Chemical characteristics and antioxidant activity of exopolysaccharide fractions from Microbacterium terregens

The Gram-positive bacterial strain isolated from soil was identified as the non-pathogenic Microbacterium terregens. The exopolysaccharide (CPS) produced from M. terregens was obtained by isopropanol precipitation (13.72 g L^?1 growth medium), The resulted exopolysaccharide was purified by chromatography on DEAE-cellulose and Sephacryl S-200 columns, when two polysaccharide fractions termed CPSI and CPSII were obtained. Structure features of CPSI and CPSII were investigated by a combination of chemical and chromatographic analyses, such as acid hydrolysis, methylation analysis, periodate oxidation–Smith degradation, HPLC, GC–MS, and IR. The results indicated that CPSI and CPSII were composed of glucose: mannose in a ratio of 2.7:1 and 3.2:1 with molecular weights 80 and 150 kDa, respectively. It has a backbone of (1 → 4)-linked β-glucose residues, which occasionally branches at O-6. The branches were composed of (1 → 4)-linked β-mannose residues. The antioxidant activity of the CPS, CPSI and CPSII was evaluated in-vitro by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay (RSA). CPSI fraction showed the highest antioxidant activity among the three fractions, with an IC₅₀ value of 230 μg mL^?1. The effect of molecular weight of the polysaccharide on the improvement of the antioxidant potential seems to be significant.     

Separation,purification and preliminary characterization of sulfated polysaccharides from Sargassum plagiophyllum and its in vitro anticancer and antioxidant activity

Sulfated polysaccharides (SPs) were identified in different portions of the thallus of Sargassum plagiophyllum C. Agardh, with TBO staining. SPs were extracted using a blade and purified by Q sepharose fast flow anion-exchange chromatography, resulting in SP fractions F1, F2 and F3, with molecular weights of 30, 35 and 20 kDa, respectively. An SP yield of 43.1% was obtained in F3, while F2 yielded a sulfate content of 21.9%. Furthermore, the in vitro anticancer and antioxidant activities of the polysaccharide fractions were evaluated. The F2 fraction showed higher anticancer activity against HepG2 and A549 cells than the other two fractions, with IC₅₀ values of 600 μg/mL and 700 μg/mL, respectively. The normal breast epithelial cell line (HBL-100) exhibited IC₅₀ concentrations of 1200 and 1400 μg/mL for crude sulfated polysaccharides (CSPs) and all SP fractions (F1–F3). These results indicated that the anticancer activity of F2 could be related to its sulfate content. However, the antioxidant activities of F1–F3 were low at their tested concentrations.     

Characterization and antioxidant activity for exopolysaccharide from submerged culture of Boletus aereus

Three polysaccharide fractions (designated as Fr-I, Fr-II and Fr-III) were successfully purified from the crude exopolysaccharide (EPS) produced from submerged culture of Boletus aereus by gel filtration chromatography on Sepharose CL-6B. The size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS) system showed that the average molecular weights (Mws) of these three fractions were 1.365 × 10⁶, 1.048 × 10⁵ and 2.471 × 10⁴ g/mol, respectively. The SEC/MALLS also revealed that the molecular conformation of the Fr-I was a random coil, with Fr-II being a rigid rod in aqueous solution. Moreover, monosaccharide composition analysis indicated that Fr-I was mainly composed of glucose, while both of Fr-II and Fr-III were mainly composed of mannose and glucose. Then, FT-IR spectral analysis of the purified EPS revealed prominent characteristic groups. Furthermore, thermo gravimetric analysis (TGA) indicated the degradation temperature of the Fr-I (170 °C) was higher than those of Fr-II (156 °C) and Fr-III (155 °C). Finally, on the basis of the antioxidant activity test in vitro, Fr-I exhibited the highest antioxidant ability among these samples, which might be attributed to the monosaccharide composition and molecular weight in the EPS fraction.     

CEP131 indicates poor prognosis and promotes cell proliferation and migration in hepatocellular carcinoma

