| Transglutaminase (EC 2.3.2.13) is an enzyme that catalyzes the transfer of acyl group, resulting the cross-linking between a γ-carboxyamide group of glutaminyl residues and an acyl acceptor, which could dramatically change the function and the property of proteins. Currently, transglutaminase has been widely used in many industrial areas, such as food engineering, textile and leather processing, material engineering, and biomedical engineering. More interests have been focused on microbial transglutaminase (MTG) because of its Ca2+-independence and broad substrate specificity for acyl donors. However, the low catalytic activity and the poor thermal stability of MTG limit its industrial applications.In this study, the zymogen gene (promtg) from Streptomyces mobaraensis (S. mobaraensis) and its five mutated genes (promtg-s2p, promtg-w69a, promtg-sl99a, promtg-s23v-y24n-k294l, and promtg-s23v-y24n-s199a-k294l) were expressed respectively in Escherichia coli (E. coli) BL21 (DE3). After induction, expression, activation and purification, six kinds of purified recombinant MTGs were obtained for further research on their enzymatic properties. Moreover, the fermentation of MTG by E. coli BL21(DE3)/pET-promtg was preliminary optimized. The main results are listed as follows:(1) Based on pET-28c(+) expression vectors, pET-28c(+)-promtg, pET-28c(+)/s2p, pET-28c(+)/w69a, pET-28c(+)/s199a, pET-28c(+)/s23v-y24n-k294l and pET-28c(+)/s23v-y24n-sl99a-k294l, each of which contains an original or a mutanted promtg gene, were successfully constructed by using S. mobaraensis genomic DNA as the template. The recombinant vector was transformed to E. coli BL21 (DE3), respectively, to obtain six expressing strains:E. coli BL21(DE3)/pET-promtg, E. coli BL21(DE3)/pET-s2p, E. coli BL21(DE3)/pET-w69a, E. coli BL21(DE3)/pET-s199a, E. coli BL21(DE3)/pET-s23v-y24n-k2941 and E. coli BL21(DE3)/pET-s23v-y24n-s199a-k2941. After induction, expression, activation and purification, MTG and its five variants were successfully obtained, which are MTG/S2P (serine at the position 2 replacing by proline), MTG/W69A (tryptophan at the position 69 replacing by alanine), MTG/S199A (serine at the position 199 replacing by alanine), MTG/S23V-Y24N-K294L (serine, tyrosine, and lysine at the position 23,24, and 294 replacing by valine, asparagine, and leucine, respectively) and MTG/S23V-Y24N-S199A-K294L (serine, tyrosine, serine, and lysine at the position 23,24,199, and 294 replacing by valine, asparagine, alanine, and leucine, respectively). The specific activity of MTG, MTG/S2P, MTG/W69A, MTG/S199A, MTG/S23V-Y24N-K294L and MTG/S23V-Y24N-S199A-K294L were 25.35±0.48 U/mg,44.05±0.75 U/mg,39.77±0.44 U/mg,54.62±0.38 U/mg,34.43±0.76 U/mg,53.40±0.87 U/mg, respectively. Among them, MTG/S199A had the highest specific activity, which was almost 2.2 times as much as that of MTG.(2) It was found that the optimal reaction pH values of all recombinant MTGs were tested to be 7.0. All recombinant MTGs have relatively good stablility in the pH range of 5.0-9.0 except for MTG/W69A. The optimal reaction temperature of MTG, MTG/S2P, MTG/S199A, MTG/S23V-Y24N-K294L and MTG/S23V-Y24N-S199A-K294L were 50℃, while for MTG/W69A, it was 40℃. All recombinant MTGs are able to stay stable in the range of 20-40℃ except for MTG/W69A. The specific activity of MTG, MTG/S2P and MTG/S199A declined rapidly at 50℃, while MTG/S23V-Y24N-K294L and MTG/S23 V-Y24N-S199A-K294L remained higher than 65% of relative activity at 50℃. All recombinant MTGs would be inactived at 60℃. Except for MTG/W69A, the half-life (t1/2) at 50℃ and 60℃ of mutated MTGs increased when compared to that of MTG, and the ti/2(50℃) of MTG/S23V-Y24N-K294L and MTG/S23V-Y24N-S199A-K294L increased by nearly 27 times and 26 times, respectively. K+, Ca2+, Mn2+, Mg2+, Ba2+ and Na+ ions slightly improved the specific activity of MTGs; Li+ and Co2+ ions slightly inhibited the specific activity of MTGs; and Zn2+, Fe3+, Sn2+, Cu2+ and Pb2+ ions had a strong inhibitory effect on the specific activity of MTGs. The Km values of MTG, MTG/S2P, MTG/W69A, MTG/S199A, MTG/S23V-Y24N-K294L and MTG/S23V-Y24N-S199A-K294L were 66.5 mmol/L,23.6 mmol/L,19.2 mmol/L,18.2 mmol/L,37.6 mmol/L and 18.6 mmol/L, respectively. Compared to MTG, the Km values of mutated MTGs were significantly decreased, which might be caused by the increasing substrate affinity of mutated MTGs.(3) The secondary structures of recombinant MTGs detected by Circular Dichroism spectrum were generally similar to each other, which contained a lot of β-sheets and random coils, while the content of a-helix and β-turn were relatively small. Homologous modeling of the crystal structure of recombinant MTGs was run by SWISS-MODEL software. It was found that the 3D structures of recombinant MTGs were basically the same, which were all disc-shaped and have a deep v-shaped cracks in the margin of enzymes.(4) The growth curve of E. coli BL21 (DE3)/pET-promtg showed that the lag phase was in 0-3 h, the logarithmic phase was in 3-8 h, the stable phase was in 8-10 h, and the bacterium entered the death decline after 10 h. The fermentation of MTG by E. coli BL21 (DE3)/pET-promtg was optimized in 250 mL shake-flasks using single-factor experiment, Plackett-Burman design and Box-Benhnken design. The optimal composition of media were tested to be 28 g/L of yeast extract,14 g/L of peptone,12.54 g/L of K2HPO4,2.31 g/L of KH2PO4,5 g/L of glycerol and 0.1 g/L of kanamycin at pH 7.0. When the bacteria were cultured in 50 mL, pH 7.0 optimized media at 34℃ for 4 h with 5%(V/V) inoculum and induced by 0.6 mmol/L IPTG at 25℃ for 14 h, the maximum specific activity of MTG was 1.51±0.03 U/mg, which was nearly 1.6 times as much as the specific activity of MTG (0.96±0.08 U/mg) produced in initial conditions. |
PDF Full Text Request