-
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 -
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 -
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