phenyl-Sepharose CL-4B Research Articles

Camel hydatidosis diagnostic kit: optimization of turnip and horseradish peroxidase conjugates using glutaraldehyde method.

Echinococcosis/hydatidosis is one of the most important parasitic zoonotic diseases in the world. Cystic echinococcosis increases public health and socio-economic concern due to considerable morbidity rates that give rise to elevated economic losses both in the public health part and in the farm animal field. The enzyme linked immunosorbent assay (ELISA) is consider the more accurate tool for diagnosis of hydatidosis in camels. In the present study, affinity purified Echinococcus granulosus (E. granulosus) antigens (APA) were purified from crude hydatide E. granulosus germinal layer proteins for detection of E. granulosus antibodies in infected camels, using affinity matrix (camel IgGs coupled to CNBr-activated Sepharose). The electrophoretic profile of the APA showed that it was separated into two bands; one major band of 130kDa and one minor band at 55kDa. These antigens were used successfully as specific coating antigenic proteins in detection of echinococcosis in camel. In a trial to prepare an anti-camel IgGs peroxidase conjugate; peroxidase enzyme was purified from turnip roots (TPOD) using ammonium sulfate precipitation and affinity chromatography on phenyl Sepharose CL-4B. The purified TPOD showed a major band at 35kDa. Rabbit anti-camel IgG antibodies (AC IgGs) were prepared then purified using affinity chromatography on Protein G-Sepharose. The TPOD, and commercial HRP for comparison, enzymes were conjugated to AC IgGs using 1%, 5% and 10% glutaraldehyde. The results revealed that the HRP was much better than TPOD in conjugation with AC-IgG antibodies and the 10% glutaraldehyde concentration was the most efficient concentration with ELISA titer 1:50.

Purification and Product Characterization of Lipoxygenase from Opium Poppy Cultures (Papaver somniferum L.).

Opium poppy (Papaver somniferum L.) is an ancient medicinal plant producing pharmaceutically important benzylisoquinoline alkaloids. In the present work we focused on the study of enzyme lipoxygenase (LOX, EC 1.13.11.12) from opium poppy cultures. LOX is involved in lipid peroxidation and lipoxygenase oxidation products of polyunsaturated fatty acids have a significant role in regulation of growth, development and plant defense responses to biotic or abiotic stress. The purpose of this study was to isolate and characterize LOX enzyme from opium poppy callus cultures. LOX was purified by ammonium sulfate precipitation and then followed by hydrophobic chromatography using Phenyl-Sepharose CL-4B and hydroxyapatite chromatography using HA Ultrogel sorbent. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting revealed that LOX from opium poppy cultures was a single monomeric protein showing the relative molecular weight of 83 kDa. To investigate the positional specificity of the LOX reaction, purified LOX was incubated with linoleic acid and the products were analyzed by high-performance liquid chromatography in two steps, firstly with reverse phase (120-5 Nucleosil C18 column) and secondly with normal phase (Zorbax Rx-SIL column). LOX converted linoleic acid primarily to 13-hydroperoxy-(9Z,11E)-octadecadienoic acids (78%) and to a lesser extent 9-hydroperoxy-(10E,12Z)-octadecadienoic acids (22%). Characterization of LOX from opium poppy cultures provided valuable information in understanding LOX involvement in regulation of signaling pathways leading to biosynthesis of secondary metabolites with significant biological activity.

Purification and characterization of an esterase from larval Diplolepis fructuum (Rübsaamen, 1895) (Hymenoptera: Cynipidae)

