DEAE-Sepharose Chromatography Research Articles

Isolation and purification of high molecular weight adiponectin from human plasma fraction

Adiponectin, a crucial protein hormone originating from adipose tissue, regulates key metabolic processes, including lipid metabolism, mitochondrial activity, and insulin sensitivity. These pleiotropic roles of adiponectin, along with its inverse correlation with metabolic disorders such as obesity, type II diabetes, and atherosclerosis, establish this protein as a potential therapeutic target. However, due to this complexity, challenges have arisen in its production with a natural conformation in bacterial or mammalian expression systems, hindering clinical translation. Furthermore, while inducers for adiponectin secretion or chemical agonists targeting adiponectin receptors have shown promise in laboratory settings, clinical studies with these agents have not yet been conducted. This study proposes a method for isolating and purifying natural high molecular weight (HMW) adiponectin from discarded plasma fractions during the conventional pharmaceutical protein manufacturing process. The process involved Cohn-Oncley fractionation, initial chromatography using reduced cellufine formyl, and subsequent purification via DEAE Sepharose chromatography. Characterization involved gel electrophoresis and biological assays on a hepatocyte cell-line. The purification process effectively captured adiponectin from the I + III paste, demonstrating that this fraction contained a significant portion of total plasma adiponectin. The two-step chromatography led to highly purified HMW adiponectin, confirmed by native-PAGE showing a 780 kDa multimeric complex. Biological assessments demonstrated normal downstream signaling, with HMW adiponectin inducing AMPK phosphorylation. This study demonstrates the feasibility of obtaining purified HMW adiponectin by repurposing plasma fractionation processes. It offers a promising avenue for the HMW adiponectin production, tapping into HMW adiponectin’s therapeutic potential against metabolic disorders while optimizing plasma resource utilization in healthcare.

GroEL Secreted from Bacillus subtilis Natto Exerted a Crucial Role for Anti-Inflammatory IL-10 Induction in THP-1 Cells.

Although diverse immunomodulatory reactions of probiotic bacteria have been reported, this effect via Bacillus subtilis natto remains unclear, despite its long consumption history in Japan and usage in Natto production. Hence, we performed a comparative analysis of the immunomodulatory activities of 23 types of B. subtilis natto isolated from Natto products to elucidate the key active components. Among the isolated 23 strains, the supernatant from B. subtilis strain 1 fermented medium showed the highest induction of anti-inflammatory IL-10 and pro-inflammatory IL-12 in THP-1 dendritic cells (THP-1 DC) after co-incubation. We isolated the active component from strain 1 cultured medium and employed DEAE-Sepharose chromatography with 0.5 M NaCl elution for fractionation. IL-10-inducing activity was specific to an approximately 60 kDa protein, GroEL, which was identified as a chaperone protein and was significantly reduced with anti-GroEL antibody. Differential expression analysis of strains 1 and 15, which had the lowest cytokine-producing activity, showed a higher expression of various genes involved in chaperones and sporulation in strain 1. Furthermore, GroEL production was induced in spore-forming medium. The present study is the first to show that the chaperone protein GroEL, secreted by B. subtilis natto during sporulation, plays a crucial role in IL-10 and IL-12 production in THP-1 DC.

Purification of MAO A and MAO B from Mammalian Tissue Sources.

Procedures are described for the purification of the mitochondrial-bound enzymes human and bovine monoamine oxidases A and B (MAO A and B) from placental and liver tissue sources, respectively. Enzyme purification follows isolation of the mitochondria and preparation of outer membrane particles. The membrane-bound enzymes are solubilized by treatment of membranes with phospholipases and detergent extraction. Functional bovine MAO B is purified by polymer fractionation and differential centrifugation. Functional human MAO A is purified by ion-exchange DEAE-Sepharose chromatography.

