Purification and characterization of a subtilisin-like proteinases secreted in the stationary growth phase of Bacillus amyloliquefaciens H2

Proteinases secreted during the early and late stationary phases have been isolated from the culture liquid of Bacillus amyloliquefaciens H2 using CM-cellulose ion-exchange chromatography with subsequent FPLC on a Mono S column. Considering the character of hydrolysis of specific chromogenic substrates and the type of inhibition, these enzymes were identified as subtilisin-like proteinases. The molecular weight of both proteinases is 29 kD. The proteolytic activity of the proteinases secreted during the early and late stationary phases towards the synthetic substrate Z-Ala-Ala-Leu-pNA was maximal at pH 8.5 and 9.0, respectively. The maximal activity of both proteinases was observed at 37°C, and the proteins were stable within the pH range of 7.2-9.5. The subtilisin-like proteinases from B. amyloliquefaciens were shown to catalyze synthesis of peptide bonds. © Nauka/Interperiodica 2007

Subtilisin-like proteinase secreted by the Bacillus pumilus KMM 62 strain at different growth stages

Proteinase secreted in the environment by bacilli on different growth stages was isolated by ion chromatography from the culture medium of Bacillus pumilus KMM 62. According to the hydrolysis character of specific chromogenic substrates and inhibition type, the enzyme belongs to subtilisin-like serine proteinases. The isolated proteinase with the molecular mass of 30 kDa displays maximum activity on hydrolysis of the peptide substrate Z-Ala-Ala-Leu-pNA at pH 8.0 - 8.5 and temperature 30°C. The protein is stable in the range of pH 7.5 - 10.0. It was shown that subtilisin-like serine proteinase from B. pumilus KMM 62 possessed thrombolytic activity. © Pleiades Publishing, Ltd., 2012

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth. © Nauka/Interperiodica 2007

Selection of cultivation medium for production of late stationary phase serine proteinases from Bacillus intermedius

B. intermedius have been shown previously to secrete two serine proteinases: glutamyl endopeptidase 2 and subtilisin 2 during the late stationary phase, with maximal levels of the enzymes activities recorded at the 40th and 44th hours of growth, respectively. In the current study, we analyzed the impact of various culture medium components on biosynthesis of these proteinases. Yeast extract and gelatin did not stimulate the enzymes biosynthesis. However, on the medium containing 0.1% casein subtilisin 2 production increased to reach 140%. Biosynthesis of both serine proteinases, produced by B. intermedius at the late stationary phase, were found to be inhibited by individual amino acids, and to be insensitive to catabolite repression. In order to maximise enzyme production, the presence of Ca 2+ and Mg2+ at concentration of 5 mM was shown to be necessary. Based on the results of this work, the composition of a complex culture media for the effective production of late stationary phase proteinases by B. intermedius was developed

Isolation and characterisation of Bacillus amyloliquefaciens H2 glutamyl endopeptidase that is secreted in stationary phase of culture growth

The culture broth of Bacillus amyloliquefaciens H2 was used for isolation by chromatography on CM-cellulose and MonoS columns of proteinase that are secreted in early and late stationary phases of culture growth. Based on their inhibition by specific serine proteinase inhibitors and their action on specific chromogenic substrates and on oxidised B-chain of insulin, the enzymes were classified as glutamyl endopeptidases. The molecular weight of secreted enzyme is 26.5 kDa. We show that although the enzyme is secreted in different phases of culture growth, both enzyme fractions have similar enzymatic properties and amino acid content, but they were distinguished in

Design of Web-Application for Emotional Classification of Social Network Texts in Russian

Описываются предварительные результаты финальной стадии проекта по разработке компьютерного классификатора интернет-текстов на русском языке по критерию ведущей эмоции, вербализованной в них. Описание собственно архитектуры и функционала приложения предваряется ремарками относительно этапов формирования датасета аннотированных текстов, специфики их разметки и использованных моделей машинного обучения.We present some results obtained on the final stage of the project in which we do the task of designing computer classifier of internet-texts in Russian according to the criterion of leading text emotion. The description of the application architecture and its functioning is anticipated by some details of data set building, specificity of its annotation and models used for machine learning.Исследование выполнено при финансовой поддержке РФФИ № 19‑012‑00205 «Разработка классификатора русскоязычных интернет-текстов по критерию их тональности на основе модели эмоций “Куб Лёвхейма”»

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

Addition of calcium ions to the Ca2+-regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (25000 cm-1) assigned as from the Ca2+-discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t1/2 2 ps) in the case of the Ca2+-discharged obelin than aequorin (t1/2 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19400 cm-1, bound to Ca2+-discharged obelin, and 21300 cm-1, bound to Ca2+-discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavit