Polysaccharide hydrolases are enzymes capable of hydrolyzing polysaccharides to generate oligosaccharides that have diverse applications in the food, feed and pharmaceutical industries. However, the detailed mechanisms governing the compositions of their hydrolysates remain poorly understood. Previously, we identified a novel neopullulase Amy117, which exclusively converts pullulan to panose by specifically cleaving α-1,4-glycosidic bonds. Yet, several enzymes with high homology to Amy117 produce a mixture of glucose, maltose and panose during pullulan hydrolysis. To explore this particular phenomenon, we compared the sequences and structures between Amy117 and the maltose amylase ThMA, and identified a specific residue Thr299 in Amy117 (equivalent to His294 in ThMA) within the product-releasing cleft of Amy117, which might be responsible for this characteristic feature. Using structure-based rational design, we have successfully converted the product profiles of pullulan hydrolysates between Amy117 and ThMA by simply altering this key residue. Molecular docking analysis indicated that the key residue at the product-releasing outlet altered the product profile by affecting the panose release rate. Moreover, we modeled the long-chain pullulan substrate G8 to examine its potential conformations and found that G8 might undergo a conformational change in the narrow cleft that allows the Amy117 variant to specifically recognize α-1,6-glycosidic bonds.
Keywords: Polysaccharide hydrolases; Product profile; Single point mutation.
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