Sephadex G-100 Research Articles

Pectinase production by fungal-based solid-state fermentation of selected agricultural wastes

Agricultural waste is the residues from growing and processing raw agricultural products. They can be properly managed using fungi to produce economical materials such as pectinase enzymes that act as biological agents which are applicable in many areas. This research was aimed at producing pectinase by fungal-based solid-state fermentation of agricultural wastes. Fungi were isolated from dump sites soil using the pour plate method. These fungal isolates were inoculated into the pretreated agricultural waste, including watermelon, mango, and pineapple peels. The degradation period was 15 days on mineral salt solid media using separate flasks for each waste and fungus. They were incubated at room temperature (28°C). The pectinase was assayed using pectin and DNSA (dinitrosalicyclic acid). Fungal isolates were identified and screened for pectinase production using standard methods. The pH and temperature were optimized to study the effect on pectinase activities using standard procedures and pectinase was purified using column sephadex G-100. Molecular identifications were carried out on the isolates using 18srRNA gene primers. The fungi were identified as Aspergillus niger strain AGF3 (OM273994), Penicillium Chrysogenum strain AGF2 (OM273982) and Cladosporium tenuissimum strain AGF1 (OM273981). Aspergillus niger strain AGF3 (OM273994) produced the highest specific pectinase activities in mango waste (2400 U/mg. protein/ mL and it can be compared to what was produced by Cladosporium tenuissimum strain AGF1 (OM273981) (1590 U/mg. protein/mL). High specific pecrtinase activity was also obtained from pineapple peels (2200 U/mg. protein/mL) produced by Cladosporium tenussimum strain AGF1 (OM273981). It can be concluded in this study that Aspergillus niger strain AGF3 (OM273994) and Cladosporium tenuissimum strain AGF1 (OM273981) were the best producers of pectinase using mango and pineapple peels, while Penicillium chrysogenum strain AGF2 (OM273982) produced substantial pectinase in all the three agricultural wastes but not at higher levels.

OPTIMUM CONDITIONS FOR PHENYLALANINE AMMONIA LYASE EXTRACTION AND PURIFICATION FROM LOCALLY CULTIVATED GRAPE SEEDS

In the present study, Phenylalanine ammonia lyase (PAL) activity was detected in different parts of grape plant (leaves, pulp, whole seeds and defatted seeds) extracts. The results revealed that the whole seed possess highest PAL activity (69.44 U), followed by defatted seed (44.22 U), whereas extracts of leaves and pulp gave the lowest activity values (22.77 and 5.11 U, respectively). The optimum conditions for crude PAL extraction achieved using 50mM potassium phosphate buffer (pH 7) at mixing ratios of (1:7) for 1 h. extraction time. PAL was shown to precipitate out at the saturation range between 20-90%. For further purification, gel filtration chromatography using Sephadex G-200 column (1.5×70) and ion exchange chromatography using DEAE- Cellulose (DE 52) column (3.2× 10) were applied. The specific activity, number of folds and yield of purified PAL was 1157 U/mg, 19.94 and 15.32 % respectively.

Structural characterization and immunomodulatory activity of a polysaccharide from finger citron extracted by continuous phase-transition extraction

FCP-2-1, a water-soluble polysaccharide rich in galacturonic acid was isolated by continuous phase-transition extraction and purified with DEAE-52 cellulose and Sephadex G-100 column chromatography from finger citron with essential oil and flavonoids removed. The structural characterization and immunomodulatory activity of FCP-2-1 were further investigated in this work. FCP-2-1 with a Mw and Mn of 1.503 × 104 g/mol and 1.125 × 104 g/mol, respectively, was predominantly composed of galacturonic acid, galactose, and arabinose in a molar ratio of 0.685: 0.032: 0.283. The main linkage types of FCP-2-1 were proved to be →5)-α-L-Araf-(1→ and →4)-α-D-GalpA-(1→ based on methylation and NMR analysis. Moreover, FCP-2-1 was demonstrated to have significant immunomodulatory effects on macrophages in vitro by improving the cell viability, and enhancing phagocytic activity and secretion of NO and cytokines (IL-1β, IL-6, IL-10 and TNF-α), indicating that FCP-2-1 could be used as a natural agent in immunoregulation functional foods.

