Isopeptidase activity of human transglutaminase 2:disconnection from transamidation and characterization by kinetic parameters

Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca2+-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins or γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the Km and the Vmax kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2 -driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of cross-linked proteins correlates with the manifestation of degenerative disorders.

Comprehensive characterization of Napier grass as a feedstock for thermochemical conversion

Study on Napier grass leaf (NGL), stem (NGS) and leaf and stem (NGT) was carried out. Proximate, ultimate and structural analyses were evaluated. Functional groups and crystalline components in the biomass were examined. Pyrolysis study was conducted in a thermogravimetric analyzer under nitrogen atmosphere of 20 mL/min at constant heating rate of 10 K/min. The results reveal that Napier grass biomass has high volatile matter, higher heating value, high carbon content and lower ash, nitrogen and sulfur contents. Structural analysis shows that the biomass has considerable cellulose and lignin contents which are good candidates for good quality bio-oil production. From the pyrolysis study, degradation of extractives, hemicellulose, cellulose and lignin occurred at temperature around 478, 543, 600 and above 600 K, respectively. Kinetics of the process was evaluated using reaction order model. New equations that described the process were developed using the kinetic parameters and data compared with experimental data. The results of the models fit well to the experimental data. The proposed models may be a reliable means for describing thermal decomposition of lignocellulosic biomass under nitrogen atmosphere at constant heating rate.