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Applied and Environmental Microbiology Feb 2000A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy...
A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.
Topics: Bacillus; Cloning, Molecular; Endopeptidases; Hydrogen-Ion Concentration; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Plasmids; Sequence Analysis, DNA
PubMed: 10653758
DOI: 10.1128/AEM.66.2.825-827.2000 -
PeerJ 2020Bacterial biofilms have become a major threat to human health. The objective of this study was to isolate amylase-producing bacteria from soil to determine the overall...
BACKGROUND
Bacterial biofilms have become a major threat to human health. The objective of this study was to isolate amylase-producing bacteria from soil to determine the overall inhibition of certain pathogenic bacterial biofilms.
METHODS
We used serial dilution and the streaking method to obtain a total of 75 positive amylase isolates. The starch-agar plate method was used to screen the amylolytic activities of these isolates, and we used morphological and biochemical methods to characterize the isolates. Optimal conditions for amylase production and purification using Sephadex G-200 and SDS-PAGE were monitored. We screened these isolates' antagonistic activities and the purified amylase against pathogenic and multi-drug-resistant human bacteria using the agar disk diffusion method. Some standard antibiotics were controlled according to their degree of sensitivity. Finally, we used spectrophotometric methods to screen the antibiofilm 24 and 48 h after application of filtering and purifying enzymes in order to determine its efficacy at human pathogenic bacteria.
RESULTS
The isolated species were (26.7%), (16%), (13.3%), (10.7%), (10.7%), (5.3%), (5.3%), (4%), (4%), and (4%). Interestingly, all isolates showed a high antagonism to target pathogens. had the highest recorded activity (48 mm) and had the lowest recorded activity (12 mm) against (MRSA) and , respectively. On the other hand, we detected no antibacterial activity for purified amylase. The supernatant of the isolated amylase-producing bacteria and its purified amylase showed significant inhibition for biofilm: 93.7% and 78.8%, respectively. This suggests that supernatant and purified amylase may be effective for clinical and environmental biofilm control.
DISCUSSION
Our results showed that soil bacterial isolates such as supernatant and its purified amylase are good antibiofilm tools that can inhibit multidrug-resistant former strains. They could be beneficial for pharmaceutical use. While purified amylase was effective as an antibiofilm, the isolated supernatant showed better results.
PubMed: 33194439
DOI: 10.7717/peerj.10288 -
Microbiological Research 2007We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689...
We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Phix174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Phix174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg2+ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 degrees C (pH 8.0) with 100 mM NaCl and 10 mM Mg2+ concentrations.
Topics: Bacillus; Chemical Fractionation; Chromatography, Affinity; Chromatography, Liquid; Coenzymes; DNA Restriction Enzymes; DNA, Bacterial; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; Magnesium; Molecular Weight; Osmolar Concentration; Plasmids; Temperature
PubMed: 16644193
DOI: 10.1016/j.micres.2006.01.008 -
RSC Advances Jan 2021Brewers' spent grain was used as a substrate to obtain protein hydrolysates with antioxidant activity. Hydrolysis was conducted in the culture using proteolytic...
Brewers' spent grain was used as a substrate to obtain protein hydrolysates with antioxidant activity. Hydrolysis was conducted in the culture using proteolytic bacteria. Hydrolysis was controlled by measurement of α-amino group concentration and with the aid of size exclusion chromatography. For each culture the degree of hydrolysis was calculated. The most efficient protein hydrolysis was observed in the cultures of (43.06%) and (41.81%). In addition, gelatin zymography was performed in order to detect bacterial proteases and their activity. The profile of secreted enzymes was heterogeneous, while the greatest variety was observed for . Brewers' spent grain protein hydrolysates exhibited high antioxidant activity. and post-cultured media displayed the highest activity, respectively 1291.97 and 1621.31 μM TEAC per g for ABTS, 188.89 and 160.93 μM TEAC per g for DPPH, and 248.81 and 284.08 μM TEAC per g for the FRAP method.
PubMed: 35424402
DOI: 10.1039/d0ra08830g -
PeerJ 2020Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme...
BACKGROUND
Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown.
METHODS
Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals.
RESULTS
The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.
PubMed: 32617193
DOI: 10.7717/peerj.9408 -
ACS Synthetic Biology Feb 2021Compartmentalization of single genes in water-in-oil emulsion droplets is a powerful approach to create millions of reactors for enzyme library selections. When these...
Compartmentalization of single genes in water-in-oil emulsion droplets is a powerful approach to create millions of reactors for enzyme library selections. When these droplets are formed at ultrahigh throughput in microfluidic devices, their perfect monodispersity allows quantitative enzyme assays with a high precision readout. However, despite its potential for high quality cell-free screening experiments, previous demonstrations of enrichment have never been successfully followed up by actual enzyme library selections in monodisperse microfluidic droplets. Here we develop a three-step workflow separating three previously incompatible steps that thus far could not be carried out at once: first droplet-compartmentalized DNA is amplified by rolling circle amplification; only after completion of this step are reagents for protein expression and, finally, substrate added via picoinjection. The segmented workflow is robust enough to allow the first evolution in droplets, improving the protease Savinase that is toxic to for higher activity and identifying a 5-fold faster enzyme.
Topics: Bacillus; Bacterial Proteins; Base Sequence; DNA, Bacterial; Directed Molecular Evolution; Emulsions; Escherichia coli; Genes, Bacterial; High-Throughput Screening Assays; Microfluidic Analytical Techniques; Microfluidics; Nucleic Acid Amplification Techniques; Plasmids; Protein Engineering; Serine Endopeptidases; Workflow
PubMed: 33502841
DOI: 10.1021/acssynbio.0c00538 -
Journal of Bacteriology Nov 1999A teichuronopeptide (TUP) is one of major structural components of the cell wall of the facultative alkaliphilic strain Bacillus lentus C-125. A mutant defective in TUP...
A teichuronopeptide (TUP) is one of major structural components of the cell wall of the facultative alkaliphilic strain Bacillus lentus C-125. A mutant defective in TUP synthesis grows slowly at alkaline pH. An upper limit of pH for growth of the mutant was 10.4, while that of the parental strain C-125 was 10.8. Gene tupA, directing synthesis of TUP, was cloned from C-125 chromosomal DNA. The primary translation product of this gene is likely a cytoplasmic protein (57. 3 kDa) consisting of 489 amino acid residues. Introduction of the tupA gene into the TUP-defective mutant complemented the mutation responsible for the pleiotropic phenotypes of the mutant, leading to simultaneous disappearance of the defect in TUP synthesis, the diminished ability for cytoplasmic pH homeostasis, and the low tolerance for alkaline conditions. These results demonstrate that the acidic polymer TUP in the cell wall plays a role in pH homeostasis in this alkaliphile.
Topics: Amino Acid Sequence; Bacillus; Base Sequence; Blotting, Southern; Cell Wall; Cloning, Molecular; Culture Media; Genes, Bacterial; Glucuronates; Homeostasis; Hydrogen-Ion Concentration; Immunoblotting; Molecular Sequence Data; Plasmids; Polyglutamic Acid; Restriction Mapping; Transformation, Bacterial
PubMed: 10542159
DOI: 10.1128/JB.181.21.6600-6606.1999 -
ACS Central Science Nov 2017Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the...
Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify"). This allowed the creation of a regioisomer of His (iso-His, His) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-His. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from and pantothenate synthetase.
PubMed: 29202018
DOI: 10.1021/acscentsci.7b00341 -
European Journal of Biochemistry Feb 1996Backbone dynamics of Savinase, a subtilisin of 269 residues secreted by Bacillus lentus, have been studied using 15N relaxation measurements derived from proton-detected...
Backbone dynamics of Savinase, a subtilisin of 269 residues secreted by Bacillus lentus, have been studied using 15N relaxation measurements derived from proton-detected dimensional 1H-15N-NMR spectroscopy. 15N spin-lattice rate constants (R1), spin-spin relaxation-rate constants(R2), and 1H-15N nuclear Overhauser effects (NOE) were determined for 84% of the backbone amide 15N nuclei. The model-free formalism [Lipari, G. & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559] was used to derive values for a generalized order parameter, S2, interpretable as a measure of the amplitude of motion on the picosecond-nanosecond timescale, for each N-H bond vector. Additional terms used to fit the data include an effective correlation time for internal motions (taue) and an exchange term (Rex) to account for exchange contributions to R2. The overall rotational correlation time (taum) is 9.59 +/- 0.02 ns; the average order parameter (S2) is 0.90 +/- 0.07, indicative of a rigid structure consistent with Savinase's high degree of secondary structure and compact tertiary fold. Residues S125-S128, located in the substrate-binding region, represent the longest stretch of protein which exhibits disorder on the picosecond-nanosecond timescale. These residues also exhibit significant exchange terms, possibly indicative of motion on the microsecond-millisecond timescale, which could also be influenced by the proximity of the phenyl ring of the substituted aryl boronic acid inhibitor used in this study. S103 and G219 in the substrate-binding region, represent the longest stretch of protein which exhibits disorder on the picosecond-nanosecond timescale. These residues also exhibit significant exchange terms, possibly indicative of motion on the microsecond-millisecond timescale, which could also be influenced by the proximity of the phenyl ring of the substituted aryl boronic acid inhibitor used in this study. S103 and G219 in the substrate-binding region also show flexibility on the picosecond-nanosecond timescale. There is also significant motion in the turn, G258-T260, of a small solvent-exposed loop region which may make the protein vulnerable autolysis at that point. Some residues in both calcium-binding sites and nearby also show mobility.
Topics: Bacillus; Binding Sites; Calcium; Magnetic Resonance Spectroscopy; Nitrogen Isotopes; Serine Endopeptidases; Serine Proteinase Inhibitors
PubMed: 8654411
DOI: 10.1111/j.1432-1033.1996.00629.x -
Microbiology (Reading, England) Aug 1997A method was established to measure the cytoplasmic pH of the facultative alkaliphilic strain, . The bacterium was loaded with a pH-sensitive fluorescent probe,...
A method was established to measure the cytoplasmic pH of the facultative alkaliphilic strain, . The bacterium was loaded with a pH-sensitive fluorescent probe, 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), and cytoplasmic pH was determined from the intensity of fluorescence of the intracellular BCECF. The activity of the organism to maintain neutral cytoplasmic pH was assessed by measuring the cytoplasmic pH of the cells exposed to various pH conditions. The cytoplasmic pH maintenance activity of C-125 increased with increasing culture pH, indicating that the activity was regulated in response to the culture pH.
PubMed: 33657729
DOI: 10.1099/00221287-143-8-2531