Maximum Activity Research Articles

Extraction and characterization of carotenoid pigments with antioxidant and antibacterial potential from marine yeast Rhodotorula sp. KSB1.

Pigments are coloring agents used widely in different industrial sectors. There is a demand for using natural pigments rather than synthetic dyes because of the health hazards caused by synthetic dyes. Many natural pigments have different medicinal activities which can contribute to the nutritional value of the product. This study was carried forward with marine yeasts which can produce pigments. A total of 4 marine yeast isolates were recovered from the mangrove area of Sundarbans, West Bengal, India. Among them, the isolate KSB1 produced 856µg/g total concentration of carotenoid pigment and the dry mass weight was 3.56g/L. The stability of the extracted pigments was checked using temperature, pH, UV light exposure time, and different saline conditions. The pigments were characterized using HPLC and FTIR analysis. All of the extracted pigments showed good antioxidant activity in DPPH, metal chelating, and reducing power assay. The pigments were also found to have good antibacterial activity against the bacterial pathogens Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli. Carotenoid pigment from KSB1 was found to have maximum activity in all the pathogens. The cytogenotoxicity using onion roots and phytotoxicity analysis indicated that the pigments were non-toxic and safe for cells. Finally, the potential marine yeast was identified using 18s rRNA sequencing and identified as Rhodotorula sp. KSB1 (Accession no. MH782232).

Radionuclide Transfer in Tea Cultivation: Assessing Radiological Risks in the Largest and First Established Tea Garden in Bangladesh

ABSTRACT To address potential health risks associated with radioactivity in tea consumption, a study was conducted utilizing high purity germanium (HPGe) γ-ray spectrometry to measure the concentrations of 226Ra, 232Th, 40K, and 137Cs radionuclides in soil and tea leaves collected from several major tea gardens (including the Malnicherra tea garden which is the largest and first established tea garden in the Indian subcontinent) situated in Sylhet, Bangladesh. Although nearly all soil samples showed the presence of 137Cs (with a maximum activity of 6.5 ± 1.31 Bq/kg), none of the analyzed tea leaf samples exhibited its presence, which can be attributed to its restricted transferability from soil to plants. The range of transfer factors for 226Ra, 232Th and 40K from soil to tea leaves was calculated to be (0.39–0.54), (0.25–0.46) and (1.39–3.03), respectively. The heightened concentrations of 40K identified in the tea leaf samples can be mainly ascribed to specific metabolic processes associated with potassium, along with the heightened utilization of potassium chloride-based fertilizers aimed at enhancing tea production. The examination of hazard parameters, including the estimated committed effective dose and the excess lifetime cancer risk associated with tea consumption, reveals values that are comparatively lower than the recommended limits. Consequently, the radiation exposure from tea consumption does not present a significant radiological health concern for the general population.

Zinc Oxide:Gold Nanoparticles (ZnO:Au NPs) Exhibited Antifungal Efficacy Against Aspergillus niger and Aspergillus candidus

AbstractFungal pathogens are a major health issue that threatens the era of antifungal drugs commonly used in the treatment of infections. An effective approach of biosynthetic nanoparticles can be used as antifungal agents owing to their intrinsic features such as their simplicity, non-toxic, and physicochemical properties. Therefore, this study was aimed to molecularly ascertain Aspergillus species known to cause aspergillosis and investigate the potency of zinc oxide:gold nanoparticles (ZnO:Au NPs) against the fungal pathogens. Two Aspergillus strains retrieved with potato dextrose agar (PDA) culture media from commercial food products in South Africa were molecularly identified using calmodulin (CaM) gene region. DNA sequence phylogeny of the gene showed that the strains were A. niger and A. candidus. ZnO:Au (1%) NPs were synthesised and characterised using X-ray diffraction (XRD), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). Two distinct peak plasmon bands for ZnO and ZnO:Au NPs were 390 nm and 565 nm, respectively. FE-SEM images demonstrated the presence of Au on the surface of ZnO nanoparticles in the ZnO:Au nanocomposites. The ZnO:Au NPs antifungal activity of 10 µg/mL and 50 µg/mL concentrations were evaluated against the two Aspergillus spp. ZnO:Au NPs at 50 µg/mL exhibited a maximum antifungal activity against A. candidus and A. niger, with zones of inhibition (ZoI) of 31.2 ± 0.15 mm and 25.0 ± 0.06, respectively. When the ZoI was observed by SEM, major morphological damages on the conidia were observed for both strains, indicating that the antifungal activity may have been enhanced by the ZnO:Au NPs. Therefore, due to these outstanding properties, ZnO:Au NPs can be utilised as potential antifungal agents to inhibit the proliferation of fungal pathogens.