Purification, structural identification, antioxidant activity, and inhibitory effects on digestive enzymes of α-glucan from Chuanminshen violaceum

Chuanminshen violaceum, as a food and folk medicine, is cultivated and grown only in China. Its polysaccharides play an important role in medicinal benefits. Herein, crude C. violaceum polysaccharides (CVPs) were purified to yield two polysaccharide fractions (CVPs-1-G and CVPs-2-G) using DEAE Cellulose-52 and Sephadex G-100 column chromatography. Structural analyses indicated that both CVPs-1-G and CVPs-2-G were glucans with average molecular weights of 8.1 kDa and 187.5 kDa, respectively. The main chain of the major component (CVPs-1-G) might be composed of (1 → 4)-linked α-Glcp, and the branched-chain might be an α-Glcp → 4)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 4)-α-D-Glcp, which connected to the main chain through the O-6 position of → 3,6)-α-D-Glcp-(1 →. In addition, CVPs-1-G displayed ABTS, DPPH, and hydroxyl radical scavenging activity, as well as competitive inhibitory activities of α-amylase and non-competitive inhibitory activities of lipase and pepsin. According to molecular docking, its inhibitory activity on digestive enzyme might be caused by hydrogen bond and hydrophobic interaction between them. Thus, CVPs exhibit strong potential for use in functional foods and pharmaceuticals.

Structural characterization and anti-inflammatory activity of polysaccharides from Astragalus membranaceus

In this study, two homogeneous polysaccharides (APS-A1 and APS-B1) were isolated from Astragalus membranaceus by DEAE-52 cellulose and Sephadex G-100 column chromatography. Their chemical structures were characterized by molecular weight distribution, monosaccharide composition, infrared spectrum, methylation analysis, and NMR. The results revealed that APS-A1 (2.62 × 106 Da) was a 1,4-α-D-Glcp backbone with a 1,4,6-α-D-Glcp branch every ten residues. APS-B1 (4.95 × 106 Da) was a heteropolysaccharide composed of glucose, galactose, and arabinose (75.24:17.27:19.35). Its backbone consisted of 1,4-α-D-Glcp, 1,4,6-α-D-Glcp, 1,5-α-L-Araf and the sidechains composed of 1,6-α-D-Galp and T-α/β-Glcp. Bioactivity assays showed that APS-A1 and APS-B1 had potential anti-inflammatory activity. They could inhibit the production of inflammatory factors (TNF-α, IL-6, and MCP-1) in LPS-stimulated RAW264.7 macrophages via NF-κB and MAPK (ERK, JNK) pathways. These results suggested that the two polysaccharides could be potential anti-inflammatory supplements.

Extraction and Compositional Analysis of Anthocyanins and Polysaccharides from Aronia Melanocarpa Fruit