Diplolepis fructuum (Rübsaamen, 1895) (Hymenoptera: Cynipidae) is one of the important insect species that causes damages on Rosaceae species. With this study commenced in 2018 at the laboratory of Department of Biochemistry, Faculty of Science, Cumhuriyet University to get a biochemical data, an esterase (EC 3.1.1.X) from the larvae of D. fructuum was purified using Q Sepharose anion exchange, phenyl Sepharose CL-4B and Sephacryl S100 HR gel filtration chromatography, respectively. The enzyme had 6.94 U/mg protein specific activity, about 29-fold purity, and 8.8% yield. Only one activity band was observed in native-PAGE studies. The molecular weight of the esterase was estimated as 60 kDa using native-PAGE and SDS-PAGE techniques. By the kinetic data, optimum temperature and pH for the enzyme was determined as 40ºC and 9.0, respectively. The enzyme was stable for 4 h at 40ºC and pH 8.0. K m and V max values were found to be 0.035 mM and 1.41 µmol/mL.min., using 4-nitrophenyl butyrate (p-NPB) as substrate. The enzyme exhibited its highest activities on p-NPB (100%) and 4-nitrophenyl acetate (52%). All of these data indicate that the enzyme might be a typical esterase with different kinetic properties and molecular weight than esterolytic enzymes reported from other insect species.

Purification and characterization of exo-β-1,4-glucosaminidase produced by chitosan-degrading fungus, Penicillium sp. IB-37-2A.

Chitosan-degrading fungal strain, Penicillium sp. IB-37-2A, produced mainly extracellular chitosanolytic enzymes under submerged agitating cultivation in presence of soluble chitosan or colloidal chitin as main carbon source. Significant N-acetyl-β-D-glucosaminidase activity (8-18 × 103 U·ml-1) was also detected in culture filtrate of the fungal strain. Alone major exo-chitosanase from culture filtrate of Penicillium sp. IB-37-2A was purified in 46-fold using ultrafiltration, affinity sorption on colloidal chitosan and hydrophobic chromatography on Phenyl-Sepharose CL 4B and characterized. Molecular weight of the exo-β-1.4-glucosaminidase is 41kDa according to SDS-PAGE. The purified enzyme has optima pH and temperature 4.0 and 50-55°C, respectively, pI 4.9; it is stable under pH 3.0-8.0 and 55°C. Activity of the enzyme is strongly inhibited by 1mM Hg2+ and Ag+, in less degree-10mM Cu2+, Zn2+, Ni+ and Fe2+, slightly activated-with 1mM Mg2+, 10mM Ca2+, tween-80 (10mM) and Triton X-100 (1mM). Viscosimetric assay confirmed reported earlier exo-splitting manner of the enzyme activity. Soluble chitosan (deacetylation degree (DD) 80-85%) is most rapidly hydrolyzed by the enzyme (Vmax = 7.635µM × min-1 × mg-1, KM ~ 0.83mg/ml). Purified exo-chitosanase also degraded laminarin, β-glucan, colloidal chitin and showed significant chitobiohydrolase activity (V ~ 50µM × ml-1 × min-1 for pNP-GlcNAc2) but no hydrolyzed CMC, cellulose, xylan and galactomannan. It is found that crude and partially purified exo-β-1.4-glucosaminidase inhibits in vitro the growth of some phytopathogenic fungi that is first report for antifungal activity of exo-chitosanase.

HPLC Analysis and Biochemical Characterization of LOX from Eschscholtzia californica Cham.