Optimization studies on laccase activity of Proteus mirabilis isolated from treatment sludge of textile industry factories : Optimization of laccase activity of Proteus mirabilis.

Laccase is an exothermic enzyme with copper in its structure and has an important role in biodegradation by providing oxidation of phenolic compounds and aromatic amines and decomposing lignin. The aim of this study is to reach maximum laccase enzyme activity with minimum cost and energy through optimization studies of Proteusmirabilis isolated from treatment sludge of a textile factory. In order to increase the laccase enzyme activities of the isolates, medium and culture conditions were optimized with the study of carbon (Glucose, Fructose, Sodium Acetate, Carboxymethylcellulose, Xylose) and nitrogen sources (Potassium nitrate, Yeast Extract, Peptone From Soybean, Bacteriological Peptone), incubation time, pH, temperature and Copper(II) sulfate concentration then according to the results obtained. Response Surface Method (RSM) was performed on six different variables with three level. According to the data obtained from the RSM, the maximum laccase enzyme activity is reached at pH 7.77, temperature 30.03oC, 0.5g/L CuSO4, 0.5g/L fructose and 0.082g/L yeast extract conditions. After all, the laccase activity increased 2.7 times. As a result, laccase activity of P. mirabilis can be increased by optimization studies. The information obtained as a result of the literature studies is that the laccase enzymes produced in laboratory and industrial scale are costly and their amounts are low. This study is important in terms of obtaining more laccase activity from P.mirabilis with less cost and energy.

Further characterisation of late somatosensory evoked potentials using electroencephalogram and magnetoencephalogram source imaging.

Beside the well-documented involvement of secondary somatosensory area, the cortical network underlying late somatosensory evoked potentials (P60/N60 and P100/N100) is still unknown. Electroencephalogram and magnetoencephalogram source imaging were performed to further investigate the origin of the brain cortical areas involved in late somatosensory evoked potentials, using sensory inputs of different strengths and by testing the correlation between cortical sources. Simultaneous high-density electroencephalograms and magnetoencephalograms were performed in 19 participants, and electrical stimulation was applied to the median nerve (wrist level) at intensity between 1.5 and 9 times the perceptual threshold. Source imaging was undertaken to map the stimulus-induced brain cortical activity according to each individual brain magnetic resonance imaging, during three windows of analysis covering early and late somatosensory evoked potentials. Results for P60/N60 and P100/N100 were compared with those for P20/N20 (early response). According to literature, maximal activity during P20/N20 was found in central sulcus contralateral to stimulation site. During P60/N60 and P100/N100, activity was observed in contralateral primary sensorimotor area, secondary somatosensory area (on both hemispheres) and premotor and multisensory associative cortices. Late responses exhibited similar characteristics but different from P20/N20, and no significant correlation was found between early and late generated activities. Specific clusters of cortical activities were activated with specific input/output relationships underlying early and late somatosensory evoked potentials. Cortical networks, partly common to and distinct from early somatosensory responses, contribute to late responses, all participating in the complex somatosensory brain processing.

Investigation on antioxidant, antimicrobial, anti-inflammatory, and neuropsychiatry potential of phyto-mediated ZnONPs using Colebrookeaoppositifolia

