Sephadex G-100 Research Articles

Production of a novel cellulase by Bacillus amyloliquefaciens OKB3 isolated from soil: Purification and characterization

Microbial cellulases have become significant biocatalysts because of their complex composition and extensive industrial applications. This study aimed to isolate an efficient cellulase-producing strain, followed by molecular identification, enzyme purification, and characterization. Among 110 isolates, Bacillus amyloliquefaciens OKB3 was selected for its significant cellulase production, with optimal activity at pH 5.0 and 34 °C. The purification using ammonium sulfate and Sephadex G-100 chromatography resulted in specific activity of 2720.76 U/mg, 2.91 fold purification, and 29.44 % yield. The purified cellulase named CelB was a dimeric macromolecule of 123 kDa consisting of 67 and 54 kDa subunits. CelB was most active at 60 °C and pH 6, and it was stable at pH 5.5 to 6.0 and 0 °C to 4 °C. CelB was unaffected by metal cofactors and inhibited in the presence of divalent cations Cu2+, Hg2+, Cd2+, and Ag2+. The CelB has higher specificity of CMC compared to other substrates. The Km, Vmax, and Kcat values were 0.037 mM, 188.67 μmole/min, and 7430 S−1 respectively. The unique attributes of CelB make it a very promising candidate for various biotechnological applications.

Improved exopolysaccharide production by Lactiplantibacillus plantarum Z-1 under hydrogen peroxide stress and its physicochemical properties

In this study, a strain with good exopolysaccharide (EPS)-producing ability was isolated from the fermented Benincasa hispida and identified as Lactiplantibacillus plantarum Z-1. Its EPS production was further improved by H2O2 stress under optimized culture conditions, increasing from 180 ± 0.45 mg/L to 409.52 ± 2.16 mg/L. Purification of EPS with DEAE-52 and subsequent Sephadex G-100 column chromatography provided three fractions, namely, EPS-0, EPS-1 and EPS-3, respectively. The molecular weight of EPS, EPS-0, EPS-1 and EPS-3 were 85.4, 25.7, 131.88 and 93.2 kDa, respectively. EPS, EPS-1 and EPS-3 were mainly composed of glucose, rhamnose, arabinose and galactose with molar ratios of 1:0.544:0.211:0.281, 1:1.279:0.807:0.704, and 1:1.459:0.759:0.75, respectively, along with small proportions of fucose, mannose and xylose. EPS-0 was composed of glucose, arabinose, galactose and xylose, with molar ratios of 1:0.618:0.206:0.275. The structural analysis indicated that EPS-3 was mainly consisted of (1,2,4)-β-Rhap, (1,2,3)-β-Araf, (1,4)-β-Galp, T-α-Glcp units. The three purified fractions showed typical characteristics of non-Newtonian fluids and good viscoelasticity. Congo red test revealed that irregular triple-helical conformation existed in EPS and EPS-3. These physicochemical properties of EPSs make them a potential candidate for the use as a health-beneficial food additive in the food processing industry.

Structure elucidation and molecular mechanism of an immunomodulatory polysaccharide from Nostoc commune

Nostoc commune Vaucher, a terrestrial and benthic blue-green alga, widely used in food and medicine worldwide. N. commune Polysaccharides (NCVP) have excellent biological activities, especially immunomodulatory, hypoglycemic and anti-tumor activities. However, the mechanism and structure-activity relationship of NCVP has been less studied. In this study, based on methylation and NMR results, a novel polysaccharide NCVP2 with 135 kDa, containing→4)-α-D-Galp-(1→, → 4)-β-D-Glcp-(1→, and →4)-α-D-Xylp-(1→ residues as the backbon, was sequentially purified from N.commune by DEAE-52 and Sephadex G-100 column. NCVP2 (50 μg/mL) exhibited the strong in vitro immunomodulatory activity by promoting the generation of nitric oxide (NO) and reactive oxygen species (ROS). A total of 2048 differentially expressed genes (DEGs) were identified by RNA-seq, including 1019 down-regulated genes and 1065 up-regulated genes. These DEGs were mainly enriched in the immune-related biological processes, involving in Mitogen-activated protein kinase (MAPK) and Toll-like receptor (TLR) signaling pathways by GO and KEGG enrichment analysis. Furthermore, Western blot results proved NCVP2 could recognize TLR2 and TLR4/MD2, and regulate TLR7/IRF7, MAPK and PI3K/AKT signaling pathways. In summary, a novel polysaccharide NCVP2 from N.commune was proposed to exhibit significant immunomodulatory effects with multiple-paths and targets, and has great potential in the development of healthy foods such as immunomodulators.

