Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1

Autor: Fatma Cebeci, Melinda J. Mayer, John T. Rossiter, Richard Mithen, Arjan Narbad
Rok vydání: 2022
Předmět:
Zdroj: The Protein Journal. 41:131-140
ISSN: 1875-8355
1572-3887
DOI: 10.1007/s10930-021-10034-5
Popis: Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. K
Databáze: OpenAIRE
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