Background: Plant lipoxygenases (LOXs, EC 1.13.11.12) are involved in lipid degradation, regulation of growth and development, senescence, and defence reactions. LOX represents the starting enzyme of the octadecanoid pathway. The aim of the work was to purify LOX from California poppy (Eschscholtzia californica Cham.), to determine its biochemical properties and to identify and quantify the products of LOX reaction with unsaturated fatty acids. Methods: LOX from California poppy seedlings was purified by hydrophobic chromatography (Phenyl-Sepharose CL-4B) and by ion-exchange chromatography (Q-Sepharose). The isolated LOX was incubated with linoleic acid used as a substrate. The HPLC experiments were performed with the Agilent Technologies 1050 series HPLC system. For the preparative separation of a mixture of hydroxy fatty acids from the sample matrix, the RP-HPLC method was used (column 120-5 Nucleosil C18). Then, the NP-HPLC analysis (separation, identification, and determination) of hydroxy fatty acid isomers was carried out on a Zorbax Rx-SIL column. Results: The purified LOX indicates the presence of a nontraditional plant enzyme with dual positional specificity (a ratio of 9- and 13-hydroperoxide products 1:1), a relative molecular mass of 85 kDa, a pH optimum of 6.5, an increasing activity stimulation by CaCl2 till 2 mM, and a high substrate reactivity to linoleic acid with kinetic values of KM 2.6 mM and Vmax 3.14 μM/min/mg. Conclusions: For the first time, the LOX from California poppy seedlings was partially purified and the biochemical properties of the enzyme were analyzed. A dual positional specificity of the LOX found from California poppy seedlings is in agreement with the results obtained for LOXs isolated from other Papaveraceaes. A 1:1 ratio of 9-/13-HODE is attractive for the simultaneous investigation of both biotic stress responses (indicated by the 9-HODE marker) and the biosynthesis of jasmonic acid and jasmonates (indicated by the 13-HODE marker).

Purification, characterization and partial primary structure analysis of rutin-degrading enzyme in tartary buckwheat seeds

Rutin-degrading enzymes (RDE) can degrade rutin into poorly water soluble compound, quercetin, and cause the bitter taste in tartary buckwheat. In the present study RDE from Yu 6-21 tartary buckwheat seeds was purified by ammonium sulphate precipitation, followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion exchange chromatography on CM-Cellulose and gel filtration chromatography on Sephadex G-150. Purified RDE showed single band with molecular weight of 66 kDa on SDS-PAGE. The optimum pH and temperature of RDE were 5.0 and 50 ℃ respectively. The Km was 0.27 mmol/L, and the Vmax was 39.68 U/mg. The RDE activity could be inhibited by Cu²⁺, Zn²⁺, Mn²⁺ and EDTA, and showed tolerance to 50% methanol (V/V). The N terminal sequence (TVSRSSFPDGFLFGL) was obtained by Edman degradation method and 15 internal peptide sequences were determined by MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). These results established the foundations for identification of the candidate gene of RDE via transcriptome data and further studying RDE biological function.

Partial Purification and Characterization of Cellulase Produced by <i>Bacillus sphaericus</i> CE-3

Cellulase is an enzyme produced by fungi, bacteria, protozoa and termite, that hydrolyze cellulose. They are known for their diverse applications in industry and medicine. The aim of this study is to purify and investigate cellulolytic properties of cellulase enzyme produced by Bacillus sphaericus CE-3 isolated from refuse dump in Nnamdi Azikiwe University, Awka, Nigeria. Enzyme was produced by submerged fermentation at 30°C for 30 h. The enzyme was purified to homogeneity by dialysis in 4M sucrose solution, ion-exchange chromatography on Q-Sepharose FF and by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The relative molecular mass of the enzyme was estimated using SDS-Polyacrylamide gel electrophoresis. Effects of temperature, pH and metals on enzyme activity and stability and the relative rate of hydrolysis of various substrates were also studied. The Purification fold for the enzyme was 7.8, with 66.4 μ/mg specific activity protein and overall yield of 35.8. The relative molecular mass range of the enzyme was estimated between 22.3 kDa - 26.3 kDa. The enzyme was optimally active at pH 9.0 and 40°C, stable at pH 9.0 and unusually retained over 90% activity between 50°C - 100°C after 30 min incubation. It was strongly activated by Mn2+ but inhibited by Ba2+, Co2+, Hg2+, Pb2+, Cu2+, Sr2+, Fe2+, Ca2+ and Zn2+. The cellulase displayed high catalytic activity with untreated sawdust, followed by carboxymethyl cellulose, while sodium hydroxide treated sawdust was the least hydrolyzed. Since the enzyme is thermo-stable, alkalophilic and could utilize natural wastes like sawdust as substrate, it is obvious that it would be of great use in textile, starch processing and pulp and paper industries.

Purification and properties of lipoxygenase from opium poppy seedlings(Papaver somniferum L.)

