Decoding the DSCC1 gene as a pan-cancer biomarker in human cancers via comprehensive multi-omics analyses.

OBJECTIVES: While dysregulation of DSCC1 (DNA Replication And Sister Chromatid Cohesion 1) has been established in breast cancer and colorectal cancer, its associations with other tumors remain unclear. Therefore, this study was launched to explore the role of DSCC1 in pan-cancer. METHODOLOGY: In this study, we investigate the biological functions of DSCC1 across 33 solid tumors, elucidating its role in promoting oncogenesis and progression in various cancers through comprehensive analysis of multi-omics data. RESULTS: We conducted a comprehensive analysis of DSCC1 expression using RNA-seq data from TCGA and GTEx databases across 30 cancer types. Striking variations were observed, with significant overexpression of DSCC1 identified in numerous cancers. Elevated DSCC1 level was strongly associated with poorer prognosis, shorter survival, and advanced tumor stages in kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), as indicated by Kaplan-Meier curves and GEPIA2 analysis. Further investigation into the molecular mechanisms revealed reduced DNA methylation in the DSCC1 promoter region in KIRP, LIHC, and LUAD, supporting enhanced RNA transcription. Protein expression analysis via the Human Protein Atlas (HPA) corroborated mRNA expression findings, showcasing elevated DSCC1 protein in KIRP, LIHC, and LUAD tissues. Mutational analysis using cBioPortal revealed alterations in 0.4% of KIRP, 17% of LIHC, and 5% of LUAD samples, predominantly characterized by amplification. Immune cell infiltration analysis demonstrated robust positive correlations between DSCC1 expression and CD8+ T cells, CD4+ T cells, and B cells, influencing the tumor microenvironment. STRING and gene enrichment analyses unveiled DSCC1's involvement in critical pathways, emphasizing its multifaceted impact. Notably, drug sensitivity analysis highlighted a significant correlation between DSCC1 mRNA expression and responses to 78 anticancer treatments, suggesting its potential as a predictive biomarker and therapeutic target for KIRP, LIHC, and LUAD. Finally, immunohistochemistry staining of clinical samples validated computational results, confirming elevated DSCC1 protein expression. CONCLUSION: Overall, this study provides comprehensive insights into the pivotal role of DSCC1 in KIRP, LIHC, and LUAD initiation, progression, and therapeutic responsiveness, laying the foundation for further investigations and personalized treatment strategies.

Microenvironment created by Plantago lagopus L. may affect cover and diversity of coexisting species in urban vegetation.

Some plant species may exhibit new microenvironments which lead to significant changes in the cover and diversity of the coexisting species. In this investigation, we evaluated the effects of Plantago lagopus L. on the cover and diversity of the associated plant species in the urban vegetation. A total of 70 plots were conducted in sites with- and without this species in urban gardens. Cover of the associated species and different diversity indices including species richness, Shannon-Wiener, evenness, and Simpson indices were measured. The allelopathic potential of P. lagopus was verified using its rhizosphere and non-rhizosphere soils on two target species existing within the same environment. Some soil criteria and seed sizes of the associated species were also determined. Most of the coexisting weeds were reduced in terms of their cover in plots with Plantago. The reduction of plant diversity depended on its cover. Besides, the aboveground biomass was reduced in sites comprising Plantago. The degree of inhibition was not related to the seed size of the species found. This species reduced the incident solar radiation and the local temperature over the soil surface. The locations exhibiting such species contained lower contents of available potassium and zinc. Rhizosphere soil of P. lagopus substantially inhibited germination and growth of Amaranthus viridis, but it didn't do so for Medicago lupulina. Reduction in cover, diversity, and biomass of the urban weeds associated with P. lagopus may be related to the reduction of received solar radiation, soil temperature, and nutrient availability. The allelopathic potential of P. lagopus may have a partial role in this reduction. These results suggest that P. lagopus may create a microenvironment of new conditions not favorable for most of the coexisting species.

Correction: Biogenic synthesis of silver nanoparticles using Rubus fruticosus extract and their antibacterial efficacy against Erwinia caratovora and Ralstonia solanacearum phytopathogens.

