Taxonomy and protease of halophilic bacteria isolated from fish sauce

Abstract

In order to isolate and screen for protease-producing halophiles, 40 isolates were isolated from fish sauce samples collected in Samutprakarn, Samutsongkram, Rayong and Singburi and from bu-du in Pattani provinces. On the basis of their phenotypic and chemotaxonomic characteristics. DNA-DNA similarity and the phylogenetic analysis using 16S rDNA sequences. 14 isolates of moderate halophiles belonged to Lentibacillus salicampi (3 isolates), L. juripiscarius sp. Nov (5 isolates), Chromohalobacter salexigens (4 isolates), Halobacillus sp. (1 isolate), and Filobacillus sp. (1 isolate). Lentibacillus and Filobacillus isolates contained meso-diaminopimelic acid in cell wall-peptidoglycan, MK-7 as predominant menaquinone, and phosphoglycerol and diphosphoglycerol as major polar lipids. The DNA G+C contents of Lentibacillus, Filobacillus, and Halobacillus isolates were 42.2 to 43.5, 36.5, and 40.3 mol%, respectively. The major cellular fatty acids of Lentibacillus and Filobacillus isolates were anteiso-C[subscript 15:0] and iso-C[subscript 15:0], respectively. Chromohalobacter salexigens isolates contained mesodiaminopimelic in cell wall-peptidoglycan, cyclo C[subscript19.0] [omega] 8c and C[subscript16.0] as the major cellular fatty acids, and phosphatidylamine as a polar lipid. The DNA G+C contents were 61.7 to 63.5 mol%. Twenty-six isolates of extreme halophiles were belonged to Halobacterium salinarum (10 isolates), Halococcus saccharolyticus (1 isolate) and Halococcus thailandensis sp. nov. (15 isolates). Halobacterium isolates comprised C[subscript20] C[subscript25] derivatives of phosphatidylglycerol and phosphatidylglycerol methylphosphate and MK -8 as the predominanat menaquinone. The DNA G+C contents were 63.4 to 64.7 mol%. Halococcus isolates contained C[subscript20] C[subscript25] and C[subscript20] C[subscript20] derivatives of phosphatidylglycerol and phosphatidylglycerol methylphosphate, and MK- 8(H[subscript2]). The DNA G+C contents were 60.2 to63.2 mol%. Two novel species of halophillic bacteria are proposed. In this study, five isolates of L. juripiscarius sp.nov. were differentiated from L. salicampi by several biochemical properties, low 16S rDNA sequence similarity less than 95% low DNA-DNA similarity (12 to 32%). Fifteen isolates of H. thailandensis sp.nov. were related to H. dombrowskii and H. morrhuae with 99.1 and 98.6% 16s rDNA sequence similarity, respectively. They were distinguished from H. dombrowskii, Halococcus morrhuae and the other Halococcus species by several phenotypic characteristics and DNA-DNA similarity ([is less than or equal to] 56.3%). Nine isolates of extreme halophiles and seven isolates of moderate halophiles corresponding to 39% of the total isolates showed caseinolytic forming colonies on the plate. SR5-3 showed the highest protease activity in the presence of 10% NaCl. This isolate was selected for purification and characterization of protease. Based on the 16S rDNA sequence similarity, SR5-3 is assumed as a new species of Halobacillus, Halobacillus sp. SR5-3. Production of protease of the SR5-3 was enhanced to 100-fold by the addition of aspartic acid, glutamic acid, and glycine into the JCM No 377 medium. The protease from this bacterium was purified and characterized. Molecular weight of the purified enzyme was estimated as 43 kDa. The enzyme showed the highest activity at 50 ํC and pH 9~10, and highly stable in the presence of 20-35% NaCl by using Suc-Ala-Ala-Phe-Pro-MCA as substrate. The protease activity was increased about 2-fold and 2.5 -fold by the addition of 5% and 20-35% NaCl, respectively using Suc-Ala-Ala-Pho-Pro-MCA as a substrate. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and [alpha]-microbial alkaline protease inhibitor (MAPI). Substrate specificity of the enzyme was determined by using fluorescence resonance energy transfer substrate (FRETS) combinatorial libraies, One of the unique features was that the enzyme absolutely preferred Ile at the P2 position of the substrates and it was found to prefer three amino acids, Leu, Gln and Ala at the P1 position.