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Significant Enhancement Catalytic Activity of Nitrile Hydratase by Balancing the Subunits Expression. | LitMetric

Significant Enhancement Catalytic Activity of Nitrile Hydratase by Balancing the Subunits Expression.

Chembiochem

School of Chemical Engineering, Laboratory of Advanced Materials and Catalytic Engineering, Dalian University of Technology, Dalian, 116024, China.

Published: December 2024

AI Article Synopsis

  • E. coli is widely used for producing recombinant proteins because it's easy to genetically modify.
  • A previous study successfully created a plasmid for a nitrile hydratase enzyme that showed good catalytic performance, but imbalanced expression of its subunits limited its effectiveness.
  • This research optimized the mRNA structure to improve subunit balance, resulting in a 12-fold increase in enzyme activity, and explored various fusion tags to enhance soluble expression, although some tags affected enzyme function negatively.

Article Abstract

Escherichia coli (E. coli) is the most commonly used bacterial recombinant protein production system due to its easy genetic modification properties. In our previous study, a recombinant plasmid expressing the Fe-type nitrile hydratase derived from Rhodococcus erythropolis CCM2595 (ReNHase) was successfully constructed and the recombinant ReNHase exerted an excellent catalytic effect on dinitrile compounds. Nevertheless, the ReNHases were confronted with imbalanced subunit expression during heterologous expression, which restricted the enzymes assemble functionally. In this study, the secondary structure of mRNA in the ribosome binding sequence region of the β-subunit was optimized to elevate the translation efficiency of the β-subunit gene and balance the expression of α- and β-subunits in ReNHase. The optimized ReNHase showed a 12-fold increase in specific enzyme activity over wild-type ReNHase. To further enhance the soluble expression of ReNHase, the ReNHase was labeled using three different fusion tags, resulting in three new recombinant ReNHases. In these recombinant ReNHases, some of the fusion tags promoted the soluble expression of ReNHase, but also affected the balance of α-/β-subunit expression and the secondary structure of the ReNHase, and reduced the enzyme activity. In conclusion, our results provide an optimized strategy for the heterologous expression of multi-subunit proteins.

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Source
http://dx.doi.org/10.1002/cbic.202400526DOI Listing

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