Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of alpha-arbutin.
- Author:
Yuejia WAN
1
;
Jiangfeng MA
;
Rong XU
;
Aiyong HE
;
Min JIANG
;
Kequan CHEN
;
Yin JIANG
Author Information
1. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
- Publication Type:Journal Article
- MeSH:
Arbutin;
biosynthesis;
Catalysis;
Enzyme Stability;
Escherichia coli;
enzymology;
genetics;
Glucosyltransferases;
biosynthesis;
genetics;
metabolism;
Hydroquinones;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
metabolism;
Sucrose;
metabolism
- From:
Chinese Journal of Biotechnology
2012;28(12):1450-1459
- CountryChina
- Language:Chinese
-
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.
