Nazenin Ahmetoğlu, F. Bekler, Ö. Acer, R. G. Guven, K. Güven
{"title":"新分离芽孢杆菌KG5耐热金属蛋白酶的制备、纯化及特性研究","authors":"Nazenin Ahmetoğlu, F. Bekler, Ö. Acer, R. G. Guven, K. Güven","doi":"10.5053/EJOBIOS.2015.9.0.L","DOIUrl":null,"url":null,"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. \nMaterial and Methods: Bacterial strain KG5 was isolated from Kos (Bingol) 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. \nResults: 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 Ca2 + and Mg2 + , while it was greatly inhibited by Cu2 + , Zn2 + , Hg2 + 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. \nConclusions: Our findings suggest the potential of this isolate for protease production and that this enzyme may be suitable for biotechnological applications.","PeriodicalId":11848,"journal":{"name":"Eurasian Journal of Biosciences","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2015-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"18","resultStr":"{\"title\":\"Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5\",\"authors\":\"Nazenin Ahmetoğlu, F. Bekler, Ö. Acer, R. G. Guven, K. Güven\",\"doi\":\"10.5053/EJOBIOS.2015.9.0.L\",\"DOIUrl\":null,\"url\":null,\"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. \\nMaterial and Methods: Bacterial strain KG5 was isolated from Kos (Bingol) 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. \\nResults: 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 Ca2 + and Mg2 + , while it was greatly inhibited by Cu2 + , Zn2 + , Hg2 + 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. \\nConclusions: Our findings suggest the potential of this isolate for protease production and that this enzyme may be suitable for biotechnological applications.\",\"PeriodicalId\":11848,\"journal\":{\"name\":\"Eurasian Journal of Biosciences\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2015-03-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"18\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Eurasian Journal of Biosciences\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.5053/EJOBIOS.2015.9.0.L\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"Agricultural and Biological Sciences\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Eurasian Journal of Biosciences","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.5053/EJOBIOS.2015.9.0.L","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Agricultural and Biological Sciences","Score":null,"Total":0}
Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5
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 Kos (Bingol) 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 Ca2 + and Mg2 + , while it was greatly inhibited by Cu2 + , Zn2 + , Hg2 + 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.
期刊介绍:
EurAsian Journal of BioSciences (Abbrev. Eurasia J Biosci or EJOBIOS) is an international, refereed electronic journal. It publishes the results of original research in the field of biological sciences restricted tomorphology, physiology, genetics, taxonomy, ecology and biogeography of both prokaryotic and eucaryotic organisms. The journal encourages submission of manuscripts dealing with plant biology, animal biology, plant physiology, microbiology, hydrobiology, ecology and environmental science, ethnobiology, biodiversity and conservation biology. EurAsian Journal of BioSciences publishes original articles in the following areas: -Agriculture, Fisheries & Food -Anatomy & Morphology -Behavioural Sciences -Biology, Biochemistry and Biotechnology -Biophysics -Biology Education -Cellular Biology and Anatomical Sciences -Ecology, Evolution & Environment -Entomology -Forestry -General Biology -Genetics & Heredity -Life Sciences - Other topics -Microbiology and Immunology -Molecular Biology -Mycology -Palaeontology -Parasitology -Pharmacology & Pharmacy -Physiology and Related Sciences -Plant Sciences -Toxicology -Veterinary Sciences -Virology -Zoology