Exchange Chromatography Research Articles

Dicationic imidazole ionic liquid stationary phase for preservative detection and its application under mixed mode of HILIC/RPLC/IEC

BackgroundFood safety has always been a great concern, and the detection of additives is vital to ensuring food safety. Therefore, there is a necessity to develop a method that can quickly and efficiently separate and detect additives in food. High performance liquid chromatography is widely used in the analysis and testing of food additives. Ionic liquids have attracted wide attention in the preparation of high performance liquid chromatography stationary phases owing to their high stability, low vapor pressure and adjustable structure. ResultsWe developed a novel dicationic imidazole ionic liquid stationary phase for the simultaneous determination of organic preservatives (sodium benzoate, potassium sorbate) and inorganic preservatives (nitrate and nitrite) in foodstuffs under mixed-mode chromatography. The method had the advantages of easy operation, high reproducibility, good linearity and precision. In the detection of these four preservatives, the limit of detection ≤0.4740 mg⋅L−1 and the limit of quantification ≤1.5800 mg⋅L−1. The intra-day and inter-day precision were less than 4.02%, and the recovery rate was 95.90∼100.19 %. At the same time, we also characterized the stationary phase, explored the mechanism and evaluated the chromatographic performance. The stationary phase was able to operate under the mixed mode of reversed phase/hydrophilic interaction/ion exchange chromatography, and it was capable of separating hydrophilic substances, hydrophobic substances, acids, and inorganic anionic substances with good separation efficiency and had high column efficiency. SignificanceIn summary, the stationary phase has a promising application in the routine analysis of organic and inorganic preservatives in food. In addition, the stationary phase has good separation ability for hydrophilic, hydrophobic, ionic substances and complex samples, making it a prospective material for chromatographic separation.

Preparation of two zwitterionic polymer functionalized stationary phases and comparative evaluation under mixed-mode of reversed phase/ hydrophilic interaction/ion exchange chromatography

Zwitterions are a promising choice to prepare separation materials because of their hydrophilicity and biocompatibility. We described the preparation of two zwitterionic polymer functionalized stationary phases and evaluation under mixed-mode chromatography. A zwitterionic monomer, S-(4-vinylbenzyl) cysteine (SVC), was synthesized and bonded to silica via reversible addition fragmentation chain transfer (RAFT) polymerization to afford a zwitterionic stationary phase, Sil-SVC. A hydrophobic monomer, N-(4-phenylbutan-2-yl) acrylamide (NPA), was copolymerized with SVC onto the stationary phase (Sil-SVCNPA) for comparison. The stationary phases were characterized with FT-IR, TGA, EA, and zeta-potential measurements. Mobile phase composition (ACN content, pH and salt concentration) was varied to study the retention property. Linear solvation energy relationship and Van't Hoff plot were used to investigate the retention mechanism and how chromatographic conditions influenced it. Both stationary phases showed a mixed-mode of RPLC/HILIC/IEC and satisfactory performance in separating hydrophobic analytes (alkylbenzenes and polycyclic aromatic hydrocarbons), hydrophilic nucleotide and bases, and anions, high column efficiency of 60,000 plates·m−1 was achieved. In summary, zwitterionic polymers are attractive options to prepare stationary phases and the retention property can be easily regulated by copolymer.

Efficient preparation of polyethylene glycol (PEG)-modified lignin from steam exploded hardwood biomass

The structures and characteristics of lignin derivatives prepared from steam exploded beech under alkali delignification and acid catalyzed polyethylene glycol (PEG) solvolysis were studied in detail. An efficient method for directly isolating PEG-modified lignin from wood was proposed to improve its thermal stability. The effects of steam explosion (SE) parameters and delignification process on lignin structure were studied by high performance anion exchange chromatograph, fourier transform infrared spectroscopy (FTIR), heteronuclear single quantum coherence (HSQC) NMR spectroscopies, ultraviolet and visible spectrophotometry, size exclusion chromatography (SEC) and thermogravimetric analysis (TGA). The results showed that SE pretreatment can greatly promote the yield of PEG-modified lignin. The PEG-modified lignin, isolated from steam exploded wood at 210 °C for 2.5 min, was ∼14% yield. The PEG moiety was located at the alpha carbon position of the β-O-4 linkage. The PEG-modified lignin exhibited improved thermal properties with a maximum weight loss temperature (Tdmax) greater than 380 ℃, and a degradation starting temperature (Tdst) in the range of 258–277 °C, met the temperature for polymer processing and contribute to improve the added value of lignin.

