EurAsian Journal of Biosciences Eurasia J Biosci 9,1-11 (2015) http://dx.doi .Org /10.5053/ejobios.2015.9.0.l Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5 Nazenin Ahmetoglu 1 , Fatma Matpan Bekler 1 *, Omer Acer 1 , Reyhan Gul Güven 2 , Kemal Güven 1 1 Department of Biology, Science Faculty, Dicle University, 21280, Diyarbakir, Turkey department of Primary Education, Ziya Gökalp Education Faculty, Dicle University, 21280, Diyarbakir, Turkey Corresponding author: fatmatpan@hotmail.com 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 CaCU 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 CaCU. 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 CaCU 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. Keywords: Bacillus sp. KG5, biotechnology, protease production and characterisation. Abbreviations: BM: Basal medium; EDTA: Ethylenediaminetetraacetic; NB: Nutrient broth; OD: Optical density; phen: 1,10-phenanthroline; PMSF: Phenylmethylsulfonyl fluoride; SDS-PAGE: sodium dodecyl sulphate- polyacrylamide gel electrophoresis; TCA: trichloroacetic acid. Ahmetoglu N, Matpan Bekler F, Acer O, Güven RG, Guven K (2015) Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5. Eurasia J Biosci 9: 1-11. http://dx.doi.Org/10.5053/ejobios.2015.9.0.1 ©EurAsian Journal of Biosciences INTRODUCTION Proteases are by far the most important groups of commercially and biotechnological enzymes, produced by various organisms such as bacteria, yeasts, moulds, plants and animal tissues, accounting for nearly 65% of the global industrial enzyme market (Anwar and Saleemuddin 1997, Banik and Prakash 2004, Annamalai et al. 2014). Microbial proteases, especially from Bacillus species, are the major industrial workhorses, and the use of proteases in several applications has increased in the last decade (Joo and Chang 2006). Proteases are used in a number of applications such as bioreme- diation, biosynthesis and biotransformation, brewing, dairy industries, detergent, diagnostics, food, meat, leather, photographic, and Received: January 2015 Received in revised form: February 2015 Accepted: February 2015 Printed: March 2015 1