The present study reports the phyto-mediated synthesis of ZnONPs using methanol extracts and n-Butanol fractions from the roots and leaves of C. oppositifolia. The synthesized nanoparticles were characterized using XRD, FE-SEM, HR-TEM, and XPS techniques to determine their crystallite structure, elemental composition, surface topography, and physio-chemical properties. Furthermore, the biological potential of ZnONPs was evaluated for antioxidant, antimicrobial, anti-inflammatory, and in vitro cytotoxic potential on SH-SY5Y neuroblastoma cell lines. Notably, LEME-ZnONPs exhibited exceptional antioxidant activity with an IC50 of 30.200 μg/mL in the DPPH assay. The antimicrobial efficacy was also remarkable, displaying high maximum activity against bacterial strains, evidenced by zones of inhibition (ZOI) measuring 15.67 ± 0.58 mm (E. coli) and 14.67 ± 0.58 mm (S. aureus) for RTME-ZnONPs, attributable to their small crystallite size. Similarly, against antifungal strains, LEBF-ZnONPs (60.94 ± 0.83 %) and RTBF-ZnONPs (60.85 ± 0.74 %) exhibited potency to inhibit both F. oxysporum and R. necatrix due to their small particle size. Furthermore, LEBF-ZnONPs demonstrated superior anti-inflammatory properties, registering an impressive 98.681 %, outperforming Diclofenac sodium (96.835 %). Importantly, the phyto-mediated ZnONPs exhibited minimal cytotoxicity on SH-SY5Y cell lines at 62.5 μg/mL, a concentration employed as an experimental model to assess neuronal function. The multifaceted applications of phyto-mediated ZnONPs extend across various fields, including pharmaceutical sciences, agriculture, and the treatment of neurological diseases.

Enhanced antioxidant activity in Imidazolium-based protic organic salts: An insight into structure activity relationship via experimental and modelling approaches

Sulfonyl hydroxide functionalized imidazolium based protic ionic liquids (PILs) have been evaluated as a new form of antioxidants. The antioxidant activity was performed using free radical quenching using UV–visible spectroscopy and cyclic voltammetry. The studies confirmed that the investigated ILs with variable anions exhibited substantial radical scavenging activity. In UV–Visible study, Glutamine based-IL ([1MeimPS][Gln]) showed least IC50 value of 95 µM indicating maximum radical quenching activity as an oxidant, while proline based-IL ([1MeimPS]Pro]) exhibited highest IC50 value of 675 µM, demonstrating least anti-oxidant activity. Moreover, cyclic voltammetry results complemented well the UV–visible findings with least IC50 value of 90 µM for [1MeimPS][Gln], showing maximum antioxidant activity, and highest IC50 value of 593 µM for [1MeimPS]Pro], exhibiting the least antioxidant activity. The presence of two primary amines moieties in glutaminate anion may be responsible for enhanced antioxidant activity by providing more active sites for radical quenching. Contrarily, lack of primary amine functionality in prolinate anion may contribute to reduced antioxidant activity of respective IL. The Density Functional Theory (DFT) calculations predicted that the trend of radical scavenging potential was comparable with the experimentally obtained IC50 values. Molecular Electrostatic Potential (MESP) Maps illustrated that [1MeimPS][Pro] has the maximum red zones indicating a poor potential against electron scavenging capacity among all the studied ILs, while blue region in [1MeimPS][Gln], corresponding to low electron density, suggested strong attraction for the incoming electrons. Also, the high electronic distribution of HOMO orbital, appreciable stability, and significant chemical potential to attack on radical species provided by [1MeimPS][Gln] with the maximum radical scavenging potential. Furthermore, the associated LUMO energy for [1MeimPS][Pro] (−1.297 eV) is quite higher than that of [1MeimPS][Gln] (−1.422 eV) which reduced the ability of [1MeimPS][Pro] to attack free radicals of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and thus have higher associated IC50 values. This is also supported by the results of chemical hardness (η) and chemical potential (μ) descriptors values indicating [1MeimPS][Pro] is least stable and reactive than [1MeimPS][Gln]. These findings further explain the least IC50 values observed experimentally and consequent enhanced antioxidant potential of [1MeimPS][Gln].

Improvement of phenolic profile and biological activities of wild mustard sprouts

The current study aimed to assess the effect of the germination process of wild mustard seeds on the phenolic profile, antioxidant, antibacterial, and antidiabetic properties, and some relevant enzyme activities. The total phenolic and flavonoid contents increased 5- and 10-fold, respectively, and were maximized on 5-days sprouts. One new phenolic compound was identified on 5-days sprout extract using HPLC. The concentrations of the identified phenolic compounds increased 1.5–4.3 folds on 5-days sprouts compared with dry seeds. The total antioxidant activity multiplied 17- and 21-fold on 5-days sprouts using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays, respectively. The activity of carbohydrate-cleaving, phenolic-synthesizing and antioxidant enzymes also increased during germination. On 5-days sprouts, there was a substantial correlation between the highest β-glucosidase and peroxidase activities with highest phenolic and flavonoid levels and maximum antioxidant activity. The phenolic extract of 5-days sprouts exhibited antimicrobial activities against Escherichia coli and Staphylococcus aureus and showed potent antidiabetic activity established by its inhibitory effect against α-amylase and α-glucosidase compared to dry seeds.