Preparation, identification and α-glucosidase inhibitory activity of a high-methoxyl HG-type pectin from Liangping pomelo (Citrus maxima cv. Liangpin Yu) peel.

The peel from Liangping pomelo (Citrus maxima cv. Liangpin Yu) is generally discarded as waste during post-harvest handling and process, resulting in environmental pollution and waste. Pectin is the major component in pomelo peels and yields significant economic advantages. Thus, developing pomelo peel pectin (PPs) might be a feasible strategy to reduce environmental pollution caused by pomelo peel. The optimized PPs yield was 156.5 ± 2.5 g kg-1 under the inoculum size of 100 mg g-1, liquid-solid ratio of 31 mL g-1, fermentation time of 64 h, and fermentation temperature of 39 °C. PPs-6Aa, a pectin fraction from PPs purified with DEAE-52 cellulose, Sephadex G-100 and Sephadex G-75 column chromatography, showed higher α-glucosidase inhibitory activity, with an IC50 of 0.12 ± 0.03 mg mL-1. It was a high-methoxyl HG-type pectin of 42.8 kDa, and its repeat unit was →4)-α-GalpA-6-OMe-(1→4)-α-GalpA-6-OMe-(1→. Additionally, its α-glucosidase inhibitory activity might be related to hydrogen bonds formed with Lys-156, Glu-277, His-280, Asp-307, Arg-315, Asn-350, Asp-352 and Glu-411, and to hydrophobic interactions formed with Ser-157, Tyr-158, Asp-233, Gln-239, Ser-240, Phe-303, Thr-306, Leu-313, Phe-314, Gln-353 and Arg-442. These findings provide structural and bioactivity information on pectin from Liangping pomelo peel, which could be beneficial for the development of functional foods and pharmaceuticals. © 2024 Society of Chemical Industry.

Identification and Purification of Cholera Like Toxin from Environmental Isolate of Vibrio cholerae

The presence and prevalence of V. cholerae were investigated in forty five water samples collected from different locations of Tiger River/ Baghdad city. Twenty one isolates were isolated by adopting a simple isolation techniques. The final identification revealed that only three isolates were confirmed as V. cholerae. They were named 1J, 1R and Dial 131 which are all serogrouped as non-O1. Toxin Coregulated Pili (TCP) and heat labile enterotoxin (LT) were determined in only the environmental isolate 1J while non of the isolates produced heat stabile toxin (ST). The purification scheme was improved, few steps were adopted to include back extraction of ammonium sulfate, saturation between 80-20%, desalting through Sephadex G25, and gel filtration using Sephadex G100 which highly increase the specific activity.

Phytochemical Screening, Nutritional Properties and Biological Activities of Sweet and Conventional Potato Planted in Morocco

This study aimed at studying phytochemical composition, nutritional properties and biological activities (antioxidant and anti-inflammatory) of two potato varieties (sweet potato (SP): Ipomoea batatas L. and conventional potato (CP): Solanum tuberosum L.) planted and consumed in Morocco. For this, potato samples (SP and CP) were firstly subjected to phytochemical and nutritional analysis, including water content, total and reducing sugars, proteins, lipids, vitamin C, carotenoids, phenolic compounds and flavonoids. Secondly, phenolic compounds extracts were subjected to biological activities namely antioxidant and invitro anti-inflammatory. Potato extracts antioxidant activity was tested by DPPH and total antioxidant capacity (TAC) methods. Proteins and lipids contents were found much higher in SP (5.21 ± 0.26 and 0.29 ± 0.02 g/100g DW) respectively than in CP (2.93 ± 0.15 and 0.11 ± 0.01 g/100g DW) respectively. Besides, total and reducing sugars contents were 16.65 and 2.53 g/100g DW in the CP, and 12.47 and 2.13 g/100g DW in the SP, respectively. The antioxidant activity of different SP extracts was found to be higher than those of CP, by using DPPH and TAC methods. These findings could be explained by SP richness in carotenoids (212 ± 10.60 mg/100g DW), phenolic compounds (130.11 ± 6.51 mg GAE/100g DW), flavonoids (85.03 ± 4.2 5mg QE/100g DW) and vitamin C (471.33 ± 23.57 mg/100g DW). Exclusion chromatography done by Sephadex G50 showed that polymeric phenolic compounds in SP were more abundant when compared to monomeric ones. This difference was consistent with the anti-inflammatory activity assessed in vitro.

