Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography

A highly purified extracellular -amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.     

Purification and Characterization of Vibrio vulnificus Protease

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone-yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl-Sephacel column chromatography, Sephacryl S-200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000-fold purification was achieved, with a yield of about 30%. The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.     

Purification and Some Properties of Chaetomium trilaterale β-Xylosidase

A β-xyloside hydrolytic enzyme of the fungus Chaetomium trilaterale was further purified by a modification of Kawaminami’s procedure (DEAE-Sephadex A-25 and Sephadex G-75 column chromatography), followed by isoelectric focusing. The purified preparation was homogeneous by polyacrylamide disc gel electrophoreses at pH 4.3 and pH 8.3. The purified enzyme hydrolyzed β-d-glucopyranosides as well as β-d-xylopyranosides, and the ratio of β-glucosidase activity against β-xylosidase activity increased about 3 fold during the purification steps. The molecular weight of this preparation was estimated to be about 240,000 by Sephadex G-200 gel filtration and 118,000 by SDS-polyacrylamide slab gel electrophoresis. The isoelectric point was 4.86 and the amino acid composition was also determined.

The optimum pH was at 4.2 for phenyl β-d-glucoside and around 4.5 for phenyl β-d-xyloside. The β-xylosidase activity was relatively stable but β-glucosidase activity was rapidly inactivated, at the alkaline pH range above 11. The heating of the preparation at 60°C didn’t show a parallel inactivation of the two activities. N-Bromosuccinimide strongly inactivated both enzyme activities. Nojirimycin and glucono-l,5-lactone showed a stronger inhibition on β-xylosidase activity than on β-glucosidase activity. The maximal velocities decreased in the order; phenyl β-d-glucoside > cellobiose > phenyl β-d-xyloside > xylobiose; the value with phenyl β-d-glucoside was about 28-fold higher than that with phenyl β-d-xyloside.     

Isolation,Purification, and Properties of Lytic Enzyme from Polysphondylium pallidum Myxamoebae

Two lytic enzymes (enzyme I and enzyme II) that lysed Micrococcus lysodeikticus were isolated from the crude extract of Polysphondylium pallidum myxamoebae grown in the presence of Klebsiella aerogenes by precipitation with protamine sulfate and by chromatography on DEAE-Sepharose CL-6B. Enzyme I was further purified by gel filtration on a Superose12 column, and enzyme II by chromatography on a MonoQ HR 5/5 column and gel filtration on a Superose12 column. Enzyme I was a basic protein, while enzyme II was acidic. The molecular weights of enzyme I and II were about 14,000 and 22,000, respectively by SDS-polyacrylamide gel electrophoresis. Optimum pHs for the activity were 5.0 for enzyme I and between 3.5 and 4.0 for enzyme II. The maximum activity of enzyme I and II was obtained at 65°C and 45°C to 55°C and at ionic strength of 0.0075 to 0.03 and 0.06, respectively. Both enzymes cleaved the glycosidic bond of β(1,4)-N-acetylmuramyl-acetylglucosamine of the cell wall peptidoglycan of Micrococcus lysodeikticus. These results indicate that the two lytic enzymes of Polysphondylium pallidum myxamoebae are N-acetylmuramidases.     

Isolation and Purification of Protein Responsible for the Conversion of Dimethylallylpyrophosphate from Poplar Leaves into Isoprene

The protein converting dimethylallylpyrophosphate (DMAPP) into isoprene in vitrowas isolated and purified 3000-fold from leaves of berry-bearing poplar (Populus deltoidesMarsh.). As the enzyme was purified, its specific activity increased and at the final stage reached 266 nmol/(min mg protein). The enzyme was eluted by anion-exchange chromatography in a 120–170 mM NaCl gradient and by chromatography on the hydroxyapatite column in 170 mM sodium phosphate. The active molecular weight of the protein determined by gel filtration was 100–110 kD. As the enzyme was purified, the K _Mvalue increased from 2 to 9 mM. A parallelism isoprene emission from DMAPP and an increase in the specific activity of the enzyme as it was purified proved that the enzyme catalyzed isoprene emission.     

灰绿曲霉β-葡萄糖苷酶的分离及特性

目的：利用灰绿曲霉EU7-22发酵产纤维素酶,从中分离到β-葡萄糖苷酶,分析其理化特性,确定其最佳活性条件。方法：灰绿曲霉EU7-22发酵液离心后,上清液经硫酸铵沉淀、Phenyl 6 Fast Flow（highsub）疏水层析和Sephacryl S-200凝胶层析,获得纯化的β-葡萄糖苷酶。结果：纯酶的比活性为5.1 IU/mg,得率为13.89%。SDS-PAGE凝胶电泳分析表明该酶是单亚基蛋白,其分子量为56.2 kDa。在pH4.0~6.0范围内,β-葡萄糖苷酶具有较高的稳定性,该酶的最适酶促反应pH为5.0。当β-葡萄糖苷酶在温度低于60℃的缓冲液中温育1 h后,酶活损失不大,表现了较好的稳定性;当该酶在温度高于60℃的缓冲液中温育1 h后,酶活迅速丧失。β-葡萄糖苷酶在70℃时具有最大催化活性。结论：灰绿曲霉EU7-22发酵产生的β-葡萄糖苷酶具有较高活性,具有分子量较小、最佳催化温度较高的特点。     

