Journal of Biosciences

, Volume 42, Issue 4, pp 537–546 | Cite as

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

Article
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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.

Keywords

Enzymatic properties microbial transglutaminase overlapping extension PCR purification 

Abbreviations

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

MTG

Microbial transglutaminase

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TG

Transglutaminase

Trx

Thioredoxin

Notes

Acknowledgements

This study was funded by the Nature Science Foundation of Hubei Province (BZY10033).

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Copyright information

© Indian Academy of Sciences 2017

Authors and Affiliations

  • Wenjie Wan
    • 1
  • Donglan He
    • 1
  • Zhijun Xue
    • 1
  • Zewen Zhang
    • 1
  1. 1.South-Central University for NationalitiesWuhanPeople’s Republic of China

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