Addition of Co2+ to culture medium decides the functional expression of a recombinant nitrile hydratase in Escherichia coli |
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Authors: | Xiaolin Pei Qiuyan Wang Chenglu Li Xiaopu Yin Rong Chen Tian Xie |
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Affiliation: | 1. Center for Biomedicine and Health, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, 311121, China
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Abstract: | A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans, cloned and expressed in Escherichia coli, gave an enzyme that efficiently hydrated 3-cyanopyridine to nicotinamide with high thermal stability. We have now found that adding Co2+ at 0.1 mM to LB medium was essential for production of an active enzyme. However, ≥0.3 mM Co2+ inhibited the growth of host cells in LB medium and decreased the production of the recombinant NHase. Furthermore, β-mercaptoethanol promoted regeneration of the Co2+-defective apoenzyme in vitro possibly by breaking a key disulfide bond thereby promoting the incorporation of Co2+ into the apoenzyme. |
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