Affinity Chromatography of Thymidine Kinase from a Rat Colon Adenocarcinoma

Thymidine kinase from a transplantable colon adenocarcinoma, induced by 1, 2-dimethylhydrazine and maintained in CDF rats, was purified by affinity chromatography using thymidine-3′(4-amino-phenylphosphate) coupled to carboxyhexyl-Sepharose. Most of the contaminating protein passed through the column; non-specifically adsorbed protein was washed from the column by 0.1 M KC1 in 0.01 M Tris-HCl, pH 7.5. Thymidine kinase was eluted with 0.1 mM thymidine, 0.1 M KC1 in 0.01 M Tris-HCl, pH 7.5. The purified enzyme accounted for about 267. of the applied activity, the specific activity of the purified material (peak fraction) was 3, 500 nmoles TMP formed per mg protein per 10 min., a 1, 800-fold purification of the applied extract. The preparation is free of nucleoside phosphotransferase, but contains other protein impurities. Purification was completed in less than 1 hour, making this a useful procedure for isolation of this unstable enzyme.     

Ribonucleic Acid-Dependent Ribonucleic Acid Polymerase in the Immune Response

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 M column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA preparation (iRNA) as template made from the spleens of immunized mice but very low activity was found with an nRNA preparation made from the spleens of normal mice. Incorporation of ³H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12–13 S was most active as a template. It was followed by a fraction corresponding to 6–7 S. Sucrose gradient analysis of the ³H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Single-step purification of a recombinant thermostable α-amylase after solubilization of the enzyme from insoluble aggregates

The expression of the gene encoding a thermostable α-amylase (EC 3.2.1.1) (optimal activity at 100°C) from the hyperthermophilic archaeon Pyrococcus woesei in the mesophilic hosts Escherichia coli and Halomonas elongata resulted in the formation of insoluble aggregates. More than 85% of the recombinant enzyme was present within the cells as insoluble but catalytically active aggregates. The recombinant α-amylase was purified to homogeneity in a single step by hydrophobic interaction chromatography on a phenyl superose column after solubilization of the enzyme under nondenaturing conditions. The enzyme was purified 258-fold with a final yield of 54%.     

Characterization of cytochrome oxidase purified from rat liver

The purpose of this study was to characterize the physical properties of cytochromec oxidase from rat liver. The enzyme was extracted from isolated mitochondria with nonionic detergents and further purified by ion-exchange chromatography on DEAE Bio-Gel A. The purified enzyme contained 9.64 nmol heme a/mg protein and one iron atom plus one copper atom for each heme a. The specific activity of the final preparation was 146 µmol of ferrocytochromec oxidized/min · mg protein, measured at pH 5.7. The spectral properties of the enzyme were characteristic of purified cytochrome oxidase and indicated that the preparation was free of cytochromesb, c, andc ₁. In analytical ultracentrifugation studies, the enzyme sedimented as a single component with anS ^20,w of5.35S. The Stokes radius of the enzyme was determined by gel filtration chromatography and was equal to 75 Å. The molecular weight of the oxidase calculated from its sedimentation coefficient and Stokes' radius was 180,000, indicating that the active enzyme contained two heme a groups. The purified cytochrome oxidase was also subjected to dodecyl sulfate-polyacrylamide gel electrophoresis in order to determine its components. The enzyme was resolved into five polypeptides with the molecular weights of I, 27,100; II, 15,000; III, 11,900; IV 9800; and V, 9000.     

An α-1, 3-Glucanase from Streptomyces sp. KI-8 Production and Purification

An α-l,3-glucanase was detected in the culture supernatant of a micro-organism, which was isolated from soil on agar medium containing α-l,3-glucan as sole carbon source. The isolated strain was characterized as a strain of Streptomyces, tentatively named KI-8. This enzyme required α-l,3-glucosidic linkage as an inducer. The optimum conditions for enzyme production were studied.

The enzyme was purified by (NH₄)₂SO₄ precipitation, column chromatography on DEAE-cellulose and P(phospho)-cellulose. To eliminate the concomitant β-l,3-glucanase activity, partially purified enzyme preparation was passed through a column packed with pachyman. Final purification was accomplished by the adsorption chromatography using Sephadex G-150 from which the α-l,3-glucanase was eluted with a solution of α-1,3-linked gluco-oligo-saccharides. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 78,000 by SDS-polyacrylamide gel electrophoresis.     

