Optimization of cultivation media for heterologous gene expression of adamalysin-like melalloendopeptidase Bacillus pumilus

© 2015 Rudakova et al. The paper is dedicated to selecting main components of the nutrient medium and additional sources of nutrients for the maximum production of adamalysin-like metalloproteinase Bacillus pumilus. For convenience, the gene of metalloproteinase MprBp was cloned in protein-deficient strain B. subtilis BG2036. During the study, optimal concentration of peptone, inorganic phosphate, and casein was selected. Besides, it was shown that the enzyme production is stimulated by kasamino acids, leucine, alanine, asparagine, glutamine, as well as calcium and magnesium ions

Cloning and Heterologous Expression of Phytase Gene from Pantoea sp. 3.5.1

© 2016, Springer Science+Business Media New York.Phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyzes the stepwise hydrolysis of phosphate groups from phytic acid (myo-inositol hexakisphosphate) or its salt phytate. These enzymes could be potentially used for the stereospecific and efficient production of different myo-inositol phosphate isomers with therapeutic features. In the present study, we cloned the 1728 bp open reading frame encoding Pantoea sp. 3.5.1 phytase into the expression vector pET28a. The recombinant Escherichia coli BL21 pLysS pET28a/agpP strain expressing Pantoea sp. 3.5.1 AgpP phytase was obtained

Peculiarities of the biosynthesis of Bacillus intermedius glutamyl endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase

We studied the biosynthesis of bacillus intermedius glutamyl endopeptidase in the recombinant bacillus subtilis strain AJ73 Δ58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and the growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During the final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different. © 2005 Pleiades Publishing, Inc

Inositol Phosphates and their Biological Effects

The paper is dedicated to data analysis on inositol phosphates nature distribution, structure and biological functions as well as enzymes - phytases - capable to hydrolyze these hardly digestible compounds and their complexes. Pharmaceutical application of inositol phosphates in treatment and prevention of various inflammatory and cancer diseases is discussed

Peculiarities of the biosynthesis of Bacillus intermedius glutamyl endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase

We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 Δ58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different

Membrane-bound forms of serine proteases in Bacillus intermedius

Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes

Membrane-bound forms of serine proteases in Bacillus intermedius

Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes

The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain AJ73 during sporulation

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th h of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different

Novel glucose-1-phosphatase with high phytase activity and unusual metal ion activation from soil bacterium Pantoea sp. strain 3.5.1

© 2015, American Society for Microbiology. Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values toward phytate, glucose-6- phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify DL-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features

Selection of Efficient Taq DNA Polymerase to Optimize T-DNA Genotyping Method for Rapid Detection of Mutant Arabidopsis thaliana Plants

© 2016, Springer Science+Business Media New York.Plants harbor homologues of various animal genes involved in phosphorus metabolism, telomere biology and other cellular processes. Compared to experiments with many other multicellular organisms, research in the model plant Arabidopsis thaliana takes advantage of short generation time and an ever increasing arsenal of genetic and transgenic tools, including large collections of T-DNA knockout and activation lines. The availability of thousands of publicly available transgenic Arabidopsis lines provides a unique opportunity to address a number of important biological questions. However, identification of individual T-DNA mutant plants from a pool of seeds provided by a biological stock distribution center remains a laborious and time-consuming procedure. Here we compared a number of commercial Taq DNA polymerases commonly used for routine PCR genotyping to identify a single polymerase most suitable for genotyping T-DNA mutant plants. Our data indicate that Emerald Amp GT PCR Master Mix provides the most reliable, quick and simple DNA genotyping tool to determine the presence of a T-DNA insertion and to establish whether an individual A. thaliana plant is heterozygous or homozygous for the mutant allele