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

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Abstract
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 < 0.05 or P < 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.
Funding Information
  • Nature Science Foundation of Hubei Province (BZY10033)

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