The mechanisms of action of AN5-1 against Gram-negative and Gram-positive bacteria were investigated by evaluations of the intracellular content leakage and by microscopic observations of the treated cells. Escherichia coli and Staphylococcus aureus were used for this investigation. Measurements of DNA, RNA, proteins, and β-galactosidase were taken, and the results showed a significant increase in the cultivation media after treatment with AN5-1 compared with the untreated cells. The morphological changes of treated cells were shown using transmission electron microscopy (TEM) and atomic force microscopy (AFM). The observations showed that AN5-1 acts against E. coli and against S. aureus in similar ways, by targeting the cell wall, causing disruptions; at a high concentration (80 AU/ml), these disruptions led to cell lysis. The 3D AFM imaging system showed that at a low concentration of 20 AU/ml, the effect of AN5-1 is restricted to pore formation only. Moreover, a separation between the cell wall and the cytoplasm was observed when Gram-negative bacteria were treated with a low concentration (20 AU/ml) of AN5-1.
Nanobiocatalysis is a new frontier of emerging nanosized material support in enzyme immobilization application. This paper is about a comprehensive review on cellulose nanofibers (CNF), including their structure, surface modification, chemical coupling for enzyme immobilization, and potential applications. The CNF surface consists of mainly -OH functional group that can be directly interacted weakly with enzyme, and its binding can be improved by surface modification and interaction of chemical coupling that forms a strong and stable covalent immobilization of enzyme. The knowledge of covalent interaction for enzyme immobilization is important to provide more efficient interaction between CNF support and enzyme molecule. Enzyme immobilization onto CNF is having potential for improving enzymatic performance and production yield, as well as contributing toward green technology and sustainable sources.
Artocarpus altilis (breadfruit) pulp, peel and whole fruit were extracted with various solvents such as hexane, dichloromethane (DCM) and methanol. The antioxidant activity of these extracts were examined using the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test. IC50 was 55 ± 5.89 μg/ml for the pulp part of methanol extract. In the β-carotene bleaching assay, the antioxidant activity was 90.02 ± 1.51 % for the positive control (Trolox) and 88.34 ± 1.31 % for the pulp part of the fruit methanol extract. The total phenolic content of the crude extracts was determined using the Folin-Ciocalteu procedure; methanol pulp part demonstrated the highest phenol content value of 781 ± 52.97 mg GAE/g of dry sample. While the total flavonoid content was determined using the aluminium chloride colorimetric assay, the highest value of 6213.33 ± 142.22 mg QE/g was indicated by pulp part of the fruit methanol extract. The antimicrobial activity of the crude extracts was tested using disc diffusion method against pathogenic microorganisms: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Salmonella typhimurium, Escherichia coli, Klebsiella pneumonia and Candida albicans. Methanol extract of pulp part was recorded to have the highest zone of inhibition against Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration (MIC) and MBC/minimal fungicidal concentration (MFC) for the extracts were also determined using the microdilution method ranging from 4000 to 63 μg/ml against pathogenic microbes. The MBC/MFC values varied from 250 to 4000 μg/ml. A correlation between antioxidant activity assays, antimicrobial activity and phenolic content was established. The results shows that the various parts of A. altilis fruit extracts promising antioxidant activities have potential bioactivities due to high content of phenolic compounds.
Bacterial cells of Enterobacter aerogenes NBO2 were entrapped in calcium alginate beads in order to enhance polygalacturonase production compared to free cells. The optimized condition of 5 % (w/v) sodium alginate concentration, agitation speed of 250 rpm, and 15 beads of calcium alginate with inoculum size of 4 % (v/v; 5.4 × 10(7) cells/ml) produced 23.48 U/mL of polygalacturonase compared to free cells of 18.54 U/ml. There was about 26.6 % increment in polygalaturonase production. However, in this study, there was 296.6 % of increment in polygalacturonase production after improvement parameters compared to before improvement parameters of calcium alginate bead immobilization cells (5.92 U/ml). This research has indicated that optimized physical parameters of calcium alginate bead immobilization cells have significantly enhanced the production of polygalacturonase.
Xylitol production by bioconversion of xylose can be economically interesting if the raw material can be recovered from a cheap lignocellulosic biomass (LCB). Meranti wood sawdust (MWS) is a renewable and low-cost LCB that can be used as a promising and economic source of xylose, a starting raw material for the manufacture of several specialty chemicals, especially xylitol. This study aimed to optimize the hydrolysis process of MWS and to determine the influence of temperature, H2SO4 concentration, and residence time on xylose release and on by-product formation (glucose, arabinose, acetic acid, furfural, hydroxymethylfurfural (HMF), and lignin degradation products (LDPs)). Batch hydrolysis was conducted under various operating conditions, and response surface methodology was adopted to achieve the highest xylose yield. Xylose production was highly affected by temperature, acid concentration, and residence time. The optimum temperature, acid concentration, and time were determined to be 124 °C, 3.26 %, and 80 min, respectively. Under these optimum conditions, xylose yield and selectivity were attained at 90.6 % and 4.05 g/g, respectively.
