Purification and Characterization of an Alkaline Protease Produced by the Bacterium Xenorhabdus nematophila BA2, a Symbiont of Entomopathogenic Nematode Steinernema carpocapsae

Xenorhabdus nematophila, a bacterium pa thogenic for insects associated with the nematode Steinernema carpocapsae, secretes p roteases during growth. As the b acterial population i ncreased, the proteolytic activities increased and r eached its maximum l evel at the stationary phase, after 48 h of inoculation. A reduction in the enzymatic activity was recorded by increasing the time of inoculation. The isoenzyme pat tern of proteolytic a ctivities re vealed t hat one slow and two f ast isoenzymes were r eleased during bacterial gr owth. The major iso enzyme, X. n ematophila PAII, was purified to homogeneity in a three-step procedure involving ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. The purified enzyme had a specific activity of 295 units/mg protein with purification fold 7.7 over crude extract. A m olecular weight of 39 kDa wa s estimated f or bot h t he native and denatured enzyme, suggesting that the enzyme is monomeric. The enzyme had characteristics of a c old-adapted protein, it was more active i n t he range of 15 to 30 C and had an optimum activity at 30 C. The oo activation energy for the hydrolysis of azocasein was determined to be 16.1 kcal/mol. Temperatures above 30 C have deleterious e ffect o n the enzyme activity and stability. The enzyme activity was totally o abolished by 1 mM EDTA and 1,10-phenanthroline, b ut no t affe cted by c ysteine, se rine and aspartyl protease inhibitors. X. n ematophila PAII had an optimum pH of 8.5 and classified as an al kaline metalloprotease. Its substrate specificty strengthens the possibility that it is involved in degradation of insect tissues for providi ng nut rients to the ass ociated nematode, which i s unable t o develope and reproduce inside the infected insect without a previous bioconversion of the insect cadavers by the symbiotic bacteria. Its biochemical characteristics were compared with those previously reported for different species of animal pathgenic bact eria.