Members of the genus Aglaia have been reported to contain bioactive phytochemicals. The genus, belonging to the Meliaceae family, is represented by at least 120 known species of woody trees or shrubs in the tropical rain forest. As some of these species are very similar in their morphology, taxonomic identification can be difficult. A reliable and definitive molecular method which can identify Aglaia to the level of the species will hence be useful in comparing the content of specific bioactive compounds between the species of this genus. Here, we report the analysis of DNA sequences in the internal transcribed spacer (ITS) of the nuclear ribosomal DNA and the observation of a unique nucleotide signature in the ITS that can be used for the identification of Aglaia stellatopilosa. The nucleotide signature consists of nine bases over the length of the ITS sequence (654 bp). This uniqueness was validated in 37 samples identified as Aglaia stellatopilosa by an expert taxonomist, whereas the nucleotide signature was lacking in a selection of other Aglaia species and non-Aglaia genera. This finding suggests that molecular typing could be utilized in the identification of Aglaia stellatopilosa.
In the world of fast fashion, textile industries are blooming rapidly to meet the consumer's demands. However, vast amounts of wastewater have been constantly produced, and it is becoming a serious environmental problem in the waterways. Although the technology for treating textile wastewater has been well reported and established, more sustainable efforts have taken the attention nowadays. Through the use of living Malaysian Ganoderma lucidum mycelial pellets (GL) and activated dolomite (AD) in the treatment system, the study explores the synergy between biosorption and physisorption as alternative treatment for textile wastewater. In the current work, mixture of GL premixed with AD (50:50; v/v) is used to treat industrial textile wastewater. The morphology, adsorption characteristics, and antibacterial activity of the adsorbents were studied. The mixture of adsorbents is capable of removing colours by 77.8% and reducing chemical oxygen demand (COD) by 75% within 48 h contact. Furthermore, the kinetic and adsorption had been studied and follow the pseudo-first-order kinetic model while both adsorption of Langmuir and Freundlich model was deduced from the treatment. In addition, antimicrobial activities from the treatment potentially reduced 10 × 101 CFU/mL after 48 h. The synergistic treatment by Ganoderma lucidum mycelial pellets and activated dolomite has immense potential in future wastewater treatment technology to obtain cleaner water.
Microalgae are the most promising sources of protein, which have high potential due to their high-value protein content. Conventional methods of protein harnessing required multiple steps, and they are generally complex, time consuming, and expensive. Currently, the study of integration methods for microalgae cell disruption and protein recovery process as a single-step process is attracting considerable interest. This study aims to investigate the novel approach of integration method of electrolysis and liquid biphasic flotation for protein extraction from wet biomass of Chlorella sorokiniana CY-1 and obtaining the optimal operating conditions for the protein extraction. The optimized conditions were found at 60% (v/v) of 1-propanol as top phase, 250 g/L of dipotassium hydrogen phosphate as bottom phase, crude microalgae loading of 0.1 g, air flowrate of 150 cc/min, flotation time of 10 min, voltage of 20 V and electrode's tip touching the top phase of LBEF. The protein recovery and separation efficiency after optimization were 23.4106 ± 1.2514% and 173.0870 ± 4.4752%, respectively. Comparison for LBEF with and without the aid of electric supply was also conducted, and it was found that with the aid of electrolysis, the protein recovery and separation efficiency increased compared to the LBEF without electrolysis. This novel approach minimizes the steps for overall protein recovery from microalgae, time consumption, and cost of operation, which is beneficial in bioprocessing industry.
Various studies showed that the suppression of α-glucosidase activity can impede the glucose absorption in our body, and therefore, it can be used to treat type 2 diabetes. Hence, the compounds with anti-α-glucosidase have gained considerable attention because of their potential application in diabetes treatment. In previous literature studies, these anti-α-glucosidase compounds were extracted from plants and fungus. Less studies are being conducted to identify the anti-α-glucosidase compounds in the microbial community. In this study, 23 marine bacterial strains were screened for their potential to suppress the α-glucosidase activity. The highest inhibitory activity was exhibited by isolated L06 which was identified as Oceanimonas smirnovii EBL6. The cultivation conditions, such as temperature and pH, were optimized to increase the production of α-glucosidase inhibitors by Oceanimonas smirnovii EBL6 strain. The result findings showed that the highest yield of α-glucosidase inhibitors can be obtained at the culture time of 120 h, fermentation temperature of 30 °C, and pH 4.6. Under these conditions, the inhibitory activity of α-glucosidase can reach 81%. The IC50 of n-butanol extract was 13.89 μg/ml, while standard acarbose was 31.16 μg/ml. Overall, these findings suggest that Oceanimonas smirnovii produces α-glucosidase inhibitors and could been applied in the biochemical and medicinal fields in the future.
