Gene Cloning and Enzymatic Characterization of an Alkali-Tolerant Endo-1,4-尾-mannanase from Rhizomucor miehei

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• 作者：Priti Katrolia ; Qiaojuan Yan ; Pan Zhang ; Peng Zhou ; Shaoqing Yang ; Zhengqiang Jiang
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2013
• 出版时间：January 16, 2013
• 年：2013
• 卷：61
• 期：2
• 页码：394-401
• 全文大小：639K
• 年卷期：v.61,no.2(January 16, 2013)
• ISSN：1520-5118

文摘

An endo-1,4-尾-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-尾-mannanases from glycoside hydrolase family 5. The purified enzyme was a monomer of 43 kDa. RmMan5A displayed maximum activity at 55 掳C and an optimal pH of 7.0. It was thermostable up to 55 掳C and alkali-tolerant, displaying excellent stability over a broad pH range of 4.0鈥?0.0, when incubated for 30 min without substrate. The enzyme displayed the highest specificity for locust bean gum (K_m = 3.78 mg mL^鈥?), followed by guar gum (K_m = 7.75 mg mL^鈥?) and konjac powder (K_m = 22.7 mg mL^鈥?). 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 尾-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.

Keywords:

Alkali tolerance; cloning; characterization; endo-1,4-尾-mannanase; Rhizomucor miehei