期刊
BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY
卷 56, 期 4, 页码 531-539出版社
INST TECNOLOGIA PARANA
DOI: 10.1590/S1516-89132013000400002
关键词
sorbitol dehydrogenase; recombinant SDH; expression and purification; Ni-NTA; enzyme assay; peach fruit
类别
资金
- National Natural Science Foundation of China [30901160]
- Natural Science Foundation of Shandong Province [ZR2010CQ039]
A gene encoding NAD(+)-dependent sorbitol dehydrogenase (SDH) in peach fruit was cloned and expressed in Escherichia coli. Recombinant SDH protein with 6xHis-tagged was localized exclusively in the cytoplasmic soluble fraction of E. coli when the strains were grown for 4-5 h at 37 degrees C. Highly pure protein was isolated by Ni2+ -resin chromatography with 150 mM imidazole in 50 mM Tris, pH 8.0, by elution. In order to ensure that the recombinant SDH could be used for further study, the fluorescence and ultraviolet spectrum of the recombinant SDH were detected. Recombinant SDH was confirmed to be capable of oxidizing sorbitol by enzymatic activity assay. The activity of the recombinant SDH was 2.73 U mg(-1) min(-1), which was similar with that directly extracted from peach fruits. The activities of SDH extracted from the fruits in different periods (30, 60, 90 days after flowing) were 7.75, 5.95, 3.26 U mg(-1) min(-1), respectively.
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