octyl-Sepharose Column Chromatography Research Articles

A Purified Alkaline and Detergent-Tolerant Lipase from Aspergillus fumigatus with Potential Application in Removal of Mustard Oil Stains from Cotton Fabric

Abstract In the present study, the lipase from Aspergillus fumigatus was purified which was found to be stable to commercial detergents and oxidising agents. A purification fold of 6.96 and yield of 11.03% were achieved when the enzyme was purified using Octyl Sepharose column chromatography. In presence of various oxidizing agents, the highest activity of lipase was 15.56 U/mg with hydrogen peroxide. Among various surfactants used, the maximum activity exhibited by lipase was with Tween 80. While studying the effect of various detergents, the highest activity of 9.3 U/mg was achieved with "Vanish" detergent. Wash performance was studied with various detergents out of which "Vanish" showed highest oil removal of 79%. Lipase from Aspergillus fumigatus possessed better stability with various surfactants and oxidizing agents. The results of this study have shown that the lipase from Aspergillus fumigatus along with detergent "Vanish" (0.7%) under optimized conditions (5 μg/ml lipase, 40°C wash temperature and 40 min wash duration) improved oil removal from cotton fabric stained with mustard oil by 84%.

Synthesis of Methyl Butyrate Catalyzed by Lipase from Aspergillus fumigatus.

Lipase is a potential biocatalyst and can be exploited for various applications such as food, pharmaceutical, oleochemistry, organic chemistry, biofuels and in detergent industries. In the present study, lipase from Aspergillus fumigatus was purified to homogeneity by SDS and Native PAGE and evaluated as biocatalyst for the synthesis of methyl butyrate which is a flavor ester. A purification fold of 6.96 was achieved by using Octyl Sepharose column chromatography. Methyl butyrate was synthesized by trans-esterification of vinyl butyrate with methanol, in a medium containing n-hexane as a solvent. The molar ratio of 2:2 (vinyl butyrate:methanol) was found to be optimum for the synthesis of methyl butyrate. The yield of methyl butyrate was maximum when reactants were incubated for 16 h at an incubation temperature of 40°C. The maximum yield (86%) of ester was obtained with 30 µg/ml of purified lipase.

Purification of lipase from Aspergillus fumigatus using Octyl Sepharose column chromatography and its characterization.

Lipases are ubiquitous biological macromolecules which have many industrial and environmental applications. The purpose of this study was to purify lipase from Aspergillus fumigatus by using Octyl Sepharose column chromatography. The enzyme was purified by ammonium sulfate precipitation and hydrophobic interaction chromatography which resulted in sevenfold purification. The molecular weight of protein using Native-PAGE was found to be 70 kDa. The apparent molecular weight by SDS-PAGE was found to be 35 kDa which indicated that the enzyme was homodimer. The optimum temperature and pH for activity of the enzyme was found to be 40 °C and 9.0, respectively. The kinetic parameters Vmax and Km of the purified lipase were 10.42 µmol min-1 mg-1 and 9.89 mM, respectively. Detergents Tween-20 and Triton X-100 inhibited enzyme activity. However, there was minimal loss of enzyme activity with SDS and Tween-80. All metal ions inhibited the enzyme activity. Among solvents, maximum loss (65%) in the enzyme activity was in hexane.

Characterization of a Low Molecular Weight Glycolipid Antigen from Cryptosporidium parvum

Cryptosporidium parvum, an Apicomplexan parasite of the mammalian gut epithelium, causes a diarrheal illness in a wide range of hosts and is transmitted by contamination of food or water with oocyst-laden feces from an infected animal. We have identified a glycosylinositol phospholipid from the sporozoite stage of the parasite that is frequently recognized by serum antibodies from human cryptosporidiosis patients. The humoral immune response is dominated by IgG1 subclass antibodies but can also include IgA and IgM antibodies. The glycosylinositol phospholipids were purified by butanol extraction of a Triton X-114-soluble fraction followed by octyl-Sepharose column chromatography and preparative high performance TLC and were shown to include at least 5 species. By using mass spectrometry and radiolabeled neutral glycan analysis, we found that the structure of the dominant glycosylinositol phospholipid antigen contained a C18:0 lyso-acylglycerol, a C16:0-acylated inositol, and an unsubstituted mannose3-glucosamine glycan core. Other diacyl species were also identified, most notably a series of glycosylinositol phospholipids having an acyl-linked C20:0 to C28:0 lipid on the inositol ring. Less abundant species having three acyl-linked fatty acids and species with an additional 1-3 hexoses linked to the mannose core were also observed. We are currently working to determine the role that these glycolipids may play in the development of disease and in the clearance of infection.

