Taxonomy and Protease of Halophilic bacteria isolated from Pla-ra

Abstract

In the isolation for protease-producing halophilic bacteria, fifty seven isolates from 46 samples of fermented fish, pla-ra collected from the markets and home made factories were isolated. These bacteria were divided into nine groups based on their phenotypic and chemotaxonomic characteristics including 16S rDNA sequences of the representative strains. Fifty-five strains were Gram-positive rods belonged to genus Virgibacillus 38 isolates, Halobacillus 6 isolates, Gracilibacillus 1 isolate and Bacillus 2 isolates. Two of Gram-negative rods were Salinivibrio and Chromohalobacter. They were identified as V. dokdonensis 10 isolates, V. halodenitrificans 13 isolates and V. marismortui 13 isolates and Virgibacillus sp. 1 isolate, Bacillus sp. 10 isolates, and C. salexigens 1 isolate. In addition, ND1-1 contained ubiquinone with 8 isoprene unit, cellular fatty acids of C16:0 and C12:0, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG). DNA G+C content was 49.0 mol%. The 16S rDNA sequence analyses indicated that strain ND1-1 was closely related to S. costicola and S. proteolytica 98.3-98.6%. Based on its low levels of DNA-DNA relatedness to the type strains of Salinivibrio, therefore it was proposed as S. siamensis sp. nov. TP2-8 contained MK-7, cellular fatty acids of anteiso-C15:0, iso-C15:0 and anteiso-C17:0, and the polar lipids of PG, DPG and unidentified glycolipid. DNA G+C content was 37.6 mol %. The 16S rDNA sequence analyses indicated that strain TP2-8 was different from Gracilibacillus (96.2-99.2%). Based on its low levels of DNA-DNA relatedness to the type strains of Gracilibacillus, therefore it was proposed as G. thailandensis sp. nov. Strain ND1-1 was selected for further study due to the high protease production. The moderately halophilic bacterium, strain ND1-1 produced extracellular protease at the middle of exponential phase. The maximum protease production of ND1-1 was at the beginning of stationary phase and could be achieved when cultivated in a JCM no.377 medium (pH 8.0) that replaced casamino acids with 0.5% skim milk and incubated at 37°C for 2 days. At the optimal condition, the crude protease produced by strain ND1-1 increased 6.25 times. The purified protease from ND1-1 was monomeric protein with the molecular mass of about 36.8 kDa. The enzyme had a maximal activity in the presence of 5% w/v NaCl, pH 8.0 at 55°C. Stability remained more than 50% in the presence of 5-30% w/v NaCl, pH 5.0-9.0 and at 30–55 °C. The ethylenediaminetetraacetic acid (EDTA) was found to inhibit the protease activity strongly, suggesting that the ND1-1 protease was metalloprotease.