DEAE-cellulose Column Chromatography Research Articles

Exploratory studies of the antidepressant effect of Cordyceps sinensis polysaccharide and its potential mechanism

Cordyceps sinensis, a traditionally prized medicinal fungus, contains polysaccharides as one of its main bioactive constituents, known for their significant immunomodulatory properties. In this study, we systematically investigated the composition and structure of Cordyceps sinensis polysaccharide, followed by an evaluation of its therapeutic effect on depression using a chronic restraint stress-induced depression model. The polysaccharide CSWP-2, extracted via hot water, precipitated with ethanol, and purified using DEAE-cellulose column chromatography from Cordyceps sinensis, is primarily composed of glucose, mannose, and galactose, with α-1,4-D-glucan as its major structural component. Behavioral tests, immunological profiling, metabolomics, and gut microbiota analyses indicated a notable ameliorative effect of CSWP-2 on depressive-like symptoms in mice. Furthermore, the action of CSWP-2 may be attributed to the modulation of the gut microbiome's abundance and its metabolic impacts, thereby transmitting signals to the host immune system and exerting immunomodulatory activity, ultimately contributing to its antidepressant effects.

Production of an Efficient Enzymatically Fab Fragment Antivenom against Cobra Snake (Naja naja oxiana) Venom.

Since around 100 years ago, the best treatment for millions of global snakebite victims has been polyclonal antivenoms. However, common antivenoms need continuous improvement to reduce rare, their side effects and get better performance. In the present study, Fab antivenom was produced through papain digestion of anti-cobra venom plasma, multi-step purification, and optimization, including ammonium sulfate precipitation and DEAE-cellulose column chromatography. Then, the existence of the corresponding Fab fragment antibody was seen and confirmed by SDS-PAGE method and double immunodifusion (Ouchterlony) test. In addition, the potency test in NIH laboratory mice revealed that each milliliter of the new Fab antivenom was able to neutralize 624 micrograms and (80LD50) of cobra venom, which is about 15% more efficient than the primary plasma of the same concentration, and 1.57 times more effective than the cobra antivenom found in commercial hexavalent antivenom of Razi Institute. According to the findings, it seems that this new Fab antivenom can be used as a new candidate for treatment of cobra snakebite victims.

Purification and Structural Characterization of Polysaccharides from Polygonum multiflorum Thunb. and Their Immunostimulatory Activity in RAW264.7 Cells.

Polygonum multiflorum Thunb. (PM) and derived products are broadly utilized in Chinese traditional medicine. According to our previous research, PM mostly contains polysaccharides, which display a wide range of biological activities. Two water-soluble polysaccharides (PMPs-1 and PMPs-2) were obtained from PM by DEAE-Cellulose and Sephadex G-100 column chromatography. Colorimetry, HPGPC-MALLS-RID, HPLC-PDA, methylation, FT-IR, NMR, and SEM were used to characterize these polysaccharides. PMPs-1 and PMPs-2 had average molecular weights of 255.5 and 55.7 kDa, respectively. PMPs-1 consisted of Man, Glc, Gal, and Ara at 0.9:78.6:1.0:1.6 and was a glucan with → 4)-Glcp-(1 → as a backbone. Meanwhile, PMPs-2, an acidic polysaccharide, comprised Rha, GalA, Glc, Gal, and Ara at 3.2:20.3:2.7:1.0:8.3. PMPs-1 and PMPs-2 significantly improved the proliferation of RAW 264.7 cells and induced NO, TNF-α, and IL-6 release. This study reveals that these two polysaccharides can be explored as novel immunomodulators and provide a basis for further development of PM in food and pharmaceutical industries.

Isolation, Purification, Fractionation, and Hepatoprotective Activity of Polygonatum Polysaccharides.