Centrosomal proteins have been implicated in the progression of human diseases. CEP131 plays important roles in centrosome duplication and genome stability, but its role in cancers remains largely unknown. Here, we showed that CEP131 expression was increased in hepatocellular carcinoma (HCC), compared to the paracarcinoma tissues, at both mRNA and protein levels. High CEP131 expression was closely associated with tumor size (P = 0.020), tumor capsule (P = 0.043), TNM stage (P = 0.007) and tumor differentiation (P = 0.019). Furthermore, patients with high expression of CEP131 were accompanied with worse overall and disease-free survivals in our and TCGA cohorts consisting of a total of 802 cases. The prognostic value of CEP131 was further confirmed by stratified survival analysis. Multivariate cox regression model indicated that CEP131 was an independent factor for overall survival (hazard ratio = 1.762, 95% confident interval: 1.443–2.151, P < 0.001). In vitro data demonstrated that nucleophosmin (NPM) physically bound to CEP131 and maintained its protein stability. Overexpression of CEP131 in HCC cell lines enhanced cell proliferation and migration, whereas the knockdown of CEP131 led to the opposite phenotypes. Further studies demonstrated that CEP131 exhibited oncogenic activity via activation of PI3K/AKT signaling pathway. Taken together, our findings suggest CEP131 serves as a potential prognostic biomarker in HCC, and functions as an oncogene in this deadly disease.     

Structural elucidation and biological activity of a novel polysaccharide by alkaline extraction from cultured Cordyceps militaris

A novel polysaccharide named CBP-1 was isolated from the fruiting body of cultured Cordyceps militaris by alkaline extraction as well as anion-exchange and gel-permeation chromatography. Its structural features were investigated by a combination of chemical and instrumental analysis approaches, including partial hydrolysis, methylation analysis, HIO₄ oxidation-Smith degradation, GC–MS, ¹³C NMR, HPAEC-PAD and FT-IR. The results indicated that CBP-1 has a backbone of (1 → 4)-α-d-mannose residues which occasionally branches at O-3. The branches were mainly composed of (1 → 4)-α-d-glucose residues and (1 → 6)-β-d-galactose residues, and terminated with β-d-galactose residues. In the in vitro antioxidant assay, CBP-1 was found to possess the hydroxyl radical-scavenging activity with an IC₅₀ value of 0.638 mg/ml.     

Carboxymethylation of a polysaccharide extracted from Ganoderma lucidum enhances its antioxidant activities in vitro

The chemical carboxylmethylated polysaccharide (C-GLP), which derived from water-insoluble crude Ganoderma lucidum polysaccharide (GLP), was prepared. Water solubility, chemical characterization, and antioxidant activities in vitro of C-GLP were determined. The solubility of C-GLP in distilled water reached 100 mg/ml, which was much higher than the solubility of GLP. Chemical analysis indicated that C-GLP was composed of Glc:Man:Gal = 33.0:1.0:3.4 with a molecular weight of 1.8 × 10⁶ Da and a carboxymethyl content of 11.07%. The signals of carboxymethyl were found in IR and ¹³C NMR spectra. Moreover, a high antioxidant activity of C-GLP was observed, especially in scavenging of hydroxyl radical (83.7% at 5 mg/ml) and hydrogen peroxide (51.6% at 10 mg/ml). This study indicates the effects of carboxymethylation on water-insoluble polysaccharide and explores a potential antioxidant in food industry and pharmaceuticals.     

Different contributions of side-chains in β-d-(1 → 3,6)-galactans on intestinal Peyer’s patch-immunomodulation by polysaccharides from Astragalus mongholics Bunge

Thirteen polysaccharides isolated from an extract of the aerial portions of Astragalus mongholics Bunge demonstrated immunomodulating activity against Peyer’s patch immunocompetent cells. Nine of the active polysaccharide fractions were composed of either arabinogalactans, pectic arabinogalactans or pectins. The activities of the arabinogalactans and pectic arabinogalactans were associated with β-d-(1 → 3)-galactan moieties branched with β-d-(1 → 6)-galactooligosaccharide side-chains having degrees of polymerization of 8 or less. Degradation of the β-d-(1 → 3)-galactan or β-d-(1 → 6)-galactosyl side-chains in the arabinogalactans significantly decreased immunomodulating activity. Rhamnogalacturonan I (RG-I) with β-d-(1 → 3,6)-galactosyl side-chains having terminal β-d-GlcA showed activity in the pectin-enriched fractions. Interestingly, the terminal GlcA was not required for activity of the arabinogalactan-enriched fractions, suggesting at least two different immunomodulating structures.     

Optimization of ultrasound-assisted extraction conditions for total phenols with anti-hyperglycemic activity from Psidium guajava leaves

The high concentration of total phenolic compounds (TPC) in Psidium guajava leaf extracts (GvEx) is correlated to its anti-hyperglycemic activity. In this study, we established the optimum ultrasound extraction conditions for maximizing TPC yield. The response surface methodology (RSM) was employed for empirical model building. The maximum value of TPC (26.12%) was obtained at solvent to solid ratio (v/w) of 12.1, extraction temperature of 59.8 °C, and extraction time of 5.1 min. The anti-hyperglycemic activity of GvEx was compared to the commonly used diabetic drug acarbose. The IC₅₀ of GvEx for α-amylase and α-glucosidase inhibition was 50.5 μg/mL and 34.6 μg/mL, respectively. However, the IC₅₀ of acarbose for α-amylase and α-glucosidase inhibition was 95.3 μg/mL and 1075.2 μg/mL, respectively. In conclusion, GvEx obtained under optimum extraction conditions had higher anti-hyperglycemic activity than acarbose. In addition, the recommended extraction procedures for GvEx save time and are environmentally friendly.     

