Crystallization and Properties of Amine Dehydrogenase from Pseudomonas sp.

An amine dehydrogenase was purified and crystallized from the cell free extract of a Pseudomonas sp., isolated from soil by means of the enrichment technique. The crystalline enzyme gave a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was estimated to be 100,000 by gel filtration on a Sephadex column. Upon SDS-gel electrophoresis, the enzyme was dissociated into two nonidentical subunits having molecular weights of 60,000 (dehydrogenase) and 39,000 (cytochrome c). The absorption spectrum of the enzyme showed absorption maxima at 550 nm, 524 nm, 411 nm and 280 nm, and a broad shoulder at around 350 nm, indicating that the enzyme was purified as a dehydrogenase-cytochrome c complex. The prosthetic group of the dehydrogenase was identified as covalently bound pyrroloquinoline quinone. The enzyme showed a broad substrate specificity toward various amines including aliphatic monoamines, aliphatic diamines, aromatic amines and polyamines.     

Purification,Crystallization and Some Properties of Extracellular Pullulanase from Aerobacter aerogenes

Extracellular pullulanase was purified and crystallized from the culture fluid of Aerobacter aerogenes. Pullulanase was purified by means of ammonium sulfate fraction, DEAE-cellulose column chromatography and Sephadex column chromatography. Crystalline pullulanase was formed when saturated ammonium sulfate solution was added to the purified enzyme solution. The crystalline enzyme appeared as colorless fine rods. On ultracentrifugation analysis, the enzyme showed a single sharp and symmetrical Schlieren peak. The sedimentation coefficient, s_20,w was 4.39S. Polyacrylamide gel electrophoresis at pH 8.4 gave a main band with two sub-bands and the molecular weight of the main enzyme was estimated to be 66,000 from Polyacrylamide gel electrophoresis and to be 58,000 from sedimentation equilibrium. The optimum pH and temperature for the enzyme action were pH 6.5 and 50°C, respectively.     

The Isolation and Physico-chemical Properties of Crystalline Quinolinate Phosphoribosyltransferase from Hog Liver

Quinolinate phosphoribosyltransferase (an intermediary enzyme in the de novo NAD biosynthetic pathway) was purified from hog liver and its crystallization from a mammal was successful for the first time. This crystalline enzyme preparation was certified to be homogeneous by ultracentrifugal analysis and polyacrylamide gel disc electrophoresis. Its molecular weight was 173,000 using the gel filtration method, and 172,000 using sedimentation velocity analysis. The subunit molecular weight was estimated at 33,500 with SDS polyacrylamide gel disc electrophoresis. Several physico-chemical parameters were also determined.     

The Structure of a New Metabolite from Aspergillus versicolor

α-Glucosidase was purified and crystallized from the mycelia of Mucor javanicus, by procedures including extraction with urea, fractionations with acetone and polyethylene glycol 6000, successive separation on columns of Sephadex G-200 and DEAE-cellulose, and crystallization by the addition of ampholine reagent. The crystalline enzyme was homogeneous in ultracentrifugal analysis and gel electrophoresis. The purified enzyme showed a sedimentation constant of 5.6 S and isoelectric point of pH 8.6. Some properties of the purified enzyme were also investigated. It was recognized that the synthesis of riboflavin α-glucoside was catalyzed by the transglucosylation activity of this α-glucosidase.     

Preparation and Properties of Crystalline Glucoamylase from Endomyces Species IFO 0111

Glucoamylase [EC 3. 2. 1. 3] of Endomyces sp. IFO 0111 was purified from the culture filtrate by chromatography on CM-cellulose, and isolated in crystalline state. The crystalline enzyme was free from α-amylase, and displayed apparent homogeneity upon ultracentrifugation and electrophoresis. The molecular weight was 55,000 by the Archibald’s method. The results of amino acid analysis revealed the absence of methionine residue in this enzyme protein. The purified enzyme acted on starch hydrolyzing up to about 80%, whereas the crude enzyme hydrolyzed the same substrate almost completely. Some other properties were also included in this paper.     

Monilidiols,Characteristic and Bioactive Metabolites of Benomyl-resistant Strains of Monilinia fructicola

A new polygalacturonase was found in a culture filtrate of Aureobasidium pullulans. The enzyme was purified and obtained in crystalline form with 10% recovery. The crystalline enzyme was a homogeneous protein by analyses by sedimentation and electrophoresis. The enzyme was most active around pH 4.5, and stable in the pH range of 4 to 6. Its molecular weight was 42,000 and its isoelectric point was pH 6.0. The enzyme was an endo-polygalacturonase, catalyzing the cleavage of glycosidic bonds of polygalacturonic acid at random.