In this study, three homogeneous fractions, PSP-N-b-1, PSP-N-b-2, and PSP-N-c-1, were obtained from an aqueous extract of Polygonatum using DEAE cellulose column chromatography, CL-6B agarose gel chromatography, and Sephadex G100 chromatography. Their monosaccharide compositions and molecular weights were analyzed. The results revealed that PSP-N-b-1, PSP-N-b-2, and PSP-N-c-1 are primarily composed of six monosaccharides: Man (mannose), GlcA (glucuronic acid), Rha (rhamnose), GalA (galacturonic acid), Glc (glucose), and Ara (arabinose), with molecular weights of 6.3 KDa, 5.78 KDa, and 3.45 KDa, respectively. Furthermore, we observed that Polygonatum polysaccharides exhibited protective effects against CCL4-induced liver damage in HepG2 cells in vitro, operating through both anti-oxidant and anti-inflammatory mechanisms. Our research findings suggest that Polygonatum polysaccharides may emerge as a promising option in the development of hepatoprotective drugs or functional foods with anti-inflammatory and antioxidant properties.

Isolation of an antitubercular protein from Staphylococcus hominis IS2 from the custard apple and evaluation of its biosafety

Tuberculosis (TB) is one of the leading causes of death throughout the world. Traditional medicine poses a serious challenge to drug resistance against Mycobacterium tuberculosis. Hence, it is an urgent need to develop, novel the anti-mycobacterial compounds. In this study, a potent anti-mycobacterial compound producing Staphylococcus hominis IS2 was isolated from the custard apple exhibiting novel anti-mycobacterial activity than other isolates. The bacterial strain was cultured and anti-mycobacterial compound was purified by the combination of ammonium sulphate precipitation and DEAE-cellulose column chromatography. The purified protein corresponding to 10 kDa presented anti-mycobacterial activity. It was further characterized and the difference in activity towards temperature and pH was observed (p < 0.01). The purified protein was analyzed using FT-IR and MALDI-TOF MS/MS analysis. Enzymatic treatment revealed the absence of carbohydrate moieties in the purified protein. The purified anti-mycobacterial protein presented anti-cancer activity against cervical cancer cell lines (HeLa) and was dose-dependent (p < 0.01). Haemolytic assay and Zebra fish embryo studies revealed that the isolated protein is safe at lower doses.

Isolation, Purification and Characterization of a-Lactalbumin from Camel Milk and Study its Antioxidant Activity in Oils

The purpose of this research was to examine the anti-inflammatory and antioxidant properties of a-lactalbumin (α-Lac) isolated from camel milk. Ion-exchange chromatography on a DEAE cellulose column and gel-filtration chromatography on a Sephadex G-100 column were used for the purification process. (α-Lac) purity was confirmed using a single-bond polyacrylamide gel-electrophoresis (SDS-PAGE) assay and a further step using a high-performance liquid chromatography (HPLC) assay. Antioxidant activity of (α-Lac) in food systems compared with chemical Antioxidants (BHT) activity at Prevent oxidation of vegetable oils (Flaxseed oil and sunflower) showed significant Ferric-reducing antioxidant power (FRAP), linoleic acid system (LAS), radical scavenging activity (DPPH).

Purification and characterization of extracellular tannase from Aspergillus fumigatus MA using Syzigium cumini leaves under solid state fermentation

This study reports the tannase purification produced by a tannery effluent-originated fungal isolate i.e., Aspergillus fumigatus MA under solid state fermentation (SSF) condition. Purification of tannase from culture filtrate was attained using ammonium sulfate precipitation with subsequent diethylaminoethyl (DEAE)-cellulose mediated ion exchange chromatographic technique. Fractional precipitation of the culture filtrate with 60–80% ammonium sulfate yielded 80.9% recovery of tannase with 6.16-fold purification. The enzyme fractions were collected and eluted as a single peak using 0.5 M NaCl-gradient concentration. DEAE-cellulose column chromatography results in overall 23-fold purification with 27.6% recovery of the enzyme. SDS-PAGE analysis of purified tannase confirmed the presence of a single band of protein with a molecular mass equivalent to 66.2 kDa. The highest activity of tannase was observed at optimum pH ranged between 5.0–6.0 whereas, the tannase stability (>80%) was observed at 4.0 to 7.0 pH ranges. The purified tannase activity was found to be optimally active at 30 °C whereas stability (>90%) was accomplished between 30–50 °C temperature. The Km and Vmax were found to be 1.61 × 10−3 M and 1.04 mM respectively. These properties suggest the potential of the enzyme to be utilized in various food, feed, and pharmaceutical sectors.

