Characteristion Of A Starch Branching Enzyme McGBE From Microvirga Sp. MC18 And Its Application In Starch Modification

Starch branching enzymes(EC 2.4.1.18)belong to the glycoside hydrolase 13 family.As a glycosyltransferase,starch branching enzymes are capable of hydrolyzingα-1,4-glycosidic bonds to produce sugar chains with non-reducing end,and then ligate the C1 end of the free sugar chain to the C6 position of the adjacent glucose unit through transferase activity to form a new α-1,6 glycosidic bond through the transferase activity,thereby change the branching structure and branching degree of starch.Starch branching enzyme has become an important enzyme for starch processing for its ability to change the properties of starch.In this study,the catalytic properties of McGBE,a high-efficiency starch branching enzyme cloned from Microvirga sp.MC18,were studied,and its application in starch modification was evaluated.McGBE was heterologously expressed and purified in Escherichia coli BL21(DE3).Enzymatic characteristics studies showed that the starch branching enzyme McGBE treatment significantly reduced the content of α-1,4-glycosidic bonds in starch,while of α-1,6 glycosidic bonds increased significantly,indicating that the amylase McGBE is a typical starch branching enzyme with high catalytic efficiency.The optimal temperature of the enzyme is 45℃,the optimal pH is 9.0,and the optimal substrate is potato starch.Under the optimal reaction conditions,the specific enzyme activity of the enzyme is 791.29 U/mg.The side chain analysis of the enzyme-treated starch showed that McGBE treatments significantly increase the branched chain content of degree of polymerization(DP)<13.Reacting with the most suitable substrate,potato starch,McGBE treated starch contains increased content with branched chains of DP<13,which shares the similar characteristics with most reported starch branching enzymes.Based on the effect of enzymatic treatments on starch structure,the role of McGBE treatment in improving the anti-aging properties of starch and the content of slowly digested starch were evaluated.Infrared spectroscopy and differential scanning calorimetry analysis showed that McGBE can change the molecular structure of starch and reduce the number of hydrogen bonds formed during starch aging,thereby delay the retrogradation of potato starch.After McGBE treatment,the content of slow-digesting starch(SDS)in starch increased from 22.53%to 33.45%.In order to further increase the content of slowly digested starch after McGBE treatment,we initially determined the optimal preparation conditions of modified starch by orthogonal test:pH 7.0,reaction temperature 30℃,the dosage is 150 mg McGBE/1 kg starch,The reaction time is 11 h.The content of SDS in the prepared modified starch under optimal conditions increased by 174%.This research has improved the efficient enzyme resources for the enzymatic preparation of modified starch,and also provided technical support for the application of starch branching enzymes in the field of starch processing.