| Glucoamylase(EC 3.2.1.3,Glucoamylases,GA),is an important enzyme in sugar industry.It can hydrolyze the a-1,4 and a-1,6 glycoside bonds in starch or oligosaccharides,and ultimately produce glucose.Glucoamylase is known to be distributed in carbohydrate GH15 family and GH97 family,but not in GH57 family.The enzymes of GH57 family come from thermophilic archaea and bacteria,and are a family of thermophilic enzymes.This study was originally intended to study a thennophilic amylopullulanase,so screened from the GH57 family and selected a hypothetical amylopullulanase derived from thermophilic bacteria Thermocrinis minervae,named GA-Themi.The highest similarity of GA-Themi protein sequence in Genebank database is only 40%,but GA-Themi sequence contains specific conserved amino acids of GH57 family.The GA-Themi gene sequence was synthesized by entrusting Biological Company,and the soluble expression was obtained in E.coli.After purifying the recombinant GA-Themi protein,its specific enzyme activity to soluble starch was determined to be 1.79 U/mg.The optimum pH was 7.0,and the optimum reaction temperature was 90 ?.The thermal stability of GA-Themi is very good.The half-lives of GA-Themi are 2.5 h,2 h and 1.5 h at 70 ?,80? and 90 ?.Three metal ions Mg2+,Co2+,and Ni+can increase the activity of GA-Themi by two times.The hydrolysis activity of soluble starch by GA-Themi was 100%,and the enzymatic activities of pullulan,amylopectin and amylose were 76.5%,71.7%and 64.2%respectively.GA-Themi also has a weak ability to hydrolyze glycogen and dextran.This indicates that GA-Themi has the ability to hydrolyze a-1,4 and a-1,6 glycoside bonds.The known amylopullulanase has stronger ability to hydrolyze pullulan than starch,and its ability to hydrolyze a-1,6 glycoside bond is much stronger than that of a-1,4 glycoside bond.Therefore,the hydrolysate of pullulan by amylopullulanase is mainly trisaccharide,which produces a small amount of glucose and maltose.However,GA-Themi hydrolysis of pullulan produced only glucose but no trisaccharide.Further verification of GA-Themi hydrolysis starch products,found that only glucose,and GA-Themi can also directly hydrolyze maltose to glucose,hydrolyzed maltose to glucose.These catalytic characteristics indicate that GA-Themi is not a amylopullulanase,but a new type of glucoamylase.This is the first discovery of a glucoamylase belonging to the GH57 family.Compared with the known glucoamylases of GH15 and GH97 families,GA-Themi catalyzed the hydrolysis of pullulan sugars,i.e.the hydrolysis of a-1,6 glycoside bonds was stronger,and the thermal stability was good.This characteristic makes GA-Themi more suitable for the hydrolysis of starch such as glutinous rice with high branched chain content.a-amylase(EC3.2.1.1,Amylase,Amy)is a kind of hydrolytic enzyme that can hydrolyze a-1,4 glycoside bonds in carbohydrates such as starch and pullulan randomly to produce maltooligosaccharide or maltodextrin.It is also an important enzyme in sugar industry.Thermophilic a-amylase is needed in sugar industry,but the pH value of high-temperature a-amylase in commerce is different from that of enzymes in subsequent processes,so it is necessary to adj ust the pH value in practical application.In order to search for a thermophilic and acidophilic a-amylase,a hypothetical a-amylase derived from thermophilic archaea Acidilobus sp.7A was screened and named AmyAC.AmyAC sequence belongs to GH13 family,but it can form an independent branch with GH13 family,which is quite different from the known a-amylase protein sequence and may have new catalytic properties.After commissioning the company to synthesize the AmyAC gene sequence,soluble expression was obtained in E.coli.However,no enzyme activity was detected with soluble starch,pullulan,glycogen,amylopectin and amylose as substrates. |
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