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Molecular and biochemical characterizations of a new low-temperature active mannanase

Zhang, Rui, Zhou, Junpei, Gao, Yajie, Guan, Yaping, Li, Junjun, Tang, Xianghua, Xu, Bo, Ding, Junmei, Huang, Zunxi
Folia microbiologica 2015 v.60 no.6 pp. 483-492
Grus nigricollis, Sphingobacterium, Sphingomonas, activation energy, active sites, amino acids, beta-mannosidase, feces, genes, half life, hydrolysis, locust bean gum, pH, polypeptides, proteins, surface area
A mannanase-coding gene was cloned from Sphingobacterium sp. GN25 isolated from the feces of Grus nigricollis. The gene encodes a 371-residue polypeptide (ManAGN25) showing less than 74 % identity with a number of hypothetical proteins and putative glucanases and mannanases. Before experiment’s performance, ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization, including (1) ManAGN25 shared the highest identity of 41.1 % with the experimentally verified low-temperature active mannanase (ManAJB13) from Sphingomonas sp. JB13; (2) compared with their mesophilic and thermophilic counterparts, ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites; (3) these increased number of amino acid residues built longer loops, more α-helices, and larger total accessible surface area and packing volume. Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase: the enzyme showed apparently optimal activity at 35–40 °C and retained 78.2, 44.8, and 15.0 % of its maximum activity when assayed at 30, 20, and 10 °C, respectively; the half-life of the enzyme was approximately 60 min at 37 °C; the enzyme presented a K ₘ of 4.2 mg/ml and a k cₐₜ of 0.4/s in McIlvaine buffer (pH 7.0) at 35 °C using locust bean gum as the substrate; and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol. This study is the first to report the molecular and biochemical characterizations of a mannanase from a strain.