Purification and Properties of Alkaline Proteinase from Aspergillus oryzae

Alkaline proteinase was purified from culture extract of a strain of Aspergillus oryzae. The process consists of the Amberlite IRC-50 adsorption, column chromatography on DEAE-cellulose and CM-cellulose and Sephadex G-100 gel filtration. The molecular weight of the enzyme was estimated to be about 23,000 by a gel filtration method. Alkaline proteinase showed neither carboxypeptidase activity nor aminopeptidase activity, but degraded 10101010 poly-l,α-glutamic acid, poly-l-lysine, 10101010 and 10101010. The enzyme was completely inhibited by diisopropylphos-phorofluoridate (10^?2 m) or potato inhibitor (250 μg/ml).     

Purification and Properties of Acid Carboxypeptidase IV from Aspergillus oryzae

Acid carboxypeptidase IV from Aspergillus oryzae was purified from the rivanol precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A–50, hydroxylapatite and P-cellulose and gel filtration through Sephadex G–100. The optimum pH is at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme activity was inhibited by sulfhydryl reagents and diisopropylphosphorofluoridate, but was not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 43,000 by gel filtration method.     

Purification and Properties of Acid Carboxypeptidase I from Aspergillus oryzae

To elucidate the constitution of peptidases from Aspergillus oryzae, systematic separation of the enzymes was carried out by batchwise treatment with Amberlite IRC-50 and precipitation with rivanol. Proteases were separated to two fractions. They were Amberlite IRC-50 adsorbed and the non-adsorbed fractions and the latter fraction was further separated to two fractions, rivanol precipitable and non-precipitable fractions.

Acid carboxypeptidase I was purified from the rivanol non-precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50 and SE-cellulose. The purified enzyme was not homogeneous on disc electrophoresis, although symmetric peaks were obtained for enzyme protein and activity in Sephadex gel filtration. The optimum pH is at pH 4.0 for carbobenzoxy-l-alanyl-l-glutamic acid. The enzyme activity was inhibited by SH reagents, but not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 120,000 by gel filtration.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

Purification and Properties of an Aminopeptidase from Rabbit Skeletal Muscle

An aminopeptidase active on l-Val-l-Val-l-Val-l-Ala was purified from rabbit skeletal muscle by the method including ammonium sulfate precipitation, DEAE-cellulose chromatography, gel-filtration on Sephadex G–200, rechromatography on DEAE-cellulose, hydroxylapatite chromatography and rechromatography on Sephadex G–200. Polyacrylamide gel disc electrophoresis showed that the enzyme thus obtained was homogeneous. The specific activity of the purified enzyme was 1500 times that of the original muscle extract. The enzyme had an optimal pH in a range of 6.0~7.6 and was stable in pH 6.1~8.1. Molecular weight of the enzyme was estimated to be 160,000 from the result of gel-filtration on Sephadex G–200. The enzyme showed specificity for tri-, tetra-, penta-, and hexapeptides. The analytical data of liberated amino acids showed that the enzyme was an aminopeptidase active on these oligopeptides. The enzyme was strongly inhibited by N-ethyl-maleimide and EDTA.     

Purification,Crystallization and Properties of Tyrosine Phenol Lyase from Erwinia herbicola

Crystalline tyrosine phenol lyase was prepared from the cell extract of Erwinia herbicola grown in a medium supplemented with l-tyrosine. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 259,000. The crystalline enzyme catalyzed the conversion of l-tyrosine into phenol, pyruvate and ammonia, in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from d-tyrosine, S-methyl-l-cysteine, 3, 4-dihydroxyphenyl-l-alanine, l- and d-serine, and l- and d-cysteine, but at lower rates than from l-tyrosine. l-Phenyl-alanine, l-alanine, phenol and pyrocatechol inhibited pyruvate formation from l-tyrosine.

Crystalline tyrosine phenol lyase from Erwinia herbicola is inactive in the absence of added pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 425 mμ. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis to be 2 moles per mole of enzyme. Addition of the substrate, l-tyrosine, or the competitive inhibitors, l-alanine and l-phenyl-alanine, to the holoenzyme causes appearance of a new absorption peak near 500 mμ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Purification and Properties of l-Arginase from Bacillus subtilis

l-Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) was purified in a crystalline form from cells of Bacillus subtilis KY 3281 with an overall yield of 23.2%. The crystalline enzyme had a specific activity of 858 i.u./mg-protein and was ultracentrifugally homogeneous. It was estimated to have a molecular weight of 115,000±5000 by the method of Yphantis.

The enzyme highly specific for l-arginine showed the maximum activity at pH 10 with Mn²⁺ ion. The Km for l-arginine was 1.35 × 10^?2 m The activity was competitively inhibited by l-lysine, but not by l-ornithine and increased by the addition of Mn²⁺ or Co²⁺ ions. The stable pH and temperature ranges became wider in the presence of Mn²⁺ ion and l-threonine.     

