Characterization Of A α-amylase From Bacillus Amyloliquefaciens BH072 And Cloning Of Its Gene

Amylase is an important enzyme for starch industry.Among that,α-amylase is one of the most significant amylases.As a ubiquitous enzyme,α-amylase is produced by many species,including animals,plants and microor-ganisms.Bacillus is genernally regard as one of the safe strains to human.They are important cell factories for producing α-amylase.We isolated a strain from honey sample and defined it as Bacillus amyloliquefaciens BH072.We found Bacillus amyloliquefaciens BH072 produced amylase with strong capacity of hydrolyzing starch.The amylase from Bacillus amyloliquefaciens BH072 is supposed to have great enzymatic characteristics.In this paper,we selected the Bacillus amyloliquefaciens BH072 to produce α-amylase.After several procedures such as ammonium sulfate fractionation,DEAE Sepharose Fast Flow anion exchange chromatography,Sephadex G-100 gel filtration chromatography and Native-PAGE gel extraction,we get the the aimed electrophoretically pure α-amylase.The purified α-amylase has an molecular weight of 68 kDa.The optimal pH and temperature are pH6.5 and 65℃,respectively.It exbit good acidstablility and thermostability.The stable range of temperature from 30℃ to 80℃ and 5.5-9.0.Surfactants have a slight inhibitory effect on the activity of the enzyme,the reducing agent has no significant effect,serine protein inhibitors have a significant role in promoting.The hydrolyzate of the hydrolyzed soluble starch of purified amylase was analyzed.By comparing it with the hydrolysis effect of the commercial α-amylase,the Bacillus amyloliquefaciens BH072 amylase was determined as a kind of α-amylase that hydrolyze soluble starch with better results.The results showed that the amylase of Bacillus amyloliquefaciens BH072 had better ability of hydrolyzing starch,and the hydrolytic effect of hydrolyzed corn,potato and wheat starch was compared.By comparing the predicted α-amylase gene in the whole genome sequence of Bacillus amyloliquefaciens BH072,the purified α-amylase was determined to be predicted α-amylase from Bacillus amyloliquefaciens BH072.In order to study the molecular basis of Bacillus amyloliquefaciens BH072 α-amylase,based on the bioinformatics analysis of the genome sequence of the strain published earlier,the α-amylase gene was found and a pair of nucleotides was designed according to its sequence The recombinant plasmid pET-28a(+)-amy072 heterologous expression system was constructed.The results of characteristics of this α-amylase showed that the recombinate α-amylase was consistent with the wild-type purified α-amylase.The result verifed the gene sequence of the purified wild type α-amylase.The genetic evolution of Bacillus amyloliquefaciens BH072 strain was further analyzed.The amino acid sequence was deduced from the nucleotide sequence,and the secondary structure and the model of the 3D structure were predicted.In this paper,the isolation and purification technology of α-amylase from Bacillus amyloliquefaciens BH072 was established.The physicochemical properties of the enzyme were systematically studied.The gene of α-amylase was cloned and analyzed.The gene sequence was analyzed and developed.These researches laid the foundation for the development and utilization of the strain.