Isolation of antioxidant constituents from Combretum apiculatum subsp. apiculatum

Species of the family Combretaceae are used extensively in traditional medicine against inflammation and infections, and although antibacterial activity has been reported in non-polar extracts, further rationale for the widespread use of the Combretaceae is expected to exist. Methanol extracts of leaves of ten different Combretum species were evaluated for antioxidant activity by spraying TLC chromatograms of each leaf extract with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Compounds with antioxidant activity were detected by bleaching of the purple DPPH colour. Leaf extracts of Combretum apiculatum subsp. apiculatum had the most antioxidant compounds. This species was consequently selected for phytochemical investigation. A DPPH assay-directed fractionation of the leaf extracts of C. apiculatum led to the isolation of four antioxidant compounds from the ethyl acetate and butanol soluble fractions. The structures of the compounds were determined by spectroscopic analyses (¹H-NMR, ¹³C-NMR and MS) and identified as: cardamonin (1), pinocembrin (2), quercetrin (3) and kaempferol (4). In a quantitative antioxidant assay, the more polar fractions (ethyl acetate and butanol) obtained by solvent–solvent fractionation had the highest antioxidant activity among the solvent fractions obtained from C. apiculatum, with EC₅₀ values of 3.91 ± 0.02 and 2.44 ± 0.02 μg/ml respectively. Of the four isolated compounds, quercetrin (4) and kaempferol (3) had the strongest antioxidant activity, with EC₅₀ values of 11.81 ± 85 and 47.36 ± 0.03 μM respectively. Cardamonin (1) and pinocembrin (2) did not demonstrate strong activity. L-ascorbic acid was used as standard antioxidant agent (EC₅₀ = 13.37 ± 0.20 μM or 2.35 μg/ml). The cytotoxicity of cardamonin and pinocembrin was evaluated on Vero kidney cells using the MTT (3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide) assay with berberine as positive control. At concentrations higher than 50 μg/ml of cardamonin or pinocembrin, the cells were not viable. Cardamonin was more toxic (LC₅₀ = 1.97 μg/ml) than pinocembrin (LC₅₀ = 29.47 μg/ml) and even the positive control, berberine (LC₅₀ = 12.35 μg/ml).     

Composition analysis and antioxidant activity of polysaccharide from Dendrobium denneanum

Three polysaccharide fractions (DDP1-1, DDP2-1 and DDP3-1) were successfully purified from the crude polysaccharide of Dendrobium denneanum by DEAE-Cellulose and Sephadex G-200 column chromatography. The average molecular weights (Mws) of these fractions were 51.5, 26.1 and 6.95 kDa, respectively. Monosaccharide components analysis indicated that DDP1-1 and DDP2-1 were composed of arabinose, xylose, mannose, glucose and galactose in a molar ratio of 1.00:2.82:57.11:140.82:7.76 and 1.00:1.62:1.18:77.5:7.79. DDP3-1 was composed of arabinose, mannose, glucose and galactose in a molar ratio of 1.00:1.03:8.84:2.00. On the basis of antioxidant test in vitro, DDP2-1 exhibited the highest antioxidant ability among these samples.     

Extraction method for increasing antioxidant activity of raw garlic using steam explosion

Novel extraction method for increasing the antioxidant activity of raw garlic was proposed using steam explosion. Raw garlic was hydrolyzed by high temperature (183–258 °C) and pressure steam (10–45 atm), and then crushed by the rapid decompression. The antioxidant activity of raw garlic treated by steam explosion was higher than that of black garlic, i.e. aging garlic. The lowest EC₅₀ value, i.e. the highest antioxidant activity, of extract from raw garlic was obtained at a steam pressure of 45 atm for a steaming time of 5 min, but the highest amount of phenolic compounds, i.e. 93.7 mg-catechin equiv./g-dry raw garlic, was obtained at a steam pressure of 30 atm for a steaming time of 5 min.     