Optimization of Ultrasonic-Assisted Extraction, Characterization and Antioxidant and Immunoregulatory Activities of Arthrospira platensis Polysaccharides.

This study aimed to enhance the ultrasonic-assisted extraction (UAE) yield of seawater Arthrospira platensis polysaccharides (APPs) and investigate its structural characteristics and bioactivities. The optimization of UAE achieved a maximum crude polysaccharides yield of 14.78%. The optimal extraction conditions were a liquid-solid ratio of 30.00 mL/g, extraction temperature of 81 °C, ultrasonic power at 92 W and extraction time at 30 min. After purification through cellulose DEAE-52 and Sephadex G-100 columns, two polysaccharide elutions (APP-1 and APP-2) were obtained. APP-2 had stronger antioxidant and immunoregulatory activities than APP-1, thus the characterization of APP-2 was conducted. APP-2 was an acidic polysaccharide consisting of rhamnose, glucose, mannose and glucuronic acid at a ratio of 1.00:24.21:7.63:1.53. It possessed a molecular weight of 72.48 kDa. Additionally, APP-2 had linear and irregular spherical particles and amorphous structures, which contained pyranoid polysaccharides with alpha/beta glycosidic bonds. These findings offered the foundation for APP-2 as an antioxidant and immunomodulator applied in the food, pharmaceutical and cosmetic industries.

Extraction, separation and efficacy of yam polysaccharide

Yam is used as common herbal remedy in traditional Chinese medicine and is grown all over Asia. According to previous research, one of the primary bioactive components of yam is yam polysaccharide. To shed light on the mechanism of yam polysaccharide in ulcerative colitis (UC), a yam heteropolysaccharide named CYP-3a was isolated and purified using ultrasonic extraction, the trichloroacetic acid technique, DEAE cellulose-52 and a Sephadex G75 column. CYP-3a comprises rhamnus: arabinose:galactose:mannose:galacturonic acid glucuronic acid, with a molar ratio of 2.25:4.17:3.30:0.09:0.13:0.26. CCK-8 and ELISA analysis results showed that CYP-3a increased the number of dextran sodium sulphate (DSS)-induced Caco-2 cells and could reduce and inhibit their inflammatory response by lowering the amounts of secreted TNF-α and IL-6. Western blot data demonstrated that CYP-3a at various doses could suppress the endoplasmic reticulum stress–mediated apoptotic pathway generated by DSS-induced UC and down-regulate the protein levels of GRP78, CHOP and NF-κB.

Isolation, Characterization, Moisturization and Anti-HepG2 Cell Activities of a Novel Polysaccharide from Cyanobacterium aponinum.

Polysaccharides from cyanobacteria are extensively reported for their complex structures, good biocompatibility, and diverse bioactivities, but only a few cyanobacterial species have been exploited for the biotechnological production of polysaccharides. According to our previous study, the newly isolated marine cyanobacterium Cyanobacterium aponinum SCSIO-45682 was a good candidate for polysaccharide production. This work provided a systematic study of the extraction optimization, isolation, structural characterization, and bioactivity evaluation of polysaccharides from C. aponinum SCSIO-45682. Results showed that the crude polysaccharide yield of C. aponinum reached 17.02% by hot water extraction. The crude polysaccharides showed a porous and fibrous structure, as well as good moisture absorption and retention capacities comparable to that of sodium alginate. A homogeneous polysaccharide (Cyanobacterium aponinum polysaccharide, CAP) was obtained after cellulose DEAE-52 column and Sephadex G-100 column purification. CAP possessed a high molecular weight of 4596.64 kDa. It was mainly composed of fucose, galactose, and galacturonic acid, with a molar ratio of 15.27:11.39:8.64. The uronic acid content and sulfate content of CAP was 12.96% and 18.06%, respectively. Furthermore, CAP showed an in vitro growth inhibition effect on human hepatocellular carcinoma (HepG2) cells. The above results indicated the potential of polysaccharides from the marine cyanobacterium C. aponinum SCSIO-45682 as a moisturizer and anticancer addictive applied in cosmetical and pharmaceutical industries.