We have examined the basic composition of anthocyanins and polysaccharides, the active components in Aronia melanocarpa fruit. To this end, microwave-assisted ethanol extraction and cellulase enzymatic hydrolysis were employed to extract anthocyanins and crude polysaccharides, respectively. Aronia melanocarpa fruit polysaccharides were separated and purified by DEAE-52 cellulose and Sephadex G-100 glucan gel column chromatography, followed by high-performance liquid chromatography. The results demonstrated that the optimal extraction parameters of anthocyanin from Aronia melanocarpa fruit were a 60% ethanol volume fraction, a liquid-to-solid ratio of 30:1, a microwave time of 60 s, and a microwave power of 210 W. The extracted anthocyanins reached 200.47 mg/100 g, while the yield of polysaccharides was 3.82%. Anthocyanins in Aronia melanocarpa fruit were mainly composed of cyanidin, delphinidin, and mallow pigment, among which cyanidin was the main component, with a content as high as 90.79%. There were two distinct fractions of polysaccharides. The fraction-I polysaccharides were mainly composed of rhamnose, galacturonic acid, glucose, galactose, and arabinose, with a molar ratio of 8.80: 8.97: 2.38: 27.46: 47.94. In contrast, the fraction-II polysaccharides were primarily composed of rhamnose, glucuronic acid, galactose, xylose, and arabinose, with a molar ratio of 1.67: 9.22: 8.47: 70.69: 6.16. The results of this study can provide a reference for the in-depth development of Aronia melanocarpa resources

Cryptococcus diffluens D44 tarafından üretilen lipaz(lar)ın saflaştırılması ve karakterizasyonuna ait ön çalışma

In the present work, preliminary purification, and characterization of lipases from Cryptococcus diffluens D44, which was isolated from petroleum sludge, were performed. In the purification steps, subsequential to acetone precipitation, lipases from C. diffluens D44 were purified by DEAE Sepharose resulting in two different peaks, named Lip1 and Lip4. Sephadex G-100 size-exclusion chromatography was also performed for further purification of Lip1 and Lip4 and resulted in three different lipases as Lip1-1 (1.0 purification fold with 2.4% recovery), Lip1-2 (0.8 purification fold with 7.2% recovery), and Lip4-1 (1.2 purification fold with 4.5% recovery). As a result of characterization studies of these three lipases resulting from different peaks, optimum temperatures were found as 60 °C, 65 °C, and 65 °C for Lip1-1, Lip1-2, and Lip4-1, respectively. Furthermore, thermal stability studies were conducted at 50 °C, 60 °C, and 70 °C, and lipases of C. diffluens D44 maintained over 70% of their initial activity at 50 °C. The optimum pH for Lip1-1 and Lip1-2 was pH 9.0 although pH 5.0 was for Lip4-1. Considering the organic solvent effect on lipase activity, 10% methanol enhanced the relative activity of Lip1-1 and Lip4-1 while 10% ethanol caused a decrease in the relative activity of lipases except for Lip1-2. According to the indicated features based on the results, these different lipases from C. diffluens D44 could be promising candidates for industrial and biotechnological applications. To the best of our knowledge, this is the first study on the purification of lipases from C. diffluens D44.

Activity of laccase enzyme extracted from Malva parviflora and its potential for degradation of reactive dyes in aqueous solution

The use of enzymes, particularly laccase as a dye-degrading biogenic agent, is a significantly low carbon footprint, bio-inspired approach towards dye wastewater remediation, which not only decomposes and decolorizes the dyes but has also been reported to transmute dyes into less toxic by-products. The work presented herein utilizes both original and purified laccase extracted from Malva parviflora for decomposing pigments that adversely affect ecosystems. Extractions of enzymes from plants were designed by changing one variable at one time and by using response surface methodology (RSM). The optimum enzyme-specific activity of 420 units/mg protein from this flora occurred with extraction for 16.1 min at pH 5.0 with an extraction ratio of 0.17 in 0.42 M sodium acetate buffer. Sephadex G-150 was utilized to filter and purify the enzymes. An ultimate purity of 42% was thus achievable, which constituted a fourfold increase over that of the original extract. The extent to which both natural and purified laccases eliminated textile dyes was assessed under optimal conditions, which were considered to be 180 min at 37 °C. The enzyme extracted from Malva parviflora exhibited high removal efficiency and was able to transmute dyes into less toxic by-products. The extraction process was optimized using response surface methodology, resulting in an optimum enzyme-specific activity. The study found that unpurified enzymes exhibited a greater removal efficiency of dyes compared to purified enzymes. Overall, the findings suggest that laccase enzyme from Malva parviflora could serve as a practical biocatalyst for continuous decolorization of textile dyes with low carbon footprint.