Purification,structural analysis and hydroxyl radical-scavenging capacity of a polysaccharide from the fruiting bodies of Russula virescens

In this study, one water-soluble polysaccharide (RVP) was isolated from the fruiting bodies of Russula virescens. Its molecular weight (Mw) was ∼3.9 ×10⁴ Da, as determined by high-performance size-exclusion chromatography (HPSEC). The RVP was composed of d-galactose and d-mannose in a ratio of 2:1, as detected by gas chromatography (GC). The structural features of RVP were investigated by partial hydrolysis with acid, periodate oxidation and Smith degradation, acetylation, methylation analysis, and NMR spectroscopy (¹H, ¹³C). The results revealed that RVP had a backbone consisting of (1 → 6)-linked-α-d-galactopyranosyl and (1 → 2, 6)-linked-α-d-galactopyranosyl residues that terminated in a single non-reducing terminal (1 → )-α-d-mannopyranosyl residue at the O-2 position of each (1 → 2, 6)-linked-α-d-galactopyranosyl residues along the main chain in the ratio of 1:1:1. Preliminary, in vitro antioxidant activity tests showed that RVP could dose-dependently enhance the scavenging effect on hydroxyl radicals at concentrations from 20 to 160 μg/ml. These results indicated that RVP was mainly composed of water-soluble galactomannan, and it had a significant effect on scavenging of hydroxyl radicals in vitro.     

Nutritionally rich marine proteins from fresh herring by-products for human consumption

Marine oils and proteins are valuable compounds, but under unfavorable conditions these components are easily oxidized and enzymatically degraded. We have designed an industrial process for producing high quality human grade proteins from fresh herring by-products. Two different processes were tested in semi-industrial scale: (i) thermal extraction to separate oil and proteins and (ii) enzymatic hydrolysis with different commercial proteases to produce fish protein hydrolysates (FPH) and separate oil. Both stick water from thermal extraction and fish protein hydrolysates, after hydrolysis, are nutritionally rich fractions and yielded approx. 18 and 25% of dry material in these fractions respectively. Neither season nor enzymes used influenced the color of hydrolysate powders, but time and temperature of the processes are important tools for controlling the color. Fishing seasons did not influence bitterness of hydrolysates and stick water samples, but both the process conditions and applied enzymes played an important role for the formation of bitterness. Insoluble fractions after both processes had significantly higher protein efficiency ratio: 3.08 and 2.93 compared to soluble fractions: 2.62 and 2.76 after hydrolysis and thermal extraction respectively. Both stick water and FPH showed antioxidative activity against both iron (15 μM of Fe³⁺) and hemoglobin (80 ppm) induced oxidation. Herring proteins (1.25 mg/ml) were able to reduce iron induced oxidation by 50–70%, while Hb induced oxidation was reduced by 70–80% using 4 mg/ml proteins concentration.     

Oxidative stress and apoptosis in preeclampsia

We aimed to determine the oxidative stress and antioxidant status in preeclamptic placenta. Also, we investigated the apoptotic index of villous trophoblast and proliferation index of cytotrophoblasts. The study included 32 pregnant with preeclampsia and 31 normotensive healthy pregnant women. Malondialdehyde (MDA) and total antioxidant status (TAS) levels were measured in the placenta. For detection of apoptosis and proliferation in trophoblast, apoptosis protease activating factor 1 (APAF-1) and Ki-67 were used. Placental MDA levels in preeclamptic women were significantly higher than normal pregnancies (p = 0.002). There was no significant difference between the groups in the TAS levels of placenta (p = 0.773). Also, the apoptotic index in villous trophoblasts increased (p < 0.001), but proliferation index did not change in preeclampsia (p = 0.850). Increased oxidative stress and apoptosis in pathological placenta are not balanced by antioxidant systems and proliferation mechanisms.     

Intestinal Peyer’s patch-immunomodulating glucomannans from rhizomes of Anemarrhena asphodeloides Bunge

During screening for intestinal Peyer’s patch-immunomodulating polysaccharides from plant resources including medicinal herbs, a potent modulating activity was observed in a crude polysaccharide fraction (AS-1) from the rhizome of Anemarrhena asphodeloides Bunge. Oral administration of AS-1 (100 mg/kg/day) to aged BALB/c mice enhanced productions of IL-10, IFN-γ and IL-6 from Peyer’s patch immunocompetent cells, and its oral administration to ovalbumin (OVA)-fed B10.A mice led to significant suppression on induction of OVA-specific IgE in systemic immune system. Further fractionation of the polysaccharides in the crude polysaccharide fraction, AS-1, yielded 4 polysaccharide fractions that were potently active, and contained glucomannans. Treatment of these polysaccharide fractions with endo-β-d-(1 → 4)-mannanase significantly decreased their activities. Mannanase digestion of the active glucomannan gave both long and short hexosyl-oligosaccharides, whereas konjac glucomannan, which was inactive, released short oligosaccharides. Structural analysis indicates that the long oligosaccharides from the active glucomannan contain mannanase-resistant complex structure comprising β-d-Man and β-d-Glc.     

