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A two-step refolding of acid-denatured microbial transglutaminase escaping from the aggregation-prone intermediate

Mototaka Suzuki1, Kei-ichi Yokoyama, Young-Ho Lee

  • 1Institute for Innovation, Ajinomoto Co, Inc, Suzuki-cho 1-1, Kawasaki-ku, Kawasaki 210-8681, Japan. mototaka_suzuki@ajinomoto.com

Biochemistry
|October 29, 2011
PubMed

Insights

Microbial transglutaminase (MTG) unfolding is pH-dependent, forming a molten globule intermediate. A two-step refolding method enhances yield, proving beneficial for larger proteins.

Area of Science:

  • Biochemistry
  • Protein Chemistry

Background:

  • Microbial transglutaminase (MTG) is a globular enzyme crucial in various biotechnological applications.
  • Understanding MTG's conformational stability is key to optimizing its use and refolding processes.

Purpose of the Study:

  • To investigate the conformational changes of MTG across a pH range of 2.0-6.0.
  • To identify conditions influencing MTG unfolding and refolding.
  • To develop an optimized refolding strategy for MTG.

Main Methods:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure.
  • 1-anilino-8-naphthalenesulfonate (ANS) binding to probe tertiary structure.
  • pH and NaCl concentration-dependent phase diagram construction.

Main Results:

  • MTG undergoes stepwise unfolding below pH 4.0 at low ionic strength, forming a molten globule intermediate.
  • High ionic strength stabilizes MTG, preventing full unfolding.
  • A two-step refolding process (pH 4.0 then pH 6.0) significantly improved refolding yield compared to direct refolding.

Conclusions:

  • The fully unfolded state of MTG is accessible only under specific low pH and low ionic strength conditions.
  • A two-step refolding strategy effectively suppresses kinetic traps and enhances refolding yields.
  • This optimized refolding approach is potentially applicable to larger proteins with high isoelectric points (pI).

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