Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i) motile ii) rod shaped iii) non spore forming iv) catalase and amylase positive v) able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7) exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp). The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

Characterization of Leptospira borgpetersenii isolates from field rats (Rattus norvegicus) by 16S rRNA and lipL32 gene sequencing

The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 %, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.

Purification and characterization of an extreme halothermophilic protease from a halophilic bacterium Chromohalobacter sp. TVSP101

An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75°C. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v) were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM), 0.1% SDS and PMSF (1mM). The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v) DMSO, DMF, ethanol and acetone.