N. P. Balaban

Glutamyl endopeptidase of Bacillus intermedius, strain 3‐19

© 1997 Federation of European Biochemical Societies.

Purification and characterization of serine proteinase 2 from Bacillus intermedius 3-19.

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.

A novel secreted metzincin metalloproteinase from Bacillus intermedius.

The mprBi gene from Bacillus intermedius 3–19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase‐deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19 kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met‐turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes.

Structural and functional characteristics and properties of metzincins

In this review the main families of endopeptidases belonging to the clan of metzincins of zinc-dependent metal-loproteinases in organisms of wide evolutional range from bacteria to mammals are considered. The data on classification, physicochemical properties, substrate specificity, and structural features of this group of enzymes are given. The activation mechanisms of metzincins, the role of these proteins in organisms, and their participation in various physiological processes are discussed.

Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth

Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers.

Isolation and characterization of glutamyl endopeptidase 2 from Bacillus intermedius 3-19.

The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth. The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis. The molecular weight of the enzyme is 26.5 kD. The Km for Z-Glu-pNA hydrolysis is 0.5 mM. The temperature and pH dependences of the stability of the proteinase were studied. The enzyme was identified as glutamyl endopeptidase 2. The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined. The enzyme hydrolyzes Glu4–Gln5, Glu17–Asp18, and Cys11–Ser12 bonds in the oxidized A-chain of insulin and Glu13–Ala14, Glu21–Arg22, Cys7–Gly8, and Cys19–Gly20 bonds in the oxidized B-chain of insulin.

Isolation and characterization of a subtilisin-like proteinase of Bacillus intermedius secreted by the Bacillus subtilis recombinant strain AJ73 at different growth stages

Two subtilisin-like serine proteinases of Bacillus intermedius secreted by the Bacillus subtilis recombinant strain AJ73 (pCS9) on the 28th and 48th h of culture growth (early and late proteinase, respectively) have been isolated by ion-exchange chromatography on CM-cellulose and by FPLC. Molecular weights of both proteinases were determined. The N-terminal sequences of the recombinant protein and mature proteinases of the original strain were compared. Kinetic parameters and substrate specificities of the early and late proteinase were analyzed. Physicochemical properties of the enzymes were studied.

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

ABSTRACT 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.

Draft Genome Sequence of Bacillus pumilus 7P, Isolated from the Soil of the Tatarstan Republic, Russia

ABSTRACT Here, we present a draft genome sequence of Bacillus pumilus strain 7P. This strain was isolated from soil as an extracellular RNase-producing microorganism. The RNase of B. pumilus 7P is considered to be a potential antiviral and therapeutic antitumor agent, and it might be appropriate for agriculture and academic synthesis of oligoribonucleotides.

Bacterial enzymes effectively digest Alzheimer's β-amyloid peptide

Aggregated β-amyloid peptides play key roles in the development of Alzheimers disease, and recent evidence suggests that microbial particles, among others, can facilitate their polymerization. Bacterial enzymes, however, have been proved to be beneficial in degrading pathological fibrillar structures in clinical settings, such as strepto-kinases in resolving blood-clots. The purpose of this study was to investigate the ability of bacterial substances to effectively hydrolyze β-amyloid peptides. Degrading products of several proteinases from Bacillus pumilus were evaluated using MALDI-TOF mass-spectrometry, and their toxicity was assessed in vitro using cell-culture assays and morphological studies. These enzymes have proved to be non-toxic and were demonstrated to cleave through the functional domains of β-amyloid peptide. By yielding inactive fragments, proteinases of Bacillus pumilus may be used as candidate anti-amyloid agents.