Enhancement effect of AgO nanoparticles on fermentative cellulase activity from thermophilic Bacillus subtilis Ag-PQ.

BACKGROUND: Cellulase is an important bioprocessing enzyme used in various industries. This study was conducted with the aim of improving the biodegradation activity of cellulase obtained from the Bacillus subtilis AG-PQ strain. For this purpose, AgO and FeO NPs were fabricated using AgNO3 and FeSO4·7H2O salt respectively through a hydro-thermal method based on five major steps; selection of research-grade materials, optimization of temperature, pH, centrifuge, sample washed with distilled water, dry completely in the oven at the optimized temperature and finally ground for characterization. The synthesized NPs were characterized by scanning electron microscope (SEM), energy dispersive X-ray (EDX), and X-ray diffraction (XRD) to confirm the morphology, elemental composition, and structure of the sample respectively. The diameter of the NPs was recorded through SEM which lay in the range of 70-95 nm. RESULTS: Cultural parameters were optimized to achieve better cellulase production, where incubation time of 56 h, inoculum size of 5%, 1% coconut cake, 0.43% ammonium nitrate, pH 8, and 37 °C temperature were found optimal. The enhancing effect of AgO NPs was observed on cellulase activity (57.804 U/ml/min) at 50 ppm concentration while FeO NPs exhibited an inhibitory effect on cellulase activity at all concentrations. Molecular docking analysis was also performed to understand the underlying mechanism of improved enzymatic activity by nanocatalysts. CONCLUSION: This study authenticates AgO NPs as better nanocatalysts for improved thermostable cellulase biodegradation activity with the extraordinary capability to be potentially utilized in bioethanol production.

Production, purification, and characterization of p-diphenol oxidase (PDO) enzyme from lignolytic fungal isolate Schizophyllum commune MF-O5.

Fungi are producers of lignolytic extracellular enzymes which are used in industries like textile, detergents, biorefineries, and paper pulping. This study assessed for the production, purification, and characterization of novel p-diphenol oxidase (PDO; laccase) enzyme from lignolytic white-rot fungal isolate. Fungi samples collected from different areas of Pakistan were initially screened using guaiacol plate method. The maximum PDO producing fungal isolate was identified on the basis of ITS (internal transcribed spacer sequence of DNA of ribosomal RNA) sequencing. To get optimum enzyme yield, various growth and fermentation conditions were optimized. Later PDO was purified using ammonium sulfate precipitation, size exclusion, and anion exchange chromatography and characterized. It was observed that the maximum PDO producing fungal isolate was Schizophyllum commune (MF-O5). Characterization results showed that the purified PDO was a monomeric protein with a molecular mass of 68 kDa and showed stability at lower temperature (30 °C) for 1 h. The Km and Vmax values of the purified PDO recorded were 2.48 mM and 6.20 U/min. Thermal stability results showed that at 30 °C PDO had 119.17 kJ/K/mol Ea value and 33.64 min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0 mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1 mM showing 12.04% activity. The enzyme showed stability against 10 mM concentration oxidizing reducing agents like DMSO, EDTA, H2O2, NaOCl, and urea and retained more than 75% of relative activity. The characterization of purified PDO enzyme confirmed its tolerance against salt, metal ions, organic solvents, and surfactants indicating its ability to be used in the versatile commercial applications.

Diversity in Elemental Content in Selected Artemisia L. (Asteraceae) Species from Gilgit-Baltistan Region of Pakistan Based on Inductively Coupled Plasma Atomic Emission Spectrophotometry (ICP-AES).