The distinctive morphology characteristics of microfold cells (M cells) allow the vaccine antigen not only to interact with immune cells directly, but also to effectively stimulate mucosal immune responses via receptors on its apical surface. Human prion protein, a transmembrane receptor for Brucella abortus Hsp60, is highly expressed on the M cell surface. Nonetheless, this protein tends to express in inclusion body in prokaryotic hosts. In this study, the shorter interacting regions of human prion protein were identified via computational methods such as docking and molecular dynamics simulations to minimize its aggregation tendency. The computational calculations revealed three novel human prion protein-interacting regions, namely PrP125, PrP174, and PrP180. In accordance with in silico prediction, the biologically synthesized peptides fusing with GST tag demonstrated their specific binding to Hsp60 protein via pull-down assay. Hence, this finding laid the groundwork for M-cell targeting candidate validation through these newly identified interacting regions.
The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.
Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.
The infection produced by the SARS-CoV-2 virus remains a significant health crisis worldwide. The lack of specific medications for COVID-19 necessitates a concerted effort to find the much-desired therapies for this condition. The main protease (Mpro) of SARS-CoV-2 is a promising target, vital for virus replication and transcription. In this study, fifty pyrazole derivatives were tested for their pharmacokinetics and drugability, resulting in eight hit compounds. Subsequent molecular docking simulations on SARS-CoV-2 main protease afforded two lead compounds with strong affinity at the active site. Additionally, the molecular dynamics (MD) simulations of lead compounds (17 and 39), along with binding free energy calculations, were accomplished to validate the stability of the docked complexes and the binding poses achieved in docking experiments. Based on these findings, compound 17 and 39, with their favorable projected pharmacokinetics and pharmacological characteristics, are the proposed potential antiviral candidates which require further investigation to be used as anti-SARS-CoV-2 medication.
This study aimed to evaluate the two-stage and one-stage anaerobic co-digestion of vinasse and spent brewer yeast cells (SBY) for biohydrogen and methane production. Optimization of the vinasse-to-SBY ratio and fly ash concentration of the two-stage and one-stage production processes was investigated. In the two-stage process, the vinasse-to-SBY ratio and fly ash concentration were optimized, and the leftover effluent was used for methane production. The optimum conditions for biohydrogen production were a vinasse-to-SBY ratio of 7:3% v/w and fly ash concentration of 0.4% w/v, in which the maximum hydrogen yield was 43.7 ml-H2/g-VSadded. In contrast, a vinasse-to-SBY ratio of 10:0% v/w and fly ash concentration of 0.2% w/v were considered optimal for methane production, and resulted in a maximum methane yield of 214.6 ml-CH4/g-VSadded. For the one-stage process, a vinasse-to-SBY ratio of 10:0% v/w and fly ash concentration of 0.1% w/v were considered optimal, and resulted in a maximum methane yield of 243.6 ml-CH4/g-VSadded. In the two-stage process, the energy yield from hydrogen (0.05-0.47 kJ/g-VSadded) was 0.62%-11.78%, and the major fraction was approximately 88.22%-99.38% gain from methane (3.19-7.73 kJ/g-VSadded). For the one-stage process, the total energy yield distribution ranged from 4.20 to 8.77 kJ/g-VSadded.
Biomolecules produced by living organisms can perform vast array of functions and play an important role in the cell. Important biomolecules such as lysozyme, bovine serum albumin (BSA), and bromelain are often studied by researchers due to their beneficial properties. The application of reverse micelles is an effective tool for protein separation from their sources due to the special system structure. Mechanisms of transferring biomolecules and factors that influence the extraction of biomolecules are reviewed in this paper. The enhancement of biomolecule extraction could be achieved depending on the properties of reverse micelles. This paper provides an overall review on lysozyme, BSA, and bromelain extraction by reverse micelle for various applications.
