Modification Of Disproportionation Characteristics And Thermal Stability Of Cyclodextrin Glucosyltransferase

Cyclodextrin glucosyltransferase(CGTase)is an extracellular enzyme belonging to theα-amylase family(glycoside hydrolase 13＿2,GH13＿2)that is produced by various microorganisms such as bacteria,fungi,and archaea,with the main production strains being different types of Bacillus.CGTase can catalyze glycosylation and hydrolysis reactions using starch,maltodextrin,and other substrates.The reactions that it can catalyze include disproportionation,cyclization,and coupling reactions.The CGTase disproportionation reaction is a transglycosylation reaction that can transfer the cut-off portion of the linear oligosaccharide to another receptor.The coupling reaction is the reverse reaction of the cyclization reaction,which can open the ring of cyclodextrin and transfer glycosides to linear malt oligomers.The CGTase enzyme has a wide range of applications,with its cyclization reaction commonly used to convert starch into cyclodextrins.The enzyme’s disproportionation reactions are often used to generate glycoside compounds with improved stability and to modify the properties of various substances such as stevioside,rhamnose,rutin,_L-ascorbic acid,and others.In this study,the researchers synthesized the gene encoding cyclodextrin glucosyltransferase from Bacillus sp.G1 to improve its disproportionation property and thermal stability.Through directed evolution,the researchers obtained a mutant N33K that has enhanced affinity for maltose receptors and can be applied to the system of trehalose synthesis by the dual enzyme method,thereby improving the yield of trehalose.In addition,the researchers obtained a mutant S211G with improved thermal stability through semi-rational modification.The researchers expressed the mutants N33K and S211G in Bacillus subtilis to achieve efficient preparation of the mutants.The primary research findings are outlined below:(1)The CGTase gene from Bacillus sp.G1 was synthesized,and the p ET28a/cgt vector was constructed for heterologous expression in Escherichia coli.Rational design was utilized to identify amino acid residue mutations that could improve maltose-specific binding,and site-directed mutagenesis was performed on CGTase.Mutants were constructed,including N33K,N33R,Y119R,Y122E,L216Q,H255Y,E258Y,E258Q,P394R,P394Q,E566H,and N33K/E258Q,N33K/P394R,E258Q/P394R,and N33K/E258Q/P394R through superposition and combination mutagenesis.These mutants were shown to achieve secretion expression,with optimal mutant N33K having a 2.10-fold increase in the activity of mutant N33K compared to the wild-type dismutase.Additionally,the optimal temperature was determined to be 55℃,and the optimal pH was 6.0.(2)It was discovered that the thermal stability of wild-type and mutant strains did not meet industrial requirements.Mutants N33K/H255W,N33K/H255F,N33K/H255Y,N33K/I212R,N33K/S211G,and N33K/S211E were obtained through directed mutation of amino acids near the calcium binding site.The optimal temperature of mutant N33K/S211G was found to be increased to 60℃,and the half-lives of the mutant N33K/S211G at 50℃,55℃,60℃,and 65℃were 2.4 h,2.0 h,55.4 min,and 38.4 min,respectively.The half-life of N33K/S211G at 60℃was 2.27 times that of the mutant N33K,and there was no significant change in dismutase activity compared to the mutant N33K.(3)Shake flask level fermentation was optimized for the mutant recombinant bacterium E.coli BL21/PET28a-CGTase-N33K/S21G.The optimal fermentation conditions were determined in terms of induction fermentation time,induction temperature,and addition amount of inducer.The optimal growth temperature was 37℃,the optimal induction temperature was 25℃,the optimal addition amount of inducer was0.2 m M,and the optimal induction fermentation time was 8 h.Under these optimal conditions,the recombinant enzyme dismutase activity produced by the recombinant bacteria reached 25.33 U/m L.(4)In the study of trehalose production using maltodextrin as substrate in a multi-enzyme system,the addition of CGTase was found to improve the trehalose conversion rate.A recombinant strain B.subtilis WB800N with mutant N33K/S211G gene was constructed based on TB medium,with a fermentation temperature of 37℃,a fermentation time of 48 hours,and a maximum enzyme activity of 21.0 U/mL.