Purification and determination of antibody drugs in bio-samples by EGFR/cell membrane chromatography method

With the rapid development of therapeutic monoclonal antibody drugs, it is increasingly difficult to meet clinical needs using traditional antibody purification techniques. In this study, epidermal growth factor receptor (EGFR)-SNAP-tag was expressed in HEK293 cells. Then we captured the EGFR-SNAP-tag from the cell membrane suspension onto a O6-benzylguanine-modified silica gel to prepare a new EGFR stationary phase as a bioactive material, which could specifically recognize its antibody in bio-samples. The EGFR stationary phase was systematically characterized via scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and fourier transform infrared spectroscopy. Then we used EGFR stationary phase to establish a new EGFR cell membrane chromatography (CMC) model. The EGFR/CMC-online-ion exchange chromatography (IEC)/high performance liquid chromatography (HPLC) was established for the efficient purification and specific identification of cetuximab, nituzumab, and panizumab from cell culture medium and human serum. The results show that the EGFR stationary phase prepared by one-step immobilized technique can maintain biological activity and stability like EGFR in cell membrane. The EGFR/CMC-online-IEC/HPLC method has a high specificity, accuracy and sensitivity. Therefore, it will present a valuable method for the purification, identification, and analysis of monoclonal antibody drugs.

Antifungal activity of chitinase produced by Streptomyces species isolated from grassland soils in Futa Area, Akure

BackgroundThis study was carried out to investigate the antifungal activity of chitinase produced by Streptomyces species isolated from grassland soils against selected pathogenic fungi, namely: Magnaporthe oryzae, Fusarium graminearum, Rhizoctonia solani, Puccinia species and Botyrtis cinerea. Chitinase produced by Streptomyces albus was purified consecutively with dialysis, gel filtration and ion- exchange chromatography, the test isolate can be exploited for large scale production of chitinase.ResultsSeven Streptomyces species (Streptomyces rimosus, S. albus, S. riseoflavus, S. fumosus, S. spiralis, S. aureofaciens and S. flavogriseus) isolated from three different grassland soils were screened based on the morphology and their ability to produce chitinase. S. rimosus, S. fumosus, S. albus and S. flavogriseus were able to produce chitinase, although the enzyme chitinase was found to be higher in Streptomyces albus. The effect of environmental factors were determined on Streptomyces albus being the best chitinolytic producing isolate. Temperature of 30οC was favourable for Streptomyces albus and pH 6.0 was a growth factor for maximum chitinase production. The activity of the enzyme was stimulated by the addition of metal ions like Mn2+, K+, Na+, Mg+, Fe2+, Ca2+ but Zn2+ inhibited chitinase activity. Chitinase activity was favoured by maltose as carbon source and NH4 (SO4)2 as nitrogen source. Culture filtrate of Streptomyces albus was able to inhibit the growth of all the selected pathogenic fungi namely Magnaporthe oryzae, Fusarium graminearum, Rhizoctonia solani, Puccinia species and Botyrtis cinerea whereas Streptomyces rimosus has the least inhibitory activity.ConclusionsStreptomyces albus has the highest ability to produce chitinase which can act as antifungal for degradation of the cell wall of many phytopathogenic fungi.

Identification of arginine kinase as an allergen of brown crab, Callinectes bellicosus, and in silico analysis of IgE-binding epitopes