A rapid method for the purification of β-lactamase from Bacillus cereus by affinity chromatography

A procedure for the purification of β-lactamase from Bacillus cereus in a single chromatographic step is described. The enzyme is isolated from the crude culture supernatant by affinity chromatography. An inhibitor, methicillin, was immobilised by covalent attachment to the insoluble column gel, Sepharose. The enzyme was adsorbed to the column ligand from the crude supernatant and was subsequently released by increasing the ionic strength of the eluting buffer. In this way the enzyme was selectively isolated from other proteins in the crude supernatant. About 98% of the original β-lactamase activity was recovered in the purified enzyme fraction.     

Complementable Fraction and Complemented Enzyme of Mutant Ml5 from Escherichia Coli: Partial Purification by Affinity Chromatography

The technique of affinity chromatography has been used in the partial purification of complementable fractions and complemented enzyme of β-galactosidase from Escherichia coli mutant M15. The crude extract of mutant ML5 was incubated with fragment CM-B. The complemented enzyme and complementable fractions were passed through a small column of p-amino-phenyl-β-D-thiogalactoside to which inhibitors had been covalently attached. A high percentage of the nonspecific protein passed directly through the affinity column while the specific enzymatic protein remained bound to the gel. Phosphate buffer with NaCl was used to elute the complementable fractions from the column. Sodium borate buffer was used to elute the bound complemented enzyme from the affinity support. The results of this study show that 100% of the complemented enzyme was bound to the column. The partially purified enzyme had the same position in disc gel electrophoresis as β-galactosidase from E. coli.     

Phytase from Aspergillus terreus

1) Aspergillus terreus No. 9A-1 was cultivated by a shaking method and the optimal cultural conditions for the phytase production were concluded as follows: Composition of medium; rice bran 30 g, ammonium sulfate 3 g, distilled water 1.0 liter; initial pH 5.5; shaking condition; 50 ml of medium/500 ml vol. flask; 120 oscil./min, 90 hr.

2) Phytase from Asp. terreus was purified by ammonium sulfate precipitation, acetone precipitation and chromatography on SE-Sephadex C-50 and Sephadex G-200 columns. The enzyme was purified about 520-folds with the yield of 20% from the broth. The purified enzyme was homogeneous by column chromatography, ultracentrifugation and electrophoresis.

3) This purified preparation of phytase showed following properties, a) Optimal pH for the reaction was 4.5; b) optimal temperature for the reaction was about 70°C; c) the enzyme was stable in the range of pH from 1.2 to 9.0     

Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum.

A β-glucosidase was isolated from $\text{Clostridium thermocellum}$; the enzyme was localized in the periplasmic space.It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing.The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity.Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.     

Purification and characterization of sweet potato cytochrome c oxidase.

Sweet potato cytochrome c oxidase (EC 1.9.3.1) was purified 45-fold with respect to its specific activity, with a high recovery by solubilization of the enzyme from the submitochondrial particles with deoxycholate, diethylaminoethyl-cellulose column chromatography, and fractionation with ammonium sulfate. Impurities, if any, could be removed by sucrose density gradient centrifugation of the purified enzyme preparation, although a considerable inactivation of the enzyme took place during centrifugation. The purified enzyme contained approximately 12 nmol of heme a per milligram of protein and about 2.5% phospholipid. The cytochrome c oxidase consisted of at least five polypeptides with molecular weights of 39,000, 33,500, 26,000, 20,000, and 5700, as determined by polyacrylamide gel electrophoresis of the purified enzyme preparation in the presence of sodium dodecyl sulfate and urea. Phosphatidylcholine and phosphatidylethanolamine stimulated the activity over 3-fold. The optimal pH of the purified enzyme was 7.0 to 7.5 in the presence of phosphatidylcholine (egg yolk or soybean) and pH 6.5 in the presence of phosphatidylethanolamine.     

Column Chromatographic Fractionation of Apricot Emulsin

Crude emulsin of apricot (Prunas armenica) kernel was prepared by the method of tannin-fractionation. It was then purified by a fractional ammonium sulfate precipitation. The purified enzyme was further fractioned by adsorbing the enzyme on a CM-cellulose column and eluting it with the diluted Mcllvaine’s buffer solution. By this chromatography, six peaks of activities of β-glucosidase and β-xylosidase were developed. From one component of these peaks, petal-like crystals were obtained. The fractions thus obtained by chromatographic fractionation and crystallization were found differ with regard to the ratio of the β-glucosidase activity to the β-xylosidase one.     

Purification of an β-D-(1→3)-Glucanase from Rhizopus niveus

An β-D-(l→3)-glucanase has been purified from the culture medium of Rhizopus niveus. The purification involves calcium acetate treatment, polyethylene glycol 6000 fractionation, CM-cellulose batch treatment, DEAE-cellulose column chromatography and gel filtration on Sephadex G–150.

The final preparation is homogenous on the basis of discelectrophoresis on acryl amide gel, sedimentation in the ultracentrifuge.

Some properties of the purified enzyme have been also tested.     

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.