A novel and efficient method for the isolation and purification of polysaccharides from lily bulbs by Saccharomyces cerevisiae fermentation

A water-soluble polysaccharide from lily bulbs was isolated and purified by Saccharomyces cerevisiae fermentation. Proteins present in lily bulb extract were removed by extracellular proteases secreted by S. cerevisiae during fermentation. This novel method differs from traditional protein removal methods. A suitable yeast strain was selected. Culture conditions were optimized. Response surface methodology (RSM) was utilized to evaluate the effects of variables on the lily polysaccharide (LP) yield and the protein removal ratio (PRR). The results of applying RSM revealed that the optimum fermentation conditions were 87.5 g L⁻¹ lily bulb powder, pH 5.6, and temperature 27.9 °C. When lily bulb extract was cultured with S. cerevisae under optimum conditions, the LP yield and the PRR were 6.56% and 91.46%, respectively. These values are in close agreement with the value predicted by the model. The resulting LP curding was further purified by DEAE Sepharose Fast Flow chromatography after isolation by alcohol precipitation post-fermentation. DEAE chromatography resulted in a fraction, LP-1 (yield: 4.46%) with a molecular weight of 65.0 kDa. LP-1 consisted of glucose and mannose in a molar ratio of 1:1.2.     

Optimization protocol for the extraction of antioxidant components from Origanum vulgare leaves using response surface methodology

In the present work, the response surface methodology (RSM) based on a central composite rotatable design (CCRD), was used to determine optimum conditions for the extraction of antioxidant compounds from Origanum vulgare leaves. Four process variables were evaluated at three levels (31 experimental designs): methanol (70%, 80%, and 90%), the solute:solvent ratio (1:5, 1:12.5, 1:20), the extraction time (4, 10, 16 h), and the solute particle size (20, 65, 110 micron). Using RSM, a quadratic polynomial equation was obtained by multiple regression analysis for predicting optimization of the extraction protocol. Analysis of variance (ANOVA) was applied and the significant effect of the factors and their interactions were tested at 95% confidence interval. The antioxidant extract (AE) yield was significantly influenced by solvent composition, solute to solvent ratio, and time. The maximum AE was obtained at methanol (70%), liquid solid ratio (20), time (16 h), and particle size (20 micron). Predicted values thus obtained were closer to the experimental value indicating suitability of the model. Run 25 (methanol:water 70:30; solute:solvent 1:20; extraction time 16 h and solute particle size 20) showed highest TP contents (18.75 mg/g of dry material, measured as gallic acid equivalents) and DPPH radical scavenging activity (IC₅₀ 5.04 μg/mL). Results of the present study indicated good correlation between TP contents and DPPH radical scavenging activity. Results of the study indicated that phenolic compounds are powerful scavengers of free radical as demonstrated by a good correlation between TP contents and DPPH radical scavenging activity.     

The study on microwave assisted enzymatic digestion of ginkgo protein

Microwave-assisted enzymatic digestion (MAED) technique was applied for ginkgo protein digestion with both free and immobilized enzyme. Under the optimized conditions of MAED (0.01 g/mL substrate concentration of bromelain, 4500 U/g enzyme/ginkgo protein, 30 min, 300 W microwave power), a higher digestion rate (7.50%) and a significant increase in antioxidant activity (72.7 mg/g) were obtained in contrast with the conventional methods. With the optimized digestion conditions (0.625% glutaraldehyde (v/v), 0.4 mg/mL initial concentration of bromelain and 4 h of immobilization), the activity and effectiveness factor of immobilized bromelain were respectively 86 U and 81.6%. The results of ginkgo digestion by applying MAED indicated that the digestion rate of immobilized bromelain obtained by MAED method (6.41%) was comparable to that of free bromelain in the conventional digestion (8.13%). In both case with immobilized and free bromelain while applying MAED, a homogeneously abundant distribution of peptide fragments (from 7.863 Da to 5856 Da) and a few different peptide profiles were found. This report brings in conclusion that applying MAED with immobilized enzyme has the potential to obtain the highest number of antioxidant activity peptides.     

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Background aimsAlloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use.MethodsWe describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells.ResultsNK cells (6.0 ± 1.2 × 10⁸) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR⁺ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML.ConclusionsThe approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.