In recent years there has been an increase in the prevalence of allergic reactions to contact with/or consumption of crustaceans by immune responses mediated by IgE antibodies. Arginine kinase (AK) is considered one of the main allergens present in marine invertebrates. Currently, the allergenic potential of the brown crab (Callinectes bellicosus), which is a crustacean of great economic importance, has not been studied. Therefore, the aim of this work was to identify C. bellicosus AK as an allergen and to predict IgE-binding epitopes through immunobioinformatic analysis. AK was purified by precipitation with ammonium sulfate and ion- exchange chromatography. AK allergenicity was evaluated by IgE reactivity against sera from crustacean-allergic and non-allergic patients in both native and denaturing conditions. Additionally, a homology model was built based on the deduced amino acid sequence. A single band (~40 kDa) was found in SDS-PAGE, which was identified as an AK by mass spectrometry. AK showed immunoreactivity against crab-allergenic sera in both native and denaturing conditions with 70% and 80% positive reactions, respectively. Additionally, a 1073 bp ORF was obtained which codes for a deduced sequence of 357 amino acids corresponding to AK with > 90% identity with other AKs. Structural homology model of AK showed two main domains with conserved / folding of phospho-guanidine kinases. BediPred and Discotope were used for epitope prediction analysis, which suggests eight possible linear epitopes and seven conformational epitopes, respectively; and shows to be similar to other crustaceans AKs. C. bellicosus AK was identified as an allergenic protein by IgE reactivity and immunobioinformatic analysis indicates that both linear and conformational epitopes could be located in the surface of C. bellicosus AK structure.

Proteoglycan profiling of human, rat and mouse insulin-secreting cells.

Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography tandem mass spectrometry. We identified CS modifications of chromogranin-A (CgA), islet amyloid polypeptide, secretogranin-1 and secretogranin-2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (CgA and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For CgA, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.

Purification, characterization and fibrino(geno)lytic activity of cysteine protease from Tabernaemontana divaricata latex.

Protease was isolated and purified from Tabernaemontana divaricata latex and its hemostatic potential was analyzed. Crude latex enzyme was purified through ion exchange and gel filtration chromatography. Purified protease was characterized and its thrombin-like (coagulant assay, fibrinogen polymerizing, and fibrinogenolytic activity) and plasmin-like (blood and plasma clot lysis) activities were assessed accordingly. The homogeneous nature of protease was confirmed with the identification of a single band approximately at 25-kDa molecular weight position. The purified enzyme showed an enhancement of 77.32% in clot inducing ability and 89.86% improvement in blood clot lysis in comparison to that by the crude enzyme. All three subunits (Aα, Bβ and γ chains) of human fibrinogen were hydrolyzed by the purified enzyme. PAGE results of the fibrinolytic activity and blood clot lytic effect by the purified enzyme indicated the plasmin-like activity. The study lays a foundation for the development of enzyme-based approaches for pharmaceutical innovations, in which plant latex proteases can be utilized as a potential natural agent for wound healing applications.

Impact of some metal ions on activity of purified human enzyme paraoxonase (hPON1)

Inhibition of enzymes has a great influence on various metabolism activity in the body. The objective of this research has been to assess the impact of some metals and the drug statin on the activity of purified enzyme paraoxonase extracted from human sera (hPON1). The enzyme was purified in consecutive four steps using Ammonium Sulphate, Dialysis, Ion exchange and Gel filtration chromatography, respectively. These four purification steps have approved their credence in obtaining a maximum purification state of the enzyme. The inhibitory impact of some metals ions, i.e. Mg+2, K+1, Mn+2, Na+1, EDTA, and Zn+2, on the activity of purified hPON1 has also been assessed. The result showed the highest inhibitory impact of 10 mMol/L of Sodium Nitrate (NaNO3) on the activity of purified hPON1 while medication atorvastatin had significantly increased the activity of hPON1. It is concluded that any subtle change in the enzyme PON-1 could consequently lead to various diseases. More assessments are required for type two (PON-2) and type three (PON-3) to be explored. Atorvastatin is recommended to regulate cholesterol metabolism by the intestine and to lower the risk of diseases for elder ages.