Production, purification and properties of xylanase from a newly isolated Fusarium proliferatum

Fusarium proliferatum (NRRL 26517), was isolated by screening soil samples surrounding decaying corn and wood using corn fibre xylan as carbon source. The extracellular xylanase from this fungal strain was purified 975-fold to homogeneity from the culture supernatant by ammonium sulphate treatment, DEAE Bio-Gel A column chromatography, octyl-Sepharose column chromatography and Bio-gel A-0.5 m gel filtration. The purified xylanase (specific activity 591 U mg −1 protein) was a monomeric glycoprotein with a molecular weight (MW) of 22 400 as determined by SDS-PAGE. The optimum pH and temperature for the action of the enzyme were at 5.0–5.5 and 55 °C, respectively. The purified xylanase was fully stable at pH 5.0–7.5 and temperature up to 55 °C. It hydrolyzed a variety of xylan substrates mainly to xylobiose and higher short-chain xylooligosaccharides. No xylose was formed. The enzyme did not require metal ions for activity and stability.

Characterization of multiple acid phosphatases in bovine liver cytosol and lysosome. Inactivation of cytosolic enzymes by disulfides and its redox regulation by thioltransferase

Cytosolic and lysosomal acid phosphatases have the ability to hydrolyze orthophosphoric monoesters below pH 5–6. However, it is thought they may have different intracellular roles. To clarify their properties, substrate specificity, inhibitor sensitivity and the modulation of enzyme by redox conditions were determined using bovine liver enzymes. DEAE-cellulose chromatography following (NH 4) 2SO 4 fractionation revealed three forms of cytosolic acid phosphatases as in the KCl gradient (0–500 mM). After Sephadex G-75 gel filtration, the enzymes appeared as single bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their activities for d-erythrose 4-phosphate co-purified with p-nitrophenylphosphatase activities in all steps. In contrast the lysosomal enzyme was purified by Octyl-Sepharose column chromatography after n-butanol treatment, (NH 4) 2SO 4 fractionation, Bio gel P-200 gel filtration and DE-52 chromatography. The relative molecular masses ( M r) determined by SDS-PAGE indicated that M r of the cytosolic enzymes (16 000) was less than that of lysosomal enzyme (160 000). The cytosolic enzymes were active against sugar phosphates and were inhibited by 1 mM Cu 2+. In addition, the cytosolic enzymes were inactivated by 5 mM oxidized glutathione and protected by 10 mM reduced glutathione (in the presence or absence of thioltransferase), suggesting that sensitive cysteinyl residue(s) existed. The lysosomal enzyme was active against various substrates and was strongly inhibited by 1 mM Cu 2+ and 2 mM fluoride. The results presented here suggest that cytosolic enzymes have different properties from those of lysosomal enzyme with respect to substrates, inhibitors and regulation of activity.

Production and characteristics of an intracellularα-glucosidase from a color variant strain ofAureobasidium pullulans

Aureobasidium pullulans produced an intracellularα-glucosidase. The enzyme was purified 124-fold by solubilization with Triton X-100, Q-Sepharose treatment, hydroxylapatite, octyl-Sepharose column chromatography, and gel filtration on Sephacryl S-200, and had a specific activity of 316.82 U/mg protein. The enzyme displayed an optimum pH for its action at 4.0 and was fully stable at pH 3.0–6.0 at 50°C. Theα-glucosidase was completely stable up to 60°C and had an optimum activity at 60°C. The partially purified enzyme preparation hydrolyzed maltose, isomaltose, sucrose, and trehalose at relative rates of 100, 60, 47, and 50, respectively, and had little or no activity on polysaccharides. TheK m value for maltose hydrolysis at pH 4.0 and 50°C was 1.85mm. The enzyme was not adsorbed onto raw corn starch and showed little raw starch degradation. Theα-glucosidase did not require any metal ion for activity. This represents the first characterization of intracellularα-glucosidase fromA. pullulans.