Structural characterization, α-glucosidase inhibitory activity and antioxidant activity of neutral polysaccharide from apricot (Armeniaca Sibirica L. Lam) kernels

Screening for α-glucosidase inhibitors and antioxidants from natural sources that could reduce postprandial glucose in diabetic patients and reduce oxidative stress had attracted considerable interest. In this study, a neutral polysaccharide (AP-1) with a triple helix structure was isolated and purified from the residue of apricot (Armeniaca sibirica L. Lam.) kernels by using DEAE-52 and Sephadex G-100 columns. The molecular weight of AP-1 was 23.408 kDa and consisted mainly of glucose with trace amounts of arabinose, galactose, and mannose, which had molar percentages of 98.48, 0.63, 0.62 and 0.27 %, respectively. The main chain of AP-1 was composed of →4)-α-D-Glcp-(1 → interlinked, and α-D-Glcp-(1 → was attached as a branched chain at the O-6 position of →4,6)-α-D-Glcp-(1→. In addition, AP-1 exhibited stronger α-glucosidase inhibition and free radical scavenging ability compared to crude polysaccharides. Therefore, AP-1 could be used as a potential natural hypoglycemic agent and antioxidant in the treatment of diabetes mellitus.

Isolation, purification, and identification of antifungal protein produced by Bacillus subtilis SL-44 and anti-fungal resistance in apple

Apple anthracnose is a fruit fungal disease that is currently recognized as one of the most severe threats to apples worldwide. In this study, antifungal protein from Bacillus subtilis SL-44 was isolated, purified, identified, and applied for Colletotrichum gloeosporioides control. The antagonistic experiment showed that SL-44 had an excellent broad spectrum against plant pathogenic fungi. The optimal fermentation conditions were as follows: initial pH was 7, inoculum volume was 2%, and rotational speed was 180 r/min. The optimized yield of antifungal protein increased by 45.83% compared with that before. The crude protein was isolated and purified by (NH4)2SO4 precipitation, DEAE-Sepharose Fast Flow, and Sephadex G-100 column chromatography. LC-MS analyzed that antifungal protein was likely to be a novel protein with a molecular weight of 42kDa. The mechanism revealed that the antifungal protein may disrupt the cell wall structure of C. gloeosporioides and function as its antifungal action. Additionally, antifungal protein significantly alleviated the size of the lesion to more than 70% in the apple infection protection test. In conclusion, antifungal protein has remarkable potential in developing fungicides for the biological control of apple anthracnose. HIGHLIGHTS: 1. B. subtilis SL-44 had broad-spectrum antagonism against plant pathogenic fungi. 2. The optimal fermentation conditions for extracting antifungal protein were optimized. 3. The antifungal protein is a novel protein with a molecular weight of 42kDa. 4. The mechanism of antifungal protein may disrupt the cell wall structure of C. gloeosporioides.

The Purification and Characterization of a Cutinase-like Enzyme with Activity on Polyethylene Terephthalate (PET) from a Newly Isolated Bacterium Stenotrophomonas maltophilia PRS8 at a Mesophilic Temperature

