Molecular mutagenesis at Tyr-101 of the amylomaltase transcribed from a gene isolated from soil DNA

Synthesis of long-chain isomaltooligosaccharides from tapioca starch and an in vitro investigation of their prebiotic properties

“…This isolated gene had a high amino acid sequence identity to amylomaltase of Thermus thermophilus [17]. The expression of both recombinant enzymes was induced by 0.8 mM IPTG when A 600 of the culture reached 0.5.…”

“…The recombinant plasmids containing WT-and Y101S-amylomaltase genes were expressed in E. coli BL21 (DE3) [17], and the obtained enzymes were purified by a Ni-NTA column. The specific activity of the mutated enzyme for the cyclization reaction showed a small increase, while significant decreases in the starch transglycosylation, disproportionation, coupling, and hydrolytic activities were observed (Table 1).…”

“…The bound proteins were eluted by 500 mM imidazole in the same buffer. The active fractions were pooled and assayed for their enzyme activity by a starch transglycosylation assay [17], and the protein concentration was determined by the Bradford method [18] using BSA as the standard.…”

In silico analysis of family GH77 with focus on amylomaltases from borreliae and disproportionating enzymes DPE2 from plants and bacteria

“…Tyr54 and Tyr101 278 (T. aquaticus amylomaltase numbering [9]), since both were identified 279 as the aromatic positions involved in the secondary glucan-binding 280 site of the enzyme[49,57,58]. While the former was determined to con-281 trol the hydrolytic activity[57,59], the latter was shown to be responsi-282 ble rather for disproportionating activity of the amylomaltase[58,60].283 Interestingly, the residues of the secondary glucan-binding site from 284 T. aquaticus amylomaltase were found to be not strictly conserved 285 among its counterparts [27,61]. A detailed inspection of the alignment 286 of all 416 4-α-glucanotransferases compared in the present study con-287 firmed that neither Tyr54, nor Tyr101 does represent a conserved posi-288 tion in the family GH77 (Fig.…”