Partial purification and characterization of feline gamma-like interferon

Feline interferon (IFN) was produced from spleen cells stimulated by Staphylococcus enterotoxin A (SEA). The IFN was purified by a three-step procedure using controlled pore glass adsorption chromatography, concanavalin A (ConA)-agarose column chromatography and gel filtration. The final product of these procedures which had activity of an IFN appeared as a single peak of activity with molecular weight of approximately 50,000. It was sensitive to acid and heat, suggesting that the isolated material was a gamma IFN. The total recovery of feline gamma IFN was 8.2%. Specific activity was 2.95 X 10(4) unit/micrograms protein and was concentrated 2.8 X 10(4) times. This preparation of purified feline gamma IFN was destroyed completely by 0.1% sodium dodecyl sulfate within 20 min. As an inducer of feline gamma IFN, SEA appeared to produce a more uniform IFN product than did ConA. Further, the presence of 10% ethylene glycol in the sample applied to ConA-agarose column as well as its absence in the elution buffer was effective in reducing contaminating acid- and heat-resistant IFNs in the preparation.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Actinomycetes

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl₂ and AgNO₃ caused complete inhibition of the enzyme activity at 2 × 10^?5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.

A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.     

Studies on Sugar-isomerizing Enzyme Purification,Crystallization and Some Properties of Glucose Isomerase from Streptomyces sp.

Glucose isomerase was purified by means of acetone fractionation, DEAE-cellulose column chromatography, DEAE-Sephadex column chromatography and crystallization. The purified enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis. The sedimentation coefficient, s_20,w, the diffusion coefficient, D_20,w, and partial specific volume of the enzyme were 8.0S, 4 × 10^?7cm²/sec and 0.69 ml/g, respectively. The molecular weight of the enzyme was estimated to be 157,000 from the sedimentation and diffusion measurements. The crystalline glucose isomerase contained cobalt and magnesium ions. The properties of the enzyme were also studied.     

Isolation of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Thienodolin, a new plant growth-regulating substance, was isolated from the fermentation broth of a streptomycete strain identified as Streptomyces albogriseolus.

The active principle was extracted with ethyl acetate and purified by silica gel column chromatography and preparative HPLC. The substance showed growth promoting activity with 1.2 × 10^?6–1.2 × 10^?5 M treatment to rice seedlings, and inhibitory activity with 4.0 × 10^?5 M treatment.     

Development of efficient method for purified recombinant bovine granulocyte-macrophage colony-stimulating factor production with baculovirus-silkworm gene expression system

Recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) was produced by the baculovirus-silkworm expression system. It was purified to 98% by (NH₄)₂SO₄, followed by a three-step column chromatography with silica gel, ion exchange resin and a metal chelate column. The specific activity of purified rboGM-CSF was 1.6–6.3 × 10⁶ ED₅₀ mg⁻¹. By this method, the specific activity was raised 160-fold and 21% of the expressed rboGM-CSF was recovered.     

Purification and characterisation of monoamine oxidase from Avena sativa

FAD-containing monoamine oxidase (MAO; EC 1.4.3.4) oxidises monoamines to their corresponding aldehydes, H₂O₂, and NH₃. It has been purified to homogeneity in mammals, but to our knowledge, there have been no reports of the enzyme in plants. MAO activity was detected in Avena sativa seedlings during germination using benzylamine as substrate. The enzyme was purified to homogeneity (as assessed by native PAGE) by Sephadex G-25, DEAE Sephacel, hydroxyapatite, Mono Q, and TSK-GEL column chromatographies. The molecular mass estimated by gel filtration using the TSK-GEL column was 220?kDa. SDS-PAGE yielded four distinct protein bands of 78, 58, 55, and 32?kDa molecular masses. The pI value of the enzyme was 6.3. The enzyme showed high substrate specificity for an endogenous amine, phenethylamine, which was oxidised to phenylacetaldehde, but not for ethylamine, propylamine, butylamine, pentylamine, dopamine, serotonin, tryptamine, or tyramine. The K _m values for benzylamine and phenethylamine were 2.7?×?10^?4 and 7.1?×?10^?4?M, respectively. Enzyme activity was not inhibited by pargyline, clorgyline, semicarbazide, or Na-diethyldithiocarbamate. Benzaldehyde, the product of benzylamine oxidation, exhibited strong competitive inhibition of enzyme activity with a Ki of 3???M. FAD was identified by ODS-column chromatography as an enzyme cofactor. The enzyme contained 2?mol of FAD per 220,000?g of enzyme.     