Biophysical Properties of Thermostable Amidase Produced by Aspergillus fumigatus in Submerged Fermentation

Amidases, also known as amidohydrolases (EC 3.5.1.4), are enzymes within the nitrilase superfamily or amidase signature family. They hydrolyze amides and nitriles into their corresponding acids while releasing ammonia. These enzymes are widely used in biotechnology and industry. However, their production is limited to a few organisms, which cannot meet industrial demand. Therefore, exploring new amidase sources is crucial. This study focuses on purifying and characterizing amidase from Aspergillus fumigatus using cold acetone precipitation and column chromatography with Sephadex G-100. The effects of temperature, pH and metal ions, on the enzyme activity were examined for both crude and purified amidase. The thermal and pH stability of the enzyme alongside the enzyme kinetics were also assessed. The purified amidase exhibited optimal activity of 13.1 U/mL at 70°C, with 65% residual activity after 5 hours at this temperature. The pH studies showed optimal activity at pH 5.0 for the purified enzyme (7.8 U/mL) and at pH 4.0 for the crude enzyme (5.5 U/mL). Metal ion effects indicated that Zn²⁺, Mg²⁺, and EDTA enhanced, while Cu²⁺ inhibited, the activities of both crude and purified amidase. Kinetic analysis showed that the enzyme followed Michaelis-Menten kinetics, with Km and Vmax values of 0.104 mM and 1.884 U/mL for crude amidase, and 0.017 mM and 1.872 U/mL for purified amidase. These findings suggest that amidase from Aspergillus fumigatus holds potential for industrial applications where amidases are essential.

Optimization and purification of L-methioninase enzyme purified from clinical samples of Pseudomonas spp.

Background: This study focused on Pseudomonas aeruginosa, which secrete important enzymes in medical and pharmaceutical applications and are isolated from clinical samples. One of these enzymes is L-methioninase, the most desirable enzyme in the medical and industrial fields today since it converted L-methionine into methanethiol, ammonia, and alpha-ketobutyrate. Objectives: Evaluated the optimum conditions of L-methioninase production and then purified it. Methods: Pseudomonas samples were identified by microscopic and biochemical tests and confirmed with the VITEK2 system. After the L-methioninase production was examined, the optimal conditions for the production of L-methioninase (MGL) were established using clinical samples of P. aeruginosa. Additionally, MGL was purified from the supernatant of P.aeruginosa using ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G150 accordingly. Results: There were 33 isolates of P.aeruginosa according to microscopic characteristics and biochemical tests, followed by the VITEK2 system to ensure identification. The optimization conditions were achieved when supplement media with sucrose 1% w\v, casein 1% w\v, pH 7, temperature 37oC, and incubation for 48hrs within the specific activity of 1.6, 3.3, 3.4, 3.4, and 3.5U\mg protein, respectively, and 4.6 fold the amount of the purified enzyme as compared to the crude enzyme. A single 55 kDa band on the SDS-PAGE indicates the enzyme is purified. Conclusions: P.aeruginosa can be an efficient source of L-methioninase, and this enzyme had better and higher specific activity after applying optimum conditions for it and purifying it.

Structural analysis, acetylcholinesterase inhibitory activity and immunoregulatory activity of two acidic polysaccharides from Chrysanthemum morifolium cv. Gongju

The structural characteristics, acetylcholinesterase inhibitory activity, and immune effects of two purified acid polysaccharides from Chrysanthemum morifolium cv. Gongju were investigated. GJP-1 and GJP-2 were isolated and purified using DEAE-sepharose and Sephadex G-100 chromatography. GJP-1 consisted of Man, Rha, Ara, Glc, Gal, and GalA, with a ratio of 1.99:3.98:81.2:2.65:7.03:3.15, while GJP-2 comprised of Man, Rha, Ara, Glc, Gal, and GalA in a ratio of 0.27:2.36:8.03:38.44:2.38:48.52. GJP-2 is a kind of pectin-type polysaccharide. GJP-1 and GJP-2 differed in average molecular weight of 5.86 kDa and 4.92 kDa. The results indicated that GJP-1 and GJP-2 significantly inhibited the activity of acetylcholinesterase and had good immunomodulatory activities. GJP-1 and GJP-2 are bioactive polysaccharides with the potential to improve cognitive function. GJP-2 promoted more proliferation, as well as the release of tumor necrosis factor-α and interferon γ. Clarifying the structures and bioactivities of polysaccharides could lay a foundation for the application of Gongju in functional foods and medicines production.

