Gene Cloning And Characterization Of Maltose-forming α-amylase

Maltose-formingα-amylase is the amylase which hydrolyzes starch and related polysaccharide to produce maltose as the main products. Maltose-formingα-amylase belongs to glycoside hydrolases family 13(GH13), mainly used in starch and food industry. It has different names, including maltogenic amylase, neopullulanase as well as cyclomaltodextrinase but shares a similar structure. Maltose-formingα-amylase exhibits distinct substrate specificity and substrate preferential selectivity. It catalyzes the hydrolysis ofα-1,4- andα-1,6-glucosidic linkages appeared in many oligosaccharides and polysaccharides, such as starch, cyclodextrine and pullulan. The main product isα-maltose.In this study, two sets of primers were designed according to the maltogenic amylase gene of Bacillus licheniformis and maltose-formingα-amylase gene conservative sequence. Two different sources maltose-formingα-amylase sequence have been successfully amplified by using the chromosome DNA of B. licheniformis ATCC14580 and Bacillus cereus CICIM-B0627 as templates. Sequencing of PCR product showed that the nucleotide sequence of amyM-Bl was 1764 bp, the deduced protein was 587 amino acids and the isoelectric point of the protein was 4.93; the nucleotide sequence of amyM-Bc was 1685 bp, the deduced protein was 561 amino acids and the isoelectric point of the protein was 5.18. Sequence blasting of two proteins revealed that they both contained a highly conservative catalytic area which specially existed inα-amylase family. AmyM-Bl sequence was the same as maltogenic amylase in B. licheniformis ATCC14580 but different from those of other strains of B. licheniformis; AmyM-Bc shared a 99.29% similarities with neopullulanase from B. cereus ATCC14579.The amyM-Bl gene and amyM-Bc gene were subcloned into expression vector pET-28a(+) and expressed in Escherichia coli BL21 (DE3) successfully after induced with 0.5 mmol/L IPTG. The recombinant AmyM-Bl had a molecular mass of 67 kDa on SDS-PAGE, while the recombinant AmyM-Bc had a molecular mass of 65 kDa. The recombinant AmyM-Bl showed the enzyme activity of 3.797 U/mL, but the recombinant form of AmyM-Bc in E. coli showed no detectable activity.The enzymatic properties and hydrolytic characters of the recombinant AmyM-Bl were analyzed systemically in the study. The maximum activity of the recombinant enzyme were at 45℃and pH 6.5. It was stable in a low temperature environment (no more than 45℃) as well as at a wide pH range (pH 4.5~8.5), and the enzyme was not sensitive to metal ions. The recombinant enzyme can catalyze the hydrolysis of cyclodextrins, soluble starch and pullulan, The cyclodextrin was the optimal substrate.