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

Journal of Biotechnology 136 (2008) 156–162 Contents lists available at ScienceDirect Journal of Biotechnology j o u rn al h omepage: www.elsevi er.c om /locate/j biotec Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants Christian K. Marx, Thomas C. Hertel, Markus Pietzsch ∗ Department of Downstream Processing, Faculty of Natural Sciences I, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, 06099 Halle (Saale), Germany a r t i c l e i n f o a b s t r a c t Article history: Recombinant microbial transglutaminase (rMTG), an enzyme useful for the cross-linking or the posttrans- Received 16 January 2008 lational modification of (therapeutic) proteins, was optimized by random mutagenesis for the first time. A Received in revised form 9 June 2008 screening method was developed which, in addition to state-of-the-art procedures, includes a proteolytic Accepted 17 June 2008 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, Keywords: isolated and characterized. After just one round of mutagenesis, nine variants with a single amino acid Random mutagenesis ◦