Screening and identification of protease-producing halophilic bacteria from fermented fish, Pla-ra

Abstract

The screening of protease-producing halophilic bacteria from 65 pla-ra (fermented fish) samples collected at the markets was carried out. Forty-six isolates exhibited caseinolytic activity and 12 strains with high activity on JCM No. 168 agar with 15% NaCI containing 1% skim milk were selected for further characterization. On the basis of the phenotypic and chemotaxonomic characteristics including DNA-DNA similarity, they were separated into 3 groups, 5 strains (A, BN1-1, HUT1-1, NB2-1, NB3-1) in Group I; 6 (BKN1-1, PL1-1, PL3-1, PR5-1, SS1-1 and TSY4-4) in Group II; and 1 (ND1-1) in Group III. All the tested strains contained meso-diaminopimelic acid in the cell wall, except ND1-1. Menaquinone with 7 isoprene units was a major component. The G+C content of the tested strains ranged from 36.2 - 49.6 mol%. The 16S rDNA sequences similarity of the representative strains, BN1-1 was almost identical (99.8%) with Virgibacillus marismortui and SS1-1 and PR5-1 was 99.7% with V. halodenitrificans. Therefore, Group I strains were identified as V. marismortui, Group II were V. halodenitrificans, and Group III was left unidentified. The strain, NB2-1 identified as V. marismortui was selected for the further optimization of protease production. This strain had an optimum growth in the medium containing 15% NaCI and produced the highest protease activity at the stationary growth phase when cells were cultivated in a modified halobacterium medium JCM. No. 168 without casamino acid but containing 0.5% yeast extract and 15% NaCI, at pH 9 and at 30 °c. The optimum pH and temperature of the crude protease was pH 10 and 50 °c Thus, the enzyme was slightly or moderately thermophilic. The protease showed the highest activity in the presence of NaCI at 5 %. The phenylmethylsulphonylfluoride (PMSF) was found to inhibit the protease activity strongly, suggesting that the crude enzyme from NB2-1 was serine type protease.