Water-extractable polysaccharide fraction PNE-P1 from Pinus koraiensis pine nut: Structural features and immunostimulatory activity

The polysaccharide fraction PNE-P1 was isolated from hot water extract (PNE) of the defatted meal of pine nuts (Pinus koraiensis) using DEAE-cellulose column chromatography. This fraction had three components of molecular masses 1251, 616, and 303 g/mol consisting mainly of arabinose, xylose, and galacturonic acid at a molar ratio of 2:1.6:1. Structural analysis with FTIR/Raman, methylation and GC-MS, and NMR revealed that PNE-P1 is a cell wall polysaccharide complex including arabinan, heteroxylan, homogalacturonan (HM) and rhamnogalacturonan I (RG-I) parts. Being nontoxic to RAW 264.7 macrophages in the concentration range of 10–200 μg/mL, PNE-P1 promoted proliferation of these cells, significantly induced the secretion of proinflammatory cytokines (TNF-α and IL-6) and chemokines (RANTES and MIP-1α) and enhanced the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nitric oxide (NO). PNE-P1 also markedly induced macrophage-mediated phagocytosis of apoptotic Jurkat T cells. These results demonstrate that pine nuts Pinus koraiensis contain a complex of water-soluble plant cell wall polysaccharides, which can stimulate innate immunity by potentiating macrophage function.

Smilax china L. Polysaccharide Alleviates Dextran Sulphate Sodium-Induced Colitis and Modulates the Gut Microbiota in Mice.

This work aimed to investigate the preventive effect of Smilax china L. polysaccharide (SCP) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. Smilax china L. polysaccharide was isolated by hot water extraction, ethanol precipitation, deproteinization, and purification using DEAE-cellulose column chromatography to yield three polysaccharides: SCP_C, SCP_A, and SCP_N. Acute colitis was induced by administering 3% (w/v) DSS in drinking water for 7 days. Sulfasalazine, SCP_C, SCP_A, and SCP_N were administered by gavage for 9 days. SCP_C, SCP_A, and SCP_N could significantly improve symptoms, as evidenced by the declining disease activity index (DAI), decreased spleen weight, increased length of the colon, and improved colonic histology. Moreover, SCP_C, SCP_A, and SCP_N increased serum glutathione and decreased the levels of pro-inflammatory cytokines, malondialdehyde, nitric oxide, and myeloperoxidase in colon tissues. Additionally, SCP_C, SCP_A, and SCP_N modulated gut microbiota via ascending the growth of Lachnospiraceae, Muribaculaceae, Blautia, and Mucispirillum and descending the abundance of Akkermansiaceae, Deferribacteraceae, and Oscillibacter in mice with UC. The results suggested that Smilax china L. polysaccharide ameliorates oxidative stress, balances inflammatory cytokines, and modulates gut microbiota, providing an effective therapeutic strategy for UC in mice.

Extraction, purification and characterization of papain cysteine-proteases from the leaves of Carica papaya