Evaluation of cytotoxic potential of L-asparaginase from Scopulariopsis brevicaulis on cell lines in vitro

Introduction and Aim: This study reports the cytotoxic potential of L-Asparaginase isolated from the fungus Scopulariopsis brevicaulis.
 Materials and Methods: Extracellular L- Asparaginase was isolated from Scopulariopsis brevicaulis and purified by ammonium sulfate precipitation, followed by dialysis, ion exchange and gel filtration chromatography. Varying concentrations (31.25, 62.5, 125, 250, 500 µg/ml) of purified L-Asparaginase was tested on MCF7, HeLa, HepG2 and 3T3L1cell lines by MTT assay. Curcumin was maintained as a positive control.
 Results: Minimum inhibition of 23.57% was observed at an enzyme concentration of 31.25 µg/ml and maximum inhibition (66.41%) was observed at 500 µg/ml against MCF7 cell line. Minimum inhibition of 2.87% was observed at an enzyme concentration 31.25 µg/ml and maximum inhibition (58.49%) was observed at 500 µg/ml against HeLa cell line. Minimum inhibition of 4.58% was shown at an enzyme concentration of 31.25 µg/ml and maximum inhibition (46.14 %) was observed at 500 µg/ml against HepG2 cell line. Minimum inhibition of 1.4% was shown by enzyme concentration 31.25 µg/ml and maximum inhibition (50.9%) was observed at 500 µg/ml against 3T3L1 cell line.
 Conclusion: We report for the first time the cytotoxic potential of L-Asparaginase from Scopulariopsis brevicaulis.

Purification of Leucine aminopeptidase and its correlation with biochemical parameters in sera of subclinical hypothyroidism and hypothyroidism

The aim of this study purification of Leucine aminopeptidase (LAP) from hypothyroidism patients sera and its relation to thyroid hormones, lipid peroxidation levels of subclinical hypothyroidism and hypothyroidism patients, blood samples were collected from (50) healthy subject and (100) patients, consisted of fifty with hypothyroidism and fifty with subclinical hypothyroidism.. The purification is done by addition of ammonium sulfate, dialysis, anion- exchange chromatography and size-exclusion chromatography. FT3, FT4, TSH, LAP, peroxynitrite, malondialdehyde were determined. Results showed the precipitate and concentrated protein appeared five peaks in ion exchange column, LAP activity located in the first and second protein peak, also, results showed single peak for both first and second peaks after eluted in gel filtration chromatography following steps by using SDS-polyacrylamide gel electrophoresis. From current study, it is concluded hypothyroidism (LAP) have two isoenzymes, also, concluded a highly significant increase in LAP, MDA and peroxynitrite levels in the subclinical hypothyroidism and hypothyroidism patients when compared to the healthy subject, and their correlation with FT3, FT4, TSH in the patient group, when compared by the control group that indicate these parameters could be play an effect role in these diseases.

Production of Recombinant Allergens Phl p 1 and Amb a 1 for Detection of Class E Immunoglobulins

Recombinant major allergens Phl p 1 from meadow timothy (Phleum pratense) and Amb a 1 from ragweed (Ambrosia artemisiifolia) were obtained in E. coli cells. Allergen purification procedures were developed using anion exchange and affinity chromatography, from which we obtain 10 mg Phl p 1 and 7.5 mg Amb a 1 from 1 L of culture medium with an electrophoretic purity of at least 90%. To analyze the ability of IgE antibodies to bind Phl p 1 and Amb a 1, sera obtained from people with IgE to various respiratory allergens, identified by the RIDA method, were used. Part of the sera bound Phl p 1 or Amb a 1, which confirmed the immunoreactivity of the obtained recombinant proteins. Antibody titers to Amb a 1 correlated with RIDA data on sensitivity to a mixture of herbs and rye pollen. Antibody titers to Phl p 1 did not correlate with RIDA data. Of the 20 positive sera selected, 73 and 89% of the sera recognizing Phl p 1 or Amb a 1, respectively, had antibodies to the cross-reactive alder/birch/hazel allergen group.

Optimization of the Production Method for Recombinant Chymosin in the Methylotrophic Yeast Komagataella phaffii

An effective recombinant strain of Komagataella phaffii for Bos taurus prochymosin production was obtained. A method for the isolation and purification of recombinant chymosin that includes two-stage purification via ion exchange and hydrophobic chromatography has been developed. A highly purified (~90%) recombinant chymosin at a concentration of 4 mg/mL (1000 IMCU/mL) was obtained with a yield of 62%. The developed method can be used in industry.