Lipoxygenase (linoleate:oxygen oxidoreductase; EC 1.13.11.12; LOX) is a key enzyme in the signaling pathway leading to the production of biologically active compounds involved in plant growth development and stress responses. In this study the novel LOX enzyme from opium poppy (Papaver somniferum L.) seedlings was purified to electrophoretic homogeneity and characterized. LOX activity during the germination of opium poppy seeds was analyzed, and the highest LOX activity was determined on the fourth day of germination. Opium poppy LOX was purified from 4-day-old poppy seedlings after ammonium sulfate precipitation followed by hydrophobic chromatography (Phenyl-Sepharose CL-4B), ion exchange chromatography (Q-Sepharose), and affinity chromatography (linoleyl-aminopropyl agarose) for the first time. The relative molecular weight of purified LOX was estimated to be 78 kDa by immunoblotting. The highest enzyme activity occurred at pH 6.5 and 0.75 mM calcium ion concentration. LOX showed preferential activity towards linoleic acid followed by linolenic acid as a substrate. To investigate the positional specificity of the LOX reaction, purified LOX was incubated with linoleic acid, and the products were analyzed by high-performance liquid chromatography. Opium poppy LOX converted linoleic acid to 13-hydroperoxy-(9Z,11E)-octadecadienoic acid (63.5%) and, to a lesser extent, 9-hydroperoxy-(10E,12Z)-octadecadienoic acid (36.5%).

English

Phenoloxidase (PO) is a key factor in insect immunity. On invasion of microorganisms and pathogens, prophenoloxidase (PPO) changes to its active form, PO. The present study has been conducted to purify and characterize the PO from the haemolymph of desert locust, Schistocerca gregaria (Forskal) following activation of immune system by invasion of bacteria, Bacillus thuringiensis kurstaki (Bt). PO is purified by a combination of ammonium sulfate precipitation, blue sepharose CL-6B and phenyl sepharose CL-4B chromatography yielded a 209.97-fold purity and 54.75% recovery of activity. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) reveals that the molecular weight of the purified PO is 70.154 kDa. The purified PO is characterized in terms of its biochemical and enzymatic properties by using L-DOPA as a specific substrate. Ca2+ and Cu2+ significantly stimulated PO activity when compared with other metals. The PO reaction was strongly inhibited by phenylthiourea and thiourea, moderately inhibited by ethylene diamine tetractic acid (EDTA) and poorly inhibited by ethylene glycol tetraacetic acid (EGTA) and diethyl dithiocarbamate (DTC). Inhibition of PO showed excellent recovery ability by addition of Ca2+ on EGTA-inhibited enzyme. Therefore, PO is most probably a kind of tyrosinase-type Ca2+-containing metalloenzyme. The content of Ca2+ is higher than other trace metal elements. The reactive intermediates yielded by PO with its specific substrate L-DOPA had a broad-spectrum bactericidal activity against Gram +ve bacteria (Bacillus cereus and Staphylococcus aureus) with a greater degree more than Gram-ve bacteria (Escherichia coli and Pseudomonas aeruginosa). From the present study, PO from S. gregaria is most probably a tyrosinase-type calcium-containing mono-phenoloxidase, which functions not only as a catalytic enzyme in melanin production in locusts, but perhaps also as a humoral factor in host defense via melaninization as in other insects. Key words: Schistocerca gregaria, phenoloxidase, purification.

Purification, characteristics and identification of chitinases synthesized by the bacterium Serratia plymuthica MP44 antagonistic against phytopathogenic fungi

