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[摘要]：An endo-1,4-beta-mannanase gene (RmMan5A) was cloned from the thermophilic fungus Rhizomucor miehei for the first time and expressed in Escherichia coli. The gene had an open reading frame of 1330 bp encoding 378 amino acids and contained four introns. It displayed the highest amino acid sequence identity (42%) with the endo-1,4-beta-mannanases from glycoside hydrolase family 5. The purified enzyme was a monomer of 43 kDa. RmMan5A. displayed maximum activity at 55 degrees C and an optimal pH of 7.0. It was thermostable up to 55 degrees C and alkali tolerant, displaying excellent stability over a broad pH range of 4.0-100, when incubated for 30 mm without substrate. The enzyme displayed the highest specificity for locust bean gum (Km = 3.78 mg mL(-1)), followed by guar gum (K-m = 7.75 mg mL(-1)) and konjac powder (K-m = 22.7 mg mL(-1)). RmMan5A hydrolyzed locust bean gum and konjac powder yielding mannobiose, mannotriose, and a mixture of various mannose-linked oligosaccharides. It was confirmed to be a true endo-acting beta-1,4-mannanase, which showed requirement of four mannose residues for hydrolysis, and was also capable of catalyzing transglycosylation reactions. These properties make RmMan5A highly useful in the food/feed, paper and pulp, and detergent industries. |
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