Characteristics Of Two Amylases Derived From GH13 Family And Evaluation Of Their Application In The Preparation Of Modified Potato Starch

China is the world’s biggest producer of starch.In 2019,the total output of starch in China was about 33 million tons,the further-processed products of starch accounted for54.06%,and about 82.63% further-processed products are starch sugar.In food processing,the involved high temperature,stirring and low temperature affect the properties of raw starch,which fail to meet the related requirements.To improve the stability and nutritional properties of starch for meeting the industry application,starch modification is required.Compared with physical,chemical and compound modification,enzymatic modification of raw starch has received the attention due to its high efficiency,green and safety.Amylases from Glycoside Hydrolases 13(GH13)family are important starch processing enzymes,which play important roles in preparation of starch sugar and resistant starch.Based on the current situations and demands of starch processing industry,the maltogenic amylase CoMA and starch branching enzyme AqGBE,two kinds of amylase with excellent characteristics from GH13 family in this study,were used to investigate their catalytic properties and practical application.In our previous studies,we have identified that maltogenic amylase CoMA converts starch into maltose with high purity through multiple molecular catalytic reactions such as hydrolysis and transglycogen.Based on these results,the effects of CoMA on the structure and retrogradation characteristics of potato starch were explored.Our results showed that CoMA exhibits simultaneous hydrolysis and turn sugar-based role during the reaction with G3 as the minimum catalytic unit.During the catalytic process,the complexes of CoMA and G1 were formed,and the G1 was regarded as the donor,the freed molecules like G1 and oligosaccharide were used as receptors,the donor was transferred to receptor with synchronous generation of malto-oligosaccharides with degree of polymerization(DP)more than 20.At the same time,CoMA showed exo-hydrolysis model to produce G2 from the nonreducing end.In addition,it has been found that CoMA modifications change the ratio of amylose to amylopectin in starch and reduce the degree of lateral amylopectin polymerization.Iodine binding assay showed that the complete structure of starch is destroyed by CoMA and the formation of insoluble blue complex increased.Scanning electron microscopy(SEM)showed that the surface of starch particles appeare pores and micropores after CoMA treatment.Dynamic rheological analysis showed that the interaction of starch network is weakened and the recovery rate of starch is reduced.Maltose produced during this process is also associated with inhibition of starch retrogradation.CoMA treatment reduced degree of polymerization of outside chain of starch grains,which made CoMA as an effective additive to inhibit starch regrowth with formation of maltose,the lowest effective dose is 0.5 mg/kg.Starch branching enzymes,as the member of glycosyltransferases from GH13 family,catalyze the breaking of α-1,4-glycosidic bonds and the formation of α-1,6-glycosidic bonds through intermolecular transfer reactions,and then form new branches on the main chain of starch molecules.The characteristics of starch branching enzyme make it valuable in starch modification and preparation of starch with high branching degree.In this paper,a starch branching enzyme AqGBE with high catalytic efficiency was obtained.The specific activity of AqGBE was 2850 U/mg.After AqGBE treatment of potato starch,the chain length distribution in starch granules was changed,which resulted in the decreased amylose content and increased amylopectin content in starch.These structural changes exhibited a significant decrease in the number of hydrogen bonds formed in the process of starch regeneration,a continuous decrease in viscosity and short-term regeneration value were also observed.The X-ray diffraction pattern showed that the aging peak of starch and the enthalpy change of aging decreased.Our results showed that AqGBE-treated starch exhibits low retrogradation properties.Otherwise,the results also showed that AqGBE treatment can effectively reduce the content of fast digested starch(RDS),increase the content of slow digested starch(SDS)and resistant starch(RS)in potato starch,which may find great potential in improving the nutritional properties of starch by enzymatic modification.