Continuous enzyme-coupled assay for microbial transglutaminase activity

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• 作者：Samuel K. Oteng-Pabi ; Jeffrey W. Keillor
• 关键词：Microbial transglutaminase ; Glutamate dehydrogenase ; Enzyme-coupled assay ; Microtiter plate ; Transamidation ; Hydroxamate assay
• 刊名：Analytical Biochemistry
• 出版年：2013
• 出版时间：15 October, 2013
• 年：2013
• 卷：441
• 期：2
• 页码：169-173
• 全文大小：410 K

文摘

Transglutaminases (protein-glutamine:amine ¦Ã-glutamyltransferase, EC 2.3.2.13) are a family of calcium-dependent enzymes that catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When a lysine residue acts as the acyl-acceptor substrate, a ¦Ã-glutamyl-¦Å-lysine isopeptide bond is formed. This isopeptide bond formation represents protein cross-linking, which is critical to several biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond formation. Furthermore, mTG¡¯s promiscuity in acyl-acceptor substrate preference highlights its biocatalytic potential. The acyl-donor substrate, however, is limited in its scope; the amino acid sequences flanking glutamine residues dramatically affect substrate specificity and activity. Here, we have developed and optimized a modified glutamate dehydrogenase assay with the intention of analyzing potential high-affinity peptides. This direct continuous assay presents significant advantages over the commonly used hydroxamate assay, including generality, sensitivity, and ease of manipulation. Furthermore, we identified 7M48 (WALQRPH), a high-affinity peptide that shows greater affinity with mTG (K_M = 3 mM) than the commonly used Cbz-Gln-Gly (K_M = 58 mM), attesting to its potential for application in biocatalysis and bioconjugation.