Papain is a globular cysteine-protease family consisting of a single polypeptide chain with three disulfide bridges and a sulfhydryl group necessary for proteolytic activity. Its application is extensive to the fields of medicine and food. The aim of this study is to extract, purify and characterize papain enzymes from the leaves of Carica papaya. Crude extracts containing the enzymes were extracted from the leaves of papaya in Mountain Top University, Nigeria, and purified in a 3-step purification using 70% NH4(SO4)2, DEAE-Cellulose column chromatography and Sephadex G-25 column chromatography to achieve a purification fold of 1.6424. The effects of temperature, pH, substrate concentration and Mg2+ concentrations on the activity of the enzymes were determined. This is a premier report on the presence of five cysteine-proteases in the leaves of papaya with different measures of binding affinity and catalytic efficiencies towards the casein-substrate. The purified enzymes from Carica papaya are optimally active from 50 °C - 59 °C and pH 4.5 – 6.6. Their activities are generally enhanced in the presence of Mg2+. The Km and Vmax values for the enzyme in the various pools ranged from 1.47mg/ml - 8.70mg/ml and 0.42µmol/ml/min - 0.4167µmol/ml/min. In this study, papain E displayed optimum activity at pH 5.5 and 50 °C in the presence of Mg2+ with the best catalytic efficiency (Kcat/Km 59.776 - pmL/mg.min) and binding affinity (Km - 0.83 mg/mL) when compared to the isolated enzymes. Values of the parameters assessed from this study could be put to use by the industry in the production of papain and also for household and medical use in storing the product.

Biochemical and molecular characterization of temperature-adapted agarase from sea urchin associated Vibrio sonorensis NIOT_SU2 from Andaman Island

A total of 143 isolates of aerobic, heterotrophic bacteria were isolated from the gut, peristomial membrane, spine, tooth and coelomic fluid of sea urchin, Diadema savignyi and Echinometra oblonga from the coastal area of Marina Park, Port Blair, South Andaman. The bacterial strain with agarolytic potential has been identified as Vibrio sonorensis NIOT_SU2 C 9(b). Agarase-NIOT_SU2 C 9(b) from the culture medium was isolated and purified with 47.78-fold from the culture fluid by a combination of ammonium sulphate precipitation, dialysis and successive purification by DEAE cellulose column chromatography. Purified protein migrated as a single band (46 kDa) on sodium dodecyl sulphate-polyacrylamide gels. The optimum pH and temperature of the agarase were 7.6 and 30–35 °C, respectively. Agarase enzyme maintained as much as 85% of the maximum activities at wide range of temperatures and this enzyme did not decompose sodium alginate. This work is the first evidence of cold and temperature-adapted agarase from sea urchin associated bacteria and the results of this study authenticated the potential for the agarase-NIOT_SU2 C 9(b) as a catalyst in medicine, food, cosmetic industries and practical application in gene technology.

EXTRACTION, PURIFICATION AND CHARACTERIZATION OF LIPASE FROM THE DIGESTIVE DUCT OF COMMON CARP CYPRINUS CARPIO L.

This study was aimed to use digestive duct of common carp Cyprinus Carpio L. as economic source for fish lipase extraction . The enzyme was extracted using potassium phosphate buffer (pH 7 , 0.2 M ). The crude extract then precipitated at the saturation range between 40 – 80 % and dialyzed . Further purified carried out through ion exchange chromatography using DEAE-Cellulose column (2.5 × 30 cm) and gel filtration chromatography by Sephadex G-100 column (1.5 x 60 cm). The purified lipase had a molecular weight of 37.150KDa ,and optimum pH and temperature for enzyme activity about 45◦C and 6.5 using olive oil as substrate .The specific activity, number of purification folds and yield of purified Lipase were 146.48U/mg, 9.76 and 40.50% respectively.

Studies on Purification and Characterization of Sweet Potato (Ipomoea Batatas) Cultivated in the Northern Part of Bangladesh