Production of thermostable xylanase using Streptomyces thermocarboxydus ME742 and application in enzymatic conversion of xylan from oil palm empty fruit bunch to xylooligosaccharides

Empty fruit bunch (EFB), the largest amount of biomass from a palm oil mill, is a promising renewable source for many value-added products. This study aimed to utilize EFB for production of xylanase by Streptomyces thermocarboxydus ME742 and applied the enzyme to the production of xylooligosaccharides (XOS) from EFB derived xylan (EFB-xylan). The strain ME742 produced the maximum xylanase (473.21 ± 6.55 U/mL) in a medium containing 50 g/L EFB supplemented with 3.0 g/L peptone at 40 °C for 4 days. Xylanase ME742 was purified 23.68 fold with specificity of 1229 U/mg by acetone precipitation and DEAE-Sepharose chromatography . It was stable in a wide range of pH (4.0–9.5) and in elevated temperatures up to 55 °C. Xylanase ME742 effectively hydrolyzed EFB-xylan. HPLC analysis revealed that the hydrolysate was composed of xylose and XOS with degree of polymerization between 2 and 5 and xylobiose was the predominant product. The XOS yield ranging in 95.01–151.19 mg/g EFB-xylan was generated from enzymatic processing for 12–72 h. Interestingly, arabinosyl-xylooligosaccharides were also predicted to be produced from EFB-xylan. XOS mixture exhibited high antioxidant activity (71.36 ± 0.57%) against DPPH at 100 μg/mL concentration and moderate activity (45.18 ± 1.56%) against ABTS radical. These findings reveal the potential use of Streptomyces thermocarboxydus ME742 and xylanase produced in a sustainable bioprocess to add value of EFB waste. • This study constituted a sustainable bioprocess to enrich value of oil palm waste. • Empty fruit bunch (EFB) was used to produce xylanase and xylan that both further used in XOS production. • Purified xylanase ME742 has characteristics favorable for industrial use in XOS production. • Purified xylanase ME742 generated arabinosyl-XOS from EFB-xylan. • XOS mixture contained arabinosyl-XOS had high antioxidant activity against DPPH radical.

A water-soluble polysaccharide from Anethum graveolens seeds: Structural characterization, antioxidant activity and potential use as meat preservative

A novel water-soluble polysaccharide named AGP1 was successfully isolated from seeds of Anethum graveolens by hot water extraction and further purified by DEAE-Sepharose chromatography. AGP1 has a relative molecular weight of 2.1 104 Da determined by Ultra-high-performance liquid chromatography (UHPLC). The AGP1 characterization was investigated by chemical and instrumental analysis including gas chromatography mass spectrometry (GC–MS), Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction. Results showed that AGP1 was mainly composed of glucose, galactose, mannose and arabinose in a molar percent of 54.3, 23.8, 14.7 and 7.2, respectively. The thermogravimetry analysis (TGA) and the differential scanning calorimetry (DSC) were used and showed that AGP1 has good thermal stability until 275 °C. Moreover, the purified polysaccharide demonstrated an appreciable in vitro antioxidant potential. The addition of the AGP1, particularly at 0.3% (w/w), in turkey sausages instead of ascorbic acid, as preservative, reduced the lipid peroxidation, preserved the pH and color and improved the bacterial stability during cold storage at 4 °C for 12 days. Overall, the results showed that the AGP1 deserves to be developed as functional and bioactive components for the food and nutraceutical industries.

Production of a novel α-amylase by Bacillus atrophaeus NRC1 isolated from honey: Purification and characterization

Different bacterial isolates with amylolytic activity were insulated from various honey samples. The most active isolate was identified by the molecular 16SrRNA sequence technique as Bacillus atrophaeus NRC1. The bacterium showed maximum amylase production under optimum culture conditions at pH 6.0, 40 °C and after 24 h incubation. Two amylase isoenzymes (AmyI and AmyII) from Bacillus atrophaeus NRC1 have been purified to homogeneity by using ammonium sulfate precipitation, Sephacryl S-200 and DEAE-Sepharose chromatography. The major isoenzyme, AmyI, had a specific activity 4635 U/mg proteins with molecular weight of 61 kDa using SDS-PAGE electrophoresis. The maximum activity of AmyI against starch was determined at pH 6.0 and 50 °C. AmyI was stable up to 50 °C after incubation for 30 min, retained 65 and 23% of its activity at 60 and 70 °C, respectively. Pre-incubation with Ca2+, Mg2+ and Ba2+ cations for 30 min enhanced the enzyme activity; while it was completely inhibited by Hg2+. Varied inhibition degree of the enzyme activity was determined with K+, Ni2+, Zn2+, Na2+ and Cu2+ ions. AmyI was inhibited by EDTA, PMSF and SDS, while it was activated by l-Cysteine-HCl and DTT. AmyI had the ability to degrade starch, amylopectin, glycogen, amylose and lacked the affinity towards β-1,4-linked xyloses.