[This corrects the article DOI: 10.1039/D3RA06723H.].

Targeting the NF-κB p65/Bcl-2 signaling pathway in hepatic cellular carcinoma using radiation assisted synthesis of zinc nanoparticles coated with naturally isolated gallic acid.

PURPOSE: Oral diethylnitrosamine (DEN) is a known hepatocarcinogen that damages the liver and causes cancer. DEN damages the liver through reactive oxygen species-mediated inflammation and biological process regulation. MATERIALS AND METHODS: Gallic acid-coated zinc oxide nanoparticles (Zn-GANPs) were made from zinc oxide (ZnO) synthesized by irradiation dose of 50 kGy utilizing a Co-60 γ-ray source chamber with a dose rate of 0.83 kGy/h and gallic acid from pomegranate peel. UV-visible (UV) spectrophotometry verified Zn-GANP synthesis. TEM, DLS, and FTIR were utilized to investigate ZnO-NPs' characteristics. Rats were orally exposed to DEN for 8 weeks at 20 mg/kg five times per week, followed by intraperitoneal injection of Zn-GANPs at 20 mg/kg for 5 weeks. Using oxidative stress, anti-inflammatory, liver function, histologic, apoptotic, and cell cycle parameters for evaluating Zn-GANPs treatment. RESULTS: DEN exposure elevated inflammatory markers (AFP and NF-κB p65), transaminases (AST, ALT), γ-GT, globulin, and total bilirubin, with reduced protein and albumin levels. It also increased MDA levels, oxidative liver cell damage, and Bcl-2, while decreasing caspase-3 and antioxidants like GSH, and CAT. Zn-GANPs significantly mitigated these effects and lowered lipid peroxidation, AST, ALT, and γ-GT levels, significantly increased CAT and GSH levels (p<0.05). Zn-GANPs caused S and G2/M cell cycle arrest and G0/G1 apoptosis. These results were associated with higher caspase-3 levels and lower Bcl-2 and TGF-ß1 levels. Zn-GANPs enhance and restore the histology and ultrastructure of the liver in DEN-induced rats. CONCLUSION: The data imply that Zn-GANPs may prevent and treat DEN-induced liver damage and carcinogenesis.

Biogenic synthesis of silver nanoparticles using Rubus fruticosus extract and their antibacterial efficacy against Erwinia caratovora and Ralstonia solanacearum phytopathogens.

In the current research, we produced green, cost-effective, eco-friendly silver nanoparticles using a single-step approach. Plants are considered highly desirable systems for nanoparticle synthesis because they possess a variety of secondary metabolites with significant reduction potential. In the current research, the dried leaf extract of Rubus fruticosus was utilized as a capping and reducing agent for the fabrication of silver nanoparticles, to prepare reliable biogenic silver nanoparticles and subsequently to investigate their potential against some common phytopathogens. The prepared silver nanoparticles were exploited to quantify the total flavonoid content (TFC), total phenolic content (TPC) and DPPH-based antioxidant activity. Different concentrations of aqueous extracts of plant leaves and silver nitrate (AgNO3) were reacted, and the color change of the reactant mixture confirmed the formation of Rubus fruticosus leaf-mediated silver nanoparticles (RFL-AgNPs). A series of characterization techniques such as UV-vis spectroscopy, transmission electron microscopy, energy dispersive X-ray analysis and X-ray diffraction revealed the successful synthesis of silver nanoparticles. The surface plasmon resonance peak appeared at 449 nm. XRD analysis demonstrated the crystalline nature, EDX confirmed the purity, and TEM demonstrated that the nanoparticles are mostly spherical in form. Furthermore, the biosynthesized nanoparticles were screened for in vitro antibacterial activity, antioxidant activity, and total phenolic and flavonoid content. The nanoparticles were used in different concentrations alone and in combination with plant extracts to inhibit Erwinia caratovora and Ralstonia solanacearum. In high-throughput assays used to inhibit these plant pathogens, the nanoparticles were highly toxic against bacterial pathogens. This study can be exploited for planta assays against phytopathogens utilizing the same formulations for nanoparticle synthesis and to develop potent antibacterial agents to combat plant diseases.