Diversity in eleven Artemisia species from northern Pakistan was assessed based on as per suitability of their elemental contents with thermal conductivity detection and ICP-AES procedures. Results indicated the presence of 13 major elements in the Artemisia species with varied concentrations including Carbon (45.7%, 45,7000 ppm-49.8%, 49,8000 ppm), Nitrogen (2.03%, 20,300 ppm-3.50%, 35,000 ppm), Phosphorus (0.168%, 1680 ppm-0.642%, 6420 ppm), Potassium (2.38%, 23,800 ppm-4.72%, 47,200 ppm), Sulphur (1920 ppm, 0.192%-4780 ppm, 0.478%), Boron (23.8 ppm, 0.00238%-71.7 ppm, 0.00717%), Calcium (0.733%, 7330 ppm-2.249%, 22,490 ppm), Magnesium (0.116%, 1160 ppm-0.267%, 2670 ppm), Zinc (27.7 ppm, 0.00277%-47.9 ppm, 0.00479%), Manganese (25.7 ppm, 0.00257%-93.8 ppm, 0.00938%), Iron (353 ppm, 0.0353%-1532 ppm, 0.1532%), Copper (14.1 ppm, 0.00141%-26.2 ppm, 0.00262%) and Sodium (105 ppm, 0.0105%-587 ppm, 0.0587%). Cluster analysis distributed the Artemisia species into two major groups (G1 and G2) on the basis of their elemental content where G1 contained species like, Artemisia herba alba Asso., A. tournefortiana Rachb., A. rutifolia Steph. ex Spreng., and A. vulgaris L., with the presence of all elements with the maximum amount of S, Zn, P, Ca, and Mg, while G2 contained species like Artemisia biennis Willd., A. chamaemelifolia Vill., A. capillaris, L., A. gmelinii Weber ex Stech., A. indica Willd., A. maritima L., and A. verlotiorum Lamotte., with all elements but significant concentrations of B, N, C, K, Mn, Fe, Cu, and Na. PCA analysis displayed maximum species diversity in the axes two, while axes one showed lower diversity. Additionally, the elevated levels of elements recorded as compared to the threshold levels recommended in the literature for medicinal plants require extraordinary precautionary measures before or during using Artemisia as medication to avoid metal toxicity.

Evaluation of Antioxidant Activity and Histopathological Changes Occurred by the Oral Ingestion of CuO Nanoparticles in Monosodium Urate Crystal-Induced Hyperuricemic BALB/c Mice.

Nanotechnology is an intensive branch of science due to the unique features of nano range particles (1-100 nm). Their nano size results in a high surface area of absorption when orally administered. Monosodium urate crystal excessive deposition causes a commonly known inflammatory disease called gout into the synovial joints. Previously it has been observed that copper oxide nanoparticles (CuONPs) had a significant effect in reducing the serum uric acid levels in BALB/c mice as well as reducing the inflammation in the ankles of mice. This study was made to investigate the antioxidant and histopathological changes in hyperuricemic BALB/c mice upon the oral administration of copper oxide nanoparticles. Different concentrations of copper oxide nanoparticles 5, 10, and 20 ppm were given orally to gouty mice. To investigate the antioxidant activity of CuONPs, various antioxidant protocols were applied. It was noted that the nanoparticle-treated group of 20 ppm showed no significant changes in superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), thiobarbituric acid reactive substances (TBARS), and ROS values while the protein estimation values of the negative control group exhibited a significant increase (0.001). When compared to negative control, no significant effect was shown on the interpretation of histopathological changes of muscles, kidney, and liver tissues.

In Vivo Effects of Orally Administered Different Concentrations of Silver Oxide Nanoparticles in Hyperuricemic Mice.

Silver oxide nanoparticles (AgO-NPs) antioxidant, anti-cancer, anti-microbial, and tissue repair properties. Gouty arthritis is the inflammation of tissues and joints caused by the deposition of monosodium urate crystals. In this experiment, we investigated the anti-hyperuricemic effectiveness of different concentrations of AgO-NPs in mice. The present study aimed to investigate the effect of administration of AgO-NPs in monosodium urate (MSU)-induced gouty mice for the very first time. Monosodium urate (MSU) crystals were administered intraperitoneal for gout induction, followed by 5, 10, and 20 µg/mL doses of AgO-NPs for 2 weeks. The positive control was provided with the commercially available drug allopurinol to compare the effects of AgO-NPs and allopurinol. The main purpose of the study was to investigate the effectiveness of the nanoparticles in comparison with commercially available drugs. AgO-NPs have been shown to improve the condition of gouty arthritis by reducing significantly (P ˂ 0.001) increased levels of ALT, AST, and total bilirubin. The total protein estimation results showed significant improvement at concentration of 20 µg/mL of AgO-NPs. The lipid profile results showed that high concentration (20 µg/mL) of AgO-NPs decrease the lipid content significantly as compared to control. It was concluded from this study that the antioxidant, anti-inflammatory, and antilipidemic properties of AgO-NPs may improve the hyperuricemic condition in gouty arthritis mice.

