Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.
The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, k(cat) and k(cat)/K(m) higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.
Bacteria belonging to the genus Novosphingobium are known to be metabolically versatile and occupy different ecological niches. In the absence of genomic data and/or analysis, knowledge of the bacteria that belong to this genus is currently limited to biochemical characteristics. In this study, we analyzed the whole genome sequencing data of six bacteria in the Novosphingobium genus and provide evidence to show the presence of genes that are associated with salt tolerance, cell-cell signaling and aromatic compound biodegradation phenotypes. Additionally, we show the taxonomic relationship between the sequenced bacteria based on phylogenomic analysis, average amino acid identity (AAI) and genomic signatures.
A novel α-amylase was isolated successfully from Glaciozyma antarctica PI12 using DNA walking and reverse transcription-polymerase chain reaction (RT-PCR) methods. The structure of this psychrophilic α-amylase (AmyPI12) from G. antarctica PI12 has yet to be studied in detail. A 3D model of AmyPI12 was built using a homology modelling approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9.9. Analysis of the AmyPI12 model revealed the presence of binding sites for a conserved calcium ion (CaI), non-conserved calcium ions (CaII and CaIII) and a sodium ion (Na). Compared with its template-the thermostable α-amylase from Bacillus stearothermophilus (BSTA)-the binding of CaII, CaIII and Na ions in AmyPI12 was observed to be looser, which suggests that the low stability of AmyPI12 allows the protein to work at different temperature scales. The AmyPI12 amino acid sequence and model were compared with thermophilic α-amylases from Bacillus species that provided the highest structural similarities with AmyPI12. These comparative studies will enable identification of possible determinants of cold adaptation.
Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
Probiotic delivery system was developed via the use of microbial transglutaminase (MTG) cross-linked soy protein isolate (SPI) incorporated with agrowastes such as banana peel (BE), banana pulp (BU), and pomelo rind (PR). Inoculums of Lactobacillus bulgaricus FTDC 1511 were added to the cross-linked protein matrix. The incorporation of agrowastes had significantly (P<0.05) reduced the strength, pH value, and the lightness of the SPI gel carriers, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the occurring cross-links within the SPI gel carriers were attributed to the addition of MTG. Scanning electron microscope micrographs illustrated that SPI carriers containing agrowastes have exhibited a less-dense protein matrix. All the SPI carriers possessed maximum swelling ratio at 4 to 4.5 within 15 min in simulated gastric fluid (SGF), whereas the maximum swelling ratios of SPI/BE, SPI/BU, and SPI/PR were higher compared to that of control in simulated intestinal fluid (SIF). Additionally, SPI carriers in SGF medium did not show degradation of structure, whereas a major collapse of network was observed in SIF medium, indicating controlled-release in the intestines. The addition of agrowastes into SPI carriers led to a significantly (P<0.0001) lower release of L. bulgaricus FTDC 1511 in SGF medium and a higher release in SIF medium, compared to that of the control. SPI carriers containing agrowastes may be useful transports for living probiotic cells through the stomach prior to delivery in the lower intestines.
A Na(+)/H(+) antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na(+)/H(+) antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCl, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na(+) extrusion at lower pH values and in pH homeostasis at pH 10 under Na(+)-limiting conditions.
The alginate lyase AlyQ from Persicobacter sp. CCB-QB2 is a three-domained enzyme with a carbohydrate-binding module (CBM) from family 32. The CBM32 domain, AlyQB, binds enzymatically cleaved but not intact alginate. Co-crystallisation of AlyQB with the cleaved alginate reveals that it binds to the 4,5-unsaturated mannuronic acid of the non-reducing end. The binding pocket contains a conserved R248 that interacts with the sugar's carboxyl group, as well as an invariant W303 that stacks against the unsaturated pyranose ring. Targeting specifically the non-reducing end is more efficient than the reducing end since the latter consists of a mixture of mannuronic acid and guluronic acid. AlyQB also seems unable to bind these two saturated sugars as they contain OH groups that will clash with the pocket. Docking analysis of YeCBM32, which binds oligogalacturonic acid, shows that the stacking of the pyranose ring is shifted in order to accommodate the sugar's axial C1-OH, and its R69 is accordingly elevated to bind the sugar's carboxyl group. Unlike AlyQB, YeCBM32's binding pocket is able to accommodate both saturated and unsaturated galacturonic acid.
Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.
Bacterial toxin-antitoxin systems usually comprise of a pair of genes encoding a stable toxin and its cognate labile antitoxin and are located in the chromosome or in plasmids of several bacterial species. Chromosomally-encoded toxin-antitoxin systems are involved in bacterial stress responses and activation of the toxins usually leads to cell death or dormancy. Overexpression of the chromosomally-encoded YoeB toxin from the yefM-yoeB toxin-antitoxin locus of the Gram-positive bacterium Streptococcus pneumoniae has been shown to cause cell death in S. pneumoniae as well as E. coli.
To study the antimicrobial effects of quaternary ammonium silane (QAS) exposure on Streptococcus mutans and Lactobacillus acidophilus bacterial biofilms at different concentrations. Streptococcus mutans and Lactobacillus acidophilus biofilms were cultured on dentine disks, and incubated for bacterial adhesion for 3-days. Disks were treated with disinfectant (experimental QAS or control) and returned to culture for four days. Small-molecule drug discovery-suite was used to analyze QAS/Sortase-A active site. Cleavage of a synthetic fluorescent peptide substrate, was used to analyze inhibition of Sortase-A. Raman spectroscopy was performed and biofilms stained for confocal laser scanning microscopy (CLSM). Dentine disks that contained treated dual-species biofilms were examined using scanning electron microscopy (SEM). Analysis of DAPI within biofilms was performed using CLSM. Fatty acids in bacterial membranes were assessed with succinic-dehydrogenase assay along with time-kill assay. Sortase-A protein underwent conformational change due to QAS molecule during simulation, showing fluctuating alpha and beta strands. Spectroscopy revealed low carbohydrate intensities in 1% and 2% QAS. SEM images demonstrated absence of bacterial colonies after treatment. DAPI staining decreased with 1% QAS (p
Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide-protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.
Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li(+), Na(+), K(+), Rb(+) and Cs(+) after 30min treatment. Heavy metal ions such as Cu(2+), Fe(3+) and Zn(2+) inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature.
In the present study, the novel Ag/cellulose nanocrystal (CNC)-doped CeO2 quantum dots (QDs) with highly efficient catalytic performance were synthesized using one pot co-precipitation technique, which were then applied in the degradation of methylene blue and ciprofloxacin (MBCF) in wastewater. Catalytic activity against MBCF dye was significantly reduced (99.3%) for (4%) Ag dopant concentration in acidic medium. For Ag/CNC-doped CeO2 vast inhibition domain of G-ve was significantly confirmed as (5.25-11.70 mm) and (7.15-13.60 mm), while medium- to high-concentration of CNC levels were calculated for G + ve (0.95 nm, 1.65 mm), respectively. Overall, (4%) Ag/CNC-doped CeO2 revealed significant antimicrobial activity against G-ve relative to G + ve at both concentrations, respectively. Furthermore, in silico molecular docking studies were performed against selected enzyme targets dihydrofolate reductase (DHFR), dihydropteroate synthase (DHPS), and DNA gyrase belonging to folate and nucleic acid biosynthetic pathway, respectively to rationalize possible mechanism behind bactericidal potential of CNC-CeO2 and Ag/CNC-CeO2.