Metagenomic evaluation of peanut rhizosphere microbiome from the farms of Saurashtra regions of Gujarat, India

The narrow zone of soil around the plant roots with maximum microbial activity termed as rhizosphere. Rhizospheric bacteria promote the plant growth directly or indirectly by providing the nutrients and producing antimicrobial compounds. In this study, the rhizospheric microbiota of peanut plants was characterized from different farms using an Illumina-based partial 16S rRNA gene sequencing to evaluate microbial diversity and identify the core microbiome through culture-independent (CI) approach. Further, all rhizospheric bacteria that could grow on various nutrient media were identified, and the diversity of those microbes through culture-dependent method (CD) was then directly compared with their CI counterparts. The microbial population profiles showed a significant correlation with organic carbon and concentration of phosphate, manganese, and potassium in the rhizospheric soil. Genera like Sphingomicrobium, Actinoplanes, Aureimonas _A, Chryseobacterium, members from Sphingomonadaceae, Burkholderiaceae, Pseudomonadaceae, Enterobacteriaceae family, and Bacilli class were found in the core microbiome of peanut plants. As expected, the current study demonstrated more bacterial diversity in the CI method. However, a higher number of sequence variants were exclusively present in the CD approach compared to the number of sequence variants shared between both approaches. These CD-exclusive variants belonged to organisms that are more typically found in soil. Overall, this study portrayed the changes in the rhizospheric microbiota of peanuts in different rhizospheric soil and environmental conditions and gave an idea about core microbiome of peanut plant and comparative bacterial diversity identified through both approaches.

A study revealing mechanisms behind the stone cell of Yali pear degradation by mixed‐culture fermentation of lactic acid bacteria and yeast

A study revealing mechanisms behind the stone cell of Yali pear degradation by mixed‐culture fermentation of lactic acid bacteria and yeast

Phytochemical screening, antibacterial, antioxidant, and cytotoxic activities of Geranium pusillum leaves.

Traditional medicinal plants play an important role in primary health care worldwide. The phytochemical screening and activities of Geranium pusillum were investigated in this research. The dried plant leaves were extracted with ethanol, n-hexane, chloroform, dichloromethane, methanol, acetone, and aqueous solvents. These extracts were qualitatively analyzed, GC-MS, antimicrobial activities by using the disc diffusion method, antioxidant activity was determined by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging, and cytotoxic activity was analyzed by the hemolytic activity of human red blood cells. The results showed phytochemicals such as flavonoids, terpenoids, steroids, phenols, saponins, tannins, and cardiac glycosides were detected in plant leaves. The ethanol extract at a concentration of 10 mg/mL showed a maximum inhibition zone 17.5 ± 0.09, 15.6 ± 0.11, 14.2 ± 0.17, 18.4 ± 0.11, 16.6 ± 0.15, 12.5 ± 0.13, 15.9 ± 0.10, and 13.1 ± 0.11 mm, and at 15 mg/mL showed 24.5 ± 0.09, 27.2 ± 0.12, 26.3 ± 0.17, 28.4 ± 0.10, 27.9 ± 0.16, 22.5 ± 0.13, 27.1 ± 0.10, and 24.1 ± 0.16 mm against Escherichia coli, Pasturella multocida (gram-negative), Staphylococcus aureus, Bacillus subtilus (gram-positive), Rhizopus solani, Aspergillus flavus, Aspergillus niger, and Alternaria alternate (fungal strain), respectively, and dichloromethane showed a minimum inhibition zone as compared to other extracts against bacterial as well as fungal strains. Chloroform extract had maximum antioxidant activity (45.00 ± 0.08%) and minimum in dichloromethane (12.20 ± 0.04%). Cytotoxic activity was found maximum in acetone extract (19.83 ± 0.07%) and minimum in ethanol extract (4.72 ± 0.04%). It is concluded that phytochemicals like phenols, flavonoids, and others may be responsible for these activities, which is why this plant is used for traditional medicine. RESEARCH HIGHLIGHTS: Geranium pusillum has therapeutic properties that exhibit various biological activities beneficial for human health. G. pusillum has significant inhibitory effects against bacterial and fungal strains. Chloroform solvent extract indicates potential free radical scavenging abilities. Acetone extract exhibits notable effects on human red blood cells and demonstrates significant cytotoxic activity.