The contributing molecular pathways underlying the pathogenesis of breast cancer need to be better characterized. The principle of our study was to better understand the genetic mechanism of oncogenesis for human breast cancer and to discover new possible tumor markers for use in clinical practice. We used complimentary DNA (cDNA) microarrays to compare gene expression profiles of treated Michigan Cancer Foundation-7 (MCF-7) with recombinant bromelain and untreated MCF-7. SpringGene analysis was carried out of differential expression followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We identified 1,102 known genes differentially expressed to a significant degree (p<0.001) changed between the treatment. Within this gene set, 20 genes were significantly changed between treated cells and the control cells with cutoff fold change of more than 1.5. These genes are RNA-binding motif, single-stranded interacting protein 1 (RBMS1), ribosomal protein L29 (RPL29), glutathione S-transferase mu 2 (GSTM2), C15orf32, Akt3, B cell translocation gene 1 (BTG1), C6orf62, C7orf60, kinesin-associated protein 3 (KIFAP3), FBXO11, AT-rich interactive domain 4A (ARID4A), COPS2, TBPL1|SLC2A12, TMEM59, SNORD46, glioma tumor suppressor candidate region gene 2 (GLTSCR2), and LRRFIP. Our observation on gene expression indicated that recombinant bromelain produces a unique signature affecting different pathways, specific for each congener. The microarray results give a molecular mechanistic insight and functional effects, following recombinant bromelain treatment. The extent of changes in genes is related to and involved significantly in gap junction signaling, amyloid processing, cell cycle regulation by BTG family proteins, and breast cancer regulation by stathmin1 that play major roles.
This study aimed to evaluate the effects of electroporation on growth, bioconversion of isoflavones, and probiotic properties of parent organisms and subsequent passages of Bifidobacterium longum FTDC 8643. Electroporation with the strength of electric field at 7.5 kV cm(-1) for 3.5 ms was applied on B. longum FTDC 8643. The viability of B. longum FTDC 8643 increased significantly upon treatment with electroporation. Such treatment also enhanced the intracellular and extracellular β-glucosidase activity, leading to enhanced production of bioactive isoflavone aglycones in mannitol-soymilk (P
Xylose reductase (XR) is an intracellular enzyme, which catalyzes xylose to xylitol conversion in the microbes. It has potential biotechnological applications in the manufacture of various commercially important specialty bioproducts including xylitol. This study aimed to prepare XR from adapted strain of Candida tropicalis and to characterize it. The XR was isolated from adapted C. tropicalis, cultivated on Meranti wood sawdust hemicellulosic hydrolysate (MWSHH)-based medium, via ultrasonication, and was characterized based on enzyme activity, stability, and kinetic parameters. It was specific to NADPH with an activity of 11.16 U/mL. The enzyme was stable at pH 5-7 and temperature of 25-40 °C for 24 h and retained above 95 % of its original activity after 4 months of storage at -80 °C. The K m of XR for xylose and NADPH were 81.78 mM and 7.29 μM while the V max for them were 178.57 and 12.5 μM/min, respectively. The high V max and low K m values of XR for xylose reflect a highly productive reaction among XR and xylose. MWSHH can be a promising xylose source for XR preparation from yeast.
In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.
Microbial pigments are gaining intensive attention due to increasing awareness of the toxicity of synthetic colours. In this study, a novel polymer-producing bacterium designated as Cupriavidus sp. USMAHM13 was also found to produce yellow pigment when cultivated in nutrient broth. Various parameters such as temperature, pH and ratio of culture volume to flask volume were found to influence the yellow pigment production. UV-Visible, Fourier transform infrared and (13)C-nuclear magnetic resonance analyses revealed that the crude yellow pigment might probably represent new bioactive compound in the carotenoid family. The crude yellow pigment also exhibited a wide spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria with their inhibition zones and minimal inhibitory concentrations ranged from 25 to 38 mm and from 0.63 to 2.5 mg/ml, respectively. To the best of our knowledge, this is the first report on the identification and characterization of yellow pigment produced by bacterium belonging to the genus Cupriavidus.
This study aimed at optimizing the production of hyaluronic acid by Lactobacillus acidophilus FTDC 1231 using response surface methodology and evaluating the effects of divalent metal ions along the production pathway using molecular docking. Among different divalent metal ions that were screened, only iron (II) sulphate and copper (II) sulphate significantly (P
Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k1', value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ∼ 22 kJ mol(-1), while energy for laccase inactivation was 18 kJ mol(-1). Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT = 40 K) as opposed to fungal biomass (ΔT = 15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.