Anti-Hyperuricemic Effect of Iron Oxide Nanoparticles against Monosodium Urate Crystals Induced Gouty Arthritis in BALB/c Mice.

Iron oxide nanoparticles (FeO-NPs) exhibit exceptional properties which can be utilized in various aspects of biological sciences. In this experiment we investigated the anti-gout effectiveness of FeO-NPs in mice. BALB/c mice were induced gouty arthritis by administering monosodium urate (MSU) crystals. These gout induced mice were treated with three different concentrations of FeO-NPs (5 ppm, 10 ppm and 20 ppm). Precipitation method was utilized for the synthesis of FeO-NPs, these synthesized NPs were of average 54 nm in size and were characterized using XRD, SEM and EDS. FeO-NPs is given orally three weeks by using FeO-NPs solution to substitute drinking water. Blood biochemical parameters including liver function tests (LFTs), renal function tests (RFTs), lipid profile and blood count have been tested. It has been found that uric acid, blood urea and creatinine have decreased significantly after three weeks of FeO-NP administration (P Value < 0.001) thus suppressing hyperuricemia and gouty arthritis. Additionally, the liver enzymes analysis showed a slight increase in AST, ALT and alkaline phosphatase levels (P Value < 0.001). Histopathological research revealed no significant abnormal changes in the liver, muscle and kidney muscles of the test groups. The findings showed that FeO-NPs can be used for the successful treatment of hyperuricemic condition and gouty arthritis in the coming future in place of commercially available medicines.

Alkaline lipase production by novel meso-tolerant psychrophilic Exiguobacterium sp. strain (AMBL-20) isolated from glacier of northeastern Pakistan.

Lipase is an important commercial enzyme with unique and versatile biotechnological applications. This study was conducted to biosynthesize and characterizes alkaliphilic lipase by Exiguobacterium sp. strain AMBL-20T isolated from the glacial water samples of the northeastern (Gilgit-Baltistan) region of Pakistan. The isolated bacterium was identified as Exiguobaterium sp. strain AMBL-20T on the basis of morphological, biochemical, and phylogenetic analysis of 16S rRNA sequences with GenBank accession number MW229267. The bacterial strain was further screened for its lipolytic activity, biosynthesis, and characterization by different parameters with the aim of maximizing lipase activity. Results showed that 2% Olive oil, 0.2% peptone at 25 °C, pH 8, and 24 h of incubation time found optimal for maximum lipase production. The lipase enzyme was partially purified by ammonium sulphate precipitation and its activity was standardized at pH 8 under 30 °C temperature. The enzyme showed functional stability over a range of temperature and pH. Hence, extracellular alkaliphilic lipase from Exiguobacterium sp. is a potential candidate with extraordinary industrial applications, particularly in bio-detergent formulations.

Surface-functionalised hybrid nanoparticles for targeted treatment of cancer.

Cancer is a leading cause of death worldwide. Despite the great advancement in understanding the pharmacology and biology of cancer, it still signifies one of the most serious human-health related problems. The current treatments for cancer may include surgery, radiotherapy, and chemotherapy, but these procedures have several limitations. Current studies have shown that nanoparticles (NPs) can be used as a novel strategy for cancer treatment. Developing nanosystems that allow lower doses of therapeutic agents, as well as their selective release in tumour cells, may resolve the challenges of targeted cancer therapy. In this review, the authors discuss the role of the size, shape, and surface modifications of NPs in cancer treatment. They also address the challenges associated with cancer therapies based on NPs. The overall purpose of this review is to summarise the recent developments in designing different hybrid NPs with promising therapeutic properties for different types of cancer.

Effect of Endoxylanase and Iron Oxide Nanoparticles on Performance and Histopathological Features in Broilers.