Phytochemical screening and antimicrobial activity of some medicinal plants

The antimicrobial activity of the aqueous and ethanolic extracts of Myrtus communis, Ammi visnaga, and Equisetum arvense was investigated against Staphylococcus aureus, Escherichia coli, and Candida albicans by using the agar well diffusion method. Serial concentrations (15%, 30%, and 50%) of the extracts of each plant were tested and compared with gentamicin (10 μg) and fluconazole (25 μg). Most of the extract concentrations showed a relatively high antimicrobial activity against all the tested microbes, and the ethanolic extract was more effective than the aque¬ous extract. The activity of plant extracts increased with the increasing extract concentration of Myrtus communis, which appeared to possess a more antimicrobial activity than the other plants assessed; in fact, its ethanolic extract exhibited the highest inhibition zone against S. aureus (32 mm). The ethanolic plant extracts at a concentration of 50% displayed the maximum activity against the herein assessed isolates. Moreover, E. coli showed a higher sensi¬tivity to most extracts, while the lowest effect being noticed on C. albicans.

Exploration of a novel protease from Phytolacca dodecandra (Endod) berries for potential application in detergent formulation

AbstractConventional detergent formulations contain either petroleum‐derived or synthetic chemicals. Hence, the replacement of these chemicals with natural products such as enzymes is considered a green technology. Among these enzymes, protease is renowned in detergent formulations. Therefore, the objective of this study was to explore and characterize a new protease from Phytolacca dodecandra, because the plant is traditionally known for detergency purposes. Enzyme extraction was performed with Glycine/NaOH buffer, pH = 10.0. Gelatin hydrolysis and agar well diffusion tests were implemented as qualitative assays, whereas the enzyme assay technique was applied for the quantitative investigation of the new protease from plant extracts. The crude enzyme was partially characterized concerning temperature and pH tolerance, washing performance, detergent compatibility, and stability. The finding of this study revealed that Phytolacca dodecandra contains protease and is characterized by 1418.84 U/mL of enzyme activity, where 1 unit (U) of enzyme activity refers to the amount of enzyme required to liberate 1 μg of tyrosine/mL/min under the defined assay conditions. The enzyme showed maximum activity at pH 10.5 and 45°C. The enzyme was stable and maintained 99.52% activity at 40°C and 91.73% at pH 10.0 after 2 h pre‐incubation. The enzyme showed excellent wash performance and could potentially remove blood stains completely within 3 min. After 2 h of pre‐incubation the enzyme maintained 69.06% and 54.88% of its activity in the presence of Azzy and Sunlight, respectively. The enzyme maintained 71.99% of its residual activity after 60 days of storage. If further studies are conducted, all these properties of the newly investigated protease can make the enzyme applicable in detergent formulations.

Ultrasonic-assisted green extraction and incorporation of Spirulina platensis bioactive components into turmeric essential oil-in-water nanoemulsion for enhanced antioxidant and antimicrobial activities

The utilization of essential oils as natural antioxidants and preservatives is limited by high volatility, poor water solubility, and long-term instability. To address this, a novel ultrasonic-assisted method was used to prepare and stabilize a nanoemulsion of turmeric essential oil-in-water, incorporating bioactive components extracted from Spirulina platensis. Ultrasonic treatment enhanced the extraction efficacy and nanoemulsion stability. Algal biomass subjected to ultrasonic treatment (30 min at 80% amplitude) yielded a dry extract of 73.66 ± 3.05%, with the highest protein, phenolic, phycocyanin, and allophycocyanin content, as well as maximum emulsifying activity. The resulting nanoemulsion (5% oil, 0.3% extract, 10 min ultrasonic treatment) showed reduced particle size (173.31 ± 2.24 nm), zeta potential (−36.33 ± 1.10 mV), low polydispersity index, and enhanced antioxidant and antibacterial properties. Rheology analysis indicated shear-thinning behavior, while microscopy and spectroscopy confirmed structural changes induced by ultrasonic treatment and extract concentration. This initiative developed a novel ultrasonic-assisted algal-based nanoemulsion with antioxidant and antibacterial properties.

