Enzymatic activity and thermoresistance of improved microbial transglutaminase variants.

Enzymatic activity and thermoresistance of improved microbial transglutaminase variants.
Amino Acids. 2019 Jul 26;:
Authors: Böhme B, Moritz B, Wendler J, Hertel TC, Ihling C, Brandt W, Pietzsch M
Abstract
Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6...

Source: Amino Acids - July 25, 2019 Category: Biochemistry Authors: Böhme B, Moritz B, Wendler J, Hertel TC, Ihling C, Brandt W, Pietzsch M Tags: Amino Acids Source Type: research