Interfacial coupling of CuFe2O4 induced hotspots over self-assembled g-C3N4 nanosheets as an efficient photocatalytic bacterial disinfectant.

Most bacterial disinfectants contain high levels of extremely toxic and environmental hazardous chemicals, which pose a significant threat to the ecosystem. Semiconductor photocatalysis exhibits attractive prospects as an emerging greener technology for waste water disinfection. However, the fast recombination of charge carriers limits its practical application. Herein, self-assembled polymeric feather-like g-C3N4 (GCN) nanosheets modified with ferromagnetic CuFe2O4 (CFO) nanospheres were successfully applied as a reusable visible light photocatalytic disinfectant. As expected, the g-C3N4/CuFe2O4 (GCF) nanohybrid displayed superior photocatalytic inactivation efficiency of 0.157log within 120 min towards Escherichia coli DH5α (E. coli) compared with pristine GCN and CFO. The characterization results revealed the synergistic heterostructure interfaces, high surface area, and the transformative self-assembly of GCN to feather-like structure providing a rich active site for improved charge separation efficiency, and wide spectral response, therefore the superior performance of GCF. The radical trapping assay proclaimed that both O2•- and •OH radical played major role in the photocatalytic inactivation among the other reactive oxygen species (ROS). Furthermore, the chemical oxygen demand (COD), protein estimation, and DNA estimation assay results validated the cell damage caused by the photocatalyst. Besides that, GCN showed applicability in real-time wastewater samples with improved efficiency than in the saline solution. The excellent magnetic characteristics facilitated the recycling of the catalyst with insignificant leaching, magnetic induction, and distinguished separation. The results of this work signify the well-designed GCF as a high-performance and reusable photocatalyst for real-world pathogenic bacterial disinfection operations.

Decolorization of Textile Azo Dye via Solid-State Fermented Wheat Bran by Lasiodiplodia sp. YZH1.

Textile dyes are one of the major water pollutants released into water in various ways, posing serious hazards for both aquatic organisms and human beings. Bioremediation is a significantly promising technique for dye decolorization. In the present study, the fungal strain Lasiodiplodia sp. was isolated from the fruiting bodies of Schizophyllum for the first time. The isolated fungal strain was examined for laccase enzyme production under solid-state fermentation conditions with wheat bran (WB) using ABTS and 2,6-Dimethoxyphenol (DMP) as substrates, then the fermented wheat bran (FWB) was evaluated as a biosorbent for Congo red dye adsorption from aqueous solutions in comparison with unfermented wheat bran. A Box-Behnken design was used to optimize the dye removal by FWB and to analyze the interaction effects between three factors: fermentation duration, pH, and dye concentration. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM) were applied to study the changes in the physical and chemical characteristics of wheat bran before and after fermentation. An additional experiment was conducted to investigate the ability of the Lasiodiplodia sp. YZH1 to remove Congo red in the dye-containing liquid culture. The results showed that laccase was produced throughout the cultivation, reaching peak activities of ∼6.2 and 22.3 U/mL for ABTS and DMP, respectively, on the fourth day of cultivation. FWB removed 89.8% of the dye (100 mg L-1) from the aqueous solution after 12 h of contact, whereas WB removed only 77.5%. Based on the Box-Behnken design results, FWB achieved 93.08% dye removal percentage under the conditions of 6 days of fermentation, pH 8.5, and 150 mg L-1 of the dye concentration after 24 h. The fungal strain removed 95.3% of 150 mg L-1 of the dye concentration after 8 days of inoculation in the dye-containing liquid culture. These findings indicate that this strain is a worthy candidate for dye removal from environmental effluents.

Laser Light Treatment Improves the Mineral Composition, Essential Oil Production and Antimicrobial Activity of Mycorrhizal Treated Pelargoniumgraveolens.