Dihydrosterigmatocystin and Dihydrodemethylsterigmatocystin,New Metabolites from Aspergillus versicolor

L-Arabinose isomerase (L-arabinose ketol-isomerase, EC 5.3.1.4) was demonstrated from the L-arabinose-grown cells of Streptomyces sp. which was isolated from sea water. The enzyme was purified by MnCl₂ treatment, fractionation by polyethylene glycol and by column chromatographies on Sephadex G-150 and DEAE-cellulose. The purified enzyme was specific only for L-arabinose and the Michaelis constant for L-arabinose was 40 mM at pH 7.5. Manganese or cobalt ions were effective for the enzyme activity after dialysis against EDTA. The enzyme activity was inhibited competitively by L-arabitoI, ribitol and xylitol, of which inhibition constants were 1.1, 1.0, and 15 mM, respectively.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

Some Properties of Three Proteolytic Enzymes Excreted by Bacillus cereus Kp 931

The crude enzyme preparation obtained from culture media of Bacillus cereus Kp 931 was fractionated into three active fractions by Sephadex G-100 gel filtration. These three enzymes had pH optima at between 10.5 and 11.0. One of them, the largest molecular weight species, the enzyme I, was purified extensively. The enzyme catalyzes the release of a number of free amino acids from casein. Large amounts of l-alanine and l-glutamic acid and small amounts of l-leucine, l-serine, glycine, l-cysteic acid and l-arginine were released from oxidized insulin B-chain by the action of the purified enzyme I. It is also suggested that the other two enzymes, II and III, belong to so-called bacterial proteninases.     

Purification and Properties of Acid Carboxypeptidase III from Aspergillus oryzae

Acid carboxypeptidase III from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The optimum activity of the enzyme occurred at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, αα′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 61,000. The enzyme hydrolyzed the peptides that possess masked or bulky N-terminal.     

An Aminopeptidase of Aspergillus sojae

An aminopeptidase was purified from Aspergillus sojae X–816. The molecular weight of the enzyme was estimated to be 220,000. The isoelectric point was at pH 5.3. The optimum pH for l-leucylglycylglycine was 7.5. The enzyme was stable up to 37°C against temperature treatment for 15 min. Some chelating agents inhibited the enzyme activity. The Km value for l-leucylglycylglycine at pH 7.5 and 37°C was 45 mm. The Km value for l-leucyl-β-naphthylamide at pH 7.0 and 37°C was 2.2 mm.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Changes of α-Glycerophosphate Dehydrogenase Activity in Fatty Liver of Rats by Amino Acid Imbalance

An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184,000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki = 1.34 μm, 0.113mm and 0.145 mm, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mm). The enzyme was also inhibited by l-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on l-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl amino-peptidase [EC 3.4.11.14].     

γ-Glutamylcysteine Synthetase from Proteus mirabilis

The distribution of γ-glutamylcysteine synthetase (l-glutamate: L-cysteine γ-ligase, EC 6.3.2.2) was investigated in bacteria, and the enzyme was purified from Proteus mirabilis approximately 9,000-fold with an over-all yield of 10%. The purification procedure included ammonium sulfate fractionation, protamine treatment, DEAE-cellulose and hydroxylapatite column chromatographies and Sephadex gel filtrations. The purified enzyme was homogeneous by the criteria of ultracentrifugation. It showed multiple bands on disc-polyacrylamide gel electrophoresis and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band with a molecular weight of 62,000 was obtained on SDS-polyacrylamide gel electrophoresis after cross-linking of the enzyme with dimethylsuberimidate. The molecular weight was determined from the sedimentation and diffusion coefficients to be 64,000 and by Sephadex G-150 gel filtration to be 62,000. The purified enzyme catalyzed the stoichiometric formation of γ-glutamylcysteine and the reaction showed a sigmoidal dependence upon l-cysteine concentration. The enzyme also catalyzed γ-glutamyl amino acid formation from l-α-aminobutyrate, l-homoserine, glycine, l-serine, dl-norvaline or dl-homocysteine, but at lower rates than from l-cysteine. The γ-glutamyl-α-aminobutyrate formation by the enzyme did not show a sigmoidal but a hyperbolic dependence upon l-α-aminobutyrate concentration.     

Purification,Crystallization and Properties of Tryptophanase from Proteus rettgeri

An inducible tryptophanase was crystallized from the cell extract of Proteus rettgeri grown in a medium containing l-tryptophan. The purification procedure included ammonium sulfate fractionation, heat treatment, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystals were obtained from solutions of the purified enzyme by the addition of ammonium sulfate.

The crystalline enzyme preparation was homogeneous by the criteria of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be approximately 210,000.

The crystalline enzyme catalyzed the degradation of l-tryptophan into indole, pyruvate and ammonia in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from 5-hydroxy-l-tryptophan, 5-methyl-l-tryptophan, S-methyl-l-cysteine and l- cysteine. l-, d-Alanine, l-phenylalanine and indole inhibited pyruvate formation from these substrates.     

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.     

Purification and Characterization of Serine Hydroxymethyltransferase from Mitochondria of Euglena gracilis z

Mitochondrial serine hydroxymethyltransferase, l-serine: tetrahydrofolate 5,10-hydroxymethyl-transferase (EC 2.1.2.1), (m-SHMT) was extracted and highly purified from Euglena gracilis z. The specific activity increased from the crude extract with 10% yield up to 580-fold through the following steps: ammonium sulfate fractionation, DEAE-cellulose column chromatography and rechromatography, and affinity chromatography with l-lysine-Sepharose 4B. The molecular weight of the purified m-SHMT was 88,000 by gel filtration through Sephadex G-200, and 44,000 by SDS-PAGE. One mol of the purified enzyme contained two mol of pyridoxal 5′-phosphate (PLP), indicating that the enzyme is a dimer. Characteristics of the enzyme were examined and compared with SHMTs of other origins. The m-SHMT of Euglena gracilis z had l-threonine aldolase activity as did s-SHMT of the same origin in addition to the usual SHMT activity.