Once isolated from the rhizosphere, the chitinolytic bacterium Serratia plymuthica MP44 showed strong antagonistic action against such phytopathogenic fungal species as: Rhizoctonia solani, Cladosporium sp., Alternaria alternata and, to a lesser degree, to the species from the genera Fusarium and Chaetomium. In contrast, no antagonism between the bacteria and Aspergillus niger was observed. S. plymuthica MP44 demonstrated the highest chitinolytic activity on the fifth day of the cultivation on a medium with 1% colloidal chitin. The analysis of zymograms indicated that the studied bacterial strain synthesized 2 chitinolytic enzymes. After purification of culture supernatant of S. plymuthica MP44 with ammonium sulphate precipitation and hydrophobic chromatography on Phenyl-Sepharose CL-4B, 2 active fractions were obtained: fraction I (FI) containing exochitinase activity and fraction II (FII) containing endochitinase activity. The optimum pH and temperature for the purified enzyme were 6.3 and 45°C and 5.4 and 50°C for FI and FII, respectively. High thermal stability was revealed for enzyme from FII after 3 h of incubation at 50°C. The purified chitinases displayed low fungistatic activity when compared to the activity of the bacteria per se.

Purification and Characterization of Peroxidase From Anthracnose Disease Infected Papaya (Carica papaya L.)

Peroxidase enzyme was isolated and purified from the pulp of disease infected ripen papaya of local variety by 90% ammonium sulphate precipitation, chromatography on DEAEcellulose followed by hydrophobic chromatography on Phenyl Sepharose CL-4B and the purifications achieved was about 7.2 fold with 2.5% recovery. The purified enzyme was homogeneous as judged by polyacrylamide slab gel electrophoresis. The purified enzyme had a Mr of about 55,000 and 50 000 as determined by gel filtration on Sephadex G-100 and SDS-PAGE, respectively. The molecular mass of the enzyme was found to be very similar under both reducing and non-reducing conditions indicating that the enzyme contains no subunit. The enzyme has the following characteristics: pH optima at 6.0, temperature optima around 38°C, enzyme activity was found to be strongly inhibited in the presence of potassium cyanide and Fe+2 while the activity was found to be remarkably increased in the presence of ammonium sulphate. The Km value for the peroxidase obtained with pyrogallol as substrate was 0.027 mM. DOI: http://dx.doi.org/10.3329/bjmb.v6i2.17643 Bangladesh J Med Biochem 2013; 6(2): 49-57

A New in Vitro Anti-Tumor Polypeptide Isolated from Arca inflata

A new in vitro anti-tumor polypeptide, coded as J2-C3, was isolated from Arca inflata Reeve and purified by diethyl-aminoethanol (DEAE)-sepharose Fast Flow anion exchange and phenyl sepharose CL-4B hydrophobic chromatography. J2-C3 was identified to be a homogeneous compound by native polyacrylamide gel electrophoresis (Native-PAGE). The purity of J2-C3 was over 99% in reversed phase-high performance liquid chromatography (RP-HPLC). The molecular weight was determined as 20,538.0 Da by electrospray-ionization mass spectrometry (ESI-MS/MS). J2-C3 was rich in Glx (Gln + Glu), Lys, and Asx (Asp + Asn) according to amino acid analysis. Four partial amino acid sequences of this peptide were determined as L/ISMEDVEESR, KNGMHSI/LDVNHDGR, AMKI/LI/LNPKKGI/LVPR and AMGAHKPPKGNEL/IGHR via MALDI-TOF/TOF-MS and de novo sequencing. Secondary structural analysis by CD spectroscopy revealed that J2-C3 had the α-helix (45.2%), β-sheet (2.9%), β-turn (26.0%) and random coil (25.9%). The anti-tumor effect of J2-C3 against human tumor cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the IC50 values of J2-C3 were 65.57, 93.33 and 122.95 µg/mL against A549, HT-29 and HepG2 cell lines, respectively. Therefore, J2-C3 might be developed as a potential anti-tumor agent.