Pelargonium graveolens, rose-scented geranium, is commonly used in the perfume industry. P. graveolens is enriched with essential oils, phenolics, flavonoids, which account for its tremendous biological activities. Laser light treatment and arbuscular mycorrhizal fungi (AMF) inoculation can further enhance the phytochemical content in a significant manner. In this study, we aimed to explore the synergistic impact of these two factors on P. graveolens. For this, we used four groups of surface-sterilized seeds: (1) control group1 (non-irradiated; non-colonized group); (2) control group2 (mycorrhizal colonized group); (3) helium-neon (He-Ne) laser-irradiated group; (4) mycorrhizal colonization coupled with He-Ne laser-irradiation group. Treated seeds were growing in artificial soil inculcated with Rhizophagus irregularis MUCL 41833, in a climate-controlled chamber. After 6 weeks, P. graveolens plants were checked for their phytochemical content and antibacterial potential. Laser light application improved the mycorrhizal colonization in P. graveolens plants which subsequently increased biomass accumulation, minerals uptake, and biological value of P. graveolens. The increase in the biological value was evident by the increase in the essential oils production. The concomitant application of laser light and mycorrhizal colonization also boosted the antimicrobial activity of P. graveolens. These results suggest that AMF co-treatment with laser light could be used as a promising approach to enhance the metabolic content and yield of P. graveolens for industrial and pharmaceutical use.

Antibacterial, Antihemolytic, Cytotoxic, Anticancer, and Antileishmanial Effects of Ajuga bracteosa Transgenic Plants.

Herbal and traditional medicines can play a pivotal role in combating cancer and neglected tropical diseases. Ajuga bracteosa, family Lamiaceae, is an important medicinal plant. The genetic transformation of A. bracteosa with rol genes of Agrobacterium rhizogenes further enhances its metabolic content. This study aimed at undertaking the molecular, phytochemical, and in vitro biological analysis of A. bracteosa extracts. We transformed the A. bracteosa plant with rol genes and raised the regenerants from the hairy roots. Transgenic integration and expression of rolB were confirmed by conventional polymerase chain reaction (PCR) and qPCR analysis. The methanol: chloroform crude extracts of wild-type plants and transgenic regenerants were screened for in vitro antibacterial, antihemolytic, cytotoxic, anticancer, and leishmanial activity. Among all plants, transgenic line 3 (ABRL3) showed the highest expression of the rolB gene. Fourier transform infra-red (FTIR) analysis confirmed the enhanced number of functional groups of active compounds in all transgenic lines. Moreover, ABRL3 exhibited the highest antibacterial activity, minimum hemolytic activity (CC50 = 7293.05 ± 7 µg/mL) and maximum antileishmanial activity (IC50 of 56.16 ± 2 µg/mL). ABRL1 demonstrated the most prominent brine shrimp cytotoxicity (LD5039.6 ± 4 µg/mL). ABRL3 was most effective against various human cancer cell lines with an IC50 of 57.1 ± 2.2 µg/mL, 46.2 ± 1.1 µg/mL, 72.4 ± 1.3 µg/mL, 73.3 ± 2.1 µg/mL, 98.7 ± 1.6 µg/mL, and 97.1 ± 2.5 µg/mL against HepG2, LM3, A549, HT29, MCF-7, and MDA-MB-231, respectively. Overall, these transgenic extracts may offer a cheaper therapeutic source than the more expensive synthetic drugs.

A novel biogenic Allium cepa leaf mediated silver nanoparticles for antimicrobial, antioxidant, and anticancer effects on MCF-7 cell line.

In the present study, Allium cepa leaf extract was utilized to reduce the silver nitrate into the nanoscale range of silver ions (Ag NPs). The biosynthesized Ag NPs were extensively characterized by X-ray diffraction analysis (XRD), Dynamic light scattering analysis (DLS), UV-Visible spectroscopy (UV-vis), Transmission electron microscopy (TEM), Energy dispersive X-ray analysis (EDX) and Fourier transform infrared spectroscopy (FTIR). The antioxidant activity of synthesized Ag NPs was verified by DPPH assay. From the results obtained from XRD and DLS studies, the size of Ag NPs was determined to be around 54.3 nm. The measured zeta potential value of -19.1 mV confirms the excellent stability of biosynthesized Ag NPs. TEM analyses reveal that the biosynthesized Ag NPs have a spherical structure of 13 nm in size. The presence of various functional groups was confirmed through FTIR studies and EDAX verifies the weight percentage of silver content in biosynthesized nanoparticles to be 30.33%. In the present study, anti-cancer activity was carried out by using breast cancer cell line MCF-7. Further, silver nanoparticles exhibited antimicrobial effectiveness against gram-positive Bacillus cereus and gram-negative Escherichia coli. The MTT assay also showed better cytotoxic activity against the MCF- 7 cell line.

