Effect of introducing a disulfide bridge on the thermostability of microbial transglutaminase from Streptomyces mobaraensis.

Microbial transglutaminase (MTG) has been used extensively in academic research and the food industry through cross-linking or posttranslational modification of proteins. In our previous paper, the activity-increased MTG mutants were obtained by means of rational mutagenesis and random mutagenesis coupled with the newly developed screening system. In addition, the improvement of heat resistance of MTG is needed to expand further its industrial applications. Here, a structure-based rational enzyme engineering approach was applied to improve the thermostability of MTG by introducing an artificial disulfide bridge. As a result of narrowing down candidates using a rational approach, we successfully engineered a disulfide bridge into the N-terminal region of MTG by substituting Thr-7 and Glu-58 with cysteine. The T7C/E58C mutant was observed to have a de novo disulfide bridge and showed an increased melting temperature (Tm value) of 4.3 °C with retained enzymatic activity. To address the benefit-gained reason, we focused on the Cß temperature factor of the amino-acid residues that might form a disulfide bridge in MTG. Introducing the disulfide bridge had no remarkable effect on the mutant aiming to stabilize the high temperature factor. On the other hand, the mutation was effective on the relatively stable region. The introduction of a disulfide bridge may therefore be effective to stabilize further the relatively stable part. This finding is considered to be useful for the rational design of mutants aiming at heat resistance of proteins.Key Points• Microbial transglutaminase (MTG) is used as a binder in the food industry.• MTG has the potential for use in the manufacturing of various commercial materials.• Enhanced thermostability was observed for the disulfide bridge mutant, T7C/G58C.

Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis.

Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. Two enzyme engineering approaches were applied to improve MTG activity. One is a novel method of rational mutagenesis, called water-accessible surface hot-space region-oriented mutagenesis (WASH-ROM). One hundred and fifty-one point mutations were selected at 40 residues, bearing high solvent-accessibility surface area, within a 15 A space from the active site Cys64. Among them, 32 mutants showed higher specific activity than the wild type. The other is a random mutagenesis of the whole region of the MTG gene, coupled with a new plate assay screening system, using Corynebacterium Expression System CORYNEX. This in vivo system allowed us to readily distinguish the change in enzymatic activity by monitoring the intensity of enzymatic reaction-derived color zones surrounding recombinant cells. From the library of 24,000 mutants, ten were finally selected as beneficial mutants exhibiting higher specific activity than the wild type. Furthermore, we found that Ser199Ala mutant with additional N-terminal tetrapeptide showed the highest specific activity (1.7 times higher than the wild type). These various beneficial positions leading to increased specific activity of MTG were identified to achieve further enzyme improvements.