Exploring the Bioactive Potential of Moroccan Lemon Grass (Cymbopogon citratus L.): Investigations on Molecular Weight Distribution and Antioxidant and Antimicrobial Potentials.

Research on lemon grass (Cymbopogon citratus L.) revealed a variety of active molecules and examined their biological characteristics. However, most of these studies were conducted on wild varieties, while cultivated plants were addressed less. This study aimed to characterize the biomolecules and biological activities of lemon grass growing under North African conditions in Morocco. Phenolic compound profiles of aqueous (AE), ethanol (EE), and methanol (ME) extracts and their fractions were obtained with steric exclusion chromatography on Sephadex G50 gel and identified by LC-MS/MS. Then, total polyphenols (TPC), flavonoids (TFC), and antioxidant activities (FRAP: scavenging value and TAC: Total Antioxidant Capacity) of the fraction were evaluated, as well as the antimicrobial activity. The obtained results showed that the ME contained eight major compounds (i.e., apigenine-7-O-rutinoside and myricitine-3-O-rutinoside). The AE showed the presence of five molecules (i.e., kaempferol-3-O-glucuronide), while EE showed the presence of three molecules (i.e., quercetine-3-O-rutinoside). Regarding the chemical characterization, the highest value of total phenolic content (TPC) was obtained in AE (25) (4.60 ± 0.29 mg/g), and the highest value of total flavonoid content (TFC) was obtained in ME (29) (0.7 ± 0.08 mg/g). Concerning the antioxidant activity, the highest FRAP was obtained in ME (29) (97.89%), and the highest total antioxidant capacity (TAC) was obtained in ME (29) (89.89%). Correlation between FRAP, TPC, and TFC was noted only in fractions of AE and ME. All tested extracts of C. citratus and their fractions showed a significant antimicrobial effect. The lowest minimum inhibitory concentration (MIC) was recorded for ME against E. coli. Extracts' biological activities and their fractions were governed by their active molecules. These data are new and clarify a novel aspect of bioactive molecules in the extracts of cultivated C. citratus. Equally, throughout this research, we clarified the relationship between identified molecules and their biological properties, including antioxidant and anti-microbial activities, which is new for the study area. This study is suggested as a reference for comparative studies and other assays of other biological activities for the study plant.

Structure identification and activity evaluation of polysaccharide from Codonopsis pilosula (C. pilosula nannf. Var. modesta (nannf.) L. T. Shen)

Codonopsis pilosula is a medicinal plant with properties related to food, and the antioxidant and hypoglycemic components of its natural polysaccharides, as well as their related mechanisms, have not been fully understood. This study examined the physicochemical properties and structure of C. pilosula polysaccharide (WCP) using an orthogonal extraction method involving hot water-alcohol precipitation, high-resolution spectroscopy, and microscopic imaging technology. The antioxidant and hypoglycemic activities of the polysaccharides were assessed. Six fractions (WCP1, WCP2, WCP3, WCP4, WCP5, and WCP6) were identified through purification using a DEAE-cellulose column and Sephadex G-100 gel column. Mannose, glucose, and arabinose were identified as the primary monosaccharide components of WCPs through Gas Chromatography (GC). The polysaccharide exhibited a crystal structure composed of irregularly entangled pyranose particles of WCPs as revealed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The polysaccharide decomposed into smoother triple-helix fibrous flocs as indicated by Congo red analysis and circular dichroism analysis (CD). The antioxidant capacity of the polysaccharides against various free radicals was evaluated, with WCP4 demonstrating a significant reducing capacity. Moreover, WCP3 and WCP5 displayed notable inhibition rates of α-amylase and α-glucosidase, respectively. Molecular docking studies indicated the interaction of WCP5 with two enzymes, forming multiple hydrogen bonds through specific amino acids. In the gromacs simulation, both composite systems maintained a steady state in terms of root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration, providing strong support for their potential as a reference for the developing novel therapeutic approaches to diabetes.

