Screening and identification of protease-producing moderately halophilic bacteria from salt fermented foods

Abstract

An isolation and screening of protease-producing moderately halophilic bacteria from 30 salt fermented foods (pla-ra, pla-jom, shrimp paste, fermented crab, nam-pla and mang-da dong) collected from markets and factories, 54 isolates which exhibited caseinolytic activity on JCM No. 377 agar containing 1% skim milk and 10% NaCl were selected and identified. On the basis of their phenotypic and chemotaxonomic characteristics including phylogenetic analysis using 16S rRNA gene sequence, they were divided into 7 groups. Ten isolates were belonged to Halobacillus, 24 Virgibaciilus., 4 Bacillus, 4 Oceanobacillus, 4 Idiomarina, 5 Salinivibrio and 3 Halomonas. The similarity of 16S rRNA gene sequence revealed that representative strains of Group 1, TP4-4 was closely related to Halobacillus trueperi KCTC 3686T(98.5%) and TSN17 was closely related to Halobacillus salinus JCM11546T (96.8%). Group 2, TKNR13-3 was closely related to Virgibcillus halodenitrificans JCM 12304T (99.3%) and TCK24 was closely related to Virgibacillus dokdonensis KCTC 3933T (99.4%). Group 3, TPR1-2 showed 98% similarity to Bacillus aquimaris KCCM 14589T. TPS12 in group 4 showed 99% similarity to Oceanobacillus picturae LGM 19492T, TPS4-2 in group 5 showed 99% similarity to Idiomarina loihiensis DSM 15497T, TM5-3 in group 6 showed 99% similarity to Salinivibrio costicola ATCC 33508Tand TKK10 in group 7 showed 97% similarity to Halomonas alimentaria KCCM 41042T. Tested strains of Halobacillus, Virgibcillus, Bacillus and Oceannobacillus contained meso-diaminopimelic in cell wall peptidoglycan and menaquinone with 7 isoprene units (MK-7) was presented in Halobacillus strain. DNA G + C content of the tested strains ranged from 35-53.73 mol%. In addition, DNA-DNA hybridization experiments have been done to confirm the identification of Halobacillus and Virgibcillus strains. Specificity protease activitiy of the 54 isolates ranged from 0.0145-4.229 U/mg protein. The highest protease producer, TKNR13-3, had maximum growth in the medium containing 10% NaCl and showed the highest protease production in stationary growth phase when cells were cultivated in JCM. No. 377 which casamino acids was replaced by 2% yeast extract and containing 5% NaCl, pH 6.5 and at 37 °C for 3 days. The optimum pH, salt concentration and temperature for crude protease activity were pH 8, 15% NaCl and 60 ºC. Thus, the enzyme was slightly or moderately thermophilic. Protein with molecular weight of 12, 21, 29, 39 and 49 KDa in crude protease exhibited caseinolytic activity.The protease activity was strongly inhibited (77.62%) by chymostatin. Therefore, the protease of strain TKNR13-3 was serine protease type chymotrypsin.