Isolation, purifi cation and characterisation of transglutaminase from rosemary (Rosmarinus officinalis L.) leaves

Abstract

Background. Rosemary (Rosmarinus offi cinalis L.) is a spice and medicinal herb widely used around the world of the natural antioxidants, and it has been widely accepted as one of the spices with the highest antioxidant activity. Transglutaminase (EC 2.3.2.13; TGase) is an enzyme capable of catalysing acyl transfer reactions by introducing covalent cross-links between proteins, as well as peptides and various primary amines. TGase activity in plants was fi rst observed in pea seedlings, and subsequently found in organs of both lower and higher plants. Recently, TGase has captured researchers' interest due to its attractive potential application in food industries. Therefore, the objectives of this study are isolation and purifi cation of TGase from new plant source rosemary (Rosmarinus offi cinalis L.) leaves at the laboratory scale. Moreover, investigation of the biochemical properties of the purifi ed TGase to provide a suitable TGase enzyme for food industry applications are in focus. Material and methods. Rosemary (Rosmarinus offi cinalis L.) leaves was used as a new plant source to TGase. The biochemical characteristics of the crude and purifi ed enzyme were determined. Results. Rosemary (Rosmarinus offi cinalis L.) TGase was purifi ed to homogeneity by successive three purifi cation steps including ammonium sulfate precipitatation, ion exchange chromatography on DEAE-Sephadex A-50 column and Size exclusion column chromatography on Sephadex G-100 column. Under experimental conditions, 20-30% of ammonium sulfate saturation in the enzyme solution had a high yield of enzyme activity could be obtained. The purifi ed enzyme from the Sephadex G-100 column had 21.35% yield with increased about 7.31 in purifi cation fold. Rosemary TGase exhibited optimum activity at pH 7.0 and 55°C for the catalytic reaction of hydroxylamine and Z-Gln-Gly. The purifi ed TGase almost maintained full activity after incubation for 15 min up to 60°C and it was completely inactivated at 85°C. The rosemary TGase was stimulated at 2-6 mM CaCl 2 concentrations while it lost about 5-20% from its activity by increasing CaCl 2 concentration. Sodium chloride (2-14%) shows no noticeable inhibition of the purifi ed TGase activity. Mg +2 , Ba +2 were acivited by the purifi ed TGase while it was strongly inhibited by Fe +2 , moderately by Cu +2 and Mn +2 . Conclusion. This paper reports on the purifi cation and characterisation of TGase from newly isolated plant, rosemary (Rosmarinus offi cinalis L.) leaves. Finding results of the TGase properties make this enzyme a good candidate for application in the food industry. However, additional work is required to increase activity yield during extraction and purifi cation for commercial scale of TGase from this plant.

Characterization of Transglutaminase Isolated from Rosemary Leaves, and Development of Chemical, Rheological and Sensory Properties of Kareish Cheese Made with Transglutaminase.

“…Transglutaminase in a crude extract was prepared by overnight soaking of 40 gm Rosmarinus officinalis L. powdered leaves in 0.2 M Tris-HCl buffer pH 7.5 including, (25mM sucrose, 0.05% β-mercaptoethanol, 3% Polyvinylpyrrolidone and 10 mM EDTA), then mixture was filtrated and centrifuged at 6000 g for 15 min at 5 °C . Resultant supernatant was gathered and placed in screw cap tube for further determinations included transglutaminase activity and protein content (El-Hofi et al, 2014).…”

“…Screw cap tubes including the enzyme extract and substrate were kept for 10 min at various temperatures ranging from 30-80 °C (El-Hofi et al, 2014).…”

“…The extract of enzyme was heated at various temperature ranging from 60-80°C for 10 and 20 min, subsequently rapid cooling to 37°C and determined for residual activity of enzyme (El-Hofi et al, 2014).…”

“…The TGase activity was also found in barley, pea, wheat (Lilley et al, 1998). TGase was recently purified and identified from leaves of rosemary (Rosmarinus officianlis L.) (El-Hofi et al, 2014).…”

Effect of Rosemary Transglutaminase on Yoghurt Fortified with Whey Protein Isolate

“…Rosemary is a spice and medicinal herb widely used around the world as the natural antioxidant; it has been widely accepted as one of the spices with the highest antioxidant activity [Genena et al, 2008]. RTGase exhibited optimum activity at pH 7.0 and 55°C for the catalytic reaction of N-carbobenzoxy-L--glutaminylglycine and hydroxylamine [El-Hofi et al, 2014].…”