Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants.

摘要: Abstract Recombinant microbial transglutaminase (rMTG), an enzyme useful for the cross-linking or posttranslational modification of (therapeutic) proteins, was optimized by random mutagenesis first time. A screening method developed which, in addition to state-of-the-art procedures, includes a proteolytic activation step expressed soluble pro-enzyme. The library 5,500 clones screened variants with increased thermostability and heat-sensitivity, respectively. Mutant enzymes were overproduced, isolated characterized. After just one round mutagenesis, nine single amino acid exchange showed remarkably at 60 °C. serine residue close N-terminus against proline resulted rMTG mutant (S2P) 270% half-life. Seven exhibited heat-sensitivity 60 °C which (G25S) retained its specific activity between 10 40 °C. mutations responsible identified assigned three-dimensional (3D) structure. All point related changed thermal properties are located N-terminal domain (i.e. left side wall active site cleft front view MTG as defined literature [Kashiwagi, T., Yokoyama, K., Ishikawa, Ono, Ejima, D., Matsui, H., Suzuki, E., 2002. Crystal structure from Streptoverticillium mobaraense. J. Biol. Chem. 277, 44252–44260] showing importance this part protein.