Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5

Abstract

Background: Due to the importance of microbial proteases in biotechnological applications, a number of microorganisms are being explored. The production, purification and characterisation of extracellular metallo-proteases by producing Bacillus sp. KG5 was studied. Material and Methods: Bacterial strain KG5 was isolated from Kös (Bingöl) hot spring. The strain KG5 was identified by morphological, physiological, biochemical and 16S rRNA gene sequencing. The effects of various parameters on protease production, such as time, temperature, pH, carbon and nitrogen sources and CaCl2 were studied. The enzyme was purified by ammonium sulphate precipitation and Sephadex G-75 gel permeation chromatography. Molecular weight was calculated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymographic analysis. The effects of some metal ions, chelators and inhibitors on enzyme activity were determined. Results: The optimum temperature, pH and incubation period for protease production were 40-45°C, 7.0 and 24 h, respectively. It was determined that the best nitrogen sources were yeast extract and urea, while the best carbon sources were lactose and galactose. However, glucose as a source of carbon was found to inhibit the production of the enzyme. The maximum enzyme production was increased in the presence of CaCl2. The molecular weight of purified enzyme was found to be approximately 48 kDa. It was found that the enzyme was fully stable in the presence of 2 mM CaCl2 at 50°C after 120 min. Purified protease was significantly activated by Ca 2+ and Mg 2+ , while it was greatly inhibited by Cu 2+ , Zn 2+ , Hg 2+ and SDS as well as by the metal ion chelators ethylenediaminetetraacetic (EDTA) and 1,10-phenanthroline. Phenylmethylsulfonyl fluoride (PMSF) had a little effect on the enzyme. Conclusions: Our findings suggest the potential of this isolate for protease production and that this enzyme may be suitable for biotechnological applications.

Production of thermotolerant, detergent stable alkaline protease using the gut waste of Sardinella longiceps as a substrate: Optimization and characterization

“…and Brevibacillus spp. isolated from various hot springs across the world were reported to produce thermostable proteases 9,10 .…”

Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications

“…Chromatographic methods, such as size exclusion, ion exchange, hydrophobic interaction, affinity, and reversed-phase chromatography, are often employed at the laboratory study [4,5,35,[78][79][80]. Preliminarily, precipitation and concentration techniques, such as the concentration of enzyme extract by tangential filtration, organic solvents [4,80], or salt gradient [35,78,79,81], are also adopted.…”

Purification and partial characterization of a thermo-halostable protease produced by Geobacillus sp. strain PLS A isolated from undersea fumaroles

“…Methods for proteases purification vary, giving different purity and yields. In most reports, the purification is done by precipitation using ammonium sulfate and dialysis, although few reports use cold organic solvents [20]. Further purification commonly involves column chromatography, using either ion exchange [20] or size exclusion techniques [21].…”

“…In most reports, the purification is done by precipitation using ammonium sulfate and dialysis, although few reports use cold organic solvents [20]. Further purification commonly involves column chromatography, using either ion exchange [20] or size exclusion techniques [21]. Occasionally, a combination of chromatography techniques is employed to increase enzyme purity [22].…”