Characterisation of a novel cold‐adapted calcium‐activated transglutaminase: implications for medicine and food processing

Abstract

Transglutaminases are a family of enzymes that catalyse the cross‐linking of proteins by forming covalent bonds between lysine and glutamine residues in various polypeptides. Cross‐linking reactions are involved in blood clots, skin formation, embryogenesis and apoptosis. Clinically, these enzymes appear to be implicated in neurodegenerative diseases, tumours and coeliac diseases. Transglutaminases have great potential for use in the food industry because of their ability to cross‐link proteins that are not normally linked. Here, a gene coding for transglutaminase from Atlantic cod was cloned into a bacterial expression vector and used to transform protein expression in a strain of Escherichia coli. The successful expression of recombinant transglutaminase protein from Atlantic cod (AcTG‐1) as a soluble protein upon induction at low temperature was confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry analysis. Biochemical characterisation demonstrated that the transglutaminase was active between 0 and 65 °C, but was completely inactivated after 20‐min incubation at 70 °C. Interestingly, the enzyme displayed cold‐adapted features, such as temperature instability combined with high catalytic efficiency at low temperatures (8–16 °C). In addition, the enzyme had optimal activity at 50 °C, a new feature for a cold‐adapted enzyme. AcTG‐1 was active in the pH range from 6 to 9, with an optimum at pH 8, and required 5 mm calcium for maximum activity. Potential calcium‐binding sites in the enzyme were predictable, making the enzyme an appropriate model for studying structure–function relationships in the calcium‐dependent transglutaminase family. In vitro gel analysis revealed that transglutaminase cross‐linked casein, collagen and gelatin. The binding of fish fillets in the presence of recombinant AcTG‐1 provided further macroscopic proof for the potential application of AcTG‐1 as a biological cross‐linker in the food industry. Once binding occurred, fish fillets withstood further processing such as frying, boiling, freeze‐thawing and chilling. The low‐temperature activity and new enzymatic properties of AcTG‐1 appear to offer advantages over commercially available enzymatic glues in the food industry.

The temperature and pH repertoire of the transglutaminase family is expanding

“…Characterizing and sequencing the cAcTG [9] represents a new potential strategy for molecular stable, resistant covalent cross‐linking in cold milieu, not only in the processed food facilities, but also in pharmacology, biotechnology, bioconjugation, and antibody–drug conjugates for diagnostic laboratories and medical therapy. The cold‐adapted enzyme expends TG family versatility, maximizes TG advantages, widens their temperature and pH repertoire of activity, and might avoid undesired cross‐linked products.…”

“…The reader might ask how all of the above is related to the cAcTG? [9]. It is related to the heavy use of mTG in processed food with the immunogenicity and potential pathogenicity of its cross‐linked protein complexes [2,5–7,10,12–14].…”

“…The newly identified cAcTG may be a candidate for colder reactions, and thermostable mTG can be used for heated/boiled/cooked food product manufacturing. While TG isolated from cold Atlantic cod fish has mammalian features, such as the need for calcium for activation [9], thermophilic mTG, to our knowledge, was not previously described. Active thermophilic bacteria from hot springs are constantly reported.…”

“…This was a topic of criticism as it was assumed that no active mTG reached the human intestinal lumen, due to its industrial temperature inactivation and inability to resist the gastric acidic pH [8]. In this regard, Alvarez et al should be congratulated for addressing the topic of temperature and pH dependency of TG, by characterizing a novel cold‐adapted TG enzyme and indicating its potential application for medicine and food processing, as described in a research article published in this issue [9]. In the present accompanying commentary, we expand discussion from the animal TG repertoire to human enteric lumen TG activity.…”

Enzyme‐catalysed polymer cross‐linking: Biocatalytic tools for chemical biology, materials science and beyond

“…This has the potential for use in the processing of chilled foods, where the higher temperatures currently required for transglutaminase activity can lead to food spoilage. [16] The adoption of fibrin-stabilising factor XIII and its eukaryotic relatives as generic cross-linking agents has, however, been limited by both its calcium dependency and challenges associated with its large-scale manufacture in recombinant form, issues that have been addressed through the use of prokaryotic transglutaminases.…”