Enzyme activity was also determined from the Red sweet potato and white sweet potato. The activities of Amylase, Cellulase, Invertase, &amp; protease were 0.093±0.01, 0.05±0.02, 0.062± 0.015, 0.043± 0.02, for Red while 0.088± 0.01, 0.073 ±0.02, 0.072 ±0.015 &amp; 0.050± 0.02 for White sweet potato. Amylase was partially purified from the extract of sweet potato using DEAE cellulose column chromatography. On the purity checking and molecular weight determination on SDS-PAGE. The molecular weight was determined by SDS-PAGE to be 64±1 kDa. The partially purified amylase from sweet potato is β-type is confirmed by HgCl2.The optimum pH &amp; temperatures of partially purified β-amylase were 7.5 and 55◦c respectively. Amylolytic activity of this enzyme is 105% and 55% for amylopectin and Amylase. This enzyme does not affect maltose and maltotetraose. Metallic ions like Fe3+, Mn2+, Mg2+, and EDTA increased amylase activity while other ions like Cu+2, Hg+2, Pb+2, and urea reduced the amylase activity. The study indicates the importance of β-amylase as a starch degrading enzyme. This enzyme showed less toxicity towards brine shrimp nauplii with the Lc50 value of 1970.288µg/ml. This enzyme did not show any antibacterial activity against Staphylococcus aureus, Escherichia coli, Salmonella enterica, Bacillus subtilis. Keywords: Sweet potato, Ipomoea Batatas, Starch, Amylase, Protease.

Cytotoxic effect of partially purified conotoxin from Conus virgo on Human Colon Cancer cell line HT-29

The present study focused on partial purification of conotoxin from the crude venom of Conus virgo and tested for its anticancer potential against human colon cancer cell line HT-29. The screening of crude venom was carried out using human cancer cell lines such as MCF-7 and HT-29 by MTT method. The result showed IC50 for HT-29 at 40 μg/ml for 24 h. incubation whereas MCF-7 cell line failed to show the decreased cell viability (71% cell viability at a maximum concentration of 140 μg/ml). The conotoxin was purified using DEAE-cellulose column chromatography; purified fraction-1 exhibited significantly decreased cell viability at 20, 40, 60, 80, 100, 120 and 140 ng/ml concentrations in HT-29cells and IC50 was found at 120 ng/ml for 24h. incubation. In Cytomorphological observation, distinct cellular changes such as cell shrinkage, loss of adherence and loss of colony formation were observed. The nuclear morphological damages were observed in propidium iodide staining. DNA fragmentation was observed in the treatment of fraction-1 on HT-29 cells when compared to control cells using agarose gel electrophoresis. Based on these findings, the fraction 1 conotoxin induced the nuclear damage and caused cell death in HT-29 cells, so it could be a good candidate for colon cancer chemotherapy.

PURIFICATION AND CHARACTERIZATION OF L-GLUTAMINASE ENZYME FROM SYMBIONT ENDOPHYTIC BACTERIA OF MARINE PLANT Sargassum sp. AS ANTIMICROBIAL ADJUVANTS

Endophytic bacterial symbiont of marine plant seaweed is a potential source of various bioactive compounds as a raw material for drugs. This research aimed to study the antimicrobial potential of L-glutaminase enzyme isolated from seaweed bacterial symbiont collected from Cikoang Island, South Sulawesi Indonesia. The production of L-glutaminase of bacterial symbiont marine plant Sargassum sp was optimum in nutrient broth using a shaker at 37 °C for 3 d. L-glutaminase was obtained from extraction and fractionation with ammonium sulphate at increased saturation levels of 0-20%, 20-40%, 40-60% and 60-80%, continued by dialysis and column chromatography. Antimicrobial activity assessment was conducted by an agar diffusion method using paper discs. Enzyme fractions were produced by purification using DEAE-Cellulose and Sephadex G-100 column chromatography with a purity of 12.23 and 16.95 times, respectively. The enzyme characterization showed that the optimum condition was at pH 8.0 and 37oC, where at this condition, enzyme activity showed a slight increase after the addition of K+ and Na+ ions and however, significant decrease in enzyme activity after the addition of Cu2+ and Fe2+ ions. The results of the antimicrobial activity evaluation revealed that the L-glutaminase of sephadex G-100 fraction had the highest activity against Escherchia coli and Staphylococcus aureus with the inhibition zone of 12.1 ± 0.29 and 17.4 ± 0.21mm, respectively. These results demonstrated that the environmentally friendly bacterial symbiont of the marine plant Sargassum binderi could be a new alternative as a source to produce the antimicrobial L-glutaminase enzyme. This work probably provides an essential finding for further research on the antimicrobial action of protein fraction from bacterial symbiont of marine plant Sargassum sp. or the other species.