Purification and Characterization of Polyphenol Oxidase From Agaricus bisporus

A polyphenol oxidase was isolated from Agaricus bisporus using ammonium sulfate precipitation, combined with DEAE–Sepharose Fast Flow chromatography and Phenyl Sepharose CL-4B chromatography. The molecular weight of the purified polyphenol oxidase was determined to be about 43 kDa with N-terminal sequence Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation. The optimum temperature and pH for the polyphenol oxidase activity was 20°C and a pH of 6.5–7.0. Moreover, it was stable at 30 and 40°C over a 60 min pre-incubation time period. The Km and Vmax values of this polyphenol oxidase for catechol were 0.67 mM and 3333 U/ml min−1, respectively. In addition, following the cross linking with polyphenol oxidase, the emulsifying activity index, foam capacity, and foam stability of the whey proteins were evaluated to be modified. This work was useful to better understand polyphenol oxidase from A. bisporus, and may lead to its practical use in the future.

Characterization and evaluation of Staphylococcus sp. strain LZ16 for the biological control of rice blast caused by Magnaporthe oryzae

Staphylococcus sp. strain LZ16 was isolated from seawater collected in the East China Sea. Both culture filtrate and cell lysate of LZ16 possessed strong growth inhibition activities against Magnaporthe oryzae. Morphological observations revealed that conidial germination, germ tube elongation, appressorium formation were significantly inhibited after the treatment with cell lysate of LZ16. The active substances remained stable at temperature from 7 to 45°C, and pH from 6 to 8. One of the fractions conferring strong fungistatic activities was separated using Phenyl-Sepharose CL-4B and Sephacryl S-200 High resolution columns, and identified as purine nucleoside phosphorylase (PNP) by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry. The gene deoD encoding PNP was cloned and expressed in Escherichia coli for the verification of the antagonistic activity against M. oryzae. This is the first report that PNP was associated with fungistatic activity. Field experiments showed that the extract of the bacterial culture prevented and alleviated the disease severity of M. oryzae infection compared to the level in the untreated control. The acute toxicity study revealed the lethal oral dose was more than 5000mg/kg, and the adverse effect was not observed on both male and female rats after treatment for 14days. All the results suggested that strain LZ16 and its bioactive substances might be promising candidates for biopesticide development, or transgenetic plant materials for the biocontrol of rice blast.

Biochemical and kinetic characterization of the digestive trypsin-like activity of the lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

The digestive trypsin-like activity of the lesser grain borer Rhyzopertha dominica was characterized in some of its biochemical and kinetic properties. The enzyme activity from insect midguts was isolated using hydrophobic interaction chromatography with phenyl-sepharose CL-4B. Eight bands (identified from A through H) with caseinolytic activity and molecular weights in the range of 22–51.3 kDa were detected by zymography in casein-polyacrylamide gels. The strongest bands were D, G, and H, and showed estimated molecular weights of 33.6, 25.4, and 22 kDa, respectively. In-gel inhibition of caseinolytic activity showed that the serine protease inhibitors TLCK and SBTI inhibited all the proteases, except E. In-vitro inhibitory assays showed that SBTI and TLCK suppressed the BApNAase activity by 92.3% and 79.2%, respectively, indicating the presence of serine proteases. Wheat hexaploid albumin extracts were highly effective in inhibiting all the proteolytic activity. The chymotrypsin inhibitor TPCK did not affect the BApNAase activity, indicating that the proteolytic activity in R. dominica belongs to the trypsin-like type. With BApNA as the substrate, the proteolytic activity was high across a broad pH range of 6–11 with two peaks of maximum activity at pH 8 and 10 with an optimum temperature of 50 °C. SBTI inhibited the BApNAase activity with IC50 and Ki values of 0.02 μM and 1.17 × 10−8 M, respectively. The kinetic constants Km and Vmax were 0.07 mM and 2.8 mM/min, respectively. The activation energy (Ea) for BApNA hydrolysis was 33.5 kJ/mol. The results of this study confirm that R. dominica rely on serine protease activity for food digestion.