Stabilization and improved properties of Salipaludibacillus agaradhaerens alkaline protease by immobilization onto double mesoporous core-shell nanospheres.

In this study, serine alkaline protease from halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R was purified and immobilized onto double mesoporous core-shell silica (DMCSS) nanospheres. Covalent immobilization of AK-R protease onto activated DMCSS-NH2 nanospheres was more efficient than physical adsorption and was applied in further studies. DMCSS-NH2 nanospheres showed high loading capacity of 103.8 µg protein/mg nanospheres. Relative to free AK-R protease, the immobilized enzyme exhibited shifts in the optimal temperature and pH from 60 to 65 °C and pH 10.0 to 10.5, respectively. While the soluble enzyme retained 47.2% and 9.1% of its activity after treatment for 1 h at 50 and 60 °C, the immobilized protease maintained 87.7% and 48.3%, respectively. After treatment for 2 h at pH 5 and 13, the immobilized protease maintained 73.6% and 53.4% of its activity, whereas the soluble enzyme retained 32.9% and 1.4%, respectively. Furthermore, the immobilized AK-R protease showed significant improvement of enzyme stability in high concentration of NaCl, organic solvents, surfactants, and commercial detergents. In addition, the immobilized protease exhibited a very good operational stability, retaining 79.8% of its activity after ten cycles. The results clearly suggest that the developed immobilized protease system is a promising nanobiocatalyst for various protease applications.

Alkaline serine protease from the new halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R: purification and properties.

Herein, we report the purification and characterization of an alkaline protease from the alkaliphilic Salipaludibacillus agaradhaerens (formerly Bacillus agaradhaerens) strain AK-R, which was previously isolated from Egyptian soda lakes. The purification procedures resulted in enzyme purification up to 13.3-fold, with a recovery yield of 16.3% and a specific activity of 3488 U/mg protein. AK-R protease was a monomeric protein with an estimated molecular weight of 33.0 kDa. The optimum pH and temperature for AK-R protease were pH 10 and 60 °C, respectively. The enzyme thermostability was significantly enhanced in the presence of CaCl2 by approximately 1.3-fold. Moreover, under optimal conditions, the K m and V max values of the enzyme were 2.63 mg/ml and 4166.7 U/mg, respectively. PMSF caused complete inhibition of the enzyme activity, suggesting that AK-R belongs to the serine protease family. In addition, the enzyme was completely inhibited by EDTA, revealing the requirement of metal ions for AK-R protease activity; hence, it can be classified as a metalloprotease. AK-R protease is a mostly thiol-independent enzyme, since thiol reductants such as ß-mercaptoethanol and dithiothreitol had no effect on the enzyme activity. AK-R protease exhibited high stability in several organic solvents, including butanol, amyl alcohol, dimethyl ether, toluene, diethyl ether and methanol. Moreover, AK-R protease showed significant stability to a variety of surfactants and commercial detergents. The features and properties of AK-R alkaline protease are favourable and suggest its potential applications in various industries, particularly in the laundry detergent industry.

Prevalence, serotyping and antimicrobials resistance mechanism of Salmonella enterica isolated from clinical and environmental samples in Saudi Arabia

