title: Molecular and biochemical characterizations of a new low-temperature active mannanase.
abstract: 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.
authors: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors: Rui
authors: R
authors: Zhang
authors: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors: Junpei
authors: J
authors: Zhou
authors: College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors: Yajie
authors: Y
authors: Gao
authors: College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors: Yaping
authors: Y
authors: Guan
authors: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors: Junjun
authors: J
authors: Li
authors: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors: Xianghua
authors: X
authors: Tang
authors: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors: Bo
authors: B
authors: Xu
authors: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China.
authors: Junmei
authors: J
authors: Ding
authors: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China. huangzunxi@163.com.|College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China. huangzunxi@163.com.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming, 650500, People's Republic of China. huangzunxi@163.com.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People's Republic of China. huangzunxi@163.com.
authors: Zunxi
authors: Z
authors: Huang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003080:Cold Temperature; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D045249:Sphingobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D044902:beta-Mannosidase