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

Q3 Agricultural and Biological Sciences
Nazenin Ahmetoğlu, F. Bekler, Ö. Acer, R. G. Guven, K. Güven
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引用次数: 18

摘要

背景:由于微生物蛋白酶在生物技术应用中的重要性,许多微生物正在被探索。研究了芽孢杆菌KG5胞外金属蛋白酶的生产、纯化和特性。材料与方法:从Bingol温泉中分离到菌株KG5。通过形态学、生理生化和16S rRNA基因测序对菌株KG5进行鉴定。研究了时间、温度、pH、碳氮源、CaCU等参数对蛋白酶生成的影响。通过硫酸铵沉淀和Sephadex G-75凝胶渗透层析纯化酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和酶谱分析计算分子量。测定了几种金属离子、螯合剂和抑制剂对酶活性的影响。结果:产蛋白酶的最佳温度为40 ~ 45℃,pH为7.0℃,孵育时间为24 h。结果表明,最佳氮源为酵母浸膏和尿素,最佳碳源为乳糖和半乳糖。然而,葡萄糖作为碳的来源被发现抑制酶的产生。在CaCU的存在下,最大产酶量增加。纯化酶的分子量约为48 kDa。实验发现,该酶在2 mM CaCU存在下,在50°C下,120 min后完全稳定。纯化后的蛋白酶被Ca2 +和Mg2 +显著激活,而被Cu2 +、Zn2 +、Hg2 +和SDS以及金属离子螯合剂乙二胺四乙酸(EDTA)和1,10-菲罗啉显著抑制。苯基甲基磺酰氟(PMSF)对酶的影响较小。结论:我们的研究结果表明,该分离物具有生产蛋白酶的潜力,该酶可能适用于生物技术应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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.
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来源期刊
Eurasian Journal of Biosciences
Eurasian Journal of Biosciences Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)
CiteScore
1.20
自引率
0.00%
发文量
0
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
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