-、γ-cyclodextrins. Due to its hydrophilic outer surface and nonpolar hole, CDs can bond a guest molecules, resulting in the change of its physical or chemical properties, such as increasing solubility or stability. As molecular capsule, cyclodextrin is widely used in food, cosmetic, medicine and environmental protection. As a key enzyme in the production of cyclodextrin, CGTase has attracted more and more attention.At present, the catalytic products of existing CGTase are a mixture of α-、 β-、 y-cyclodextrins, The poor product specificity has adverse effects on the yield and the subsequent purification and separation of the single cyclodextrin. Therefore, it is one of the important research directions to improve the product specificity of CGTase by using the genetic engineering method.In view of the above questions, this paper was based on the recombinant plasmid pET22b/cgt which constructed easier and the key amino acid was changed by saturation mutagenesis method to analyses the internal relationship between the amino acids and CGTase product specificity. The main results are as follows:Through multiple sequence alignment and related literature, residue 43 plays an important role in product specificity of CGTase. We mutant 43 histidine of CGTase derived from Bacillus cereus Using recombinant plasmid pET22b/cgt as template by whole plasmid mutation method. PCR products were then transformed into Escherichia coli DMT after enzymatic hydrolysis. Positive clone strains were screened in the presence of 100 μg/mL in LB plate, and the mutant enzyme H43P, H43Q, H43L, H43I, H43F, H43K, H43A, H43C, h43y, H43E, H43N, H43R, H43S, H43T, H43G, H43M, H43W were constructed successfully.Using the crystal structure of 1CXK as a template, the CGTase spatial structure was constructed by homology modeling. Afterwards, to get the reliable three-dimensional models of proteins, the structure of CGTase was optimized using molecular dynamics software. Through the molecular docking with the malt nine sugar inhibitor, the related situation of the substrate binding groove was analyzed, the -3 subunit and 43 residue were analyzed.By site-directed mutagenesis,43 histidine turned into a phenylalanine, tryptophan and tyrosine, CGTase enzyme activity increased from 6570U/mL to 6667U/mL, 7814U/mL,15609U/mL, suggesting aromatic amino acids are conducive to enhance the ability of CGTase on corn starch degradation; Mostly mutation enzyme result in the proportion of β-CD reduced in the product, while y-CD ratio increased. With short residue side chain amino acid instead of the original location of histidine, such as mutation of H43P, H43A, H43G, y-CD percentage was 30.2%,28.3%,28.1% in the CGTase catalyzed products, while y-CD accounted for 20% in the wildtype enzyme catalyzed products; when histidine was insteaded by strongly hydrophobic amino acids, such as leucine and isoleucine, y-CD ratio were 28.4% and 28.0% in the product when catalyzed by H43I,H43L mutant enzyme, with regard to mutant enzyme H43T, the percentage of y-CD was 29.2% in the catalytic products.H43P H43I H43T mutant enzyme, which owns better γ-CD product specificity, the enzymatic properties were analysis. The result showed the mutant enzymes have the similar properties with the initial enzyme. The optimization temperature and pH of the mutant enzymes were 60℃ and pH9.0. they were quite stable in wide pH range of 6.0 to 9.0. H43P,H43I enzyme have better thermostability, when comed to 80℃, there were still 60% and 62% residual activity; with respect to enzyme kinetic properties, the Km value of H43P mutant enzyme became smaller, while the Km value of H43I and H43T mutant enzymes became larger, indicating that the affinity of H43P mutant and soluble starch grew stronger, result in the catalytic efficiency of H43P was 14.8% higher than that of the original enzyme.