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28th October 2015

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Development of a High Purity Recombinant Enzyme for a Biosensor Application

The customer used a wild type bacterial dehydrogenase in one of their biosensors for a medical application. The challenge was to produce a recombinant version of their existing enzyme with exactly the same activity specification. The recombinant enzyme was to have a higher specific activity and the production method scalable to accommodate increased order sizes.

Application: The customer used a wild type bacterial dehydrogenase in one of their biosensors for a medical application.

Customer Challenge: “To produce a recombinant version of the existing enzyme with exactly the same activity specification. The recombinant enzyme should have a higher specific activity and the production method must be scalable to accommodate increased order sizes.”

Approach: A codon-optimised version of the gene was synthesised and cloned into a E. coli expression vector (IP free). After transforming into a suitable E. coli host, positive clones were identified and expression of soluble, active enzyme was confirmed. The key parameters for optimal expression of soluble, active enzyme were determined at 1 mL scale using 48-well parallel microfermentations.

The resulting fermentation yielded 20-fold more active, soluble enzyme than the non-induced control.

Standard enzyme characterisation test showed that the recombinant enzyme was indistinguishable from the wild type enzyme. Expression levels were confirmed at 3 L in one of Biocatalysts’ high yielding, scalable E. coli fermentations and acceptability of the recombinant enzyme was confirmed by the customer after application tests.

Timescales

  • Cloning and optimisation of enzyme expression levels – 5 weeks
  • Enzyme characterisation – 1 week
  • Production of samples – 2 weeks
  • Customer application tests – 6 weeks

Benefits to customer

  • Cost effective production methods
  • Additional profit
  • No batch-to-batch variation