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.

A new acidophilic thermostable endo-1,4-β-mannanase from Penicillium oxalicum GZ-2: cloning, characterization and functional expression in Pichia pastoris

“…For most fungal endo-1,4-β-mannanases the optimal temperature and pH are between 40-70°C and pH 4.0-6.0, respectively [25], such as endo-1,4-β-mannanase from Rhizomucor miehei (optimal temperature 55°C and pH 7.0) [26], MANI and MANII from Aspergillus fumigatus IMI 385708 (60°C and pH 4.5) [27], endo-1,4-β-mannanase from P. oxalicum SO (60°C and pH 5.0) [28], endo-1,4-β-mannanase from A. niger BK01 (80°C and pH 4.5) [5], and rMan5C1 from P. pinophilum C1 (70°C and pH 4.0) [16]. However, the rPoMan5A possesses some superior properties compared to these fungal endo-1,4-β-mannanases, such as a long half-life (58 h) at high temperatures (60°C) and a wide range of pH adaptability (pH 2.0-7.0) and stability (pH 3.0-7.0).…”

Purification and characterization of β-Mannanase from Reinekea sp. KIT-YO10 with transglycosylation activity

“…18) Recombinant β-mannanase from A. niger yields mannobiose and other mannnooligosaccharides as major and minor products, respectively. 19) Although transglycosylation by fungal β-mannanase has been reported, 20,21) those by bacteria has rarely been reported. 22) We have recently isolated several marine bacteria from seawater at the seashore of Kanazawa Port in Japan.…”

A thermophilic β-mannanase from Neosartorya fischeri P1 with broad pH stability and significant hydrolysis ability of various mannan polymers

“…Until now, the thermophilic fungi Talaromyces leycettanus JCM12802 (Wang et al, 2014a), Aspergillus fumigatus IMI 385708 (Duruksu, Ozturk, Biely, Bakir, & Ogel, 2009), Aspergillus nidulans XZ3 (Lu et al, 2014), Humicola insolens Y1 (Luo et al, 2012), Thielavia arenaria XZ7 (Lu et al, 2013), and Rhizomucor miehei (Katrolia et al, 2013) have been reported be the ideal microbial sources of excellent b-mannanases.…”