Biochemical Studies on Cycasin

Purification of β-glucosidase from the seeds of Japanese cycad, and properties of the purified preparation are described. The enzyme activity was determined by colorimetry using ONPG as substrate. Crude preparation was obtained easily by adsorption on fibrous CMC pulp. It was further purified by chromatography on CMC powder, and a preparation which showed an activity of 135-folds of the original extract was obtained. Influences of pH, temperature, and substrate concentration upon the enzyme activity were examined. Michaelis constant of the enzyme for ONPG was 3.3×10^–3M.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

Large scale production of transfer ribonucleic acids from E. coli K-12 MO7

An engineering-scale procedure for the recovery of 300–400 g batches of mixed transfer ribonucleic acids is described. Semicontinuous growth of E. coli K-12 MO7 yielded 77 kg of harvested cells in four days. Phenol extraction and ethanol precipitation recovered a crude tRNA material that was further purified by DKAE-cellulose chromatography in runs of 1 × 10⁶ A₂₆₀ units each on a 6 × 30 in. column using a 240 1, gradient elution. The purified tRNAs were partially concentrated and resolved into three groups.     

Purification of cassava linamarase

Linamarase was purified from parenchymal tissue of cassava by extraction with acetate buffer, fractional precipitation with ammonium sulphate, followed by column chromatography on DEAE-cellulose and Sepharose-6-B gel filtration. The specific activity is increased 350 fold with 35% recovery. The K_ms for linamarin and p-nitro-phenyl β-D-glucoside are 1.45 × 10^?3 M and 0.46 × 10^?3 M, respectively. The pH optimum in 50 mM NaPi is pH 6 and the specific activity is 26.5 nkat/mg. The enzyme can be prepared from cassava peel using the same procedure and has similar properties.     

Purification and properties of d-xylulose reductase from Pachysolen tannophilus

d-Xylulose reductase (EC 1.1.1.9) from Pachysolen tannophilus IFO 1007 was purified by Sephadex G-100 gel chromatography with three columns and DEAE cellulose chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. It was most active at pH 9.1–10.0 and 55°C, and stable at pH 7–9 and below 25 °C. Its activity was stimulated by NH₄Cl,NaCl,MgCl₂,KCl, glutathione, cysteine and glycine, and inhibited remarkably by SH inhibitor such as lead acetate, HgCl₂ and AgNO₃. It oxidized xylitol, sorbitol, ribitol and glycerine but not mannitol, inositol, arabitol and erythritol. Its K_m values of enzyme against xylitol, sorbitol and ribitol were 1.1 × 10⁻² M, 3.0 × 10⁻² M and 5.0 × 10⁻² M, respectively. Its molecular weight was determined to be 120,000 by Sephadex G-200 column chromatography, and that of its subunit was 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

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.     

Konjac-mannanase from the Tubers of Amorphophallus konjac C. Koch

A crude enzyme preparation hydrolyzing konjac mannan was extracted from germinating konjac tubers, and purified by chromatography with DEAE-cellulose and alkali-swollen cellulose, and by gel-filtration on Sephadex G-100. The purified enzyme preparation showed optimal activity at pH 4.7, optimum temperature at 40°C. It was considerably stable at pH’s between 4.0 and 8.0, but inactivated rapidly by temperaters above 50°C. Hydrolysis of the mannan by this enzyme proceeded by typical random mechanism, and the rate was in agreement with an empirical equation, p=0.43 E^0.77 to^0.5. As the Km and V_max values for mannan, 7.14×10^-2(%)and 23.8×10^-3 (ΔOD_500nm) were obtained, respectively.     