Effect of purified alkaline phosphatase from Bacillus licheniformis on growth of Zea mays L.

Some soil microbes have the capability to solubilize mineral phosphate into organic phosphorous and used as biofertilizer to improve crop productivity in agricultural field. In this study, phosphate solubilization assay was carried out onto media plates containing calcium phsophate precipitated nutrient agar media for bacterial strains like Bacillus megaterium MTCC 453, Bacillus subtilis MTCC 1134, Bacillus licheniformis MTCC 2312, Pseudomonas aeruginosa MTCC 424, Escherichia coli MTCC 570. Among these bacterial strains, B. licheniformis MTCC 2312 showed largest clear zone of phosphate solubilzation and maximum activity of alkaline phosphatase. The enzyme alkaline phosphatase was purified from B. licheniformis MTCC 2312 with purification fold 3.52 and specific activity 295.89 Unit/mg protein using DEAE-sepharose chromatography. This enzyme showed molecular weight as 60 KD, thermostability upto 50?C, pH stability up to 8.5 and Michaelis constant (Km) and maximum activity (Vmax) as 2.30 mM and 2223 U/ml respectively. The lyophilized powder of this enzyme was further supplemented with media components for the growth of Zea mays for carrying tissue culture experiment. The sterilized soil supplemented with alkaline phosphatase improved the total height, dry weight, % phosphate content in the stem and root of Zea mays by 3.07, 3.15, 2.35 and 1.76 fold respectively compared to control set. This enzyme could be used at large extent as effective biofertilizer for the agricultural industry.

Purification and characterization of an extracellular lipase from Trichosporon sp. and its application in enrichment of omega-3 polyunsaturated fatty acids

Lipase catalysis has become the most promising technique for enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) due to its mild and green conditions. In this study, Trichosporon sp. F1-2 was newly isolated from oil-contaminated sludge, and its extracellular lipase was purified using ammonium sulfate precipitation and DEAE-Sepharose chromatography. The molecular weight of the lipase was estimated to be 32.6 kDa. The optimal temperature and pH for lipase activity were 50 °C and 8.0, respectively, and the optimal storage conditions were found to be temperatures below 45 °C and pH 7.0–9.0. The lipase did not require metal ions as cofactors and was significantly inhibited by Zn2+. It was stable in various organic solvents with different log P values, and n-hexane, toluene, dichloromethane, diethyl ether and glycerol even enhanced its activity. It had sn-1,3 regioselectivity and the highest activity towards a medium-chain fatty acid (C8) ester. This study demonstrated the lipase has good potential as a catalyst in enrichment of EPA and DHA in fish oil. The content of EPA and DHA reached 22.9 and 25.4 mol%, respectively, using 100 U of the lipase within 24 h, and the enrichment efficiency of DHA was higher than that of EPA.

Purification and characterization of a novel thermophilic β-galactosidase from Picrophilus torridus of potential industrial application.

Intracellular β-galactosidase (E.C 3.2.1.23) produced by the thermoacidophilic archeon Picrophilus torridus DSM 9790 was purified to homogeneity using a combination of DEAE Sepharose, gel filtration, hydroxyapatite and chromatofocusing chromatographies. LC-MS/MS analysis was used to confirm the identity of the purified protein. The enzyme was found to be a homotrimer, with a molecular mass of 157.0kDa and an isoelectric point of 5.7. To our knowledge, this enzyme has the lowest pH optimum of any intracellular β-galactosidase characterized to date. Maximal activity was exhibited at acidic pH values of 5.0-5.5 and at 70°C. The enzyme retained > 95% activity after heating to 70°C for 1h, or after incubation at pH 5.5 for 1h. The enzyme may be of interest for high-temperature bioprocessing, such as in the production of lactulose. This investigation suggests that the β-galactosidase activity produced by P. torridus is potentially more useful than several enzymes already characterized for such an application.

