RESUMEN
A cold-active transglutaminase (TGase, EC 2.3.2.13) that catalyzes the reaction of protein glutamine + protein lysine â protein with γ-glutamyl-ε-lysine cross-link + NH3 at low temperatures was reported previously. This study verified the thermal stability of the TGase from 0-80 °C. Fluorescence and CD spectra studies confirmed tertiary structural damage at 40 °C, α-helix reduction at 60 °C, and refolding during cooling to 20 °C. The TGase sequence was obtained by transcriptomics and used to build its structure. Its catalytic triad was Cys333-His403-Asp426 and its catalytic process was inferred from the model. Molecular dynamics simulation illustrated that its cold activity resulted from its flexible active site, while high thermostability was conferred by an overall rigid structure, a large amount of stable Val and Lys, and strong electrostatic interactions at the N- and C- terminals. This study fills gaps in the correlation of conformational changes with stability and activity of TGase.
Asunto(s)
Proteínas de Artrópodos/química , Euphausiacea/enzimología , Transglutaminasas/química , Secuencias de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Biocatálisis , Dominio Catalítico , Frío , Estabilidad de Enzimas , Euphausiacea/química , Euphausiacea/genética , Calor , Cinética , Simulación de Dinámica Molecular , Replegamiento Proteico , Electricidad Estática , Transglutaminasas/genética , Transglutaminasas/metabolismoRESUMEN
The aim of this study was to investigate the effects of three different natural preservatives on the microbial profile, the total volatile base nitrogen (TVB-N), and biogenic amine contents of vacuum-packed chilled pork during storage at 4°C. Solution A comprised of tea polyphenols, chitosan, spice extract, propolis, and nisin. Solution B comprised of clove extract, cassia bark extract, ginger juice, garlic juice, and lactobacillus fermentation solution. Solution C consisted of only lactobacillus fermentation solution. The results indicated that solution A was a good natural preservative with higher bacteria inhibitory effect and higher sensory score than B and C. Besides the effect on appealing color, solution B could inhibit microbial activity although its inhibition effect was not as good as solution A. Thus, solution A could be used as a good preservative in industry. Solution C could inhibit the initial growth of Pseudomonas and partially inhibited the growth of Enterobacteriaceae; however, the content of putrescine in the pork treated with solution C was as high as 30.14 ± 2.89 mg/kg after 21 days of storage at 4°C. Hence, solution C is not an ideal preservative for vacuum-packed chilled pork.
RESUMEN
The inhibitory activities of buckwheat trypsin inhibitor (rBTI) towards trypsin were compared with soybean trypsin inhibitor (SBTI) in terms of their sensitivities to temperature, pH, salt ions and organic solvents. Both rBTI and SBTI were stable over a broad pH range of 2.0-12.0. rBTI exhibited higher thermal stability than SBTI. The inhibitory activity of rBTI was decreased by Zinc ions (Zn2+), KSCN, vitamin C and urea, while that of SBTI remained unchanged. However, H2O2, Mg2+ and Cu2+ ions had no significant effects on the inhibitory activities of rBTI and SBTI. Acetonitrile enhanced the inhibitory activity of rBTI, but had no effect on SBTI, while dimethylacetamide (DMAC) increased the inhibitory effect of both rBTI and SBTI. On the contrary, the inhibitory activities of rBTI and SBTI were reduced by isopropyl alcohol and methanol. The inhibition constants Ki of rBTI and SBTI were calculated to be 7.41 × 10-9 M and 6.52 × 10-9 M, respectively.