Screening of sweet potatoes varieties, physicochemical and biological activity studies of glycoproteins of selected varieties

In order to meet the nutritional and health needs of different consumers, the main nutrients of eight new sweet potatoes (SPs) varieties were to characterize by principal component analysis. The watermelon red (WR) variety is proving to be the preferred SPs variety because of its high nutritional value and unique sensory features. Three homogeneous fractions SPFG-1, SPFG -2 and SPFG-3 with yield of 53.02% ± 3.20%, 10.50% ± 0.61% and 5.20% ± 0.35% were obtained from the WR by multifrequency ultrasonic extraction (MUE), isolated and purified with DEAE Sepharose CL-6B and Sephadex G-100 gel chromatography. Physiochemical and structural characteristics of three fractions were studied with chemical components analysis, HPLC, HPGPC, and β-elimination, UV, FT-IR and CD spectrum analysis. SPFG-1, SPFG-2 and SPFG-3 were all conjugates with O-glycosylation with the molecular weight of 30.42 ± 2.12, 391.15 ± 25.12 and 6.85 ± 0.78 kDa, including seven monosaccharides and 17 amino acids with quite different mole percentages, that they were predominantly α-helix, β-folding and irregularly curled with a relatively small β-turning content. Meanwhile, the three conjugates exhibited excellent antioxidant and hypoglycemic activities by radical scavenging, α-glycosidase and α-amylase inhibition assays. These findings are helpful to promote the breeding, cultivation and promotion of new SPs varieties with higher nutritional value, as well as the development and application of glycoproteins.

Novel glucomannan-like polysaccharide from Lycium barbarum L. ameliorates renal fibrosis via blocking macrophage-to-myofibroblasts transition

The macrophage to myofibroblasts transition (MMT) has been reported as a newly key target in renal fibrosis. Lycium barbarum L. is a traditional Chinese medicine for improving renal function, in which its polysaccharides (LBPs) are the mainly active components. However, whether the role of LBPs in treating renal fibrosis is related to MMT process remain unclear. The purpose of this study was to explore the relationship between the regulating effect on MMT process and the anti-fibrotic effect of LBPs. Initially, small molecular weight LBPs fractions (LBP-S) were firstly isolated via Sephadex G-100 column. Then, the potent inhibitory effect of LBP-S on MMT process was revealed on bone marrow-derived macrophages (BMDM) model induced by TGF-β. Subsequently, the chemical structure of LBP-S was elucidated through monosaccharide, methylation and NMR spectrum analysis. In vivo biodistribution characteristics studies demonstrated that LBP-S exhibited effectively accumulation in kidney via intraperitoneal administration. Finally, LBP-S showed a satisfactory anti-renal fibrotic effect on unilateral ureteral obstruction operation (UUO) mice, which was significantly reduced following macrophage depletion. Overall, our findings indicated that LPB-S could alleviate renal fibrosis through regulating MMT process and providing new candidate agents for chronic kidney disease (CKD) related fibrosis treatment.

Robust peroxidase from Bacillus mojavensis TH309: Immobilization on walnut shell hydrochar and evaluation of its potential in dye decolorization

Peroxidases have received considerable attention as a cost-effective and environmentally friendly catalyst for bioremediation. Their rapid activity loss under harsh environmental conditions and inability to be used repetitively limit their exploitation in real-world wastewater treatment. First, a peroxidase was produced extracellularly by Bacillus mojavensis TH309 and purified 8.12-fold with a final yield of 47.10 % using Sephadex G-100 superfine resin. The pure peroxidase (BmPer) possessed a relatively low molecular weight of ∼21 kDa and was active against L-DOPA on acrylamide gel after electrophoresis. BmPer was immobilized by adsorption functionalized walnut shell hydrochar (WsH) with 61.99 ± 1.34 % efficiency and 37.07 ± 4.16 % activity loss. BmPer and its immobilized form (WsH-BmPer) exhibited maximum activity at 50 °C and pH 9. WsH-BmPer exhibited 3.23-, 2.37-, 1.65-, and 2.25-fold longer half-life than BmPer at 50, 60, 70, and 80 °C, respectively. Immobilization significantly enhanced the stability of the enzyme under acidic conditions. BmPer and WsH-BmPer showed maximal activity in the presence of 1 % salt and retained more than 85 % of their activity even after pre-incubation with 2.5 M salt for 60 min at 50 °C. Their catalytic efficiency was significantly stimulated by pre-incubation with Triton X-100 (1 mM), Tween20 (1 mM), and Mg2+ (1 and 10 mM). Immobilization strongly reduced the loss of activity caused by inhibitors including Ba2+, Hg2+, and Cu2+. Moreover, both forms of the enzyme were compatible with solvents. The Michaelis constant (Km) values of BmPer and WsH-BmPer were 0.88 and 2.66 mM for 2,4 DCP, respectively. WsH-BmPer peroxidase maintained about 82 % and 85 % of its activity when stored at 4 °C for 30 days and reused for up to 10 cycles, respectively. Furthermore, it decolorized Cibacron red (CR), Poly R-478 (PR), Remazol Brilliant Blue R (RBBR), and Methyl red (MR) dyes by 60.13 %, 91.34 %, 86.41 %, and 50.51 % within 60 min, respectively.