Studies on continuous enzyme reactions. IV. Preparation of a DEAE-sephadex–aminoacylase column and continuous optical resolution of acyl-DL-amino acids

Conditions for the preparation of an aminoacylase column using DEAE-Sephadex as a carrier were investigated. The aminoacylase column having the highest activity was obtained when 7500 μmoles/hr. of partially purified aminoacylase was charged into a column packed with 9 ml. of DEAE-Sephadex A-25 (bead type, hydroxy form). By employing a DEAE-Sephadex–aminoacylase column, conditions for continuous optical resolution of acyl-DL -amino acids were investigated. When a solution of 0.2M acetyl-DL -methionine (pH 7.0, containing 5 × 10^?4M Co²⁺) or 0.2M acetyl-DL -phenylalanine (pH 6.0, containing 5 × 10^?4M Co²⁺) was passed through the aminoacylase column at the flow rate of SV = 2.5 or 2.0, respectively, at 50°C., the highest rate of hydrolysis of both substrates was attained. From the column effluents, enzymatically hydrolyzed L -methionine and L -phenylalanine were isolated in a good yield.     

Production and Purification of a New Chillproofing Enzyme and Its Properties

Chillproofing enzyme was obtained from broth cultures of Serratia marcescens B–103. This extracellular enzyme, tentatively, named the S-enzyme was highly purified from the culture supernatant by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G–200 and column chromatography on DEAE-Sephadex A–50.

The purified preparation appeared homogeneous on a ultracentrifugation with a sedimentation coefficient of 3.14 S and a molecular weight of 38,000~45,000 determined by the method of Whitaker.

The S-enzyme hydrolyzed various proteins at pH 4~6 and at low temperature hydrolyzed nitrogenous substances which may cause chill haze in beer. So the chillproofing activity of the S-enzyme may be due to its proteolytic activity.

The S-enzyme was stable at 4°C at pH 5~10.5. It was completely inactivated by heating at 60°C for 10 min, and was inactivated by Hg²⁺ and Pb²⁺ and activated by Mn²⁺, Ca²⁺. Mg²⁺ and Zn²⁺     

Purification of Human Placental Trophoblast Interferon by Two-Dimensional High Performance Liquid Chromatography

Abstract

Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the β-type (75%) and relatively low levels of the α-type (25%). A two-step high performance liquid chromatographic procedure (“two-dimensional HPLC”) has been developed for the complete purification of the placental trophoblast interferon beta (tro-IFN-β) from serum-containing culture supernatant. The method involved a combination of high performance liquid affinity chromatography (HPLAC) on Cibacron Blue 3GA immobilized on an activated pressure stable macroporous synthetic polymer, 2-hydroxyethyl methacrylate vinyl sulphone (HEMA-BIO 1000 VS), as the first dimension and reversed-phase high performance liquid chromatography (RP-HPLC) on Separon SGX C-18 as the second. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments showed that the tro-IFN-β was present as a 24 kDa protein. Densitometric scanning analysis of Coomassie-stained gel revealed the purity of the final preparation to be greater than 99%. The purified tro-IFN-β had a specific activity of 1.03 × 10⁸ IU/mg of protein and the overall recovery was 81% of the total IFN-β activity in the crude preparation and 61% of the total IFN activity.     

Two-Step Purification of Mouse Kidney Ornithine Decarboxylase

Abstract

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4. 1. 1. 17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1. 0 × 10⁶?1. 4 × 10⁶ nmol/h. mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54, 000 and a minor band of Mr 51, 000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [¹⁴C)difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Purification and Some Properties of a Fungal Lipase Preparation Used for Milk Flavouring

Intracellular lipase of a strain of Rhizopus fungus which is effective for producing a milk flavor was purified and fractionated into two components, I and II, by DEAE Sephadex A-50 column chromatography. They both proved homogeneous by electrophoresis and ultracentrifugal analysis. The sedimentation coefficient was respectively calculated to be 5.8×10^?13 for lipase I, and to be 2.2×10^?13 for lipase II. From substrate specificity, it was found that lipase I was an ordinary lipase hydrolyzing olive oil and tributyrin favourably, while, II, rather, a special lipase having a high affinity towards tricaprylin. They, also, respectively had an apparent phospholipase activity on soy-lecithin and, clearing activity on chylomicron prepared from olive oil and human serum. Their mode of action, and the effect of metals and emulsifying agents on their activity are also presented.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.