Abstract Salmonella is recognized as a common foodborne pathogen, causing major health problems in Saudi Arabia. Herein, we report epidemiology, antimicrobial susceptibility and the genetic basis of resistance among S. enterica strains isolated in Saudi Arabia. Isolation of Salmonella spp. from clinical and environmental samples resulted in isolation of 33 strains identified as S. enterica based on their biochemical characteristics and 16S-rDNA sequences. S. enterica serovar Enteritidis showed highest prevalence (39.4%), followed by S. Paratyphi (21.2%), S. Typhimurium (15.2%), S. Typhi and S. Arizona (12.1%), respectively. Most isolates were resistant to 1st and 2nd generation cephalosporin; and aminoglycosides. Moreover, several S. enterica isolates exhibited resistance to the first-line antibiotics used for Salmonellosis treatment including ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. In addition, the results revealed the emergence of two S. enterica isolates showing resistance to third-generation cephalosporin. Analysis of resistance determinants in S. enterica strains (n = 33) revealed that the resistance to β-lactam antibiotics, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline, was attributed to the presence of carb-like, dfrA1, floR, tetA gene, respectively. On the other hand, fluoroquinolone resistance was related to the presence of mutations in gyrA and parC genes. These findings improve the information about foodborne Salmonella in Saudi Arabia, alarming the emergence of multi-drug resistant S. enterica strains, and provide useful data about the resistance mechanisms.

Prevalence, serotyping and antimicrobials resistance mechanism of Salmonella enterica isolated from clinical and environmental samples in Saudi Arabia.

Salmonella is recognized as a common foodborne pathogen, causing major health problems in Saudi Arabia. Herein, we report epidemiology, antimicrobial susceptibility and the genetic basis of resistance among S. enterica strains isolated in Saudi Arabia. Isolation of Salmonella spp. from clinical and environmental samples resulted in isolation of 33 strains identified as S. enterica based on their biochemical characteristics and 16S-rDNA sequences. S. enterica serovar Enteritidis showed highest prevalence (39.4%), followed by S. Paratyphi (21.2%), S. Typhimurium (15.2%), S. Typhi and S. Arizona (12.1%), respectively. Most isolates were resistant to 1st and 2nd generation cephalosporin; and aminoglycosides. Moreover, several S. enterica isolates exhibited resistance to the first-line antibiotics used for Salmonellosis treatment including ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. In addition, the results revealed the emergence of two S. enterica isolates showing resistance to third-generation cephalosporin. Analysis of resistance determinants in S. enterica strains (n=33) revealed that the resistance to ß-lactam antibiotics, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline, was attributed to the presence of carb-like, dfrA1, floR, tetA gene, respectively. On the other hand, fluoroquinolone resistance was related to the presence of mutations in gyrA and parC genes. These findings improve the information about foodborne Salmonella in Saudi Arabia, alarming the emergence of multi-drug resistant S. enterica strains, and provide useful data about the resistance mechanisms.

Alkaline protease from a new halotolerant alkaliphilic Bacillus agaradhaerens strain ak-r isolated from egyptian soda lakes / Protease alcalina de uma nova estirpe ak-r halotolerante e alcalófila de Bacillus agaradhaerens isolada a partir dos lagos alcalinos egípcios

Alkaline proteases are hydrolytic enzymes that cleave peptide bonds in proteins and peptides in alkaline conditions, which occupy a pivotal importance with respect to their industrial applications. This study aimed to isolate new alkaline protease producing alkaliphilic bacteria from Egyptian soda lakes and optimize the fermentation process to enhance the enzyme production. The extensive screening process of the samples collected from Egyptian soda lakes resulted in isolation of a potent alkaline protease producing alkaliphilic strain AK-R. The isolate was identified as Bacillus agaradhaerens strain AK-R based on 16S rRNA gene analysis (99%). Wheat bran and gelatin supported maximum alkaline protease production as carbon and nitrogen sources, respectively. Strain AK-R is halo-tolerant thermotolerant alkaliphilic bacterium in nature, as it can grow over a wide range of NaCl concentrations (up to 25%) and up to 55 °C, with maximal growth and enzyme production at 2.5-5%, and pH 11 at 35 °C. Among the tested cations, only Mg2+ and Ca2+ ions significantly enhanced the enzyme production by about 1.2, and 1.3 fold compared to control, respectively. Alkaline protease secretion was coherent with the growth pattern, reaching maximal yield after about 32 h (mid stationary phase). In conclusion a new halo-tolerant thermo-tolerant alkaliphilic alkaline protease producing Bacillus agaradhaerens strain AK-R was isolated from Egyptian soda lakes. Optimization of the nutritional and cultivation conditions resulted in increase of enzyme yield by 20 fold. Strain AK-R and its extracellular alkaline protease with salt, pH and temperature, tolerance signify their potential application in laundry and pharmaceuticals industries.