Statistical Approach to Enhance α-Amylase Production from Bacillus licheniformis and Purification of the Enzyme

Background: The most widely used thermostable enzymes are the amylases in the starch industry. These are among the most important enzymes and are of great significance in present day biotechnology. Objective: The main objective of the present study was to enhance α-amylase production from Bacillus licheniformis using Response Surface Methodology (RSM) and the purification of the enzyme to homogeneity. Method: Bacterial culture producing α-amylase isolated from hot spring (Himachal Pradesh) was identified as Bacillus licheniformis using 16S rDNA gene sequencing (NCBI Accession No.: KR340466). Medium components and physical culture parameters viz. pH, temperature, inoculum size, peptone concentration and starch concentration were optimized using RSM. Among these five factors, three factors (starch concentration, peptone concentration and inoculum size) had a positive effect on amylase production. A 4.09-fold increase in the production of α-amylase from B. licheniformis was achieved using RSM as compared to One Factor At a Time. The enzyme was purified by using Diethylaminoethyl Cellulose column chromatography and subsequently by Sephadex G-75 gel filtration chromatography. Result: A purification fold of 23.39 and a yield of 12.12% were observed. A single band of 33 kDa was obtained using Sodium Dodecyl Sulphate (SDS) and native-Poly Acrylamide Gel Electrophoresis (PAGE), which indicated that the enzyme was purified to homogeneity and was a monomer. The enzyme showed stability at 50 and 65°C temperatures and at alkaline pH. Conclusion: The stability of purified enzyme at high temperatures and alkaline pH suggested its wide application in textile, detergent and paper industries.

Exopolysaccharides from the Energy Microalga Strain Botryococcus&nbsp;braunii: Purification, Characterization, and Antioxidant Activity.

Botryococcus braunii, a prestigious energy microalga, has recently received widespread attention because it can secrete large amounts of exopolysaccharides (EPS) with potential applications in food, cosmetics, and nutraceuticals. Unfortunately, the insufficiency of research on the bioactivity and structure–activity relationship of B. braunii EPS has impeded the downstream applications. In the present study, alcohol precipitation, deproteinization, and DEAE-cellulose column chromatography were used to extract and purify B. braunii SCS-1905 EPS. It was found that B. braunii SCS-1905 EPS were high-molecular-weight heteropolysaccharides containing uronic acid (7.43–8.83%), protein (2.30–4.04%), and sulfate groups (1.52–1.95%). Additionally, the EPS primarily comprised galactose (52.34–54.12%), glucose (34.60–35.53%), arabinose (9.41–10.32%), and minor amounts of fucose (1.80–1.99%), with the presence of a pyranose ring linked by a β-configurational glycosidic bond. Notably, the antioxidant activity of crude exopolysaccharides (CEPS) was stronger, and the half maximal inhibitory concentration (IC50) for ABTS and hydroxyl radicals was significantly lower than that of deproteinized exopolysaccharides (DEPS). Overall, this study indicated a potential application of B. braunii SCS-1905 EPS as a natural antioxidant. In summary, B. braunii EPS could be used as a potential feedstock for the production of antioxidant health foods.

Production of extracellular lipase from psychrotrophic bacterium Oceanisphaera sp. RSAP17 isolated from arctic soil.