A polyethylene terephthalate (PET)-degrading bacterium identified as Stenotrophomonas maltophilia PRS8 was isolated from the soil of a landfill. The degradation of the PET bottle flakes and the PET prepared as a powder were assessed using live cells, an extracellular medium, or a purified cutinase-like enzyme. These treated polymers were analyzed using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The depolymerization products, identified using HPLC and LC-MS, were terephthalic acid (TPA), mono(2-hydroxyethyl)-TPA (MHET), and bis(2-hydroxyethyl)-TPA (BHET). Several physicochemical factors were optimized for a better cutinase-like enzyme production by using unique single-factor and multi-factor statistical models (the Plackett–Burman design and the central composite design software). The enzyme was purified for homogeneity through column chromatography using Sephadex G-100 resin. The molecular weight of the enzyme was approximately 58 kDa. The specific activity on para nitrophenyl butyrate was estimated at 450.58 U/mg, with a purification of 6.39 times and a yield of 48.64%. The enzyme was stable at various temperatures (30–40 °C) and pH levels (8.0–10.0). The enzyme activity was significantly improved by the surfactants (Triton X-100 and Tween-40), organic solvent (formaldehyde), and metals (NiCl2 and Na2SO4). The extracellular medium containing the cutinase-type enzyme showed a depolymerization yield of the PET powder comparable to that of Idonella skaiensis IsPETase and significantly higher than that of Humicola insolens thermostable HiCut (HiC) cutinase. This study suggests that S. maltophilia PRS8 is able to degrade PET at a mesophilic temperature and could be further explored for the sustainable management of plastic waste.

A survey of elastase-producing bacteria and characteristics of the most potent producer, Priestia megaterium gasm32.

Ninety-one elastase-producing bacterial isolates were recovered from different localities of the Eastern Province of Saudi Arabia. Elastase from the best isolate Priestia megaterium gasm32, from luncheon samples was purified to electrophoretic homogeneity using DEAE-Sepharose CL-6B and Sephadex G-100 chromatographic techniques. The recovery was 17.7%, the purification fold was 11.7x, and the molecular mass was 30 kDa. Enzymatic activity was highly repressed by Ba2+ and almost completely lost by EDTA, but it was greatly stimulated by Cu2+ ions, suggesting a metalloprotease type. The enzyme was stable at 45°C and pH 6.0-10.0 for 2 hours. Ca2+ ions considerably enhanced the stability of the heat-treated enzyme. The Vmax and Km against the synthetic substrate elastin-Congo red were 6.03 mg/mL, and 8.82 U/mg, respectively. Interestingly, the enzyme showed potent antibacterial activity against many bacterial pathogens. Under SEM, most bacterial cells showed loss of integrity, damage, and perforation. SEM micrographs also showed a time-dependent gradual breakdown of elastin fibers exposed to elastase. After 3 hours, intact elastin fibers disappeared, leaving irregular pieces. Given these good features, this elastase may be a promising candidate for treating damaged skin fibers with the inhibition of contaminating bacteria.

Structural characterization and induction of tumor cell apoptosis of polysaccharide from purple sweet potato (Ipomoea batatas (L.) Lam)

The carbohydrate is the main ingredient of purple sweet potato. A polysaccharide, named PSP, was separated and purified from purple sweet potato by extraction with hot water, precipitation with ethanol, deproteinization with Sevag reagent and column chromatography with Sephadex G-100. The purity and structure were studied with HPLC, UV–Vis, GC–MS and NMR. The PSP is a neutral polysaccharide with Mw of 470 kDa. The monosaccharide composition of PSP contained D-xylose, d-glucose, D-galactose with ratio of 1.0: 8.3: 1.3. The backbone of PSP was composed of the residues of →6)-D-Glcp-(1 → and →2, 6)-D-Glcp-(1→. The branches of PSP contained the residues of →3)-D-Galp-(1→, and D-Xylp-(1→. The antitumor activity in vitro of PSP was analyzed with HT-29 cells. And the SEM, AO staining, MDC staining and hoechst 33342 staining were performed to study the effect on apoptosis of HT-29 cells by PSP. The results revealed that the PSP can significantly inhibit the proliferation of HT-29 cells from induction apoptosis. The manuscript provided valuable knowledges on structural characteristics of the polysaccharides from purple sweet potato.

