title: [Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of alpha-arbutin].
abstract: Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 degrees C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 degrees C and 6.7, respectively. The K(m) of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 micromol/(min x mg). Besides, alpha-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of alpha-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 degrees C for 21 h. Under these conditions, alpha-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of alpha-arbutin was about 31 g/L.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Yuejia
authors.initials: Y
authors.lastname: Wan
authors.forename: Jiangfeng
authors.initials: J
authors.lastname: Ma
authors.forename: Rong
authors.initials: R
authors.lastname: Xu
authors.forename: Aiyong
authors.initials: A
authors.lastname: He
authors.forename: Min
authors.initials: M
authors.lastname: Jiang
authors.forename: Kequan
authors.initials: K
authors.lastname: Chen
authors.forename: Yin
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D001104:Arbutin / Q000096:biosynthesis*; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D006873:Hydroquinones / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism
chemical_list: D006873:Hydroquinones; D011994:Recombinant Proteins; D013395:Sucrose; D001104:Arbutin; D005964:Glucosyltransferases; C025901:sucrose phosphorylase; C031927:hydroquinone