Amylolytic enzymes produced by a color variant strain ofAureobasidium pullulans

A color variant strain ofAureobasidium pullulans (NRRL Y-12974) produced amylase and α-glucosidase activities when grown at 28°C for 4 days in liquid culture on a wide variety of carbon sources such as starch, pullulan, glucose, maltose, cyclodextrins, sucrose, xylose, and xylan. An α-glucosidase was separated by Q-Sepharose adsorption from the cell-free culture broth and partially purified by hydroxylapatite and octyl-Sepharose chromatography. After ammonium sulfate treatment of the culture supernatant (obtained after Q-Sepharose adsorption), the amylase fraction was separated into three active fractions by hydroxylapatite column chromatography, which were identified as α-amylase, glucoamylase A, and glucoamylase B. The glucoamylase A was further purified by octyl-Sepharose column chromatography. The pH optima for the action of α-amylase, glucoamylase A, glucoamylase B, and α-glucosidase were 5.0, 4.5, 4.0–4.5, and 4.5, respectively. The α-amylase and glucoamylase B were fully stable at pH 3.0–6.0, glucoamylase A at pH 4.5–5.5, and α-glucosidase at pH 3.5–7.0 for 1 h at 50°C. The optimum temperatures for the action of these enzymes were 55°, 50°–60°, 65°, and 65°C, respectively. The α-amylase, glucoamylase A, and glucoamylase B were adsorbed onto raw corn starch and degraded it. Glucoamylase B readily cleaved pullulan. The α-glucosidase was not adsorbed onto raw starch and did not degrade it at all. It hydrolyzed both α-1,4 and α-1,6 linkages in oligosaccharides. All four enzymes did not require any metal ion for activity and were inhibited by cyclodextrins (α-and β-, 10mm).

Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30.

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.

New thermostable α-amylase-like pullulanase from thermophilic Bacillus sp. 3183

The utility of extracellular pullulanase preparation from thermophilic Bacillus sp. 3183 was evaluated. It (3.2 U g −1 DS) did not increase the saccharification yield of glucose from liquefied starch (35%, w/w, maltodextrin DE 10) with glucoamylase (12.5 U g −1 DS) and also did not decrease the reaction time. However, the enzyme preparation itself was sufficient to saccharify the liquefied starch into mainly DP1 to DP4. The pullulanase and α-amylase were separated and the pullulanase was purified to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE-Sepharose CL-6B, S-Sepharose, and Octyl-Sepharose column chromatography and finally by preparative disc gel electrophoresis. The purified enzyme cleaved pullulan in α-1,6 linkages making only maltotriose, but it cleaved starch in α-1,4 linkages producing various saccharides (DP2 and higher). The pullulanase of thermophilic Bacillus sp. is a new type of enzyme having an abnormal ability to cleave starch readily at α-1,4 linkages.

Glycyrrhizin beta-D-glucuronidase of Eubacterium sp. from human intestinal flora.

A bacterial strain capable of hydrolyzing glycyrrhizin (GL) to glycyrrhetic acid (GA) was isolated from human feces. This bacterium was identified as Eubacterium sp. The GL-hydrolyzing activity increased in parallel with the growth of this bacterium, which also produced β-D-glucuronidase (EC 3.2.1.31) acting on β-D-glucuronides of phenolic compounds such as phenolphthalein mono-β-D-glucuronide. GL-hydrolyzing activity was recovered in the supernatant fraction after disruption of this bacterium with a French press and was partially purified by means of ammonium sulfate fractionation, and Sephadex G-200 and octyl-Sepharose column chromatographies.GL-hydrolyzing enzyme was separated from the β-D-glucuronidase which hydrolyzes β-D-glucuronides of phenolic compounds by octyl-Sepharose column chromatography, indicating that the GL-hydrolyzing enzyme is a novel type of β-D-glucuronidase.

3 beta-Hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestinal bacteria.