The synergistic effects of antimicrobial nanostructures with antibiotics present a promising solution for overcoming resistance in methicillin-resistant Staphylococcus aureus (MRSA). Previous studies have introduced iron as a novel coating for silver nanoparticles (AgNPs) to enhance both economic efficiency and potency against S. aureus. However, there are currently no available data on the potential of these novel nanostructures to reverse MRSA resistance. To address this gap, a population study was conducted within the MRSA community, collecting a total of 48 S. aureus isolates from skin lesions. Among these, 21 isolates (43.75%) exhibited cefoxitin resistance as determined by agar disk diffusion assay. Subsequently, a PCR test confirmed the presence of the mecA gene in 20 isolates, verifying them as MRSA. These results highlight the cefoxitin disk diffusion susceptibility test as an accurate screening method for predicting mecA-mediated resistance in MRSA. Synergy tests were performed on cefoxitin, serving as a marker antibiotic, and iron-coated AgNPs (Fe@AgNPs) in a combination study using the checkerboard assay. The average minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) of cefoxitin were calculated as 11.55 mg/mL and 3.61 mg/mL, respectively. The findings indicated a synergistic effect (FIC index
Fusarium oxysporum f. sp. cubense is one of the most severe and threatening pathogens of bananas, causing "Panama wilt" worldwide. Confrontation assay of Foc antagonistic bacterial endophyte, Bacillus velezensis YEBBR6, with the Foc and GC-MS profiling of excised agar from the zone of inhibition, led to the unveiling of secondary metabolites produced by the endophyte. To refine the probable antifungal compounds among the numerous biomolecules formed during their di-trophic interaction with the pathogen, fungal protein targets were modeled, and docking studies (AutoDock Vina module of the PyRx 0.8 server) were done with all the compounds. Triamcinolone acetonide exhibited the most excellent affinity for the protein targets among the compounds studied. It had a maximum binding affinity of 11.2 kcal/mol for XRN2 (5' → 3'). Further, the protein-ligand complex formation kinetics was done through Molecular Dynamic Simulation studies. Graphs for the RMSD, RMSF, Rg, potential energy, and SASA were generated, and the values during the simulation period suggested the stability of the biomolecule as a complex with the protein. This indicated Triamcinolone acetonide's potential ability to act as a functional disrupter of the target protein and likely an antifungal molecule. Further, the biomolecule was tested for its activity against Foc by screening in the wet lab through the poisoned plate technique, and it was found to be fully inhibitory to the growth of the pathogen at 1000 ppm.
New pandemic infection of coronaviridae family virus spread to more than 210 countries with total infection of 1,136,851 and 62,955 (4.6%) deaths until 5th April 2020. Which stopped the regular cycle of humankind but the nature is consistently running. There is no micro molecule remedy found yet to restore the regular life of people. Hence, we decided to work on natural biophores against the COVID proteins. As a first step, major phytoconstituents of antiviral herbs like Leucas aspera, Morinda citrifolia, Azadirachta indica, Curcuma longa, Piper nigrum, Ocimum tenuiflorum, and Corallium rubrum collected and performed the lock and key analysis with major spike protein of COVID-19 to find the best fitting lead biophore using computational drug design platform. The results of protocol run showed, phytoconstituents of Morinda citrifolia and Leucas aspera were found lower binding energy range of - 55.18 to - 25.34 kcal/mol, respectively and compared with Hydroxychloroquine (HCQ) (- 24.29 kcal/mol) and Remdesivir (- 25.38 kcal/mol). The results conclude that, core skeletons chromen, anthracene 9, 11 dione and long-chain alkyl acids/ester-containing biophores showen high stable antagonistic affinity with S-protein. Which leads the breakdown of spike protein and ACE2 receptor complex formation and host mechanism of corono virus. In addition, the dynamic trajectory analysis confirmed the complete denaturation of spike protein by the molecule 4-(24-hydroxy-1-oxo-5-n-propyltetracosanyl)-phenol from Leucas aspera and stability of spike-ligand complex. These biophores will aid the researcher to fabricate new promising analogue and being recommended to assess its COVID-19 treatment.
