Isolation and characterization of a new bacillary phytase

From cell lysates of a recombinant Escherichia coli strain, Bacillus ginsengihumi phytase has been isolated and studied for the first time. The enzyme has been purified to homogeneity, and its primary structure has been determined. It has been concluded that phytase belongs to the class of β-propeller phosphatases. The molecular weight of the protein is 41 kDa, and pI is 4.8. Some physicochemical properties of the enzyme have been investigated.     

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 activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.     

Evaluation of diagnostic efficiency of the recombinant protein modeling immunodominant epitope V3 of envelope gp120 for immunoenzyme detection for HIV-1 infection antibodies

The third hypervariable domain V3 of the human immunodeficiency virus type 1 gpl20 envelope glycoprotein contains neutralizing epitopes and plays an important role in the diagnosis of HIV infection . Neutralizing antibodies bind to conserved epitope with sequence GPG of V3 loop. The effect of sequence variation on the antigenic properties of the V3 epitope gp120 was studied using five synthetic peptides. The amino acid sequence of the peptide corresponding to the V3 region gp120 of HIV-1 subtype C showed the highest immunoreactivity. The DNA fragment encoding V3-C region gp120 was synthesized by polymerase chain reaction and cloned into pET41b vector. The recombinant plasmid was expressed in the E. coli cells, and recombinant protein was purified using glutathione-S sepharose affinity chromatography. The serological activity of the recombinant protein was tested using ELISA and compared to activity of similar synthetic peptide. The results of this study showed that most immunoreactive agent was the amino acid sequence of V3 region gp120 of HIV-1 subtype C. The recombinant antigen comprising this sequence was more antigenic than synthetic peptide with the same sequence. The evaluation of this antigen shows that this protein is a good candidate for the immunoassay development.     

Diversity in the swimming motility and flagellar regulon structure of uropathogenic Morganella morganii strains

International Microbiology - In current times, the opportunistic pathogen Morganella morganii is increasingly becoming a cause of urinary tract infections. The condition has been further...     

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1

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 K_m 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.     

Comparing assemblages of Asteraceae and their insect herbivores under different land‐use regimens

Accelerated tropical landscape changes occurring over recent decades have produced environmental mosaics comprising remaining isolated green areas and mixed land‐use types. Our objective was to study the effects of alterations in the natural landscape on the species composition and structure of assemblages of Asteraceae and their endophagous insects through comparisons between cerrado (savanna), pastures and Eucalyptus stands. We first investigated whether similarities between assemblages of Asteraceae and their insects varied among land uses or localities. Secondly, we asked whether assemblages of Eucalyptus stands and pastures are subsets of those within the cerrado. We sampled within randomly deployed transects in 15 areas. Land use was found to be an important factor in determining plant composition similarity; however, locality did not exert any significant influence. Pastures were less similar to one another, suggesting high beta diversity. Similarities among insect assemblages were correlated with plant assemblage composition, but not with land use or locality. Species of Tephritidae were distributed along localities independently of land use. High beta diversity in Asteraceae assemblages among cerrados and pastures was supported by nestedness analysis. Plant assemblages in Eucalyptus stands were subsets of cerrado, but pasture assemblages were only partial subsets. A higher degree of nestedness in insect assemblages than in plant assemblages indicated lower beta diversity within these herbivores. Our data indicate that many herbivores are specialized on widely distributed plant genera. Conservation of Asteraceae species and their flower head insects depends not only on maintenance of landscape fragments but also on the correct matching of management form and land use. Such management may contribute to reducing isolation of plant and insect species by increasing the connectivity of remaining cerrado tracts, allowing population maintenance even at low abundances.     

Subtilisin-like proteinase secreted by the <Emphasis Type="Italic">Bacillus pumilus</Emphasis> 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.     

The novel ADAMs-like microbial metalloendopeptidase

Heterologous gene expression of the extracellular minor metalloendopeptidase of Bacillus pumilus 3?C19 (AN EU678894) in the protease-deficient B. pumilus 3?C19 strain has been studied. The enzyme fraction in the total pool of B. pumilus 3?C19 proteases is less than 8%. The enzyme was isolated from the culture liquid of the recombinant strain and purified to a homogeneous state; its primary structure was determined and its physicochemical properties were investigated. The secreted metalloendopeptidase of B. pumilus 3?C19 is the first prokaryotic homologue of the eukaryotic adamalysin/reprolysin protein family.     

Biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius under salt stress conditions

The biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius 3–19 by the recombinant strain Bacillus subtilis AJ73(pCS9) was found to be enhanced under salt stress conditions (growth in a medium containing 1 MNaCl and 0.25 M sodium citrate). In a recombinant strain of B. subtilis deficient in the regulatory proteins DegS and DegU, which control the synthesis of degradative enzymes, the expression of the proteinase gene was inhibited. In contrast, in the strain B. subtilis degU32(Hy), which provides for the overproduction of proteins positively regulated by the DegS-DegU system, the biosynthesis of the subtilisin-like proteinase of B. intermedius 3–19 increased by 6–10 fold. These data suggest that the DegS-DegU system is involved in the positive regulation of the expression of the subtilisin-like B. intermedius proteinase gene in recombinant B. subtilis strains.