Endoxylanase enzyme is used as poultry feed additive to degrade anti-nutritional factors like non-starch polysaccharides. Moreover, iron is one of the most important trace elements, and its deficiency can lead to various pathological conditions and stunted growth. In the current study, a combination of xylanase and iron nanoparticles is used to assess the overall effect on poultry growth. Endoxylanase enzyme was obtained from Aspergillus awamori SAIB-17 (identified on the basis of internal transcribed spacer (ITS) sequence analysis). The characterization of purified endoxylanase revealed that the Km and Vmax were 0.25 g/ml and 833.33 nkat/ml/s, respectively. Effect of pH and temperature showed that pH 4.5 and temperature 45 °C was best for enzyme activity. Iron nanoparticles were synthesized by co-precipitation of ferric chloride and ferrous chloride. Characterization of nanoparticles using X-ray diffraction (XRD) and SEM revealed that the mean diameter of synthesized iron nanoparticles was around 50 nm. These nanoparticles have no inhibitory effect on endoxylanase up to concentration of 20 ppm. Iron oxide nanoparticles along with endoxylanase were used as additives in different concentrations and were fed to the groups of broiler chicks. It was observed that the group fed with 40 nkat of endoxylanase and 15 ppm of iron nanoparticles showed 54.5% (2010 ± 103.58) more weight gain by the fifth week as compared with the control group. The iron analysis in the muscles showed no increase in iron concentration while histopathology slides showed no morphological changes in liver cells. The combination of iron oxide nanoparticles and xylanase proved to have great potential to be used in poultry feed for large-scale meat production without any toxicological effects.

Inhibition of Hyperuricemia and Gouty Arthritis in BALB/c Mice Using Copper Oxide Nanoparticles.

Nanoparticles are known for their unique properties and are being utilized in various disciplines of sciences. Their nanosize enables them to higher exposure and higher availability when given orally. Gout is an inflammatory disease caused by deposition of monosodium urate (MSU) crystal deposition into the joints. The objective of this study was to evaluate the effects of copper oxide nanoparticles on hyperuricemia and gouty arthritis in mice. In this research, synthesized copper oxide nanoparticles of size ranging from 30 to 50 nm were administered orally to mice having gouty arthritis and hyperuricemia. Various biochemical markers were conducted to determine the effects of copper oxide nanoparticles. It was observed that the mice treated with CuO NPs at various concentrations showed a significant (0.001) decrease in the serum uric acid levels in comparison with the negative control. Furthermore, creatinine levels were also normal in comparison with the control mice. Measurement of synovial joints also revealed that mice administered with CuO NPs had reduced inflammation of synovial joints in comparison with the negative control. From this research, it was concluded that copper oxide nanoparticles have potential in the treatment of hyperuricemia and gouty arthritis by decreasing serum uric acid and inflammation in synovial joints.

Antioxidant and anti-gout effects of orally administered zinc oxide nanoparticles in gouty mice.

BACKGROUND: Zinc is an essential trace element which is involved in controlling oxidative stress, growth and immune system by regulating inflammatory cytokines. Gouty arthritis is the inflammation of joints and tissues caused by the accumulation of monosodium urate crystals. METHOD AND OBJECTIVE: This study involved the oral administration of zinc oxide nanoparticles at a various concentration (5 ppm, 10 ppm, and 20 ppm) and study their antioxidant and anti-gout effects on Balb/C mice. Various parameters such as ROS, superoxide, peroxide, catalase, TBARS, RFTs, LFTs, lipid profile and blood count were studied. RESULTS: ZnO nanoparticles at the concentrations of 10 and 20 ppm were significant (P < 0.001) in reducing serum uric acid concentration thus treating gouty arthritis. Reactive oxygen species and thiobarbituric acid reactive substances were significantly increased in comparison to zinc oxide nanoparticles treated groups. Furthermore, blood count and LFTs also showed the effectiveness of zinc oxide in the reduction of hyperuricemia. Histopathological analysis showed no apparent changes in liver, kidney and muscles tissues. CONCLUSION: Zinc oxide nanoparticles can be effective in reducing oxidative stress and the treatment of gouty arthritis.

