Document Detail


Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis.
MedLine Citation:
PMID:  21863232     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.
Authors:
Karin Buettner; Thomas C Hertel; Markus Pietzsch
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-8-24
Journal Detail:
Title:  Amino acids     Volume:  -     ISSN:  1438-2199     ISO Abbreviation:  -     Publication Date:  2011 Aug 
Date Detail:
Created Date:  2011-8-24     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9200312     Medline TA:  Amino Acids     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Department of Downstream Processing, Institute of Pharmacy, Faculty of Sciences I, Martin Luther University Halle-Wittenberg, 06099, Halle (Saale), Germany.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  The non-protein amino acid ?-N-methylamino-L: -alanine in Portuguese cyanobacterial isolates.
Next Document:  Induction of multiple reinstatements of ethanol- and sucrose-seeking behavior in Long-Evans rats by ...