Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)

• Published in: Preparative biochemistry & biotechnology Vol. 47; no. 8; pp. 768 - 775
• Main Authors: Liu, Changqing, Zheng, Kai, Xu, Ying, Stephen, Lacmata Tamekou, Wang, Jiming, Zhao, Hongwei, Yue, Tongqing, Nian, Rui, Zhang, Haibo, Xian, Mo, Liu, Huizhou
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 14-09-2017; Taylor & Francis Ltd
• Subjects: ABTS; Analytical chemistry; Chemical reactions; Cloning, Molecular - methods; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Food processing; Food processing industry; Gel electrophoresis; Glycine max - enzymology; Glycine max - genetics; Glycine max - metabolism; Hemin; heterologous expression; Industrial applications; Isopropyl thiogalactoside; Kinetics; Molecular weight; Peroxidase; Peroxidases - genetics; Peroxidases - metabolism; pH effects; Plasmids - genetics; Proteins; Reaction kinetics; recombinant protein; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Seed coats; Sodium lauryl sulfate; soybean seed coat peroxidase; Substrate Specificity; Sulfonic acid; Temperature; soybean seed coat peroxidase; Escherichia coli; recombinant protein; pH; temperature; heterologous expression; ABTS
• ISSN: 1082-6068; 1532-2297
• DOI: 10.1080/10826068.2017.1342258

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39 kDa. The effects of induction temperature, concentration of isopropyl-β-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23 U/mL (8.64 U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.