USE OF ETHOXYLATED ALCOHOLS WITH ANIONIC SURFACTANTS FOR ENHANCED RECOVERY

The article is devoted to the development of surfactant compositions, including a number of known anionic surfactants, in formulations with new types of nonionic surfactants - fatty alcohols with an increased degree of ethoxylation. The solubilizing properties of new types of fatty ethoxylated alcohols have been exploited during formulation development. A conductometric method was used to control the assembly of the formulations. After evaluating the stability of the resulting compositions, physicochemical properties were evaluated, including thermal stability and corrosiveness. The freezing point of the compositions was evaluated at temperatures down to minus 40 °C. The search for stable compositions was carried out for aggressive application conditions: at 90 ° C for models of mineralized formation waters having a total amount of dissolved salts in the range of 100-300 g/l. Ranges including critical concentrations of formulations at which colloidal structures are stable and exhibit maximum surface activity were evaluated. When determining the interfacial tension at the surfactant-hydrocarbon liquid solution boundary, a number of developed compositions showed ultra-low interfacial tension (less than 0.01 mN/m). Biodegradability was evaluated for compositions showing the best properties. According to the results of the assessment, biodegradability was more than 98 %, which reduces environmental risks. The results obtained by the authors show that a number of compositions collected by conductometric method are stable under aggressive conditions and can be used in the field of chemical methods for increasing oil recovery. The resulting compositions were adapted to reduce human factors in subsequent applications. The results reflect the potential of these approaches in adapting and selecting surfactant compositions for chemical waterflooding to enhance oil recovery. The development of technologies for the ethoxylation of higher fatty alcohols allows the manufacturability of processes for the production, collection and treatment of oil and gas. The development of practices for the use of new chemical technologies will expand the capabilities of subsoil users and reduce the risks associated with the use of large-scale chemical products.

Maximization of uricase production in a column bioreactor through RSM based optimization.

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme which is widely exploited for diagnostic and treatment purposes in medicine. This study has been focused on producing recombinant uricase from E. coli BL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through Plackett-Burman design from 8 different variables and were further optimized by central composite design of RSM. The selected variables included the inoculum size (%v/v), IPTG (Isopropyl β-d-1-thiogalactopyranoside) concentration (mM) and the initial pH of culture medium. The activity of uricase, final OD600 (Optical Density at 600nm wave-length) and final pH were considered as the responses of this optimization and were modeled. As a result, the activity of 5.84 U.ml-1 and final OD600 of 3.42 were obtained at optimum conditions of 3 %v/v of inoculum size, IPTG concentration of 0.54 mM and pH=6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165±1.5 mg uricase was obtained from a 600 mL cell culture. The results of this study show that bubble column bioreactors can be a highly effective option for large scale uricase production.

Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation

BackgroundHydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers.ResultsIn this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts.ConclusionsThe recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

A novel antifungal nanoemulsion based on reuterin-assisted synergistic essential oils: Preparation and in vitro/in vivo characterization

A novel antifungal nanoemulsion based on reuterin-assisted synergistic essential oils: Preparation and in vitro/in vivo characterization

Potential elevation of exopeptidase activity of Glu-specific endopeptidase I/GluV8 mediated by hydrophobic P1′-position amino acid residue

