Cloning,Expression And Analysis Of Alpha-Amylase Gene From Bacillus Subtilis

Alpha-amylase are one of important enzymes in industry field, and the activest fields in enzyme studying.They are widely applied in the starch processing,ethanol producing,spinning and weaving and other industrial fields.A genome library of Bacillus subtilis was established by "shotgun" method and alpha- amylase gene was cloned,expressed and analysed. The total DNA of Bacillus subtilis and pUC18 plasmid were extracted respectively by different methods. The genome DNA was partially digested by 0.5U Sau3AI restriction enzyme in 20 minutes. Pieces of 2-7 Kb DNA were extracted,pUC18 plasmid was totally digested with BamH1 enzyme and purified by gel purification. To prevent the self ligation, CIP was used to dephosphorylate totally. 2-7 Kb DNA and pUC18 plasmid were mixed,then T4 DNA ligase , ligase buffer and deioned water were added to 20μl reaction volume . The ligation product was transformed with high efficiency competent cells after ligation for 16-18hr at 14℃~16℃, and nearly 20000 transformants were got. Screening for alpha-amylase activity on LBSP plate, and positive cloning of alpha-amylase gene were identified. After checking the length of positive cloning plasmid, digesting it with the restriction enzymes and testing of its function, alpha-amylase gene of about 2.9Kb was cloned and its restriction enzyme map was made.The piece of DNA sequenced by Sangon was a 2860 bp fragment. Sequence analysis revealed a potential ORF encoding a protein of 649 amino acid residues. Using the SignalP3.0 Server-prediction on line, we found the typical signal peptine sequence at the N end, having 32 amino acid residues. The deduced amino acid sequence is highly homologic to that of the alpha-amylase amino acid sequence from B.S published in NCBI by CLUSTALX1.81 analysing. The highly conserved sequence lies in the N end and in the middle part of the alpha-amylase gene, but there is much difference in the C end.The expression of the Amy gene in Escherichia coli was poor, and some of the expression were secreted. So we established the high-effective expressing vector with pET-30a(+) and transformed with BL21 competent cells.To extract the total protein, the cells were broken, SDS-PAGE (12%W/V) showed that the molecular weight of alpha-amylase was 61 KD, which was the same as the result deduced from the nucleotide sequence.