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

• Published in: Eurasian journal of biosciences Vol. 2015; no. 9; pp. 1 - 11
• Main Authors: Ahmetoglu, Nazenin, Bekler, Fatma Matpan, Acer, Omer, Guven, Reyhan Gul, Guven, Kemal
• Format: Journal Article
• Language: English
• Published: Izmir EurAsian Journal of BioSciences 01.01.2015; Foundation for Environmental Protection and Research, Çevre Koruma ve Araştırma Vakfı (ÇEVKOR)
• Subjects: Bacillus; Bacteria; Biochemistry; Coğrafya; Proteases; İzmir; Türkiye; Bacillus sp. KG5; protease production and characterisation; biotechnology
• ISSN: 1307-9867; 1307-9867
• DOI: 10.5053/ejobios.2015.9.0.1

Background: Due to the importance of microbial proteases in biotechnological applications, anumber of microorganisms are being explored. The production, purification and characterisation ofextracellular metallo-proteases by producing Bacillus sp. KG5 was studied.Material and Methods: Bacterial strain KG5 was isolated from Kös (Bingöl) hot spring. The strainKG5 was identified by morphological, physiological, biochemical and 16S rRNA gene sequencing.The effects of various parameters on protease production, such as time, temperature, pH, carbonand nitrogen sources and CaCl2 were studied. The enzyme was purified by ammonium sulphateprecipitation and Sephadex G-75 gel permeation chromatography. Molecular weight was calculatedby sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymographicanalysis. The effects of some metal ions, chelators and inhibitors on enzyme activity weredetermined.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 extractand urea, while the best carbon sources were lactose and galactose. However, glucose as a sourceof carbon was found to inhibit the production of the enzyme. The maximum enzyme productionwas increased in the presence of CaCl2. The molecular weight of purified enzyme was found to beapproximately 48 kDa. It was found that the enzyme was fully stable in the presence of 2 mM CaCl2at 50°C after 120 min. Purified protease was significantly activated by Ca2+ and Mg2+, while it wasgreatly inhibited by Cu2+, Zn2+, Hg2+ and SDS as well as by the metal ion chelatorsethylenediaminetetraacetic (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 thisenzyme may be suitable for biotechnological applications.