Protein hydrolysate from Anabaena sp. cultured in an optimized condition designed by RSM; insight into bioactive attributes

The aim of the current study was to optimize the cultivation conditions of Anabaena sp. which was designed by response surface methodology (RSM) executing central composite designs (CCD). The effect of some parameters such as pH, light intensity, and the level of phosphorus and nitrogen was stimulated and 13 conditions were suggested for Anabaena cultivation. After cultivation, their proteins were extracted according to the suggested conditions. Then, the extracted protein of Anabaena was enzymatically hydrolyzed and classified based on the degree of hydrolysis (DH). Based on the results, the highest DH (19.28 %) was found in the hydrolysate obtained from cultivation treatment number 11 (N/P = 4, pH = 7, temperature = 45 °C). Sephadex G25 filtration gel chromatography was then used to purify this hydrolysate. 5 fractions with high absorbance were separated for antioxidant and antibacterial activity evaluation, which were named F14, F15, F16, F17, and F-18. According to the results, F15 showed the strongest antioxidant activity (90.66 %) against DPPH free radicals (p < 0.05). These fractions also showed dose-dependent antibacterial properties against Escherichia coli and Staphylococcus aureus pathogens. A significant difference was seen against E. coli, but not against S. aureus, at the highest concentrations used. This investigation contributes to introducing Anabaena as a cost-effective rich source of protein. Furthermore, these hydrolyzed proteins have antibacterial and antioxidant properties for industrial use.

Structural properties and anti-inflammatory activity of purified polysaccharides from Hen-of-the-woods mushrooms (Grifola frondosa).

Grifola frondosa is an edible medicinal mushroom that has been proven to have a variety of health benefits. The main active ingredients of this mushroom are polysaccharides. In this study, ultrasonic-assisted extraction was used to obtain crude Grifola frondosa polysaccharides (GFPs). Then, purified GFP was obtained after purification. The optimum extraction conditions were an extraction time of 71 min, an extraction temperature of 90°C in a solid-to-liquid ratio of 1:37 g/mL, and an ultrasonic power of 500 W. GFP was purified using DEAE-52 and Sephadex G-100. The structural characterization of GFP was performed using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), ion chromatography (IC), and ultraviolet (UV) visible photometry. The morphology of GFP was analyzed by scanning electron microscopy (SEM), thermogravimetric differential scanning calorimetry (TG-DSC), and Congo red testing. In addition, the administration of GFP in oxazolone (OXZ)-induced ulcerative colitis (UC) in mice was found to prevent weight loss. Different doses of GFP (80, 160, and 320 mg/kg body weight) were used, and sulfapyridine (SASP) was used as a positive control (370 mg/kg body weight) for the treatment of OXZ-induced UC. After treatment, the mice were killed, and blood and colon tissue samples were collected. GFP was found to prevent decreases in colon length and the levels of leukocytes, platelets, and neutrophils in UC mice. Moreover, GFP also decreased the expression of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1 β], increased IL-10, and reduced colon injury in UC mice. The results showed that Under these conditions, the predicted polysaccharide yield was 21.72%, and the actual extraction rate was 21.13%. The polysaccharide composition (molar ratio) was composed of fucose (0.025), glucosamine hydrochloride (0.004), galactose (0.063), glucose (0.869), and mannose (0.038). GFP was also found to have a typical absorption peak, and the GFP extracted using the ultrasound-assisted extraction protocol was mainly β-glucan. These results indicate that ultrasound-assisted extraction of GFP could reduce OXZ-induced intestinal inflammation as a promising candidate for the treatment of UC, with the potential for development as a food supplement to improve intestinal diseases.