Analytical Validation and Stability Indicating Studies for Simultaneous Estimation of Benidepine and Metoprolol by Strong Cation Exchange (SCX) Chromatography

Analytical Validation and Stability Indicating Studies for Simultaneous Estimation of Benidepine and Metoprolol by Strong Cation Exchange (SCX) Chromatography

Purification and Characterization of Alpha Toxin Clostridium perfringens Type A

Clostridium perfringens is a toxin-producing anaerobe with the ability to cause multiple diseases in humans and animals. One of the toxins produced by all strains of this bacterium is an alpha toxin (phospholipase C). This study aimed to purify and characterize alpha-toxin. C. perfringens type A was grown in an optimized medium, the culture supernatant was separated, and ammonium sulfate precipitated protein was subjected to ion exchange and gel filtration chromatography methods. The purification steps were monitored by SDS-PAGE and hemolysin activity for each sample and performed by the hemolysin assay method. The purified alpha-toxin appeared in a single band in 43 KDa. The enzymatic efficiency yield was about 88% with a specific activity of 69170 and 654 fold of purification. According to the results, alpha-toxin is Zn and Mg metal-enzyme dependent on the temperature and pH optimum of 40 °C and 6, respectively. The enzyme Km and Vmax values of 3.8 mg/ml and 0.19 were calculated as the enzyme in the presence of lecithin. The partially purified alpha toxin with specific characters can be used for different applications such as antibody production and determination of the anti-toxin rate in animal serum.

Purification and biochemical characterization of a novel secretory dipeptidyl peptidase IV from porcine serum.

Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtrationfollowed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160kDa. Distinct single band was observedafter PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37°C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC50 value of 8.473µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia, ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25µg.

Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain

Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZ? plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture.

Isolation and Purification of Lipoxygenase from the Serum of Bronchial Asthma Patient and Studying the Effect of Natural Products of Horse Tail ( Equisetum arvense.L) Plant on it's Activity

This study includes the isolation and purification of the Lipoxygenase (LOX) enzyme from the serum of an Asthma patient by using different techniques. The result showed the Ion – Exchange chromatography which occurred in the column containing the ion exchange (DEAE- cellulose) diethylamino ethyl – cellulose of the proteinous precipitateth which is produced after dialysis. Two bands of protein and the first band (A) showed the highest activity of the enzyme with the specific activity (2.68 U/mg) and the number times of the purification was (10.30) once. Then, the optimum conditions of LOX enzyme showed the highest activity which it was at the fifth minute at pH = 8 and at (40°C), substrate concentration was about (0.5 mmol/ L) and the maximum Velocity (Vmax) was (168 unit / L), the value of (Km) by using Mikael’s Menten and Linwifer – Burk plot was (0.13) and (0.14) molar respectively. In addition, the determining of the approximate molecular weight of LOX enzyme (the first protein band A) by electrical migration technology (Electrophoresis) using SDS-PAGE and molecular weight and it was (69000) Dalton. Also the study includes the isolation of th the natural products of the horse tail plant (oils) and identificated by GC (Gas-Liquid chromatography) and the flavonoids isolated and identificated by (HPLC) to study their effect on the activity of the partially purified enzyme, the results showed that oils contain an Undecanoic and Lauric acid, while flavonoids contain Catechine, Qurectine, Ferulic acid and Rutin, and it’s effect on the activity of partially purified enzyme showed that flavonoids reduced high-volume lipoxygenase enzyme and then sabonin followed by oils.

Constraints in Adoption ofModern Farm Machines by Tribal Farmers in Ramgarh District of Jharkhand

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) andamp; the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method andamp; it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column andamp; the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , andamp; one normal cell line Ref ( Rat embryonic fibroblast ). The cancer andamp; normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8andamp; 0.16 mg/ml) then incubated for additional 48h at 37C 0 andamp; the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andamp;slight toxicity ( 37.12% )on normal cell line (Ref) in a concentration (0.8mg/ml).

Cytotoxic Effect of Pseudomonas aurogenosa Protease on Cancer Cells

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) and the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method and it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column and the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , and one normal cell line Ref ( Rat embryonic fibroblast ) . The cancer and normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8and 0.16 mg/ml) then incubated for additional 48h at 37C0 and the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andslight toxicity ( 37.12% ) on normal cell line (Ref) in a concentration (0.8mg/ml).