Characterization of a novel amylomaltase from corynebacterium glutamicum ATCC 13032

Abstract

To search for a novel amylomaltase and to identify residue involved in formation of cyclic oligosaccharide product. The amylomaltase gene from Corynebacterium glutamicum ATCC 13032 was cloned and expressed in Escherichia coli BL21(DE3) using the expression vector pET-19b. The ORF of amylomaltase gene, including His-tag sequence was 2,190 base pairs. The deduced amino acid sequence showed only 28-45% similarity with reported amylomaltases. The enzyme was purified by HisTrap FFTM column. The size was 84 kDa and the conditions for optimal catalysis were at pH 6.0 and 30 ºC. The large-ring cyclodextrin (LR-CDs) products were from CD19 up. After purification, removal of his-tag residues resulted in total loss of enzyme activity. The amylomaltase clone was reconstructed using pET-17b. The enzyme was expressed and purified by DEAE FFTM and Phenyl FFTM columns. The basic characters obtained were similar to the enzyme with his-tag. The recombinant plasmid was then used as a template for site-directed mutagenesis at Tyr-172. Both wild-type and mutated enzyme, Y172A, had the same physical characteristics. The Y172A mutant showed lower disproportionation, cyclization and hydrolytic activity than the wild-type enzyme. In disproportionation reaction with maltotriose, kinetic parameters were determined. The Km were 19.6 and 12.9 mM and kcat were 9,374 and 2,165 min-1 in the wild-type and Y172A enzyme, respectively. When the cyclization products were investigated, the smallest LR-CD was CD19 from both enzymes and the product profile was dependent on the incubation time and the enzyme concentration. Shorter incubation time gave larger LR-CDs as principal products. Interestingly, the wild-type enzyme showed different product pattern from that of Y172A mutant. This might be due to higher transglucosylation and hydrolytic activity of the wild-type enzyme. These results suggested that Tyr-172 should be involved in determining LR-CDs product profile.