Proteases alcalinas são enzimas hidrolíticas que quebram ligações peptídicas em proteínas e peptídeos em condições alcalinas, o que ocupa uma importância fundamental em relação às suas aplicações industriais. Este estudo teve como objetivo isolar novas proteases alcalinas e produzir bactérias alcalófilas a partir dos lagos salgados alcalinos egípcios e otimizar o processo de fermentação para aumentar a produção de enzimas. O extensivo processo de triagem das amostras coletadas dos lagos salgados alcalinos egípcios resultou no isolamento de uma protease alcalina potente produzindo uma estirpe alcalófila AK-R. O isolado foi identificado como sendo a estirpe AK-R de Bacillus agaradhaerens baseado na análise de genes 16S rRNA (99%). O farelo de trigo e a gelatina suportaram a produção máxima de protease alcalina como fontes de carbono e nitrogênio, respectivamente. A estirpe AK-R é uma bactéria alcalófila halotolerante e termotolerante, pois pode crescer dentro de uma vasta gama de concentrações de NaCl (até 25%) e até 55ºC, com crescimento e produção de enzimas máximos a 2.5-5% e pH 11 a 35ºC. Dentre os cátions testados, somente os íons Mg2+ e Ca2+ aumentaram significativamente a produção de enzimas em cerca de 1.2 e 1.3 em comparação ao controle, respectivamente. A secreção de protease alcalina foi coerente com o padrão de crescimento, atingindo o rendimento máximo após 32h (fase estacionária média). Pode-se concluir que uma nova estirpe AK-R de Bacillus agaradhaerens halotolerante, termotolerante e alcalófila produtora de protease alcalina foi isolada a partir dos lagos salgados alcalinos egípcios. A otimização das condições de nutrição e cultivo resultou num aumento da produção de enzima em 20 vezes. A estirpe AK-R e a sua protease alcalina extracelular com tolerância ao sal, pH e temperatura tornam significantes as suas potenciais aplicações nas indústrias farmacêutica e de lavanderia.

Development of novel robust nanobiocatalyst for detergents formulations and the other applications of alkaline protease.

Alkaline protease from alkaliphilic Bacillus sp. NPST-AK15 was immobilized onto functionalized and non-functionalized rattle-type magnetic core@mesoporous shell silica (RT-MCMSS) nanoparticles by physical adsorption and covalent attachment. However, the covalent attachment approach was superior for NPST-AK15 protease immobilization onto the activated RT-MCMSS-NH2nanoparticles and was used for further studies. In comparison to free protease, the immobilized enzyme exhibited a shift in the optimal temperature and pH from 60 to 65 °C and pH 10.5-11.0, respectively. While free protease was completely inactivated after treatment for 1 h at 60 °C, the immobilized enzyme maintained 66.5% of its initial activity at similar conditions. The immobilized protease showed higher k cat and K m , than the soluble enzyme by about 1.3-, and 1.2-fold, respectively. In addition, the results revealed significant improvement of NPST-AK15 protease stability in variety of organic solvents, surfactants, and commercial laundry detergents, upon immobilization onto activated RT-MCMSS-NH2nanoparticles. Importantly, the immobilized protease maintained significant catalytic efficiency for ten consecutive reaction cycles, and was separated easily from the reaction mixture using an external magnetic field. To the best of our knowledge this is the first report about protease immobilization onto rattle-type magnetic core@mesoporous shell silica nanoparticles that also defied activity-stability tradeoff. The results clearly suggest that the developed immobilized enzyme system is a promising nanobiocatalyst for various bioprocess applications requiring a protease.

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres.

The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS-NH2 nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 µg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V(max), k(cat) and k(cat)/K(m), than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

Detergent-, solvent- and salt-compatible thermoactive alkaline serine protease from halotolerant alkaliphilic Bacillus sp. NPST-AK15: purification and characterization.