We recently reported that the activities of dipeptidyl-peptidase (DPP)7 and DPP11, S46-family exopeptidases were significantly elevated by the presence of prime-side amino acid residues of substrates caused by an increase in kcat [Ohara-Nemoto Y. et al., J Biol Chem 298(3):101585. doi: 10.1016/j.jbc.2022]. In the present study, the effects of prime-side residues on Glu-specific endopeptidase I/GluV8 from Staphylococcus aureus were investigated using a two-step cleavage method with tetrapeptidyl-methycoumaryl-7-amide (MCA) carrying P2- to P2′-position residues coupled with DPP11 as the second enzyme. GluV8 showed maximal activity toward benzyloxycarbonyl (Z)-LLE-MCA, while the effects of hydrolysis of substrates one residue shorter, such as acetyl (Ac)-Val-Glu- and Leu-Glu-MCA, were negligible. Nevertheless, activity towards Ac-VE-|-ID-MCA, a substrate carrying P1′ and P2′ residues, emerged and reached a level 44 % of that for Z-LLE-MCA. Among 11 Ac-HAXD-MCA (X is a varied amino acid), the highest level of activity enhancement was achieved with P1′-Leu and Ile, followed by Phe, Val, Ser, Tyr, and Ala, while Gly and Lys showed scant effects. This activation order was in parallel with the hydrophobicity indexes of these amino acids. The prime-side residues increased kcat/KM primarily through a maximum 500-fold elevation of kcat as well as S46-family exopeptidases. The MEROPS substrate database also indicates a close relationship between activity and hydrophobicity of the P1′ residues in 93 N-terminal-truncated substrates, though no correlation was observed among all 4328 GluV8 entities examined. Taken together, these results are the first to demonstrate N-terminal exopeptidase activity of GluV8, considered to be prompted by hydrophobic P1′ amino acid residues.

A third generation PSMA-targeted agent [211At]YF2: Synthesis and in vivo evaluation

IntroductionTargeted α-particle therapy agents have shown promising responses in patients who have developed resistance to β-particle emitting radionuclides, albeit off-target toxicity remains a concern. Astatine-211 emits only one α-particle per decay and may alleviate the toxicity from α-emitting daughter radionuclides. Previously, we developed the low-molecular-weight PSMA-targeted agent [211At]L3-Lu that showed suitable therapeutic efficacy and was well tolerated in mice. Although [211At]L3-Lu had good characteristics, we now have evaluated a closely related analogue, [211At]YF2, to determine the better molecule for clinical translation. MethodsThe tin precursors and unlabeled iodo standards for [211At]YF2 and [211At]L3-Lu each were synthesized and a new one-step labeling method was developed to produce [211At]YF2 and [211At]L3-Lu from the respective tin precursor. RCY and RCP were determined using RP-HPLC. Cell uptake, internalization and in vitro cell-killing (MTT) assays were performed on PSMA+ PC-3 PIP cells in parallel experiments to compare [211At]YF2 and [211At]L3-Lu directly. A paired-label biodistribution study was performed in athymic mice with subcutaneous PSMA-positive PC-3 PIP xenografts as a head-to-head comparison of [131I]YF2 and [125I]L3-Lu. The tissue distribution of [211At]YF2 and [211At]L3-Lu were determined individually in the same animal model. Finally, the effect of co-injecting unlabeled iodinated YF2 on the biodistribution of [211At]YF2 was evaluated in the same animal model. ResultsThe syntheses of tin precursors and unlabeled iodo standards were accomplished in reasonable yields. A streamlined and scalable radiolabeling method (1 h total synthesis time) was developed for the radiosynthesis of both [211At]YF2 and [211At]L3-Lu with 86 ± 7 % (n = 10) and 87 ± 5 % (n = 7) RCY, respectively, and > 95 % RCP for both. The maximum activity of [211At]YF2 produced to date was 666 MBq. An alternative method that did not involve HPLC purification was developed that provided similar RCY and RCP. Significantly higher cell uptake, internalization and cytotoxicity was seen for [211At]YF2 compared with [211At]L3-Lu. Significantly higher uptake and longer retention in tumor was seen for [131I]YF2 than for co-administered [125I]L3-Lu, while considerably higher renal uptake was seen for [131I]YF2. The biodistribution of [211At]YF2 was consistent with that of [131I]YF2. Co-injection of 2 nmole iodinated YF2 reduced renal uptake of [211At]YF2 by 6-fold without significantly changing tumor uptake. Conclusion[211At]YF2 exhibited higher cellular uptake, internalization and cytotoxicity than [211At]L3-Lu on PSMA-positive PC3 PIP cells. Likewise, higher uptake and longer retention in tumor was seen for [211At]YF2. Experiments to evaluate the dosimetry and therapeutic efficacy of [211At]YF2 are under way.