Astaxanthin, a carotenoid pigment found in several aquatic organisms, is responsible for the red colour of salmon, trout and crustaceans. In this study, astaxanthin production from freshwater microalga Chlorella sorokiniana and marine microalga Tetraselmis sp. was investigated. Cell growth and astaxanthin production were determined spectrophotometrically at 620 and 480 nm, respectively. Astaxanthin was extracted using acetone and measured subsequent to biomass removal. Aerated conditions favoured astaxanthin production in C. sorokiniana, whereas Tetraselmis sp. was best cultured under unaerated conditions. C. sorokiniana produced more astaxanthin with the highest yield reached at 7.83 mg/l in 6.0 mM in nitrate containing medium compared to Tetraselmis sp. which recorded the highest yield of only 1.96 mg/l in 1.5 mM nitrate containing medium. Production in C. sorokiniana started at the early exponential phase, indicating that astaxanthin may be a growth-associated product in this microalga. Further optimization of astaxanthin production was performed using C. sorokiniana through a 2(3) full factorial experimental design, and a yield of 8.39 mg/l was achieved. Overall, the study has shown that both microalgae are capable of producing astaxanthin. Additionally, this research has highlighted C. sorokiniana as a potential astaxanthin producer that could serve as a natural astaxanthin source in the current market.
Poly(3-hydroxybutyrate) [P(3HB)], a polymer belonging to the polyhydroxyalkanoate (PHA) family, is accumulated by numerous bacteria as carbon and energy storage material. The mobilization of accumulated P(3HB) is associated with increased stress and starvation tolerance. However, the potential function of accumulated copolymer such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] remained unknown. In this study, Delftia acidovorans DS 17 was used to evaluate the contributions of P(3HB) and P(3HB-co-3HV) granules during simulated exogenous carbon deprivation on cell survival by transferring cells with PHAs to carbon-free mineral salt medium supplemented with 1% (w/v) nitrogen source. By mobilizing the intracellular P(3HB) and P(3HB-co-3HV) at 11 and 40 mol% 3HV compositions, the cells survived starvation. Surprisingly, D. acidovorans containing P(3HB-co-94 mol% 3HV) also survived although the mobilization was not as effective. Similarly, recombinant Escherichia coli pGEM-T::phbCAB(Cn) (harboring the PHA biosynthesis genes of Cupriavidus necator) containing P(3HB) granules had a higher viable cell counts compared to those without P(3HB) granules but without any P(3HB) mobilization when exposed to oxidative stress by photoactivated titanium dioxide. This study provided strong evidence that enhancement of stress tolerance in PHA producers can be achieved without mobilization of the previously accumulated granules. Instead, PHA biosynthesis may improve bacterial survival via multiple mechanisms.
Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates the cellular process of cryopreserved protocorm-like bodies (PLBs) was different comparative to non-cryopreserved PLBs. The cellular process was not only modified by the freezing and thawing effect but also due to the dehydration process itself during the cryopreservation procedure. Histological observation in Dendrobium Bobby Messina in encapsulation-dehydration method indicated that the degree of plasmolysis causes more cellular changes to the cryopreserved PLBs comparative to non-cryopreserved and stock culture PLBs. These results revealed higher amount of homogenous cell population and denser cytoplasm in cryopreserved PLBs. Histological analysis also revealed more voluminous nucleus in cryopreserved PLBs comparative to non-cryopreserved PLBs and PLBs stock culture. In contrast, scanning electron microscope analysis showed severe damages in cryopreserved PLBs and non-cryopreserved PLBs comparative to the PLBs stock culture which in return could be the possible reason of no regrowth in encapsulation-dehydration method. Damages incurred were on top part, side part, and at the stomata of the PLBs. Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates that the degree of plasmolysis causes changes in the cellular process of PLBs from cryopreserved PLBs was different comparative to non-cryopreserved PLBs.
The effect of cultivation condition of two locally isolated ascomycetes strains namely Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 were compared in submerged and solid state fermentation. Physical evaluation on water absorption index, solubility index and chemical properties of lignin, hemicellulose and cellulose content as well as the cellulose structure on crystallinity and amorphous region of treated oil palm empty fruit bunch (OPEFB) (resulted in partial removal of lignin), sago pith residues (SPR) and oil palm decanter cake towards cellulases production were determined. Submerged fermentation shows significant cellulases production for both strains in all types of substrates. Crystallinity of cellulose and its chemical composition mainly holocellulose components was found to significantly affect the total cellulase synthesis in submerged fermentation as the higher crystallinity index, and holocellulose composition will increase cellulase production. Treated OPEFB apparently induced the total cellulases from T. asperellum UPM1 and A. fumigatus UPM2 with 0.66 U/mg FPase, 53.79 U/mg CMCase, 0.92 U/mg β-glucosidase and 0.67 U/mg FPase, 47.56 U/mg and 0.14 U/mg β-glucosidase, respectively. Physical properties of water absorption and solubility for OPEFB and SPR also had shown significant correlation on the cellulases production.
