title: Gene cloning and enzymatic characterization of an alkali-tolerant endo-1,4-β-mannanase from Rhizomucor miehei.
abstract: An endo-1,4-β-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-β-mannanases from glycoside hydrolase family 5. The purified enzyme was a monomer of 43 kDa. RmMan5A displayed maximum activity at 55 °C and an optimal pH of 7.0. It was thermostable up to 55 °C and alkali-tolerant, displaying excellent stability over a broad pH range of 4.0-10.0, when incubated for 30 min without substrate. The enzyme displayed the highest specificity for locust bean gum (K(m) = 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 β-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.
authors.affiliation: Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People's Republic of China.
authors.forename: Priti
authors.initials: P
authors.lastname: Katrolia
authors.forename: Qiaojuan
authors.initials: Q
authors.lastname: Yan
authors.forename: Pan
authors.initials: P
authors.lastname: Zhang
authors.forename: Peng
authors.initials: P
authors.lastname: Zhou
authors.forename: Shaoqing
authors.initials: S
authors.lastname: Yang
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000378:metabolism; D020103:Rhizomucor / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase