| Cyclodextrin glycosyltransferases?EC 2.4.1.19,CGTase?can catalyze the conversion of starch or starch derivates into cyclodextrins.Usual cyclodextrins are?-cyclodextrin,?-cyclodextrin and?-cyclodextrin,containing 6,7 or 8 glucose units.Cyclodextrins can improve the properties of guest molecules by forming enclosed complex.Generally,single-ingredient cyclodextrin is preferable in formation of complex.Since the natural wild-type CGTases produce mixtures of?-,?-and?-cyclodextrin,time-consuming and expensive procedure is needed to purify the desired product.This added cost would restrict the large-scale preparation and application of clodextrins.In the present research,molecular engineering was conducted in substrate biding subsites to address the issue that CGTases have low specificity in cyclodextrin preparation.In addition,the preparation process of?-cyclodextrin and?-cyclodextrin and the fermentation process of?-CGTase were optimized.1.The effect of mutation in subsite-7 on cyclodextrin specificity was investigated.The subsite-7 of?-CGTase from Peanibacillus macerans was identified by analyzing and studying the sequences and structures of CGTases from various sources.Single point mutation and combined mutation were designed and conducted.Among these mutations,the cyclization?-cyclodextrin forming activity of D147A,R146P,D147,R146A/D147P,R146P/D147A and R146P/D147P dropped significantly.The ratio of the principal product?-cyclodextrin increased from 63.2%to 68.8%,71.4%,73.0%,75.1%,76.1%and 76.8%,when incubated with soluble starch.In addition,a proposal that enhancing?-cyclodextrin specificity by mutagenesis masking the subsite-7 of CGTases was proposed,which was supported by the mutation of?/?-CGTase from Bacillus stearothermophilus NO2.In the case of?/?-CGTase mutants E142P and T143P,the ratio of the product?-cyclodextrin increased from 39.5%to 53.0 and 48.7%,2.The effect of mutation in subsite-3 on cyclodextrin specificity was investigated.Mutations of residue 47and 94 in subsite-3?based on B.circulans 251?-CGTase?were designed and conducted by analyzing and studying the sequences and structures of CGTases from various sources.In residue 47,the cyclization?-cyclodextrin forming activity of P.macerans?-CGTase mutant K47T decreased significantly,the ratio of?-cyclodextrin increased from 12.9%to 17.0%;the cyclization?-cyclodextrin forming activity of B.circulans 251?-CGTase mutant R47T decreased significantly,the ratio of?-cyclodextrin increased from 16.2%to 19.3%,when incubated with soluble starch.Results indicates that when K or R with long side chain were mutated to T with short side chain,the principal cyclization?-cyclodextrin forming activity of K47T and the principal cyclization?-cyclodextrin forming activity of R47T decreased significantly,while the minor cyclization?-cyclodextrin forming activity of both mutants increased prominently.In residue 94,the cyclization?-,?-and?-cyclodextrin forming activity of B.clarkia?-CGTase mutant F91N and F91L increased significantly.The ratio of?-cyclodextrin decreased from 77.1%to 61.4%and 62.9%,when incubated with soluble starch.In the other side,the cyclization activity of mutants N94W of?-and?-CGTase decreased significantly.The ratio of?-cyclodextrin increased from 12.9%and 16.2 to 23.4%and 25.7%respectively,when incubated with soluble starch.These results indicated that when the residue in 94 was N or L with small side chain,it is favorable for cyclization.Inversely,when the residue in 94 was F or W with large side chain,it is not favorable for cyclization,but favorable for?-cyclodextrin specificity.3.The effect of mutation in central subsite and subsite-7 as well as the combination mutations with subsite-3 on cyclodextrin specificity was investigated.Mutations were designed and conducted by analyzing and studying the sequences and structures of CGTases from various sources.In the central subsite,the cyclization activity of mutants Y195W for P.macerans?-CGTase and B.circulans 251?-CGTase decreased significantly.The ratio of?-cyclodextrin increased from 12.9%and 16.2%to 21.4%and 33.7%,when incubated with soluble starch.In addition,the ratio of the?-cyclodextrin increased from 77.6%to 94.6%for the?-CGTase mutant Y186W,which made it possible to produce single-ingredient cyclodextrins without organic reagent.In the case of mutation combination subsite-7,-3 and central subsite,the ratio of?-cyclodextrin increased from 12.9%and 16.2%to 40.3%and 45.1%by mutant N94W/??145-151?D/Y195W of?-and?-CGTase.4.The effect of organic reagent and specificity of CGTases in the process of cyclodextrin preparation was investigated.The CGTases and mutants were employed to produced cyclodextrins through optimizing addition of alcohols and annular reagents.In the case of?-cyclodextrin preparation,when n-octanol was empolyed,the conversion rates were 56.6%and53.5%,and the ratios of?-cyclodextrin were 73.8%and 89.7%for P.macerans?-CGTase and mutant R146R/D147A.The result indicated when n-octanol with lower boiling point was adopted,the mutant R146R/D147A had an advantage over the wild?-CGTase.In the case of?-cyclodextrin preparation,when cyclododecanone was empolyed,the conversion rates were74.4%and 72.8%and the ratios of?-cyclodextrin were 88.3%and 96.6%for B.clarkia?-CGTase and mutant Y186W.The result indicated that mutant Y186W had an advantage over the wild?-CGTase.5.The effect of chemical chaperones on CGTase expression by E.coli.was investigated.The expression of?-CGTase by E.coli was enhanced through chemical chaperone addition and fermentation optimization.In shake flask cultivation,when 7.5 mM?-cyclodextrin was added,the total?-CGTase activity was 5.51 U·mL-1which was 1.95 fold of that in control.In 3-L fermenter cultivation,when 7.5 mM?-cyclodextrin was added and induced by a lactose feed rate of 0.3 g·L-1·h-1,the the total?-CGTase activity was 50.29 U·mL-1,which was 1.71 fold of that in control. |
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