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.
Highlights
Transglutaminases (TGs; protein–glutamine y-glutamyltransferases, EC 2.3.2.13) were first described in 1959 and found to form extensively cross-linked, generally insoluble protein polymers [1,2]
Recombinant expression of the construct, pETAcTG1, in E. coli BL21 cells at 13 °C yielded a recombinant protein with a molecular weight of about 80 kDa upon protein purification and Coomassie Brilliant Blue staining after SDS/PAGE (Fig. 1, lane 3)
We have succeeded in producing recombinant AcTG-1 from Atlantic cod in E. coli
Summary
Transglutaminases (TGs; protein–glutamine y-glutamyltransferases, EC 2.3.2.13) were first described in 1959 and found to form extensively cross-linked, generally insoluble protein polymers [1,2]. These structures were important for organisms in the formation of skin barrier, hair growth, wound healing and blood clotting [3]. Transglutaminases are a family of enzymes that catalyse the cross-linking of proteins by forming covalent bonds between glutamine (Q) and lysine (K) residues in different polypeptides Such iso-peptide bonds created by TG are highly resistant to proteolysis and increase the mechanical stability of the modified proteins [4]. TGs have been linked to the development of chloroplasts [12], while TGs in fish seem to be involved in immune defence [13]
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