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Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants

Paper ID Volume ID Publish Year Pages File Format Full-Text
24536 43524 2008 7 PDF Available
Title
Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants
Abstract

Recombinant microbial transglutaminase (rMTG), an enzyme useful for the cross-linking or the posttranslational modification of (therapeutic) proteins, was optimized by random mutagenesis for the first time. A screening method was developed which, in addition to state-of-the-art procedures, includes a proteolytic activation step of the expressed soluble pro-enzyme. The library of 5,500 clones was screened for variants with increased thermostability and heat-sensitivity, respectively. Mutant enzymes were overproduced, isolated and characterized. After just one round of mutagenesis, nine variants with a single amino acid exchange showed a remarkably increased thermostability at 60 °C. The exchange of a serine residue close to the N-terminus against proline resulted in an rMTG mutant (S2P) with 270% increased half-life. Seven variants exhibited an increased heat-sensitivity at 60 °C of which one mutant (G25S) retained its specific activity between 10 and 40 °C. The mutations responsible for the increased thermostability and the heat-sensitivity were identified and assigned to the three-dimensional (3D) structure. All single point mutations related to changed thermal properties of rMTG are located in the N-terminal domain (i.e. the left side wall of the active site cleft of the front view of the MTG as defined by the literature [Kashiwagi, T., Yokoyama, K., Ishikawa, K., Ono, K., Ejima, D., Matsui, H., Suzuki, E., 2002. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J. Biol. Chem. 277, 44252–44260] showing the importance of this part of the protein.

Keywords
Random mutagenesis; Recombinant microbial transglutaminase; pro-MTG-His6; Streptomyces mobaraensis; High throughput screening; Thermostability
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Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 136, Issues 3–4, 10 September 2008, Pages 156–162
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us