Characterization Of A Novel 4-?-glucanotransferase And Directed Evolution Of Maltose-producing Amylase Come From Corallococcus Sp.EGB

As one of the most abundant carbohydrates in the natural world,starch is mainly composed of 20%amylose with linear polymer of ?-1,4 linked glucose residues,and 80%amylopectin,a branched polymer of ?-1,4 linked glucose residues with about 5%?-1,6-glycosidic bonds.Starch and its derivatives are important agricultural raw materials that widely used in industrial products.Considering the potential values of diverse starch products,including dextrin and maltooligosaccharides,numbers of physicochemical and enzymatic methods have been applied in the conversion,utilization and modification of starch.4-?-glucanotransferases from GH families 13,57,and 77 catalyze the transfer from one ?-1,4-glucan to another ?-1,4-glucan or glucose with a free 4-hydroxyl group,which have been received considerable attention recently due to the development of a number of new commercial products,such as cyclodextrin,maltodextrin and functional oligosaccharides.We previously isolated Corallococcus sp.strain EGB,which abundantly produced extracellular amylolytic enzymes and also accumulated large amounts of maltooligosaccharides in the starch medium.Therefore,we were interested in understanding the functions of excretive enzymes in starch conversion.In this study,a gene encoding a putative 4-?-glucanotransferase(ccGtase)from strain EGB was cloned and expressed in Escherichia coli BL21(DE3).CcGtase exhibited low sequence identity(9-35%)compared with the reported GH774-?-glucanotransferases,whereas,three catalytic residues Asp305,Glu353 and Asp404,as well as 250s loop and 460s loop were traditionally conserved.Product analysis showed that CcGtase converted maltooligosaccharides and amylose into linear ?-1,4-maltooligosaccharides with a low degree of polymerization(DP<12),and no cycloamylose was produced during the intermolecular transglycosylation.Otherwise,the transfer reaction was only observed in the presence of glucose with starch as the substrate,and delayed production of maltose was also observed.In the transfer process,no apparent hydrolytic activity was detected.Based on the low sequence identity and distinct characteristics,CcGtase could be regarded as a new member of the GH77 family.In our previous research,a novel maltogenic amylase CoMA,which can catalyze the conversion of maltooligosaccharides(G3)and soluble starch to maltose as the sole hydrolysate,has been identified from Corallococcus sp.strain EGB.CoMA exhibited both hydrolysis and transglycosylation activities toward ?-1,4-glycosidic bonds but not to ?-1,6-linkages,and high level of maltose without the attendant production of glucose was identified by transglycosidation reaction.However,CoMA showed low hydrolysis activity and expression level,and thermolability,which is a barrier for its wide-scale industrial applications.Hence,in this study,we attempted to improve the enzymatic characteristics by use of directed evolution,site-directed mutagenesis and saturation mutagenesis.For screened facilitation,we constructed the expressed vector pMD19-T(PLacz-coMA)by error-prone PCR,the vectors were then expressed in Escherichia coli DH5?(?amyA)on LB medium for screening,which contained 0.3%starch(w/v).The transformants were further verified in E.coli BL21(DE3).We screened five mutants from 8000 variants,which exhibited 70%decreased activity,whereas,no variant was obtained with improved thermostability.Considering the three-dimensional structure prediction,seven sites were selected for site-directed mutagenesis from the obtained variants,including F314L,M318V,V50M,F55S,Y110H,R137C and F158L.The relative activity of mutants F314L and M318V decreased by 74%and 50%compared with wild type,respectively,whereas,the mutants showed similar enzymatic properties and hydrolysis products formation.Meantime,mutants Y110H,R137C and F158L showed dramatic loss of enzymatic activity,which retained less than 10%activity of the wile type.In a summary,three key amino acid sites,Y110,R137 and F158,were selected based on their importance in enzymatic activity,which provide alternative sites for further studies on the catalytic mechanism of the maltogenic amylase CoMA.