Development and characterization of nanobody against envenomation by Naja naja oxiana

Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 μg of recombinant nanobody could neutralize the toxic effects of 20 μg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.

Date nawah powder as a promising waste for β-mannanase production from a new isolate Aspergillus niger MSSFW, statistically improving production and enzymatic characterization

β-Mannanase producing fungus was isolated from coffee powder waste and identified as Aspergillus niger MSSFW (Gen Bank accession number OR668928). Dates nawah powder as industrial and agricultural waste was the most inducer of β-mannanase production. The Plackett–Burman and Central Composite designs were used to improve β-mannanase titer. Optimization studies enhanced the enzyme yield with approximate 13.50-times. β-Mannanase was purified by Sephadex G-150 gel filtration column and the molecular weight was estimated to be 60 kDa by SDS–PAGE. Crude and purified β-mannanase displayed maximum activity at temperature 60 °C and 50 °C, respectively. Crude β-mannanase showed an activation energy value 2.35-times higher than the purified enzyme. Activation energy for thermal denaturation of the purified β-mannanase was 1.08-times higher than that of the crude enzyme. Purified β-mannanase exhibited higher deactivation rate constant (Kd) and lower half-life (t0.5) and decimal reduction time (D-value) compared with the crude enzyme. Thermodynamic parameters of enthalpy, entropy, and free energy values for crude and purified β-mannanase were calculated. Substrate kinetic parameters suggested that the purified β-mannanase had a strong affinity toward locust bean gum by showing 3.44-times lower Km and 1.99-times higher Vmax compared to the crude enzyme.

Purification and characterization of a thermostable Galium aparine β-galactosidase: A competent agent with enhanced cytotoxic activity against MCF-7 cell line

β-galactosidases (EC 3.2.1.23) are capable of catalysing two distinct types of reactions: transgalactosylation and hydrolysis. It is very important for the senescence of cancer cells. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from various sources including plants, animals, bacteria, yeasts, and filamentous fungi. Purification and thermodynamic characterization of β-galactosidase from Galium aparine seeds, and in vitro efficacy assessment of a breast cancer cell line. The enzyme was purified by using ammonium sulphate precipitation, dialysis, DEAE cellulose, and Sephadex G-100 chromatography. The enzyme was purified to 6.4 fold with a yield of 14 % and specific activity of 11.6 U/mg of protein. SDS-PAGE and MALDI-TOF analysis revealed that β-galactosidase is a monomer with a molecular weight of 35 kDa. The optimum pH and temperature for the purified enzyme were 6.0 and 50 ºC. The enzyme had Km and Vmax values of 0.25 mM and 32 µmol/min, respectively. The thermodynamic parameters (ΔHº, ΔGº, ΔSº) for the irreversible denaturation of the enzyme were also investigated at different temperatures (50–75 ºC). Lactose in milk was successfully hydrolyzed using a purified enzyme at a rate of 0.0121 min−1 and 12.12 min. This is better than other studied β-galactosidases. These results suggest the use of β-galactosidase from G. aparine for producing delactosed milk for the lactose-intolerant population and prebiotic synthesis. pH and optimum temperature and its activation by Ca2+ shows that it is suitable for milk processing. As a result, the enzyme was stable even at higher temperatures and had the potential to undergo catalysis, making it economically viable, primarily in the food and pharmaceutical industries. The present study is aimed to purification and thermodynamic characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.