Screening of Postharvest Agricultural Wastes as Alternative Sources of Peroxidases: Characterization and Kinetics of a Novel Peroxidase from Lentil (Lens culinaris L.) Stubble

Aqueous crude extracts of a series of plant wastes (agricultural, wild plants, residues from sports activities (grass), ornamental residues (gardens)) from 17 different plant species representative of the typical biodiversity of the Iberian peninsula were investigated as new sources of peroxidases (EC 1.11.1.7). Of these, lentil (Lens culinaris L.) stubble crude extract was seen to provide one of the highest specific peroxidase activities, catalyzing the oxidation of guaiacol in the presence of hydrogen peroxide to tetraguaiacol, and was used for further studies. For the optimum extraction conditions found, the peroxidase activity in this crude extract (110 U mL(-1)) did not vary for at least 15 months when stored at 4 °C (k(inact) = 0.146 year(-1), t(1/2 inact) = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL(-1)) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k(inact) = 2.2 × 10(-3) day(-1), t(1/2 inact) = 315 days). Using guaiacol as an H donor and a universal buffer (see above), all crude extract samples exhibited the highest peroxidase activity in the pH range between 4 and 7. Once semipurified by passing the crude extract through hydrophobic chromatography on phenyl-Sepharose CL-4B, the novel peroxidase (LSP) was characterized as having a purity number (RZ) of 2.5 and three SDS-PAGE electrophoretic bands corresponding to molecular masses of 52, 35, and 18 kDa. The steady-state kinetic study carried out on the H(2)O(2)-mediated oxidation of guaiacol by the catalytic action of this partially purified peroxidase pointed to apparent Michaelian kinetic behavior (K(m)(appH(2)O(2)) = 1.87 mM; V(max)(appH(2)O(2)) = 6.4 mM min(-1); K(m)(app guaicol) = 32 mM; V(max)(app guaicol) = 9.1 mM min(-1)), compatible with the two-substrate ping-pong mechanism generally accepted for peroxidases. Finally, after the effectiveness of the crude extracts of LSP in oxidizing and removing from solution a series of last-generation dyes present in effluents from textile industries (1) had been checked, a steady-state kinetic study of the H(2)O(2)-mediated oxidation and decolorization of Green Domalan BL by the catalytic action of the lentil stubble extract was carried out, with the observation of the same apparent Michaelian kinetic behavior (K(m)(appGD) = 471 μM; V(max)(appGD)= 23 μM min(-1)). Further studies are currently under way to address the application of this LSP crude extract for the clinical and biochemical analysis of biomarkers.

Purification and characterization of a new alkali-thermostable lipase from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere

An extracellular lipase (EC 3.1.1.3), SAL-PP1, from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere was purified and characterized. The enzyme was purified using PALL'S Microsep centrifugal device (10kD cut off), hydrophobic interaction (phenyl sepharose CL-4B column) and Superose-12 gel filtration chromatography and found to have a molecular mass of around 49kDa. The gene fragment encoding the part of the catalytic site of the SAL-PP1 lipase was sequenced and the deduced amino acid sequence shows 93% identity with that of SEL3. SAL-PP1 showed activity against long acyl-chain triglycerides, various p-nitrophenyl esters and phospholipids. The enzyme shows high stability and activity after incubation with various metal ions (retained >90% activity in presence of Ca2+, Na+, Cu2+, Mg2+, Fe2+, or Hg2+ at 10mM), organic solvents (retained >80% activity in presence of acetonitrile, ethanol, DMSO, methanol, isopropanol, toluene, or ethylene glycol at 10mM), detergents (retained >70% activity in Triton X-100, Tween 80, or sodium deoxycholate at 10mM) and irreversible inhibitors (retained >77% activity in presence of PMSF, leupetin, or β-mercaptoethanol, at 1mM). Thermal inactivation studies revealed a temperature dependent unfolding of secondary structure of protein. SAL-PP1 showed maximal activity and stability at pH 8.0 and pH 9.0, respectively. The alkali-thermostability, organic solvent-tolerance and broad substrate specificity of this enzyme may have potential implications in detergent formulations, biotransformation, industries, and medicine.

