Molecular Modification Of The Thermophilic Cyclodextrin Glucosetransferase

Cyclodextrin (Cyclodextrin, CD) because of the unique structure of a hydrophobic cavity, to the inclusion of hydrophobic guest molecules, thereby changing the solubility of the guest molecules and other physical and chemical properties, so cyclodextrins in food, medicine, environmental protection and many other fields it has a very wide range of applications. Enzymatic synthesis is the main method of industrial production of cyclodextrin, namely the use of cyclodextrin glycosyltransferase cyclization reaction of starch or related matrix synthesis cyclodextrin. In this paper, from the thermophilic Geobacillus sp.CHB1 cyclodextrin glycosyltransferase for the study, to be rehabilitated by different methods, increase their soluble starch substrate generated cyclodextrin conversion rate, improve enzymatic big campestris expression of extracellular soluble expression of the system.The main work is as follows:(1) Improving soluble expression of Geobacillus sp.CHB1 CGTase by error-prone PCRThis study was aimed to enhance the extracellular enzymes activities and soluble expression of CGTase from Geobacillus sp.CHB1 by directed evolution. A library of CGTase mutants was constructed by introducing random mutagenesis using error-prone PCR to screen mutant enzymes with improved the extracellular enzymes activities and soluble expression. After induction, expression and purification, the mutant enzyme was characterized. After screening, two optimum mutants ds-6 and ep-9 with extracellular alpha-cyclization activity are respectively 1.72 times and 2.18 times of the original enzyme. The sequence of ep-9 cgt gene showed that three nucleotides substitution, g2005a, a2037g and t2081g have observed, and two of them caused amino acid changes. The wild-type CGTase or ep-9 genes was ligated with pET-28(a)-OmpA vector, and expressed in E. coli BL21 (DE3). After induced by lactose, the CGTases were purified and characterized. The results showed that the specific β-cyclization activity of the evolved CGTase was 1.31-fold than that of the wild-type CGTase, and the Km decreased from 4.3 to 3.74 g/L. The pH stability of the evolved CGTase was better than wild-type CGTase. Site-directed mutagenesis demonstrated the key to improve the soluble expression level and extracellular enzyme activity was g2005a. Directed evolution by error-prone PCR of Geobacillus sp.CHB1 CGTase gene is effective to improve the the extracellular enzymes activities and soluble expression, in particular mutation occured in the g2005a.(2) Saturated mutation effects on catalytic efficiency and product specificity of starch binding site N623 of cyclodextrin glucanotransferase from Geobacillus sp. CHB1In order to improve catalytic efficiency and product specificity of cyclodextrin glucanotransferase (CGTase) from Geobacillus sp. CHB1, we analyzed amino acid sequences and simulation structure model, found out that the 623th amino acid residues of starch binding sites II probably affected its catalytic efficiency. Using overlapping PCR method, we built 19 kinds of mtuants on the 623th amino acid residues (N623) of starch binding sites II of CGTase. The mutant CGTase genes were respectively linked with pET-28a(+)-ompA and expressed in Escherichia coli BL21(DE3). The recombinant pure enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild-type CGTase, mutant N623T increases catalytic efficiency of CGTase, the total cyclization activity increased 58.6%, α-cyclization activity increased 64%, β-cyclization activity increased 80.5%, while γ-cyclization activity was reduced by 35.3%. In terms of product specificity, compared to wild-type CGTase, the total starch conversion rate by mutant N623T increased from 11.3% to 39.7%, of which α-cyclodextrin, y-cyclodextrin proportion reduced to 32.8% and 7.7%,β-cyclodextrin increased to 59.5%. The possible mechanism was that, compared to wild-type CGTase, mutant N623T Threonine residue in place of asparagine caused conformation of starch binding site II was changed, the conformation optimized substrate acting direction, in favor of the reaction is carried out, thereby improving the catalytic efficiency of the enzyme.(3) Fusion of family 20 carbohydrate-binding module increases the catalytic efficiency of cyclodextrin glycosyltransferase from Geobacillus sp.CHBl by in silico designCyclodextrin glycosyltransferase (CGTase) is an important industrial enzyme for production of cyclodextrins (CDs) from starch by intramolecular transglycosylation. CGTase consists of five domains labeled A to E. To optimize enzymatic properties of the CGTase, we achieved the efficient synthesis of CDs from soluble starch by fusing a family 20 carbohydrate-binding module (CBM) from the Bacillus circulans Strain 251 CGTase to the CGTase from Geobacillus sp.CHB1. The CBMbc251 that has a low binding free energy with maltohexaose,was selected by in silico design. Then the fusion enzyme, CGTAE-CBMbc251, was constructed by fusing the CBMbc251 to the C-terminal region of CGTAE. Under the optimum reaction conditions, the total cyclization activity increased 50% compared to the wild-type CGTase. In particular, the fusion enzyme displayed an even greater enhancement of total a-cyclization activity (40.2%) and y-cyclization activity (181.58%). Compared with the wild-type CGTase, the fusion enzyme showed a marked decrease in Km and a slight alteration in Vmax. In conclusion, it was postulated that the enhancement of soluble starch catalytic efficiency may be due to the changes of substrate binding ability in the critical substrate binding sites between the CBM and starch granule.(4) Effects of molecular chaperone co-expression on heterologous solubility expression of thermophilic CGTaseTo improve the soluble expression level and enzyme activity of CGTase in prokaryotic expression system, the expression conditions were optimized. The cgt gene was cloned from Geobacillus sp.CHB1 and cloned into expression vector pET-28a(+). The optimum induction temperature was selected by enzyme activity assay and SDS-PAGE. The molecular chaperone co-expression system was consturcted and the optimum molecular chaperone vector was screened. The results was described as follows:the cgt gene was amplified and cloned into vector pET-28a(+) successfully, the enzyme activtity and soluble expression level of CGTase was highest when induced at 25 ℃; in the molecular chaperone co-expression system, the five vectors containing different molecular chaperones (pKJE8, pKJE7, pGro7, pTf16 and pG-Tf2) improved and the enzyme activity and soluble expression level of CGTase in varying degrees when co-expression with recombinant plasmid pET-28a(+)-ompA-cgt, and pKJE8 was confimed to contain the optimum molecular chaperones combination and the enzyme activity of CGTase improved 48.6%. When L-Arabinose concentration of 0.5 g/L, molecular chaperone plasmid pKJE8 made extracellular enzyme activity increased by 68.5%. These results can provide a potential value for further studies of CGTase.