Alkaline protease produced by the halotolerant alkaliphilic Bacillus sp. strain NPST-AK15 was purified to homogeneity by the combination of ammonium sulfate precipitation, anion-exchange and gel permeation chromatography. The purified enzyme was a monomeric protein with an estimated molecular weight of 32 kDa. NPST-AK15 protease was highly active and stable over a wide pH range, with a maximal activity at pH 10.5. The enzyme showed optimum activity at 60 °C and was stable at 30-50 °C for at least 1 h. Thermal stability of the purified protease was substantially improved by CaCl2 (1.1- to 6.6-fold). The K m, V max and k cat values for the enzyme were 2.5 mg ml(-1), 42.5 µM min(-1) mg(-1), and 392.46 × 10(3) min(-1), respectively. NPST-AK15 protease activity was strongly inhibited by PMSF, suggesting that the enzyme is a serine protease. The enzyme was highly stable in NaCl up to 20 % (w/v). Moreover, the purified enzyme was stable in several organic solvents such as diethyl ether, benzene, toluene, and chloroform. In addition, it showed high stability and compatibility with a wide range of surfactants and commercial detergents and was slightly activated by hydrogen peroxide. These features of NPST-AK15 protease make this enzyme a promising candidate for application in the laundry and pharmaceutical industries.

Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

Background Alkaline proteases are among the most important classes of industrial hydrolytic enzymes. The industrial demand for alkaline proteases with favorable properties continues to enhance the search for new enzymes. The present study focused on isolation of new alkaline producing alkaliphilic bacteria from hyper saline soda lakes and optimization of the enzyme production. Results A new potent alkaline protease producing halotolerant alkaliphilic isolate NPST-AK15 was isolated from hyper saline soda lakes, which affiliated to Bacillus sp. based on 16S rRNA gene analysis. Organic nitrogen supported enzyme production showing maximum yield using yeast extract, and as a carbon source, fructose gave maximum protease production. NPST-AK15 can grow over a broad range of NaCl concentrations (0-20%), showing maximal growth and enzyme production at 0-5%, indicated the halotolerant nature of this bacterium. Ba and Ca enhanced enzyme production by 1.6 and 1.3 fold respectively. The optimum temperature and pH for both enzyme production and cell growth were at 40°C and pH 11, respectively. Alkaline protease secretion was coherent with the growth pattern, started at beginning of the exponential phase and reached maximal in mid stationary phase (36 h). Conclusions A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.

Cyclodextrin glucanotransferase immobilization onto functionalized magnetic double mesoporous core-shell silica nanospheres

Background Cyclodextrin glucanotransferase (CGTase) from Amphibacillus sp. NPST-10 was covalently immobilized onto amino-functionalized magnetic double mesoporous core-shell silica nanospheres (mag@d-SiO2@m-SiO2-NH2), and the properties of the immobilized enzyme were investigated. The synthesis process of the nanospheres included preparing core magnetic magnetite (Fe3O4) nanoparticles, coating the Fe3O4 with a dense silica layer, followed by further coating with functionalized or non-functionalized mesoporous silica shell. The structure of the synthesized nanospheres was characterized using TEM, XRD, and FT-IR analyses. CGTase was immobilized onto the functionalized and non-functionalized nanospheres by covalent attachment and physical adsorption. Results The results indicated that the enzyme immobilization by covalent attachment onto the activated mag@d-SiO2@m-SiO2-NH2, prepared using anionic surfactant, showed highest immobilization yield (98.1%), loading efficiency (96.2%), and loading capacity 58 µg protein [CGTase]/mg [nanoparticles]) which were among the highest yields reported so far for CGTase. Compared with the free enzyme, the immobilized CGTase demonstrated a shift in the optimal temperature from 50°C to 50-55°C, and showed a significant enhancement in the enzyme thermal stability. The optimum pH values for the activity of the free and immobilized CGTase were pH 8 and pH 8.5, respectively, and there was a significant improvement in pH stability of the immobilized enzyme. Moreover, the immobilized CGTase exhibited good operational stability, retaining 56% of the initial activity after reutilizations of ten successive cycles. Conclusion The enhancement of CGTase properties upon immobilization suggested that the applied nano-structured carriers and immobilization protocol are promising approach for industrial bioprocess for production of cyclodextrins using immobilized CGTase.