Purification and characterisation of an acid protease from Aspergillus carbonarius

The aim of this study is to purify and characterize an acid protease from Aspergillus carbonarius. The protease was purified to apparent homogeneity by 4 M sucrose concentration, ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl Sepharose CL-4B and gel filtration chromatography through Sephadex G-100. A 10-fold purification with specific activity of 485.47 Umg-1 protein was achieved. SDS-PAGE and zymogram analysis of the protease indicated an estimated molecular mass of 72 kDa. The optimum temperature and pH for the proteolytic activity were 40°C and pH 3.0, respectively. The enzyme was active over a wide range of temperature from 40 to 80°C. The enzyme retained about 70% of its original activity (40°C) at 90 and 100°C. The enzyme was most stable at pH of 3.0 and temperature of 50°C. The enzyme has a relatively broad pH range of 4.0 to 10.0 after 30 min. The enzyme was slightly significantly (P < 0.001) stimulated by K+, Ba2+ while Ca2+ and Zn2+ moderately enhanced it. Mn2+, Fe2+ and Cu2+ strongly significantly (p<0.001) enhanced the enzyme activity while it was significantly (p<0.001) inhibited by Hg2+. The enzyme was not affected by Sr2+ and Mg2+ but Co2+ and Na+ elicited slight repression of the enzyme activity. The protease was strongly significantly (p < 0.001) inhibited by E-64, IAA and DIMSO while EDTA and 2-ME appreciably enhanced the activity of the protease. The enzyme is a cysteine protease as indicated by its inhibition studies. The protease may find potential applications in food and brewing industries as well as in meat tenderization. Key words: Aspergillus carbonarius, protease, purification, characterization, kinetic properties.

Purification and characterization of novel extracellular cholesterol esterase from Acinetobacter sp.

CHE4-1, a bacterial strain that belongs to the genus Acinetobacter and expresses high level of inducible extracellular cholesterol esterase (CHE), was isolated from feces of carnivore Panthera pardus var. The cholesterol esterase of the strain CHE4-1 was purified by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography, and then by Sephadex G-50 gel filtration. Different from other known microbial cholesterol esterase, the purified CHE from CHE4-1 strain is a monomer with molecular weight of 6.5 kD and has high activity to both long-chain and short-chain cholesterol ester. Enzymatic activity was enhanced in the presence of metal ion Ca(2+), Zn(2+) and boracic acid, and was not significantly affected by several detergents including sodium cholate, Triton X100 and Tween-80. The enzyme was found to be stable during long-term aqueous storage at 4 °C, indicating its potential as a clinical diagnostic reagent. To the best of our knowledge, this is the first report regarding purification and characterization of CHE from Acinetobacter sp. The results demonstrated that this particular CHE is a novel cholesterol esterase.

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Peroxidase (EC 1.11.1.7) from horseradish ( Armoracia rusticana ) roots was purified using a simple, rapid, three-step procedure: ultrasonication, ammonium sulfate salt precipitation, and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The preparation gave an overall yield of 71%, 291-fold purification, and a high specific activity of 772 U mg(-1) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme was homogeneous and had a molecular weight of approximately 40 kDa. The isolated enzyme had an isoelectric point of 8.8 and a Reinheitszahl value of 3.39 and was stable when stored in the presence of glycerol at -20 degrees C, with >95% retention of original enzyme activity for at least 6 months. Maximal activity of purified horseradish peroxidase (HRP) was obtained under different optimized conditions: substrate (guaiacol and H(2)O(2)) concentrations (0.5 and 0.3 mM, respectively), type of buffer (50 mM phosphate buffer), pH (7.0), time (1.0 min), and temperature of incubation (30 degrees C). In addition, the effect of HRP and H(2)O(2) in a neutral-buffered aqueous solution for the oxidation of phenol and 2-chlorophenol substrates was also studied. Different conditions including concentrations of phenol/2-chlorophenol, H(2)O(2), and enzyme, time, pH, and temperature were standardized for the maximal activity of HRP with these substrates; under these optimal conditions 89.6 and 91.4% oxidations of phenol and 2-chlorophenol were obtained, respectively. The data generated from this work could have direct implications in studies on the commercial production of this biotechnologically important enzyme and its stability in different media.