Production, optimization, and purification of alkaline thermotolerant protease from newly isolated Phalaris minor seeds

The present work aims to purify and perform a preliminary analysis on a thermostable serine alkaline protease from a recently identified P. minor. The enzyme was purified 2.7-fold with a 12.4 % recovery using Sephadex G-100 chromatography, DEAE-cellulose, and ammonium sulphate precipitation. The isolated enzyme has a specific activity of 473 U/mg. The purified protease had a molecular mass of 29 kDa, and just one band was seen, which matched the band obtained using SDS-PAGE. High thermostability was demonstrated by the enzymes, which had half-lives of 31.79 and 6.0 min (a 5.3-fold improvement), enthalpies of denaturation (ΔH°) of 119.53 and 119.35 KJ mol−1, entropies of denaturation (ΔS°) of 32.96 and 41.11 J/mol·K, and free energies of denaturation (ΔG°) of 108.87 and 105.58 KJ mol−1 for the protease enzyme. Studies on the folding and stability of alkaline proteases are important since their use in biotechnology requires that they operate in settings of extreme pH and temperature. According to the kinetic and thermodynamic properties, the protease produced by P. minor is superior to that produced by other sources and previously described plants, and it might find utility in a variety of industrial fields.

Extracellular β-d-fructofuranosidase from a novel Aspergillus sp. DHE1 with high potential for biotechnological applications: Purification and biochemical characterization

The current work focused on the purification and biochemical characterization of an extracellular β-d-fructofuranosidase from a novel fungus identified genetically using 18 S rRNA molecular sequencing, as Aspergillus sp. DHE1. The fungus was cultured under solid state fermentation using the Cotton (Gossypium herbaceum) seed waste as substrate. β-d-fructofuranosidase was purified through fractionation by ammonium sulphate salt, ion-exchange chromatography on DEAE-cellulose, and Sephadex G-100 gel filtration, yielding a 13.3-purification fold, 22.6% recovery and 192.9 U/mg protein specific activity. The apparent molecular mass of Aspergillus sp. DHE1 β-d-fructofuranosidase was 78 kDa as estimated on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Optimal pH and temperature were observed at 6.0 and 50 °C, respectively, and the enzyme was quite thermostable at neutral/alkalophilic conditions, retaining 72% of its original activity after 1 h of incubation at 60 °C. The activity of the purified Aspergillus sp. DHE1 β-d-fructofuranosidase was stimulated by 1 mM of KCl (138.5%), NaCl (125.9%), CoCl2 (121.2%), and CaCl2 (112.3%), whereas the same concentration of AgCl, FeCl3, HgCl2, and CuCl2 inhibited the activity by 48.7%, 30.5%, 29.9%, and 26.2%, respectively. The kinetic studies using d-sucrose as the substrate were investigated using a Lineweaver-Burk plot, with Km and Vmax of 0.85 mM and 47.62 U/mL, respectively, indicating the high substrate affinity of β-d-fructofuranosidase. The ability of Aspergillus sp. DHE1 β-d-fructofuranosidase to tolerate ethanol suggests that it may have numerous uses in the food industry, including the production of alcoholic beverages.

Purification and Characterization of Phytase from Fruit Bodies of Local mushroom Pleorotus ostreatus Grown by Solid State Fermentation

Phytase produced from an edible local mushroom P.ostreatus11L was purified in three steps, in precipitation with ammonium Sulfate (saturation ratio70%), the specific activity of phytase increased from 0.38u/mg protein in crude extract to 0.77 u/mg protein with purification of 2.03 fold and the enzyme yield was 92.05%, in ion exchange chromatography(DEAE-cellulose) step, specific activity was 3.6 u/mg protein with purification of 9.47 fold with a yield of 71. 82%. In the last step, with gel filtration chromatography using Sephadex G75 which gave the highest specific activity reached 7.5 U/mg with the purification folds arrived to 19.74 and enzyme yield was 42.61%. The results showed that there is only one band appeared in SDS-PAGE technique indicating a high purity of phytase enzyme , the purified phytase behaved as a monomeric protein with a molecular mass of about 25.12 kDa . The phytase was active over a broad range of incubation temperature (20 - 50ºC), maximal activity was 0.83 unit/ml when phytase incubated at 30ºC while the enzyme has thermal stability over a broad range of temperatures (20-60ºC) for one h since more than 50 % of the relative enzyme activity was retained after incubation. Phytase was active over a broad range of pH (4-8), maximal activity was 0.81unit/ml when phytase incubated at pH 6 followed by 0.79 unit/ml when phytase incubated at pH 5 without significant differences between them. In addition , the enzyme was full stable at pH 5 and 6 for 1 h of incubation at 37oC. phytase activity didn’t reach 70% at the other pH values. Hence it is inferred that the phytase was active over a broad range of pH 4-8. Among various metal ions, only MgSO4 has a syregenic effect with phytase activity in which activity increased as residual activity was 117.36% , while CuSO4 and ZnCl were the most inhibitory agents for P.ostreatus phytase.

