l-Asparaginase from E. coli

l-Asparaginase [EC 3.5.1.1], antitumor enzyme, was purified to a crystalline form from the cell free extract of Escherichia coli A-l-3 KY3598, by ethanol fractionation and chromatographies on DEAE cellulose and CM Sephadex. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation: s_{20, w} was 7.87S.

The molecular weight was estimated to be 141,000 by the short column method. The pI of the enzyme protein was 4.75 according to isoelectric electrofocusing.

Amino acid analysis revealed the absence of cysteine or cystine residues in the molecule.

The enzyme exhibited optimal activity between pH 6 and 8. It was stable in the pH range 5.5 ~ 9.0.

The enzyme activity was cleared very slowly in the plasma of dog. Intravenous administration of the enzyme caused a complete regression of the Gardner lymphoma implanted in the C3H mice.     

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.     

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,Crystallization, and Properties of an Endo β-1,3-Glucanase from Rhizopus chinensis R-69

An endo β-l,3-glucanase was purified in crystalline form from a culture filtrate of Rhizopus chinensis R-69. Molecular weight of the enzyme was determined to be 23,000 by molecular sieve chromatography and the mode of action of the enzyme was suggested to be a less random type of β-1,3-glucanase. Km and V_max of the enzyme for laminarin are 3.4 g/liter and 1541. U., respectively. The enzyme does not decompose the cell walls of living yeast; it decomposes, however, the preparation of yeast glucan.     

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.     

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.     

Adsorption of major endoglucanase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> on cellulosic substrates

A thermostable endoglucanase (EndoI) was produced by the thermophilic fungus Thermoascus aurantiacus when grown on cellulosic materials under submerged culture (SC) and solid-state fermentation (SSF). In both cultivation techniques a considerable amount of enzyme activity remained adsorbed onto solid particles, and this was taken into consideration when modeling enzyme production. The results were compatible with the assumption that, following its synthesis, an amount of EndoI was bound on substrate and gradually released into the liquid medium. Adsorption of the enzyme on crystalline cellulose was confirmed in vitro by experiments with purified endoglucanase, which was isolated by anion exchange chromatography. The Langmuir isotherm could efficiently describe the adsorption kinetics, and the estimated A _max and K _ad values compared with those obtained for cellulases bearing a binding domain. EndoI displayed high affinity for crystalline cellulose and low binding capacity, which could be beneficial in textile processing.     

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.     

Bacterial Monoamine Oxidases

A procedure for obtaining crystalline preparations of tyramine oxidase of Sarcina lutea has been developed. The procedure included fractionation with ammonium sulfate, treatment with protamine sulfate and separation by column chromatographies on DEAE-cellulose, hydroxylapatite and sephadex G-150. The specific activity of enzyme was increased 5,700~ 6,000-fold through the procedure, over the crude cell extract. Crystals were prepared from solutions of the purified enzyme by adding solid ammonium sulfate. The crystals appeared as minute, highly refractive needles, with a bright yellow color.

With the use of crystalline preparations of tyramine oxidase of Sarcina lutea, substrate and inhibitor specificities of the enzyme were investigated. The enzyme oxidized tyramine and dopamine at almost the same rates. Other monoamines, diamines, polyamines and amino acids were not oxidized at all. The oxidation of tyramine proceeded as follows: Tyramine+O₂+H₂O→p-Hydroxyphenylacetaldehyde +NH₃+H₂O₂. Ammonia and hydrogen peroxide were formed in stoichiometric amounts.

The enzyme was not inhibited by carbonyl reagents, such as hydroxylamine, hydrazine, semicarbazide and isoniazid, but was inhibited by p-CMB and iproniazid.     

Crystallization and Properties of N-Benzoylglycine Amidohydrolase from Pseudomonas putida

N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.     

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.     

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.     

Partial Hydrolysis of a Synthetic Heptapeptide Related to Gramicidin S

Galactomyces reessii L, isolated as a protopectin-solubilizing enzyme-producing strain, produced protopectin-solubilizing enzyme in the culture filtrate. The enzyme was purified by repeated CM-Sephadex C-50 column chromatography, and isolated as a crystalline form with a yield of 16% of the initial activity. The enzyme was a glycoprotein containing about 2.6% carbohydrate (as pentose). Its isoelectric point was around pH 8.4, and the sedimentation coefficient (s_20,w) was determined to be 3.83 S. The molecular weight was determined to be 30,000 by gel filtration on Sephadex G-75 and 29,300 by ultracentrifugal analysis. The enzyme catalyzed the release of highly polymerized pectin from various protopectins. The enzyme also catalyzed the depolymerization of pectic acid or galacturonic acid oligomers, and was confirmed to be an endo- polygalacturonase.     

Studies on Vitamin B6 Metabolism in Microorganisms

A large number of bacteria were searched for the activity of the synthesis of pyridoxine 5′-phosphate by the transphosphorylation between pyridoxine and p-nitrophenyl phosphate. Several properties of the transphosphorylation by the partially purified enzyme prepared from one of the isolated bacteria, Escherichia freundii K–1, were investigated accompanying with phosphatase activity. The behavior of the phosphotransferase and phosphatase activities in various reaction conditions were almost parallel. It was pointed out that the transphosphorylation might be catalyzed by the function of acid phosphatase. The phosphoryl donor specificity for the enzyme system was found to be broad.