The enzyme had less protopectinase activity than those of the endo-polygalacturonases that were isolated as protopectin-solubilizing enzymes from Trichosporon penicillatum, Kluyveromyces fragilis, and Galactomyces reessii. Some characteristics were compared with the endo-polygalacturonases, which have potent protopectinase activity.     

Purification and Properties of a Lytic Enzyme from Streptomyces griseus H-402

A lytic enzyme which was capable of lysing cells of Streptococcus mutans was purified from the culture filtrate of Streplomyces griseus H–402 by Amberlite CG–50 treatment, CM-cellulose and hydroxylapatite column chromatographies, and Sephadex G–150 gelfiltration. The lytic enzyme was obtained in a crystalline form which was homogeneous in polyacrylamide gel electrophoresis. The molecular weight was estimated to be 2×10⁴ by the thin-layer gel-filtration method on Sephadex G–75, and 2.3 × 10⁴ by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was found to be a N-acetylmuramidase whose activity was lost by N-bromosuccinimide as an inhibitor.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

Crystallization and Characterization of Polyamine Oxidase from Penicillium chrysogenum

Polyamine oxidase was purified and crystallized with an overall yield of 35% from mycelial extract of Penicillium chrysogenum by a procedure involving ammonium sulfate fractionation, and DEAE-cellulose and Sephadex G-200 column chromatographies. The crystalline enzyme was homogeneous, as judged by disc gel electrophoresis and ultracentrifugation. The sedimentation coefficient (s_{20, w}⁰) of the enzyme was determined to be 6.9S, and diffusion coefficient (D_{20, w}) to be 4.2 × 10^?7 cm² sec^?1. The enzyme showed a molecular weight of about 160,000 by gel filtration method and ultracentrifugal analysis, and it was composed of two identical subunits. The enzyme was a flavoprotein with absorption maxima at 275, 375 and 450 nm. The prosthetic group was identified to be FAD. The enzyme oxidized spermine, and slightly oxidized spermidine. Diamines and monoamines were not oxidized.     

A Crystalline Aminopeptidase from Streptomyces peptidofaciens: Physico-chemical Properties and Characteristics as a Ca-Metalloprotease

A crystalline aminopeptidase obtained from the culture filtrate of Streptomyces peptidofaciens KY 2389 appeared to be homogeneous on ultracentrifugation and acrylamide gel electrophoresis. The sedimentation coefficient, s_{20, w}., was determined to be 2.6 S. The molecular weight was estimated to be approximately 19,000 by sedimentation equilibrium studies. The amino acid analyses indicated that the enzyme was composed of 147 amino acid residues and contained no sulfhydryl group. The isoelectric point was found to be around pH 7.4 by isoelectric focusing on ampholites.

The enzyme required Ca²⁺ for its maximal activity and was strongly inhibited by some metal-chelating agents such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline. The EDTA-inactivated enzyme restored its activity almost completely by the addition of Ca²⁺ The crystalline preparation of aminopeptidase contained 1 g-atom of calcium and about 2 g-atoms of magnesium per mole of enzyme protein, and the calcium was essential for the activity of the enzyme.     

Multicomplex cellulase-xylanase system of Clostridium papyrosolvens C7.

The cellulase system of Clostridium papyrosolvens C7 was fractionated by means of ion-exchange chromatography into at least seven high-molecular-weight multiprotein complexes, each with different enzymatic and structural properties. The molecular weights of the complexes, as determined by gel filtration chromatography, ranged from 500,000 to 660,000, and the isoelectric points ranged from 4.40 to 4.85. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the complexes showed that each complex had a distinct polypeptide composition. Avicelase, carboxymethyl cellulase, and xylanase activity profiles differed from protein complex to protein complex. Three of the complexes hydrolyzed crystalline cellulose (Avicel). Activity zymograms of gels (following electrophoresis under mildly denaturing conditions) revealed different carboxymethyl cellulase-active proteins in all complexes but xylanase-active proteins in only two of the complexes. The xylanase specific activity of these two complexes was more than eightfold higher than that of the unfractionated cellulase preparation. A 125,000-M(r) glycoprotein with no apparent enzyme activity was the only polypeptide present in all seven complexes. Experiments involving recombination of samples eluted from the ion-exchange chromatography column indicated that synergistic interactions occurred in the hydrolysis of crystalline cellulose by the cellulase system. We propose that the C. papyrosolvens enzyme system responsible for the hydrolysis of crystalline cellulose and xylan is a multicomplex system comprising at least seven diverse protein complexes.     