Purification and characterization of a heterodimeric mycelial lectin from Penicillium proteolyticum with potent mitogenic activity

In the present study, Penicillium proteolyticum lectin was purified by DEAE-Sepharose chromatography followed by Sephadex G-100 gel filtration chromatography. A sequence of ion-exchange and gel filtration chromatography resulted in 52.30 fold purified lectin with a high yield of 84.21%. The purified P. proteolyticum lectin is a glycoprotein with 2.83% linked carbohydrates. A single band in Native-PAGE, whereas two bands (25.1 kDa and 22.9 kDa) in SDS-PAGE confirmed the heterodimeric nature of the purified lectin. The apparent molecular weight (48 kDa) of P. proteolyticum lectin was further confirmed by gel filtration analysis. Heterodimeric P. proteolyticum lectin was stable within a pH range of 6.5-7.5 and upto a temperature of 30 °C. The lectin activity was strongly inhibited by complex glycoproteins and denaturants. Metal ions are not required for agglutination activity of purified lectin. The lectin showed significant mitogenic response towards mice splenocytes. It exhibited highest mitogenic activity at a concentration of 50 μg/mL. This is a first report on characteristics of purified P. proteolyticum lectin having potent mitogenic potential.

Structural characterization, antioxidant and antibacterial activities of a novel polysaccharide from Periploca laevigata root barks

A novel water-soluble polysaccharide named PLP1 was successfully isolated from root barks of Periploca laevigata by hot water extraction and further purified by DEAE-sepharose chromatography. PLP1 has a relative molar mass of 5.57 105 g/mol determined by Ultra-high performance liquid chromatography (UHPLC). The PLP1 structure was investigated by chemical and instrumental analysis including gas chromatography mass spectrometry (GC–MS), methylation analysis, Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic spectroscopy (1H NMR and 13C NMR). Results showed that PLP1 was mainly composed of glucose, galactose, mannose and glucuronic acid in a molar percent of 62.9, 19.4, 11.8 and 5.9, respectively. The backbone of PLP1 was composed of 1,3-β-d-Glcp, 1,3,4-α-d-Glcp, 1,4-α-d-Manp, 1,6-α-d-Manp, 1,3-α-d-Galp and 1,6-β-d-Galp. The thermogravimetry analysis (TGA) and the differential scanning calorimetry (DSC) were used and showed that PLP1 has good thermal stability under 229 °C. Moreover, the purified polysaccharide demonstrated an appreciable in vitro antioxidant potential and high antibacterial activity against several Gram (+) and Gram (−) strains. These finding suggested that PLP1 might be suitable for use as functional foods and as potential therapeutic agents.

Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana

Butyrylcholinesterase (BChE) is a glycosylated serine hydrolase found in human serum that has been shown to protect against various cholinesterase-inhibiting organophosphate nerve agents. The supply of plasma-derived butyrylcholinesterase (hBChE) is constrained by the availability of human blood and a complex purification process and its high cost. This constraints necessitates the development of expression platforms capable of large-scale, low-cost production of an active recombinant BChE (rBChE). Traditionally, procainamide affinity chromatography has been used to purify BChE. Recently, an effective affinity chromatography resin based on a potent cholinesterase inhibitor (tacrine-huperzine A hybrid; huperine X termed as Hupresin®) was developed. Here, we describe a purification scheme of rBChE from Nicotiana benthamiana plants. Different extraction buffers were screened for their ability to extract rBChE and native plant proteins. Citrate buffer at pH 4 was selected to minimize extraction of host plant proteins and showed a 4.5-fold enhancement in rBChE specific activity compared to Tris buffer, pH 8. DEAE-Sepharose chromatography increased the purity of rBChE by 70% by removing major host plant protein impurities. The rBChE was then adsorbed to Hupresin® and purified to homogeneity for an overall process yield of 34%. The purification process represents a threefold higher product yield over the established process. Mass spectrometry confirmed the protein sequence and site-specific N-glycosylation analysis was performed.

Cloning of Oryza sativa N-glycanase gene (OsPNGase A) and its expression in Pichia pastoris