Purification, Characterization and Thermodynamic Assessment of an Alkaline Protease by Geotrichum Candidum of Dairy Origin.

BACKGROUND: Alkaline proteases is the important group of enzymes having numerous industrial applications including dairy food formulations. OBJECTIVES: The current study deals with the purification and characterization of an alkaline serine protease produced by Geotrichum candidum QAUGC01, isolated from indigenous fermented milk product, Dahi. MATERIAL AND METHODS: In total twelve G. candidum strains were screened for their proteolytic activity by using standard protease assay. The protease production from G. candidum QAUGC01 was optimized by varying physio-chemical conditions. The protease was purified by using two-step method: ammonium sulfate precipitation and gel filtration chromatography. Protease was further characterized by studying various parameter like temperature, pH, modulators, metal ions and organic solvent. A thermodynamic study was also carried out to explore the half-life of protease. RESULTS: The G. candidum grew profusely at 25 °C and at an initial pH of 4.0 for 72 h of incubation producing 26.21 U/ml maximum extracellular protease. Protease revealed that Vmax and Km was 26.25 U.ml-1.min-1 and 0.05 mg.mL-1, respectively using casein as substrate. The enzyme was stable at a temperature range (25-45 °C) and pH (8-9). Residual enzyme activity was strongly inhibited in the presence of PMSF (7.5%). The protease could hydrolyze proteinaceous substrates, casein (98%) and BSA (95%). The thermodynamic studies explored that the half-life of the enzyme that was 106.62 min, 38.72 min and 15.71 min at 50, 60 and 70 °C, respectively. CONCLUSIONS: Purified protease from G. candidum GCQAU01 is an ideal candidate for industrial application.

Evaluation of Turmeric Nanoparticles as Anti-Gout Agent: Modernization of a Traditional Drug.

Background and objectives: Turmeric has assisted in the control of inflammation and pain for decades and has been used in combination with other nutraceuticals to treat acute and chronic osteoarthritis pain. Recently, the effect of turmeric, turmeric extract, or curcuminoids on musculoskeletal pain, either by themselves or in conjunction with other substances, has been reported. The aim of this study was to develop and characterize turmeric nanoparticles (T-NPs) for various parameters, both in vitro and in vivo. Materials and Methods: The T-NPs were successfully synthesized and characterized using particle size analysis, solubility improvement, SEM, EDX, X-ray diffraction, and in vivo antigout activity in mice model. Results: The T-NPs were of about 46 nm in size with a positive zeta potential +29.55 ± 3.44 and low polydispersity index (PDI) (0.264). Furthermore, the diseased mice, with induced gout via monosodium urate crystals, were treated with 5, 10, and 20 ppm T-NPs, administered orally, and the anti-gout potential was observed through measurement of joint diameter and changes in biochemical parameters, including lipid profile, renal function test, and liver function tests which significantly reduced the levels of these biochemical parameters. Conclusions: Uric acid levels were significantly reduced after the treatment with T-NPs. indicating that T-NPs show superior potential against gout management. Thus, T-NPs can be developed as an efficient antigout agent with minimum toxicities.

Kinetics of high-level of ß-glucosidase production by a 2-deoxyglucose-resistant mutant of Humicola lanuginosa in submerged fermentation / Cinética de produção de ß-glucosidase por um mutante de Hemicola lanuginosa resistente a 2-deoxiglucose em fermentação submersa

A 2-deoxyglucose-resistant mutant (M7) of Humicola lanuginosa was obtained by exposing conidia to γ-rays and permitting expression in broth containing 0.6 percent 2-deoxyglucose (DG) and cellobiose (1 percent) before plating on DG esculin-ferric ammonium citrate agar medium from which colonies showing faster and bigger blackening zones were selected. Kinetic parameters for enhanced ß-glucosidase (BGL) synthesis by M7 were achieved when corncobs acted as the carbon source. The combination between corncobs and corn steep liquor was the best to support higher values of all product formation kinetic parameters. Effect of temperature on the kinetic and thermodynamic attributes of BGL production equilibrium in the wild organismand M7was studied using batch process at eight different temperatures in shake-flask studies. The best performance was found at 45ºC and 20 g L-1 corncobs in 64 h. Both growth and product formation (17.93 U mL-1) were remarkably high at 45ºC and both were coupled under optimum working conditions. Product yield of BGL from the mutant M7 (1556.5 U g-1 dry corncobs) was significantly higher than the values reported on all fungal and bacterial systems. Mutation had thermo-stabilization influence on the organism and mutant required lower activation energy for growth and lower magnitudes of enthalpy and entropy for product formation than those demanded by the wild organism, other mesophilic and thermo-tolerant organisms. In the inactivation phase, the organisms needed lower values of activation energy, enthalpy and entropy for product formation equilibrium, confirming thermophilic nature of metabolic network possessed by the mutant organism.