Conventionally, increasing the yield of microalgal biomass has been the primary focus of research, while the significant protein reserve within this biomass has remained largely unexplored. This protein reserve possesses substantial value and versatility, offering a wide range of prospective applications and presenting an enticing chance for innovation and value enhancement for various sectors. Current study employed an innovative research approach that focused solely on the LCA of protein production potential from microalgal biomass, a lesser-explored aspects within this domain. Most environmental impact categories were shown to be significantly affected by cultivation phase because of the electrical obligation, followed by the harvesting and protein extraction phase. Still, the environmental aspect was seen to yield a minimal impact on global warming potential, i.e., 4 × 10-3 kg CO2, underscoring the ecologically favorable nature of the process. Conversely, the overall energy impact was seen to intensify with NEB of - 39.33 MJ and NER of 0.49, drawing attention to the importance of addressing the energy aspect to harness the full potential of microalgal protein production.
Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile-Thr-Arg-Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein-solvent interaction.
The application of native enzymes may not be economical owing to the stability factor. A smaller protein molecule may be less susceptible to external stresses. Haloalkane dehalogenases (HLDs) that act on toxic haloalkanes may be incorporated as bioreceptors to detect haloalkane contaminants. Therefore, this study aims to develop mini proteins of HLD as an alternative bioreceptor which was able to withstand extreme conditions. Initially, the mini proteins were designed through computer modeling. Based on the results, five designed mini proteins were deemed to be viable stable mini proteins. They were then validated through experimental study. The smallest mini protein (model 5) was chosen for subsequent analysis as it was expressed in soluble form. No dehalogenase activity was detected, thus the specific binding interaction of between 1,3-dibromopropane with mini protein was investigated using isothermal titration calorimetry. Higher binding affinity between 1,3-dibromopropane and mini protein was obtained than the native. Thermal stability study with circular dichroism had proven that the mini protein possessed two times higher Tm value at 83.73 °C than the native at 43.97 °C. In conclusion, a stable mini protein was successfully designed and may be used as bioreceptors in the haloalkane sensor that is suitable for industrial application.
The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris-HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris-HCl pH 8, while for L2 lipase it was at 70 °C in Glycine-NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.
The present study focused on preparing and characterizing magnetite-polyvinyl alcohol (PVA) hybrid nanoparticles using Acanthophora spicifera marine algae extract as a reducing agent. Various analytical techniques, including UV-Visible spectrometry, Fourier-transform infrared (FTIR) analysis, energy-dispersive X-ray (EDX), scanning electron microscopy (SEM), and X-ray diffraction (XRD) analysis, were used to characterize the nanoparticles. The results showed the successful synthesis of nanoparticles with a characteristic color change and absorption peak at 400 nm in UV-Visible spectrometry. FTIR analysis indicated an interaction between the carboxyl group and magnetite-polyvinyl alcohol hybrid ions. SEM analysis revealed spherical nanoparticles with sizes ranging from 20 to 100 nm. EDX analysis confirmed the presence of strong magnetite peaks in Acanthophora spicifera, validating successful preparation. XRD analysis indicated the crystalline nature of the nanoparticles. Furthermore, the antimicrobial potential of As-PVA-MNPs was evaluated, demonstrating a significant zone of inhibition against tested bacterial and fungal samples at a concentration of 100 µg. These findings suggest the promising antimicrobial activity of the synthesized nanoparticles for potential applications in combating pathogenic microorganisms.
As the world population grows, the demand for food increases. Although vegetable oils provide an affordable and rich source of energy, the supply of vegetable oils available for human consumption is limited by the "fuel vs food" debate. To increase the nutritional value of vegetable oil, metabolic engineering may be used to produce oil crops of desirable fatty acid composition. We have isolated and characterized β-ketoacyl ACP-synthase II (KASII) cDNA from a high-oleic acid palm, Jessenia bataua. Jessenia KASII (JbKASII) encodes a 488-amino acid polypeptide that possesses conserved domains that are necessary for condensing activities. When overexpressed in E. coli, recombinant His-tagged JbKASII was insoluble and non-functional. However, Arabidopsis plants expressing GFP-JbKASII fusions had elevated levels of arachidic acid (C20:0) and erucic acid (C22:1) at the expense of stearic acid (C18:0) and oleic acid (C18:1). Furthermore, JbKASII failed to complement the Arabidopsis KASII mutant, fab1-2. This suggests that the substrate specificity of JbKASII is similar to that of ketoacyl-CoA synthase (KCS), which preferentially elongates stearic and oleic acids, and not palmitic acid. Our results suggest that the KCS-like JbKASII may elongate C18:0 and C18:1 to yield C20:0 and C22:1, respectively. JbKASII may, therefore, be an interesting candidate gene for promoting the production of very long chain fatty acids in transgenic oil crops.