Abstract
The aim of this work was to study the composition and functions of intracellular and extracellular proteases of the production Vibrio cholerae strains 569B serovar Inaba and M41 serovar Ogawa using zymographic and proteomic analysis.Materials and methods. Samples of intracellular proteases were obtained from cell lysates by ultrasonication of bacterial cells in a 9 M urea solution. The extracellular protease fraction was precipitated from the culture liquid by adding 50 % trichloroacetic acid to a final concentration of 10 % and incubating on ice. Lyophilized preparations of proteinase K and proteovibrin enzyme complex were used as a control of proteolytic activity. Proteases were detected by substrate gel electrophoresis in 12.5 % polyacrylamide gel impregnated with 0.1 % gelatin, followed by identification of the composition of protein fractions of lysates and exoproteins of both strains using molecular mass spectrometric scanning.Results and discussion. A comparative study of the production strains of V. cholerae 569B serovar Inaba and M41 serovar Ogawa using zymographic and proteomic analysis showed that the greatest enzymatic activity was detected in the fraction of extracellular proteases sample of V. cholerae M41 strain, where five major and four minor zones of gelatin hydrolysis were identified, and high-intensity zones with MW 20–23 and 37–40 kDa were also found in the preparation of proteovibrin isolated from the culture fluid of that strain. As a result of proteomic analysis of the studied strains, 66 enzymes of V. cholerae with different functional activity were reliably identified, among which 15 enzymes had protease activity. The high information content of the complex of modern methods provided for the possibility of identifying qualitative and quantitative differences in the composition of intracellular and extracellular proteases in production strains of V. cholerae, which offers an effective means of screening inter-strain differences in the protease spectrum in production strains.
Highlights
The extracellular protease fraction was precipitated from the culture liquid by adding 50 % trichloroacetic acid to a final concentration of 10 % and incubating on ice
Lyophilized preparations of proteinase K and proteovibrin enzyme complex were used as a control of proteolytic activity
Proteases were detected by substrate gel electrophoresis in 12.5 % polyacrylamide gel impregnated with 0.1 % gelatin, followed by identification of the composition of protein fractions of lysates and exoproteins of both strains using molecular mass spectrometric scanning
Summary
Целью работы является применение зимографического и протеомного анализа для изучения состава и функций внутриклеточных и экстрацеллюлярных протеаз производственных штаммов Vibrio cholerae 569В серовара Инаба и М41 серовара Огава. Сравнительное исследование производственных штаммов V. cholerae 569В серовара Инаба и М41 серовара Огава с помощью зимографического и протеомного анализа показало, что наибольшая ферментативная активность выявлена в образце фракции экстрацеллюлярных протеаз штамма V. cholerae М41, где идентифицировано пять основных и четыре минорных зоны гидролиза желатина, причем зоны высокой интенсивности с МW 20–23 и 37‒40 кДа обнаружены также в препарате протеовибрина, выделенного из культуральной жидкости этого штамма. Высокая информативность комплекса современных методов показала возможность выявления качественных и количественных различий состава внутриклеточных и экстрацеллюлярных протеаз производственных штаммов V. cholerae, что предлагает эффективное средство скрининга межштаммовых различий протеазного спектра у производственных штаммов.
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