Ruminococcus sp. PO1-3 obtained from human intestinal flora is able to reduce dehydrocholate as well as 3-ketoglycyrrhetinate. From this bacterium dehydrocholate- and 3-ketoglycyrrhetinate-reducing activities were purified one thousand-fold together with 3-ketocholanate-reducing and 3-beta-hydroxyglycyrrhetinate (glycyrrhetic acid) oxidizing activities by means of Matrex Red A, Sephadex G-200 and Octyl-Sepharose column chromatography. The purified enzyme catalyzed the reduction of dehydrocholic acid to 3 beta-hydroxy-7,12-diketocholanic acid and of 3-ketocholanic acid to 3 beta-hydroxycholanic acid. Studies on substrate specificity revealed that the enzyme had absolute specificity for the beta-configuration of a hydroxyl group at the 3 position of bile acid and steroids having no double bond in the A/B ring. This enzyme was neither beta-hydroxysteroid dehydrogenase [EC 1.1.1.51] nor 3 beta-hydroxy-delta 5-steroid dehydrogenase [EC 1.1.1.145], but a novel type of enzyme, defined as 3 beta-hydroxysteroid dehydrogenase.

Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing αβ dimer

Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.

Prevention of the agonist binding to gamma-aminobutyric acid B receptors by guanine nucleotides and islet-activating protein, pertussis toxin, in bovine cerebral cortex. Possible coupling of the toxin-sensitive GTP-binding proteins to receptors.

Using the membranes treated with Triton X-100, we studied the interaction between gamma-aminobutyric acid (GABA)B receptors and the GTP-binding proteins which are the substrates for ADP-ribosylation by the islet-activating protein (IAP), pertussis toxin. The addition of guanine nucleotides to the membranes markedly decreased the binding of GABA to GABAB receptors. Preincubation of the membranes with IAP plus NAD caused ADP-ribosylation of the 41,000- and 39,000-Da proteins selectively and decreased GABA binding to GABAB receptors in a time- and dose-dependent manner. This decrease of binding appeared to be due to the reduction of receptor affinity for agonist. The GTP-binding proteins which are ADP-ribosylated by IAP were purified from the membrane fraction of bovine cerebral cortex. The addition of the purified GTP-binding proteins to IAP-treated membranes restored the high affinity binding of GABA to GABAB receptor. The two GTP-binding proteins which were resolved by octyl-Sepharose column chromatography showed similar efficacy in restoring GABA binding. Thus, GABAB receptors are coupled to GTP-binding proteins, IAP-specific substrates, in the brain membranes.

Absorption spectral properties of purified halorhodopsin.

Halorhodopsin in the membrane fragments of Halobacterium halobium Y1 showed an absorption band at 576 nm, the intensity of which decreased on irradiation with red light at 0 degrees C (Ogurusu, T., Maeda, A., Sasaki, N., & Yoshizawa, T. (1981) J. Biochem. 90, 1267-1273). Using this photobleachable property as the basis for an assay of halorhodopsin, we purified halorhodopsin by octyl-Sepharose column chromatography after extracting it from the membrane with Triton X-100. In NaDodSO4-polyacrylamide gel electrophoresis, hR appeared as a major band with an apparent molecular weight of 22,000, but the preparation still showed several other faint bands. The purified halorhodopsin showed a main absorption band at 576 nm and a small band at around 415 nm in 1 M NaCl. The photoreactions of the purified halorhodopsin at 0 degrees C and at -75 degrees C were similar to those of halorhodopsin in membrane fragments. Irradiation of the purified halorhodopsin with red light at 0 degrees C resulted in a decrease of absorbance at around 576 nm with a concomitant increase of absorbance at around 410 nm. A hypsochromic photoproduct was obtained on irradiation with 650 nm light at -75 degrees C. The dependency of the absorption spectrum of halorhodopsin on the concentration of chloride indicates that halorhodopsin has a single chloride binding site, occupation of which is responsible for modifying the spectrum.

Purification of highly radioactive mouse interferon produced by Ehrlich ascites tumour cells induced by Sendai virus.

Mouse interferon (IFN) was produced to high titres after induction of Ehrlich ascites tumour cells with Sendai virus by using an improved procedure. The IFN molecules were labelled during their synthesis by the incorporation of [3H]leucine and [3H]lysine. Electrophoretically homogeneous labelled IFN with a molecular weight of 34000 was obtained after a two-step purification procedure using poly(I)-agarose and octyl-Sepharose column chromatography followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The specific radioactivity of this IFN was about 10 ct/min/IFN unit.