Characterization of halophilic bacterium nb2-1 from pla-ra and its protease production

A halophilic proteolytic bacterium, NB2-1, isolated from Pla-ra, fermented fish in Thailand, was identified as Virgibacillus marismortui on the basis of the genetic and phenotypic characteristics. NB2-1 produced a substantial level of extracellular proteolytic activity, corresponding with the growth and reaching the maximum at the end of the exponential phase (3 days). The optimum conditions required for maximum protease production were 15% (w/v) NaCl, pH at 9 and temperature at 30C. Of the media tested, maximum protease production occurred in a medium containing 15% NaCl, 0.5% yeast extract, 0.1% glutamic acid, 0.2% KCl, 0.3% trisodium citrate, 2% MgSO₄·7H₂O, 0.0036% FeCl₂·4H₂O and 0.000036% MnCl₂·4H₂O. The protease activity was optimally active at pH 10, 50C and 5% (w/v) NaCl when casein was used as a substrate. The activity was strongly inhibited by phenylmethane sulfonyl fluoride. Activity-stained substrate gel indicated the presence of several proteases with the estimated molecular weights of 17, 19, 24, 29 and 35 kDa in the crude NB2-1 extracellular protease. The results of the present investigation showed that the strain NB2-1 could be a potential source of thermoactive alkaline protease production. Halophiles are the most likely source of enzymes that not only are salt-tolerant but also thermotolerant. They constitute a heterogeneous group of microorganisms including species belonging to different genera. The isolation of halophiles able to produce extracellular enzymes will provide the possibility to have optimal activities at different salt concentrations. Virgibacillus marismortui NB2-1, a moderately halophilic bacterium isolated from Pla-ra, produced a substantial level of extracellular proteolytic activity that was active in extreme conditions. Using casein as a substrate, the protease exhibited maximal activity at pH 10, 50C in the presence of 5% (w/v) NaCl. The results of the present investigation showed that the strain NB2-1 could be a potential source of thermoactive alkaline protease production.