N-glycanase is a class of deglycosylation enzymes, widely used in the study of N-glycosylation modification of glycoprotein. In this study, an N-glycanase gene (OsPNGase A, XM_015775832) with high GC content (69.48%) was cloned from rice and then the yeast secretory expression vector pPICZ(α)A-OsPNGase A was constructed for the purpose of transformation to Pichia pastoris. After induction in Pichia pastoris SMD1168H, the target protein was purified by DEAE Sepharose and HisTrap HP chromatography, with a yield of 12.3 mg OsPNGase A from 1 L fermentation medium, showing a specific activity of 258 U/mg. SDS-PAGE revealed that the purified OsPNGase A was a single band and showed consistentcy with the expected molecular weight. OsPNGase A could act on transferrin recombinantly expressed in rice, avidin recombinantly expressed in corn and horseradish peroxidase. Furthermore, OsPNGase A showed higher activity than commercial PNGase F towards avidin. OsPNGase A displayed the highest digestion activity at pH 6.0 and 40 °C, and was also active in the neutral and alkaline environment. Despite the fact that OsPNGase A was inhibited by reducing agents and surfactants, it still maintained partial enzymatic activity in 100 mmol/L β-ME or DTT. Therefore, the successful expression of rice OsPNGase A provides a new tool for the study of plant glycoproteins and the establishment of yeast secretion expression system lays the foundation for the preparation of PNGase A.

Isolation, purification and characterization of a pH tolerant and temperature stable proteinaceous protease inhibitor from marine Pseudomonas mendocina.

An extracellular protease inhibitor (BTPI-301) of trypsin was purified and characterized from an isolate of Pseudomonas mendocina. BTPI-301was purified to homogeneity by (NH4)2SO4, precipitation, DEAE Sepharose and CNBr-activated Sepharose chromatography. Homogeneity was proved by native PAGE and SDS-PAGE. The intact molecular mass was 11567Da by MALDI-TOF analysis. BTPI-301was a competitive inhibitor with a Ki of 3.5×10-10M. It was stable and active at pH 4-12 and also at 4-90°C for 1h. Peptide mass fingerprinting by MALDI revealed that the BTPI-301 is a new inhibitor not reported so far with protease inhibitory activity. The pI of the inhibitor was 3.8. The stoichiometry of trypsin-BTPI-301 interaction is 1:1. The inhibitor was specific towards trypsin. A pH tolerant and thermostable protease inhibitor BTPI-301 active against trypsin was purified and characterized from P. mendocina that could be developed and used as biopreservative as well as biocontrol agent.

Identification and kinetic characterization of a novel superoxide dismutase from Avicennia marina: An antioxidant enzyme with unique features

A novel Cu/Zn-superoxide dismutase was extracted from Avicennia marina and purified. The sample was collected from Khamir port located in the north shore of Persian Gulf. The purification procedure comprised of (NH4)2SO4 precipitation followed by CM-Sephadex C-50 and DEAE-Sepharose chromatography, and gel filtration chromatography (Sephadex G-75). The enzyme with a characteristic molecular weight of 31kDa, measured by SDS-page, showed its highest catalytic efficiency at pH 8.0 and 50°C. Its activity was greatly inhibited by cyanide and hydrogen peroxide. The pH profile showed that the enzyme could maintain most of its activity at pH values ranging from 5 to 10. The temperature profile of this enzyme showed a broad range of activity compared with other superoxide dismutases.Catalytic hydrolysis rate followed Michaelis–Menten equation. The values of kcat and Km were obtained from Michaelis–Menten plot as 107000s−1 and 11.5μmol respectively. The evidences from kinetic and thermodynamic parameters suggest that Avicennia marina superoxide dismutase (AmSOD) can be used as a suitable enzyme for biotechnological and pharmacological applications.

Preparative method for obtaining recombinant human interferon α2b from inclusion bodies of Escherichia coli

A simple, easily reproducible, and scalable method for obtaining recombinant human interferon α2b from Escherichia coli inclusion bodies has been elaborated. It involves the following steps: preparation of producer cell biomass, isolation and washing of inclusion bodies, their dissolution with protein refolding, SP Sepharose chromatography, and DEAE Sepharose chromatography. According to the results of gel electrophoresis and reversed-phase HPLC, the purity of the protein obtained exceeds 95%.

Partial Purification and Characterization of Xylanase from Bacillus weihenstephanensis Strain ANR1 using Watermelon Rind

Aim: The aim of this study is to partially purify and characterize xylanase from Bacillus weihenstephanensis strain ANR1 using watermelon rind as a substrate. Materials and Methods: The soil bacterium, B.weihenstephanensis strain ANR1 was grown at 37°C for 72 h on rotary incubator shaker at 150 rpm in a flask containing 250 ml of medium. The xylanase enzyme was partially purified using ammonium sulfate precipitation and dialysis. It was further purified using DEAE-sepharose chromatography. Molecular weight of the xylanase enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results and Discussion: The xylanase enzyme was partially purified to a fold of 8.15 and a yield of 83.56%. Molecular weight of the protein was found to be 45 kDa. The purified enzyme was found to be optimally active at pH of 7.0 and at a temperature of 37°C. Conclusion: Based on the results obtained, the purified xylanase enzyme from B. weihenstephanensis strain ANR1 using watermelon rind as a substrate can be effectively used in various food industrial applications

