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标题：Heterologous Expression of Dehydroascorbate Reductase from Rice and Its Application to Determination of Dehydroascorbate Concentrations
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作者：Katsumi AMAKO ; Takashi USHIMARU ; Ayami ISHIKAWA 等
期刊名称：Journal of Nutritional Science and Vitaminology
印刷版ISSN：0301-4800
电子版ISSN：1881-7742
出版年度：2006
卷号：52
期号：2
页码：89-95
DOI：10.3177/jnsv.52.89
出版社：Center for Academic Publications Japan
摘要：We constructed an expression vector for rice dehydroascorbate reductase (DHAR) (EC 1.8.5.4) with a polyhistidine tag at the amino terminus and introduced the vector into several strains of Escherichia coli . On conventional induction treatment with isopropylthiol-β-D-galactoside, E. coli harboring rice DHAR cDNA produced doublet polypeptides of about 27 kDa. Induction duration or growth temperature did not affect the ratio of these polypeptides. Only the larger polypeptide, corresponding to full-length recombinant DHAR, was produced in E. coli supplemented with tRNAs for several minor codons. Most of the enzymatic characteristics of the recombinant DHAR were similar to those of the native one, although the recombinant protein showed increased heat susceptibility. Using recombinant DHAR, we developed a method for simple and precise determination of dehydroascorbate concentrations in tissue extracts by spectrophotometry, and we successfully applied the method to several fruit juices and vegetables.
关键词：ascorbic acid;dehydroascorbate;dehydroascorbate reductase;quantification