Biochemical and Kinetic Study for the Partial Purified Thioredoxin Reductase from Healthy Human Serum

The research included the separation of thioredoxin reductase (TrxR) from human blood serum using different biochemical techniques. Two proteinous peaks had been isolated by gel filtration using sephadex)G-50) and three proteinous peaks isolated from sephadex (G-100) that produced by ammonium sulphate precipitation (65%). The approximately molecular weight of the isolated protein as a source of enzyme using gel filtration chromatography (G-100) was (118059 + 925) Dalton.&#x0D; The results showed two peaks of enzyme when using ion exchange-type DEAE-cellulose and when using electrophoresis technique type SDS-PAGE indicated the presence of two bands from peak of separation column type sephadex G-100 and one band of each peak, resulting from the ion exchange process with molecular weights of 58231 +490 Dalton.&#x0D; The results showed that the optimum conditions of purified enzyme from human serum was at (200 µg/ml) of protein as a source of the enzyme using (0.4 mol/l) citric acid-Na2HPO4 buffer solution at pH (7) act for (25) minutes at (40°C). Using Line Weaver-Burk plot, the values of maximum velocity (Vmax) and Michaelis constant (Km) were found to be (0.182 µmol/ min) and (0.013 mol/l) respectively using 5,5-- dithio bis (2-nitrobenzoic acid) (DNTB) as a substrate. Finally, this was, also, involved the study of the effect of some chemicals and drugs on the enzyme activity. The results showed that gundinium - HCl is uncompetitive inhibitor, while bromoacetic acid is noncompetitive inhibitor and zinc sulphate is a competitive inhibitor for the enzyme at different concentrations of inhibitors, but dexathamazone was an activator to the enzyme.

Extraction and Purification of Pullulanase from Local Mushrooms

Pullulanase is defined as an extracellular carbohydrase enzyme responsible for the hydrolysis of pullulan into maltotriose. Pullulanase can be extracted from Agaricus bisporus edible mushroom and concentrated by dialysis tube using Tris-HCl buffer. This enzyme was purified by different ratios of ammonium sulphate; the optimal ratio was 30%. It can be purified by ion exchange using a DEAE-cellulose column with a final yield of 5.25 and a purification fold of 4.38. Following Sephadex G-100 gel filtration with a yield of 2.60 and purification fold of 5.0, the total activity of the enzyme reached 160IU and the specific activity was 14.5 U/mg. This study was concerned with estimating the optimum pH and temperature of pullulanase, and it recorded an optimal pH of 7, and the optimal temperature was 60°C. The enzyme was more stable at pH 8 and 70°C; the incubation period was also determined, and it appeared the most appropriate period was 30 minutes. CaCl2 and ZnCl2 were activator metal ions. Mercaptoethanol in different concentrations was the greatest inhibitor of pullulanase. One of the problems arising in the surrounding area is that much of the environmental pollution consists of starchy food waste. Pullulanase can degrade the glycosidic linkage of pullulan or starch present in the starch waste. The objective of this study is to produce enzymes like pullulanases to participate in minimizing environmental pollution through the degradation of starch waste as a biotechnological application. The conclusion of this study is that pullulanase can be produced from readily available, safe, and low-cost sources such as mushrooms.