Probing the Role of Threonine‐221 and Lysine‐397 in ADP‐Glucose Pyrophosphorylase from Deinococcus radiodurans

The knowledge gained from structure‐function studies of Adenosine Diphosphate Glucose Pyrophosphorylase (ADP‐glucose PPase), that serves as the rate‐limiting step in production of starch in plants and glycogen in bacteria, can inform protein engineering to generate a highly active enzyme. This enzyme is allosterically regulated by metabolites which serve as activators or inhibitors in accord with the organism's carbon metabolism pathways. An ADP‐glucose PPase form that was insensitive to feedback inhibition could enhance the biomass production of starch in plants, allowing for the efficient and economic use of starch as a renewable energy and food source. The enzyme of interest is derived from Deinococcus radiodurans (D. radiodurans), a microorganism that can withstand extreme growth conditions and ionizing radiation. Although studies on the enzyme from D. radiodurans are limited, the enzyme's characteristics of stability and altered regulation would be desirable to incorporate into an altered enzyme expressed in transgenic crops. The wild‐type D. radiodurans ADP‐glucose PPase is activated by fructose‐bisphosphate (FBP) and fructose‐6‐phosphate (F6P). The K397A and T221A enzymes were generated to further elucidate the role of amino acids in these regions on activity and regulation. Both enzymes were purified to near homogeneity by a procedure that included DEAE and phenyl sepharose chromatography. Preliminary studies indicate lower apparent binding affinity for activators and overall lower enzyme activity when alanine was substituted for lysine at position 397 (K397A), a position postulated to be near the allosteric site. These preliminary results demonstrate the importance of a positively charged amino acid at this position. The D. radiodurans enzyme harbors a threonine in a loop near the G1P binding site that is occupied by alanine in most ADP‐glucose PPases (amino acids 220–226: LTSMGIY). The T221A enzyme exhibited similar aparent affinity for the substrates ATP and G1P and co‐factor Mg but displayed a 50% reduction in activity in the absence of the activator FBP. However, the presence of the activator restores activity to the T221A enzyme, which is also more sensitive to other metabolites such as F6P, G6P, 3‐PGA, and sulfate. The T221 position appears to play a role in fold activation by metabolites as well as effector specificity. Complete kinetic and physical characerization of these enzymes is underway.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30.

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of- flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80°C in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50 °C and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni(2+), Mn(2+), and Cu(2+) to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The Km and Vmax values were estimated to be 35.7 mg/ml, and 5 × 10(4) U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

Structural features and immunostimulating effects of three acidic polysaccharides isolated from Panax quinquefolius

Three acidic polysaccharides (PPQA2, PPQA4 and PPQA5) were successfully purified from the water-extracted crude polysaccharides of Panax quinquefolius by combination of DEAE Sepharose and Sephacryl S-300 chromatography. The average molecular weights (Mws) of PPQA2, PPQA4 and PPQA5 were 2.3×104Da, 1.2×105Da, and 5.3×103Da, respectively. Monosaccharides components analysis indicated that PPQA2 and PPQA5 were composed of arabinose, rhammose, mannose, galactose, glucose, galacturonic acid and glucuronic acid in a molar percent of 8.0:4.0:2.9:7.2:12.5:26.6:38.8 and 8.5:3.2:5.3:10.8:32.4:15.5:24.4. PPQA4 was composed of arabinose, rhammose, mannose, galactose, glucose and glucuronic acid in a molar percent of 19.7:5.1:8.1:23.9:41.3:2.0. The 13C NMR spectra indicated the existence of O-acetyl groups in PPQA2 and PPQA5, while absence in PPQA4. The in vitro study showed that PPQA2, PPQA4 and PPQA5 were all able to stimulate the production of secretory molecules (NO, TNF-α, and IL-6) of RAW264.7 murine macrophages in a dose-dependent manner. These findings suggest that the three acidic polysaccharides isolated in our study have immunopotentiating effects on macrophages and should be used as a beneficial health food.