Molecular Modification Of Bacillus Stearothermophilus NO2 Cyclodextrin Glucosyltransferase And Preparation Of α-cyclodextrin

Cyclodextrins（CDs）are cyclic oligosaccharides consisting of glucopyranose units linked byα-1,4 glycosidic bonds.The common cyclodextrins areα-,β-andγ-cyclodextrins.Cyclodextrins have a hydrophilic external surface and a hydrophobic internal cavity and can form inclusion complexes with many guest molecules,thus changing their physical or chemical properties.Therefore,it can be used as a drug carrier and solubiliser in food and pharmaceutical applications.α-cyclodextrin has a small internal cavity and high resistance to enzymatic degradation and can be used as a soluble dietary fibre in the food industry,but its application is greatly limited by the low yield and high price.Cyclodextrins are mainly made from starch or starch derivatives,which are catalyzed by cyclodextrin glucosyltransferase（CGTase）.In addition,CGTase can also catalyze hydrolysis,disproportionation,and coupling reactions.The ability of CGTase to produce cyclodextrins is mainly related to the ratio of cyclisation/hydrolysis and the product specificity.In this study,Bacillus stearothermophilus NO2 CGTase with high expression level and good stability,was used for molecular modification through site-directed mutagenesis to improve the ratio of cyclisation/hydrolysis and the product specificity.Finally,the conversion rate of cyclodextrin and the ratio ofα-cyclodextrin product were increased.The main results are as follows:（1）Molecular modification based on E142 to improve the product specificity.The E142amino acid residue at the-7 subsite was selected as the mutation site and the mutant E142P was constructed by sequence alignment and structural analysis.Theβ-cyclodextrin-forming activity of mutant E142P decreased from 337.79 U·mg^-1 of wild type to 183.16 U·mg^-1,and the ratio ofα-cyclodextrin-forming activity toβ-cyclodextrin-forming activity increased from 1.14 of wild type to 1.98;During the preparation ofα-cyclodextrin from soluble starch,the proportion ofα-cyclodextrin increased from 38.05%of the wild type to 47.70%.According to the analysis of structure,mutant E142P disrupted the hydrogen bonding interaction between the E142 amino acid residue and the sugar unit at the-7 subsite,thereby improving the specificity of theα-cyclodextrin and facilitating its preparation.（2）Molecular modification based on N353 and L277 to improve the reaction specificity of CGTase.Based on multiple sequence alignment and the analysis of hydrophobic cluster near the-1/+1 subsite,the N353 amino acid residue at the+2 subsite was selected as the mutation site and mutant N353A was constructed.Mutants L277A,L277I,L277M and L277F at the+1subsite,which were designed and constructed in our laboratory earlier,were also selected.The results showed that mutants N353A,L277M and L277F could increase the ratio of cyclization/hydrolysis,which were 3.25,1.91 and 2.62 times higher than those of the wild type,and were conducive to reducing the amount of maltooligosaccharide during the preparation ofα-cyclodextrin,thereby reducing the degradation ofα-cyclodextrin by the coupling reaction.They were further combined with the mutant E142P,and then the combined mutant E142P/N353A was obtained.Finally,the proportion ofα-cyclodextrin increased by 15.62%compared with the wild type.It was shown that mutant E142P/N353A had the effect of increasing the ratio of cyclization/hydrolysis and the specificity ofα-cyclodextrin,indicating that mutant E142P and N353A had a synergistic effect.Structural analysis showed that the mutant N353A affected the charge balance near the catalytic amino acid residue E253,resulting in a larger distance between E253 and the nucleophilic amino acid residue D225,thereby increasing the ratio of cyclization/hydrolysis.（3）Recombinant expression of CGTase in Bacillus subtilis and the optimization of recombinant enzyme to prepareα-cyclodextrin.p HY300PLK was used as an expression vector to construct the recombinant plasmid p HY300PLK-P_{amy E}-SP1-E142P/N353A,which containing the promoter P_{amy E} and the signal peptide SP1,and finally it was expressed in Bacillus subtilis.The activity of recombinant strain after shake flask fermentation and 3-L bioreactor fermentation were 16.60 U·m L^-1and 110.03 U·m L^-1,respectively.The reaction of mutant E142P/N353A to prepareα-cyclodextrin was optimized by the substrate of 15%(w·v^-1)potato starch.The optimal reaction conditions were as follows:the reaction temperature was50℃,the reaction p H was 5.5,isoamylase with 48 U·g^-1 substrate,CGTase with 5 U·g^-1substrate,5%(v·v^-1)of decyl alcohol.Under these conditions,the conversion rate of cyclodextrin was 61.08%,and the proportion ofα-cyclodextrin was 80.05%.