title: Cold-active and NaCl-tolerant exo-inulinase from a cold-adapted Arthrobacter sp. MN8 and its potential for use in the production of fructose at low temperatures.
abstract: An exo-inulinase gene was cloned from Arthrobacter sp. MN8, a cold-adapted bacterium isolated from lead-zinc-rich soil. The gene was expressed in Escherichia coli BL21(DE3). The resultant 505-residue polypeptide (InuAMN8) showed the highest identity (81.1%) with the putative levanase from Arthrobacter phenanthrenivorans Sphe3 (ADX73279) and shared 57.8% identity with the exo-inulinase from Bacillus sp. snu-7 (AAK00768). The purified recombinant InuAMN8 (rInuAMN8) showed an apparently optimal activity at 35°C, and 75.3%, 39.4%, and 15.8% of its maximum activity at 20°C, 10°C, and 0°C, respectively. After pre-incubation for 60 min at 50°C and 55°C, the rInuAMN8 exhibited 69.8% and 17.7% of its initial activity, respectively. The apparent Km values of rInuAMN8 towards inulin were 2.8, 1.5, 1.2, 5.3, and 8.2 mM at 0°C, 10°C, 20°C, 30°C, and 35°C, respectively. Inulin and Jerusalem artichoke tubers were effectively hydrolyzed to release fructose by rInuAMN8 at 0°C, 10°C, and 35°C. Compared with its hyperthermophilic and thermophilic counterparts, the exo-inulinase had less aromatic amino acid F and more hydrophobic amino acid A. In addition, the purified rInuAMN8 retained 127.9%-88.4% inulinase activity at 3.5%-15.0% (w/v) NaCl concentrations. Zn(2+) and Pb(2+) at 10 mM exhibited little or no effect on the enzyme activity. This paper is the first to report a cold-active and/or NaCl-tolerant exo-inulinase from the genus Arthrobacter. The exo-inulinase rInuAMN8 shows a potential for use in the production of fructose at low temperatures.
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, PR China; College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China; Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, PR China.
authors.forename: Junpei
authors.initials: J
authors.lastname: Zhou
authors.affiliation: College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Lu
authors.affiliation: College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China.
authors.forename: Mozhen
authors.initials: M
authors.lastname: Peng
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, PR China; College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China; Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, PR China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming 650091, PR China.
authors.forename: Minghe
authors.initials: M
authors.lastname: Mo
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, PR China; College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China; Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, PR China.
authors.forename: Xianghua
authors.initials: X
authors.lastname: Tang
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, PR China; College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China; Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, PR China.
authors.forename: Junjun
authors.initials: J
authors.lastname: Li
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, PR China; College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China; Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, PR China; College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China; Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, PR China.
authors.forename: Junmei
authors.initials: J
authors.lastname: Ding
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, PR China; College of Life Sciences, Yunnan Normal University, Kunming 650500, PR China; Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, PR China. Electronic address: huangzunxi@163.com.
authors.forename: Zunxi
authors.initials: Z
authors.lastname: Huang
mesh_terms: D000064:Acclimatization; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000145:classification / Q000201:enzymology* / Q000502:physiology; D001407:Bacillus / Q000201:enzymology; D003001:Cloning, Molecular; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics; D005632:Fructose / Q000096:biosynthesis*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007444:Inulin / Q000378:metabolism; D008969:Molecular Sequence Data; D012965:Sodium Chloride / Q000494:pharmacology*
chemical_list: D005632:Fructose; D012965:Sodium Chloride; D007444:Inulin; D006026:Glycoside Hydrolases; C021512:levanase; C023086:inulinase
keywords: Arthrobacter; Cold-active; Exo-inulinase; Fructose; NaCl