Effects Of Maltose Binding Site 2 Region On Product Inhibition Of Cyclodextrin Glycosyltransferase

Cyclodextrin has been widely used in various fields because of its ability to encapsulate many hydrophobic compounds for improving their performance.In industry,cyclodextrin is usually prepared by the reaction of cyclodextrin glucosyltransferase(CGTase)on starch.However,with the accumulation of product,obvious product inhibition will occur,which is an important factor causing low production of cyclodextrin.Therefore,how to reduce product inhibition in the production of cyclodextrin has become an urgent problem to solve.In this study,two CGTases with different product inhibition types(?-CGTase from Paenibacillus macerans JFB05-01,?-CGTase from Bacillus circulans STB01)were chosen as the research objects.The relationship between enzyme structure and product inhibition was analyzed by means of bioinformatics.Single mutation,double mutation and fragment-replaced mutation were conducted for studying the effects of maltose binding site 2(MBS2)region on product inhibition of CGTases,and their related mechanisms were analyzed.Meanwhile,ideal mutants with decreased product inhibition and industrial value were expected to obtain.The main results are listed as follows:(1)The reasons for the product inhibition of CGTases were analyzed from the primary structure by means of bioinformatics.The regions and their amino acid residues related to the product inhibition of CGTases were confirmed.According to the difference of product inhibition between ?-CGTase and ?-CGTase and the particularity of MBS2,MBS2 was selected as the research target.The structures of 600 or 603 sites in this region and the whole region were analyzed,which provided guidance for the construction of subsequent mutants.(2)Position 600 in MBS2 region was chosen for research.Four single ?-CGTase mutants(Y600L,Y600 E,Y600I and Y600R)and four single ?-CGTase mutants(L600Y,L600 E,L600I and L600R)were designed and compared.The results showed that the ?-CGTase mutation at 600 site changed neither its product inhibition type(still competitive inhibition)nor its product inhibition strength;the ?-CGTase mutation at 600 site also did not change the product inhibition type(still mixed-type product inhibition),but the strength of product inhibition was significantly affected.Therefore,it can be inferred that the competitive inhibition of ?-CGTase caused by cyclodextrin is not related to the 600 site of MBS2 in the inactive region,while the mixed-type product inhibition of ?-CGTase,especially the noncompetitive inhibition,is related to the 600 site.The mechanism analysis showed that Leu600 mutation of the ?-CGTase could weaken the interaction with cyclodextrin and thus reduce product inhibition.(3)Position 603 in MBS2 region was chosen for research.Six single ?-CGTase mutants(N603E,N603 D,N603K,N603 H,N603R and N603I)and six single ?-CGTase mutants(N603E,N603 D,N603K,N603 H,N603R and N603I)were designed and compared.The results showed that single mutation at the 603 site had similar effects on both CGTases to the 600 site,but the competitive inhibition of N603 E and N603 D changed significantly.Therefore,it can be inferred that the 603 site in MBS2 region is also closely related to the noncompetitive inhibition of ?-CGTase,and the changes of competitive inhibition strength of N603 E and N603 D of two CGTases may be related to another competitive region(MBS1).The mechanism analysis showed the asparagine of the wild-type ?-CGTase at the 603 site could bind to cyclodextrin via water-mediated hydrogen bond.When it was replaced by negative charged Glu,Asp or hydrophobic Ile,the hydrogen bond could be destroyed and the product inhibition reduced.However,when it was replaced by Lys,His or Arg,the presence of side chain amino groups still led to the formation of hydrogen bonds,even stronger,and the product inhibition would be aggravated.(4)Based on the results above,two ?-CGTase double mutants(N603D/L600 R and N603D/L600E)and fragment-replaced mutants(MBS2-Bc DF9R)were designed for further study.The results showed that N603D/L600 R,N603D/L600 E and MBS2-Bc DF9 R had better effects on the decrease of product inhibition.When 5% maltodextrin(DE=4)was used as substrate for cyclodextrin production,the main products ?-cyclodextrins of N603D/L600 R and N603D/L600 E were 43.7% and 27.9% higher than that of the wild-type,respectively.Therefore,the two ?-CGTase mutants N603D/L600 R and N603D/L600 E have better application value.