Characterization And Application Evaluation Of Starch-branching Enzyme CcGBE From Corallococcus Sp. Strain EGB

Glucan-branching enzyme(BE;EC 2.4.1.18)is a kind of involved in starch biosynthesis of glycosyl transferase,cutting amylose in α-1,4 glycosidic bond is not the end of the reducing sugar chain,and through the transfer of enzyme activity will be the free C1 end of sugar chain transfer to the same or nearby C6 location on the starch chains to form new α-1,6 glycosidic bond,through this continuous process of "cutting-transfer-connection",which has high degree of branching and anti-aging properties of modified starch.High branched starch food is considered as a kind of dietary fiber because it can slow down the digestion rate of starch in the human body,make the human body feel full,and have the advantages of reducing the peak level of blood sugar in the body after meal.Therefore,starch branching enzyme has become an important enzyme for starch processing.In this paper,the catalytic characteristics of CcGBE,a highly efficient starch branching enzyme from Corallococcus sp.strain EGB,were studied,and its application in starch modification was evaluated Escherichia coli BL21(DE3)recombinant expression system was used to heterologously express CcGBE,and finally purified recombinant protein CcGBE was obtained.CcGBE treatment decreased the content of α-1,4 glycosidic bonds in tapioca starch,but significantly increased the content of α-1,6 glycosidic bonds,indicating that CcGBE is a kind of typical starch branching enzymes with high catalytic efficiency.The optimal catalytic temperature of the enzyme was 40℃,the optimal buffer system was Tris-HCl(p H 9.0),and the optimal substrate was tapioca starch.Under the optimal reaction conditions,the specific enzyme activity of the enzyme was as high as19439.9 U/mg.After enzyme treatment of tapioca starch side chain polymerization Degree analysis showed CcGBE processing within 5 min polymerization Degree(DP)of polymerization,is greater than the content of long chain branched chain up 21,afte 15 min,long chain(DP 24 or higher)content decreased gradually,DP 6-15 increased the content of short chain,show that early main catalytic DP 21-40 CcGBE processing starch long chain transfer,and the late main catalytic DP 6-15 short chain transfer.The catalysis of CcGBE is significantly different from that of starch branching enzymes which only catalyze long or short chain transfer.The unique cleavage of CcGBE can significantly improve the efficiency of starch branching.Based on the changes of starch structure by starch branching enzyme,this study evaluated the effect of CcGBE treatment on the anti-aging properties of starch and the content of resistant starch.The results showed that CcGBE significantly reduced the molecular weight of tapioca starch(25417.9 k Da to 131.2 k Da),changed the molecular structure of tapioca starch granules,and reduced the number of hydrogen bonds formed in the aging process of tapioca starch,thus delayed the regeneration behavior of tapioca starch.The content of Resistant starch(RS)increased from 3.31% to 12.8% after CcGBE treatment.This study provides an efficient enzyme resource for the enzymatic preparation of modified starch,and also provides a technical support for the application of starch branching enzyme in starch processing.