Cold-active extracellular lipases produced by different psychrotrophs are important for various industrial applications. We have isolated a Gram-negative, rod-shaped, aerobe, non-pigment producing psychrotrophic bacterial strain RSAP17 (MTCC 12991, MCC 4275) from the unexplored Arctic soil sample of NyAlesund, Svalbard, Norway (78° 55″ N, 11° 54″ E). The detailed morphological, biochemical, and molecular characteristics were investigated to characterize the isolate RSAP17. Analyses of the 16S rDNA sequence of strain RSAP17 (Accession no. MK391379) shows the closest match with Oceanisphaera marina YM319T (99.45%) and Oceanisphaera sediminis TW92 JCM 17329T (97.40%). The isolate is capable of producing extracellular lipase but not amylase, cellulase or urease. The optimal parameters for lipase production have been found in tributyrin based (10mL/L) agar media supplemented with 3% (w/v) NaCl after 2-3days of incubation at 20-22°C temperature and pH 9 at shaking condition. We have purified the extracellular lipase from the RSAP17 grown culture supernatant through 75% ammonium sulfate precipitation followed by dialysis and DEAE cellulose column chromatography. The invitro lipolytic activity of the purified lipase enzymes has been done through zymogram analysis. The molecular weight found for the lipase is 103.8 kD. The optimal activity of the purified lipase has been found at 25°C and pH 9. MALDI-TOF-MS study of the purified lipase showed the highest match with the sequence of prolipoprotein diacylglyceryl transferase with 44% sequence coverage. Further study on large-scale production, substrate utilization and enzymatic kinetics of this lipase could unravel its possibility in future biotechnological applications.

Structural characterization and immunomodulatory activity of mycelium polysaccharide from liquid fermentation of Monascus purpureus (Hong Qu)

Alkaline extracted endopolysaccharides (MPS) from Monascus purpureus (Hong Qu) mycelium were successfully separated into four sub-fractions, namely MPS-1 (18.0 %), MPS-2 (27.1 %), MPS-3 (12.6 %) and MPS-4 (14.7 %), by DEAE-Cellulose column chromatography. By combining monosaccharide composition analysis, methylation analysis and 1D & 2D NMR, the structure of sub-fractions was systematically characterized. Both MPS-1 and MPS-2 were comprised of mannose, glucose and galactose in the molar ratio of 1.5:1.6:1.0 and 10.6:1.0:13.8, respectively. The backbone of them both consisted of 2-α-Manp with several different branched chains. However, MPS-1 contained glucose based sugar residues such as 3-Glcp and 4-Glcp which were not shown on MPS-2. The proposed structures of MPS-3 and MPS-4 were not obtained due to the fairly complex molecular structure and relatively low yield. Moreover, based on the RAW 264.7 cells model, MPS-2 could significantly promote cytokines secretion including IL-6, TNF-α, and IL-10 and improve expression levels of the related mRNA.

Purification, characterization and partial amino acid sequences of thermo-alkali-stable and mercury ion-tolerant xylanase from Thermomyces dupontii KKU\u2013CLD\u2013E2\u20133

We investigated the properties of the low molecular weight thermo-alkali-stable and mercury ion-tolerant xylanase production from Thermomyces dupontii KKU-CLD-E2-3. The xylanase was purified to homogeneity by ammonium sulfate, Sephadex G–100 and DEAE–cellulose column chromatography which resulted 27.92-fold purification specific activity of 56.19 U/mg protein and a recovery yield of 2.01%. The purified xylanase showed a molecular weight of 25 kDa by SDS–PAGE and the partial peptide sequence showed maximum sequence homology to the endo-1,4-β-xylanase. The optimum temperature and pH for its activity were 80 °C and pH 9.0, respectively. Furthermore, the purified xylanase can maintain more than 75% of the original activity in pH range of 7.0–10.0 after incubation at 4 °C for 24 h, and can still maintain more than 70% of original activity after incubating at 70 °C for 90 min. Our purified xylanase was activated by Cu2+ and Hg2+ up to 277% and 235% of initial activity, respectively but inhibited by Co2+, Ag+ and SDS at a concentration of 5 mM. The Km and Vmax values of beechwood xylan were 3.38 mg/mL and 625 µmol/min/mg, respectively. Furthermore, our xylanase had activity specifically to xylan-containing substrates and hydrolyzed beechwood xylan, and the end products mainly were xylotetraose and xylobiose. The results suggested that our purified xylanase has potential to use for pulp bleaching in the pulp and paper industry.