Recombinant expression of black sesame polyphenol oxidase and its enzymatic properties.

Caiyan YE; Guang Liu; Nian DU; Ruifen Zhang; Lei Liu; Xuchao Jia; Mingwei Zhang

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Recombinant expression of black sesame polyphenol oxidase and its enzymatic properties.

Author: Caiyan YE ¹ ; Guang LIU ² ; Nian DU ² ; Ruifen ZHANG ² ; Lei LIU ² ; Xuchao JIA ² ; Mingwei ZHANG ¹
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1. College of Food Science, South China Agricultural University, Guangzhou 510640, Guangdong, China.
2. Key Laboratory of Functional Foods, Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory of Agricultural Products Processing, Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences, Guangzhou 510610, Guangdong, China.
Publication Type:Journal Article
Keywords: Escherichia coli; affinity purification; black sesame polyphenol oxidase; enzymatic properties; prokaryotic expression
MeSH: Catechol Oxidase/genetics*; Cloning, Molecular; Escherichia coli/metabolism*; Recombinant Proteins/genetics*; Sesamum/genetics*
From: Chinese Journal of Biotechnology 2021;37(12):4395-4405
CountryChina
Language:Chinese
Abstract: To investigate the enzyme properties of the black sesame polyphenol oxidase (BsPPO), a synthesized Bsppo gene was cloned into the vector pMAL-c5x and expressed in E. coli. Subsequently, the MBP fusion label in the recombinant protein was removed by protease digestion after affinity purification. The synthesized Bsppo gene contained 1 752 bp which encodes 585 amino acids with a deduced molecular weight of 65.3 kDa. Transformation of the recombinant vector into E. coli BL21(DE3) resulted in soluble expression of the fusion protein MBP-BsPPO. The enzymatic properties of the recombinant BsPPO was investigated after MBP fusion tag excision followed by affinity purification. The results demonstrated that the optimal temperature and pH for BsPPO was 25°C and 4.0, respectively. BsPPO exhibited a good stability under low temperature and acidic environment. Low-intensity short-term light exposure increased the activity of BsPPO. Cu²⁺ could improve the activity of BsPPO while Zn²⁺ and Ca²⁺ showed the opposite effect. BsPPO could catalyze the oxidation of monophenols, diphenols, and triphenols, and exhibited good catalytic activity on l-tyrosine and vanillic acid. Moreover, BsPPO exhibited high catalytic activity on black sesame metabolites, including 2-methoxy cinnamic acid, indole-3-carboxylic acid and phloretin. These results may serve as a basis for further characterization of BsPPO.