Specific mutation of transglutaminase gene from Streptomyces hygroscopicus H197 and characterization of microbial transglutaminase.

Microbial transglutaminase (MTG) gene (mtg) from Streptomyces hygroscopicus H197 strain was cloned by PCR and mutated by deleting a specific 84 bp fragment using overlapping extension PCR. The mutant MTG and the wild MTG genes expressed by recombinant plasmid pET32a+- mutant mtg and pET32a+ -mtg, respectively, and were harvested by alternating freeze-thaw steps and purified by Ni column. The purified mutant MTG and the wild MTG exhibited 0.22 U/mg and 0.16 U/mg activity, respectively, and 0.69 U/mg and 0.54 U/mg activity, respectively, after activated by trypsin. The molecular weight of mutant MTG was estimated as 67 kDa by SDS-PAGE. Both MTGs showed optimum activity at pH 6-8 for hydroxamate formation from N-CBZ-Gln-Gly and hydroxylamine, and exhibited higher stability at 40°C and 1-3% salinity. The two types of MTG were not stable in the presence of Zn(II), Cu(II), Hg(II), Pb(II), Fe(III), and Ag(I), suggesting that they could possess a thiol group. In addition, the mutant MTG and the wild MTG were strongly affected by ethanol. Furthermore, the mutant MTG was obviously (P less than 0.05 or P less than 0.01) more stable than the wild MTG at 50°C and 60°C, at pH 4, 5, and 9, at 7 % and 9 % salinity, 30 % and 35 % ethanol concentration, and in the presence of Li(I) and Ag(I). The polyhydroxy compounds as protein stabilizers could elevate MTG stability.