| Cyclodextrin glycosyltransferases（EC 2.4.1.19,CGTase）can catalyze the conversion of starch or starch derivates into cyclodextrins.Usual cyclodextrins areα-cyclodextrin,β-cyclodextrin andγ-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 ofα-,β-andγ-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 ofα-cyclodextrin andγ-cyclodextrin and the fermentation process ofγ-CGTase were optimized.1.The effect of mutation in subsite-7 on cyclodextrin specificity was investigated.The subsite-7 ofα-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 cyclizationβ-cyclodextrin forming activity of D147A,R146P,D147,R146A/D147P,R146P/D147A and R146P/D147P dropped significantly.The ratio of the principal productα-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 enhancingα-cyclodextrin specificity by mutagenesis masking the subsite-7 of CGTases was proposed,which was supported by the mutation ofα/β-CGTase from Bacillus stearothermophilus NO2.In the case ofα/β-CGTase mutants E142P and T143P,the ratio of the productα-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 251β-CGTase）were designed and conducted by analyzing and studying the sequences and structures of CGTases from various sources.In residue 47,the cyclizationα-cyclodextrin forming activity of P.maceransα-CGTase mutant K47T decreased significantly,the ratio ofγ-cyclodextrin increased from 12.9%to 17.0%;the cyclizationβ-cyclodextrin forming activity of B.circulans 251β-CGTase mutant R47T decreased significantly,the ratio ofγ-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 cyclizationα-cyclodextrin forming activity of K47T and the principal cyclizationβ-cyclodextrin forming activity of R47T decreased significantly,while the minor cyclizationγ-cyclodextrin forming activity of both mutants increased prominently.In residue 94,the cyclizationα-,β-andγ-cyclodextrin forming activity of B.clarkiaγ-CGTase mutant F91N and F91L increased significantly.The ratio ofγ-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 ofα-andβ-CGTase decreased significantly.The ratio ofγ-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 forγ-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.maceransα-CGTase and B.circulans 251β-CGTase decreased significantly.The ratio ofγ-cyclodextrin increased from 12.9%and 16.2%to 21.4%and 33.7%,when incubated with soluble starch.In addition,the ratio of theγ-cyclodextrin increased from 77.6%to 94.6%for theγ-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 ofγ-cyclodextrin increased from 12.9%and 16.2%to 40.3%and 45.1%by mutant N94W/Δ（145-151）D/Y195W ofα-andβ-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 ofα-cyclodextrin preparation,when n-octanol was empolyed,the conversion rates were 56.6%and53.5%,and the ratios ofα-cyclodextrin were 73.8%and 89.7%for P.maceransα-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 wildα-CGTase.In the case ofγ-cyclodextrin preparation,when cyclododecanone was empolyed,the conversion rates were74.4%and 72.8%and the ratios ofγ-cyclodextrin were 88.3%and 96.6%for B.clarkiaγ-CGTase and mutant Y186W.The result indicated that mutant Y186W had an advantage over the wildγ-CGTase.5.The effect of chemical chaperones on CGTase expression by E.coli.was investigated.The expression ofγ-CGTase by E.coli was enhanced through chemical chaperone addition and fermentation optimization.In shake flask cultivation,when 7.5 mMβ-cyclodextrin was added,the totalγ-CGTase activity was 5.51 U·mL-1which was 1.95 fold of that in control.In 3-L fermenter cultivation,when 7.5 mMβ-cyclodextrin was added and induced by a lactose feed rate of 0.3 g·L-1·h-1,the the totalγ-CGTase activity was 50.29 U·mL-1,which was 1.71 fold of that in control. |
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