title: Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase.
abstract: A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0-9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30-32.5 °C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20 °C, respectively, and the Ea, ΔG(*) (30 °C), Km (30 °C) and kcat (30 °C) values for hydrolysis of sucrose by rInvHJ14 was 47.6 kJ mol(-1), 57.6 kJ mol(-1), 62.9 mM and 746.2 s(-1), respectively. The enzyme also showed strong sucrose tolerance. rInvHJ14 preserved approximately 50% of its highest activity in the presence of 2045.0 mM sucrose. Furthermore, potential factors for low-temperature-active and alkaline adaptations of rInvHJ14 were presumed. Compared with more thermostable homologs, rInvHJ14 has a higher frequency of glycine residues and a longer loop but a lower frequency of proline residues (especially in a loop) in the catalytic domain. The catalytic pockets of acid invertases were almost negatively charged while that of alkaline rInvHJ14 was mostly positively charged.
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Junpei
authors.initials: J
authors.lastname: Zhou
authors.affiliation: College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Limei
authors.initials: L
authors.lastname: He
authors.affiliation: College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Yajie
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Nanyu
authors.initials: N
authors.lastname: Han
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of 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, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of 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, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of 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, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of 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, People's Republic of China.|College of Life Sciences, Yunnan Normal University, Kunming, 650500, People's Republic of China.|Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming, 650500, People's Republic of China.|Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500, People's Republic of China.
authors.forename: Zunxi
authors.initials: Z
authors.lastname: Huang
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000737:chemistry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D013395:Sucrose; D043324:beta-Fructofuranosidase; D005998:Glycine