The enzyme which catalyzed the transphosphorylation of pyridoxine accompanying with the hydrolyzation of phosphoryl donor substrates was purified and crystallized from the cell free extract of Escherichia freundii K–1. The purification procedures involved heat treatment, ammonium sulfate fractionation and DEAE-cellulose, hydroxylapatite, and CM-sephadex column chromatographies. The crystalline enzyme showed the sedimentation coefficient of 7.5 S and the diffusion coefficient of 6.15 × 10^?7 cm²/sec. The molecular weight was calculated to be 120,000. Several properties of the purified enzyme were also investigated. It was recognized that the transphosphorylation of pyridoxine might be catalyzed by the action of acid phosphatase.     

Isolation of Crystalline Quinolinate Phosphoribosyltransferase from Alcaligenes eutrophus nov. subsp. quinoiinicus and Its Behavior on Polyacrylamide Gel Disc Electrophoresis

Quinolinate phosphoribosyltransferase (EC 2.4.2.19) was purified and crystallized from cell-free extracts of Alcaligenes eutrophus nov. subsp. quinoiinicus, IAM 12305 which was isolated in our laboratory from soil. The enzyme was labile in the cold, and all purification steps were performed at room temperature (10~15°C). The crystalline enzyme was certified to be homogeneous by ultracentrifugal analysis and starch-gel electrophoresis. On polyacrylamide gel disc electrophoresis, the crystalline enzyme showed a multiple profile, but it showed a single band by addition of a certain amount of glycerol and 2-mercaptoethanol. The adding effect of these compounds was discussed.     

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.     

Crystallization and Some Properties of Mannanase

A mannan-hydrolyzing enzyme produced by a certain strain of Bacillus subtilis was purified from the culture broth and isolated in a crystalline state by being treated with several ion-exchangers. The optimal pH of the enzyme was 6.0. The enzyme was stable in a pH region of 5.0 to 9.5 and at temperatures less than 55°C. The enzyme attacked only β-1,4-mannosidic linkages in the main chain of galactomannan of soybean seed coat, guar gum and coffee bean, and of glucomannan of konjak (Amorphophalus konjac). Investigation of the hydrolysis mode revealed that the enzyme attacked coffee bean galactomannan endowise to form mannobiose, mannotriose and mannotetraose. The action patterns on several mannohomooligomers prepared from a partial hydrolysate of coffee bean galactomannan were also investigated, indicating that the enzyme preferentially attacked the β-1,4-mannosidic linkages that were present apart three to four mannose residues from the non-reducing end of the mannose chain.     

Degradation of Barley Glucan and Lichenan by a Bacillus pumilus Enzyme

A bacterial strain was isolated from soil, which rapidly degraded purified barley β-glucan as well as lichenan. The strain belonged to Bacillus pumilus, and some authentic strains of this species were also shown to hydrolyze the gluean. An enzyme active on the above substrates but not on laminaran and on CM-cellulose was partially purified from the culture fluid. This enzyme, about 27,000 in molecular weight, was found to cleave a β-(1 → 4) linkage adjacent to a β-(1 → 3) in the polymers. It was suggested that only an enzyme of this type should be called a ‘lichenanase’ and discriminated from cellulases and laminaranases.     

Evidence implicating a novel thiol methyltransferase in the detoxification of glucosinolate hydrolysis products in Brassica oleracea L.

The physiological relevance of a novel thiol methyltransferase from cabbage, and its possible role in sulphur metabolism have been investigated. The enzyme was absent from the chloroplast, the site of sulphate reduction, and was localized in the cytosol. Potential substrates were initially screened on the basis of their ability to inhibit the methylation of iodide, a previously known substrate for the enzyme. Thiocyanate, 4,4 ′ ‐thiobisbenzenethiol, thiophenol, and thiosalicylic acid were identified as possible substrates. Methylation of these thiols by the purified enzyme using [Methyl‐³H]S‐adenosyl‐ L ‐methionine confirmed their nature as substrates. The purified enzyme strongly preferred thiocyanate as a methyl acceptor. The enzyme had K_m values of 11, 51, 250 and 746 mmol m ^{? 3} for thiocyanate, 4,4 ′ ‐thiobisbenzenethiol, thiophenol and thiosalicylic acid, respectively. The identity of methylthiocyanate as the product of thiocyanate methylation by the purified enzyme was confirmed by mass spectrometry. The enzyme was strictly associated with glucosinolate‐containing plants. Thiol substrates of the enzyme are known products of glucosinolate hydrolysis. Our observations indicate that this enzyme could be involved in the detoxification of reactive thiols produced upon glucosinolate degradation in these plants.     

Properties of an Intracellular Poly(3-hydroxybutyrate) Depolymerase (PhaZ1) from <Emphasis Type="Italic">Rhodobacter spheroides</Emphasis>

The gene of an intracellular poly(3-hydroxybutyrate) (iPHB) depolymerase from Rhodobacter sphaeroides was cloned and sequenced. The nucleotide sequence of the cloned gene was homologous to that of the iPHB depolymerase gene from Ralstonia eutropha H16 (phaZ1 _Reu) and the gene was designated phaZ1 _Rsh. PhaZ1_Rsh was purified from E. coli harboring an expression vector containing phaZ1 _Rsh and its properties were examined. PhaZ1_Rsh degraded amorphous PHB granules, and the 3-hydroxybutyrate tetramer and pentamer, but not crystalline PHB granules. The enzyme activity was inhibited by p-chloromercuribenzoate and Triton X-100. Diisopropylfluorophosphate, phenylmethylsulfonylfluoride, and dithiothreitol had no effect on the activity. A mutant having alanine instead of cysteine at 178 lost the activity. These results show that PhaZ1_Rsh is a quite similar enzyme to PhaZ1_Reu.