Klebsiella pneumoniae exhibits extensive glycohydrolase activity in the gut microbiota. However, there are few studies on glucomannanase of Klebsiella pneumoniae. This study cloned and characterized a bifunctional mannanase/glucanase (GH8-3995) of K.pneumoniae MY2023. GH8-3995 exhibited excellent pH and salt tolerance, maintaining over 95 % activity in 5 M NaCl and over 80 % activity at pH 10. The results of oligosaccharides hydrolysis showed that GH8-3995 requires substrates to contain at least four glucose residues. The three-dimensional (3D) protein structure showed that GH8-3995 had a large catalytic cleavage on its surface, which was beneficial for binding enzymes and substrates. Molecular docking simulations and point mutation experiments demonstrated that D43 and E53 were the key binding sites of GH8-3995. Meanwhile, Alphafold3 predicted that E53 was also a key site for GH8-3995 to bind with Na+. In the application of oligosaccharide preparation, GH8-3995 predominantly produced oligosaccharides with DP > 4 from barley β-glucan and konjac glucomannan. This study investigated the enzymatic properties of GH8-3995 and analyzed the composition of its enzymatic hydrolysis products. The enzyme digestion characteristics and tolerance of GH8-3995 provided more possibilities for its application in producing cellooligosaccharides and mannanoligosaccharides.
Keywords: Barley β-glucan; Bifunctional; Klebsiella pneumoniae; Konjac glucomannan; Salt-adapted.
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