Purification and characterization of a novel transglutaminase from filarial nematode Brugia malayi

A transglutaminase (pTGase) was purified from filarial nematode, Brugia malayi. The steps used for purification were thermoprecipitation, ammonium sulfate precipitation, gel filtration on Superose 12 HR 10/30, ion‐exchange chromatography on a Mono‐Q column and further gel filtration on Superose 12 HR 10/30. The last step yielded an electrophoretically homogenous enzyme protein with 2200‐fold purification and a reproducible yield of approximately 20%. The purified enzyme had a molecular mass of 56 kDa, specific activity of 2.25 U/mg protein and an isoelectric point of 7.2. The enzyme was active in the basic pH range with an optimum activity at pH 8.5. The pTGase activity was Ca²⁺‐dependent and was inhibited by ammonia, primary amines, EDTA, and ‐SH group blocking reagents. The enzyme activity was also inhibited by high salt (NaCl and KCl) concentrations, detergents, metal ions, and organic solvents. Ampholine (pH 6–8) at 1% (by vol.) caused about 20% inhibition of pTGase activity but at 3% (by vol.) the inhibition increased up to 80%. Similarly, the micromolar concentrations of GTP inhibited the enzyme activity only moderately but at millimolar concentration a significant inhibition was observed. The stability of the pTGase was not affected by 0.1% SDS or other physical parameters such as freezing and thawing. Further, the pTGase was found to be highly thermostable (stable at 60°C for several hours) with optimum activity observed at 55°C. The distinct substrate specificity, unique N‐terminal sequence along with the other physico‐chemical properties studied, suggested that pTGase is a novel member of transglutaminase family.