Um mutante de Hemicola lanuginosa resistente a 2-deoxiglucose(M7) foi obtido através de exposição de conídios a raios γ, permitindo a expressão em caldo contendo 0,6 por cento de 2-deoxiglucose (DG) e celobiose (1 por cento) antes da semeadura em ágar DG esculina citrato de ferro amoniacal, da qual foram selecionadas as colônias com halo negro. Os parâmetros cinéticos para produção aumentada de ß-glucosidase (BGL) foram obtidos empregando-se sabugo de milho como fonte de carbono. A combinação de espiga de milho com água de maceração de milho foi a que forneceu os valores mais altos nos parâmetros cinéticos de formação de todos os produtos. O efeito da temperatura na cinética e atributos termodinâmicos da produção de BGL pelas cepas selvagem e M7 foi avaliado empregando-se processo de batelada em oito temperaturas diferentes in frascos em agitação. O melhor desempenho foi observado a 45ºC e 20g.l-1 de espiga de milho em 64h. Tanto a multiplicação quanto a formação do produto foram muito altas a 45ºC e ambas estavam ligadas em condições ótimas de trabalho. O rendimento de BGL produzido pelo mutante M7 (1556 U.g-1 de espiga seca) foi significativamente superior aos valores reportados para todos os sistemas fúngicos e bacterianos. A mutação influenciou a termoestabilização no microrganismo, sendo que o mutante necessitou de energia de ativação mais baixa para multiplicação e valores mais baixos de entalpia e entropia para a formação do produto quando comparado à cepa selvagem e a outros microrganismos mesofilicos e termotolerantes. Na fase de inativação, os microrganismos necessitaram valores mais baixos de energia de ativação, entalpia e entropia para o equilíbrio da formação de produto, confirmando a natureza termofílica da máquina metabólica do mutante.

Kinetics of high-Level of ß-glucosidase production by a 2-deoxyglucose-resistant mutant of Humicola lanuginosa in submerged fermentation.

A 2-deoxyglucose-resistant mutant (M7) of Humicola lanuginosa was obtained by exposing conidia to γ-rays and permitting expression in broth containing 0.6% 2-deoxyglucose (DG) and cellobiose (1%) before plating on DG esculin-ferric ammonium citrate agar medium from which colonies showing faster and bigger blackening zones were selected. Kinetic parameters for enhanced ß-glucosidase (BGL) synthesis by M7 were achieved when corncobs acted as the carbon source. The combination between corncobs and corn steep liquor was the best to support higher values of all product formation kinetic parameters. Effect of temperature on the kinetic and thermodynamic attributes of BGL production equilibrium in the wild organism and M7 was studied using batch process at eight different temperatures in shake-flask studies. The best performance was found at 45°C and 20 g L(-1) corncobs in 64 h. Both growth and product formation (17.93 U mL(-1)) were remarkably high at 45°C and both were coupled under optimum working conditions. Product yield of BGL from the mutant M7 (1556.5 U g(-1) dry corncobs) was significantly higher than the values reported on all fungal and bacterial systems. Mutation had thermo-stabilization influence on the organism and mutant required lower activation energy for growth and lower magnitudes of enthalpy and entropy for product formation than those demanded by the wild organism, other mesophilic and thermo-tolerant organisms. In the inactivation phase, the organisms needed lower values of activation energy, enthalpy and entropy for product formation equilibrium, confirming thermophilic nature of metabolic network possessed by the mutant organism.