Tailoring activity and selectivity of microbial transglutaminase

Abstract

Microbial transglutaminase (mTG), a protein-glutamine γ-glutamyltransferase from Streptomyces mobaraensis, is an enzyme capable of forming isopeptide bonds between the nearly inert (from the chemical point of view) γ-carboxamides present in the side chain of glutamine residues and primary amines. Its high substrate tolerance, compared to other bond-forming enzymes, makes it a versatile tool for numerous applications including food manufacturing, material science, and biotechnology. Although an mTG-mediated bioconjugation is a well-established technique, some major drawbacks of this approach need to be bypassed, with the poor substrate specificity being among the most essential ones. Especially biopharmaceutical methodologies require high subsite specificity of the utilized biocatalyst, which is often not warranted by mTG. Therefore, access to tailor-made transglutaminases is strongly desired. Herein, we describe a protocol for the generation of mTG libraries based on yeast surface display, which allow for the isolation of mutants with altered properties. Moreover, methods for cloning of respective expression vectors, recombinant expression, and in vitro procession are provided.