web analytics

Novel Platform for Allosterically Modifying Enzyme Specificty

Published:
Lead Inventor: Shohei Koide
Print or Save to PDF

SUMMARY

  • Fine tuning enzyme specificity is desired for industrial catalysis. Enzymes lacking structural characterization or favorable recombinant expression are not amenable to traditional directed evolution or de novo design approaches.
  • “Monobodies” are synthetic binding proteins based off a human FN3 scaffold. When Monobodies bind allosterically near the enzyme active site, they can block undesired side reactions, thereby fine-tuning enzyme specificity.
  • This platform for specificity enhancer generation uses a yeast or phage displayed Monobody library that can be biopanned against a target enzyme of interest. Screening for Monobodies that compete with substrate competitive inhibitors, but that are non-inhibitory themselves, provide an initial lead that can be further affinity matured.
  • In a proof-of-concept, the platform was used to generate a Monobody that fine-tuned the specificity of a B-galactosidase so that it no longer catalyzed the formation of galacto-oligosaccharides (GOS) longer than tri-saccharides without impacting substrate conversion. Specifically, the Monobody reduced the amount of 4-saccharides produced from 8% to 2% of total sugar weight and GOS longer than 5-saccharides were reduced from 3% of total sugar weight to negligible weight.

 

FIGURE

(A)Screening for Monobodies that are in the same bin as inhibitors, but that are not inhibitory themselves, yields specificity modifiers. (B)Affinity matured proof of concept Monobody, Mb(L23), significantly limits B Galactosidase catalysis of GOS longer than tri-saccharides as compared to control Monobodies without altering the overall substrate conversion.

 

ADVANTAGES

ADVANTAGES

  • The enzyme does not need to be structurally or mechanistically characterized.
  • The enzyme does not need to be amenable to recombinant expression.

 

APPLICATIONS

  • Industrial chemical reaction engineering (Food, Biotechnology, Pharmaceuticals)

 

PUBLICATIONS

 

  • PCT/US16/42516 with counterparts in Japan and the EU
  • Tangible Property
  • (Monobody libraries) For engineered Monobody variants with increased thermostability, see also: Thermonobodies (16-T-034)