Cloning, Expression, And Characterization Of α-amylase From Exiguobacterium Sp. DAU5

a-Amylases (EC3.2.1.1) are enzymes that catalyze the hydrolysis of a-D-1,4-glucan linkage in starch and related carbohydrates to produce dextrins and reducing sugars. The enzymes play an important role in industry and are widely applied in water improvement, grain processing, food industry, brewing, fermentation, medicine, and so on. In this study, a-amylase producing bacterial strain was identified by the method of iodine-kalium iodide (I-KI) staining, and one strain DAU5with high a-amylase activity was screened. Based on analyses of16S rDNA sequence homology and phylogenetic tree, strain DAU5belongs to Exiguobacterium but not belongs to any known species. Accordingly, the strain was named as Exiguobacterium sp. DAU5.Exiguobacterium sp. DAU5genomic DNA library was constructed by shotgun approach to clone the a-amylase gene. One clone with a-amylase activity was screened from the genomic DNA library. Through analysis of the sequence of the inserted DNA fragment, partial sequence of AmyH was obtained. According to this sequence and its highly homologous sequence in GenBank to design primer, after PCR amplification, complete sequence of AmyH was obtained. The AmyH contained an open reading frame (ORF) of1545bp and encoded514amino acids. The a-amylase belonged to glycoside hydrolase (GH) family13, N-terminal contained a signal peptide of28amino acids (aa).Utilizing plasmid pET-32a(+), the expression vector with target gene (pET-AmyH-sp) was successfully constructed. E. coli BL21(trxB) was used as host bacteria and induced expression of AmyH. The fusion proteins were mainly expressed as inclusion bodies, through these steps of denaturation with8M urea, His-tag affinity purification and dialysis refolding, the purified proteins with a-amylase activity were obtained. The result of SDS-PAGE showed that the purified fusion proteins were approximately77kDa. After the His-tag was removed with enterokinase, its approximate molecular mass was57kDa, and it is consistent with the predicted size of amino acid sequence.The study of the characterization of the a-amylase showed that AmyH exhibited its maximal activity at40℃in pH8.5Glycine-NaOH buffer, and the enzyme displayed good thermo-stability and pH stability at<40℃for1h and in pH8.0, respectively. The AmyH activity was strongly inhibited by Zn2+, Cu2+, Fe2+, SDS, and EDTA; and was partially inhibited by Li+, K+, Zn2+, and Mg2+. However, Ca2+, Ni2+and urea didn't affect the AmyH activity. In addition, AmyH exhibited good tolerance to organic solvents, among them the enzyme retained93%,94%and97%of its original activity in the presence of20%methanol, acetone and ethanol, respectively. AmyH had a high affinity and maximum activity toward starch (Km=0.94mg/mL, Vmax=208x103U/mg). According to the value of Kcat/Km, AmyH had higher catalytic efficiency than that of other reported a-amylases from Nostoc sp.PCC7119, Candida Antarctica, and Pyrococcus furiosus. The result of TLC analysis of the products of the hydrolytic activity of the AmyH protein to maltooligosaccharides and soluble starch revealed that, AmyH protein can not efficiently hydrolyze oligosaccharides smaller than maltoheptaose; the first product in the reaction of hydrolyzing soluble starch was maltotriose, and final products were various maltooligosaccharides. The results of this study demonstrated that AmyH was a typical endoamylase.