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.     

Studies on new intracellular proteases in various organs of rat. 1. Purification and comparison of their properties.

1. Specific proteases which inactivate the apo-proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared. 2. The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified. 3. They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N-acetyl-L-tyrosine ethyl ester, but not with that of trypsin, p-toluenesulfonyl-L-arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center. 4. The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM-Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N-acetyl-L-tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities. 5. The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.     

Purification of an autocatalytic protein-glycosylating enzyme from cell suspensions of Daucus carota L.

A glycosyltransferase was identified in the 174 000 · g membrane pellet and the supernatant from extracts of cell suspensions of Daucus carota L. The enzyme from the supernatant was enriched 475-fold, and sodium dodecyl sulfate-gel electrophoresis and fluorography of this purified sample showed that the only enriched protein band (40 000 Da) was simultaneously an enzyme and a glucose-acceptor. Gel filtration and electrophoresis under non-denaturing conditions proved that in vivo this protein provides the subunits for a very large molecule. Radio-gas-liquid chromatography demonstrated that only one glucosyl moiety was transferred from UDP-glucose to the protein.Abbreviations DEAE diethylaminoethyl - GT IsU glycosyltransferase I, soluble, substrate UDPglucose - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Functional and biophysical characterization of a hyperthermostable GH51 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Thermotoga petrophila</Emphasis>

A hyperthermostable glycoside hydrolase family 51 (GH51) α-l-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/β barrel proteins analogously observed for other GH51 α-l-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.     

Quinolizine Alkaloids from the African Medicinal Plant Calpurnia aurea: Molluscicidal Activity and Structural Study by 2D-NMR

l-Phenylalanine ammonia-lyase was crystallized for the first time from a cell-free extract of Rhodosporidium toruloides IFO 0559. Heat treatment at 50°C for 5 min was a smart step for enzyme purification. Column chromatographies with DEAE-cellulose and hydroxyapatite, and gel filtration on a Sephadex G-200 column were used in the subsequent purification. The enzyme was purified to a homogeneous state and crystallized as fine needles with ammonium sulfate. The crystalline enzyme was pure by both analytical ultracentrifugation and polyacrylamide gel electrophoresis. The enzyme had a 8.2 s sedimentation velocity. The molecular weight of the enzyme was 165,000 by the dual methods of sedimentation equilibrium and gel filtration. The enzyme was composed of two identical subunits with a molecular weight of 80,000.     

The aconitase of yeast. II. Crystallization and general properties of yeast aconitase.

Yeast aconitase [citrate (isocitrate) hydro-lyase, ED 4.2.1.3], inductively formed by Candida iipolytica in the presence of fluoroacetate, was purified approximately 100-fold by Sephadex G-100 gel filtration and DEAE-Sephadex column chromatography, yielding dark-brown needle crystals. The crystalline aconitase was homogenious as judged by polyacrylamide gel electrophoresis and sedimentation by ultracentrifugation. The enzyme showed maximal activity at pH 8.0 and at 55 degrees. It has an S20, W of 5.03 S, a molecular weight of 68,500 and an isolectric point of pH 4.2. The presence of 2.10 moles of iron per mole of the enzyme was demonstrated by atomic absorption spectroscopy.     

Microbial Utilization of Benzenesulfonic Acid

An enzyme which degrades yeast glucan and yeast cells in the logarithmic phase of growth (log yeast cells) and produces protoplasts from log yeast cells has been crystallized from the culture filtrate of a strain belonging to Fungi Imperfecti.

Analyses by ultracentrifugation and disc gel electrophoresis showed the crystalline enzyme to be homogeneous. Its molecular weight was found to be 24,500. The hydrolysis of laminarin, pachyman and yeast glucan was catalysed by the enzyme to produce a mixture of laminaridextrins. The conversion of log yeast cells to protoplasts was obtained by the addition of only this enzyme, the addition of mercaptoethanol or phosphomannanase to the enzyme promoted the conversion.     

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.     

Human blood group glycosyltransferase. II. Purification of galactosyltransferase

A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.