Cryopreservation is an alternative, safe, and cost-effective method for long-term plant genetic resource conservation. This study was conducted to optimize the conditions for cryopreserving the protocorm-like bodies (PLBs) of Brassidium Shooting Star orchid with the PVS3 vitrification method. Five parameters were assessed in this study: PLB size, sucrose concentration, preculture duration, PVS3 duration, and unloading duration. The viability of the cryopreserved PLBs was determined using the triphenytetrazolium chloride assay and growth recovery assessments. The optimum condition for the cryopreservation of the PLBs of Brassidium Shooting Star orchid is based on the size range between 3 and 4 mm precultured with half-strength semi-solid MS media supplemented with 0.25 M sucrose for 24 h, followed by treatment with loading solution mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min. The PLBs were then dehydrated with PVS3 at 0 °C for 20 min prior to immersion in liquid nitrogen; finally, the PLBs were immersed with half-strength liquid MS media supplemented with 1.2 M sucrose for 30 min. Histological analyses displayed denser cytoplasm and voluminous nucleus in the cryopreserved PLBs of Brassidium Shooting Star orchid.
The ability of non-living biomass of an arsenic-hypertolerant Bacillus cereus strain SZ2 isolated from a gold mining environment to adsorb As (III) from aqueous solution in batch experiments was investigated as a function of contact time, initial As (III) concentration, pH, temperature and biomass dosage. Langmuir model fitted the equilibrium data better in comparison to Freundlich isotherm. The maximum biosorption capacity of the sorbent, as obtained from the Langmuir isotherm, was 153.41 mg/g. The sorption kinetic of As (III) biosorption followed well the pseudo-second-order rate equation. The Fourier transform infrared spectroscopy analysis indicated the involvement of hydroxyl, amide and amine groups in As (III) biosorption process. Field emission scanning electron microscopy-energy dispersive X-ray analysis of the non-living B. cereus SZ2 biomass demonstrated distinct cell morphological changes with significant amounts of As adsorbed onto the cells compared to non-treated cells. Desorption of 94 % As (III) was achieved at acidic pH 1 showing the capability of non-living biomass B. cereus SZ2 as potential biosorbent in removal of As (III) from arsenic-contaminated mining effluent.
Lafora disease (LD) is an autosomal recessive, progressive form of myoclonus epilepsy which affects worldwide. LD occurs mainly in countries like southern Europe, northern Africa, South India, and in the Middle East. LD occurs with its onset mainly in teenagers and leads to decline and death within 2 to 10 years. The genes EPM2A and EPM2B are commonly involved in 90 % of LD cases. EPM2A codes for protein laforin which contains an amino terminal carbohydrate binding module (CBM) belonging to the CBM20 family and a carboxy terminal dual specificity phosphatase domain. Mutations in laforin are found to abolish glycogen binding and have been reported in wet lab methods. In order to investigate on structural insights on laforin mutation K81A, we performed molecular dynamics (MD) simulation studies for native and mutant protein. MD simulation results showed loss of stability due to mutation K87A which confirmed the structural reason for conformational changes observed in laforin. The conformational change of mutant laforin was confirmed by analysis using root mean square deviation, root mean square fluctuation, solvent accessibility surface area, radius of gyration, hydrogen bond, and principle component analysis. Our results identified that the flexibility of K87A mutated laforin structure, with replacement of acidic amino acid to aliphatic amino acid in functional CBM domain, have more impact in abolishing glycogen binding that favors LD.
Regrowth of the cryopreserved protocorm-like bodies (PLBs) of Dendrobium Bobby Messina was assessed based on the plant vitrification solution 2 (PVS2) optimisation conditions. The optimized protocol obtained based on TTC spectrophotometrical analysis and growth recovery were 3-4 mm of PLBs size precultured in 0.2 M sucrose for 1 day, treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min and subsequently dehydrated with PVS2 at 0 °C for 20 min prior to storage in liquid nitrogen. Following rapid warming in a water bath at 40 °C for 90 s, PLBs were treated with unloading solution containing half-strength liquid MS media supplemented with 1.2 M sucrose. Subsequently, the PLBs were cultured on half-strength semi-solid MS media supplemented with 2 % (w/v) sucrose without any growth regulators and resulted in 40 % growth recovery. In addition, ascorbic acid treatment was used to evaluate the regeneration process of cryopreserved PLBs. However, growth recovery rates of non-cryopreserved and cryopreserved PLBs were 30 and 10 % when 0.6 mM ascorbic acid was added. Scanning electron microscopy analysis indicates that there are not much damages observed on both cryopreserved and non-cryopreserved PLBs in comparison to PLBs stock culture.