Leupeptins Produced by the Marine Alteromonas sp. B-10–31

Endo-1,4-β-D-mannanase (1,4-β-D-mannanohydrolase, EC 3.2.1.78) was purified from viscera of a mud snail, Pomacea insularus (de Ordigny). The purified enzyme gave a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was estimated to be 44,000. The amino-terminal sequence was H· Gly-X-Leu-Arg-Arg-Gln-Gly-Thr-Asn-Ile-Val-Asp-Ser-His-Gly-His-Lys-Val-Phe-Leu-Ser-Gly-Ala-Asn-Thr-Ala-Trp-Val-Ala-Tyr-Gly-Tyr-Asp-. The enzyme was stable from pH about 5.0 to about 10.5 and had its maximum activity at pH about 5.5. The purified enzyme produced M2, M3, M4,and M5 from β-1,4-mannan. Enzyme activity was greatly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and dithiothreitol at 1 mM concentration. In addition, N-bromosuccinimide completely inhibited the enzyme activity.     

Structure of a New Opine,Mikimopine, in Hairy Root Induced by Agrobacterium rhizogenes

A enzyme that catalyzed the specific formation of ascorbic acid-2-phosphate (AsA2P) from ascorbic acid (AsA) and adenosine-5′-triphosphate (ATP), was purified 3,200-fold to homogeneity from a cell extract of Pseudomonas azotocolligans. The purified enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and consisted of a single polypeptide with a molecular weight of about 30,000. Of phosphoryl donors tested, p-nitrophenylphosphate (p-NPP) and pyrophosphate (PPi) were as effective as ATP. Optimal pHs for the phosphorylating activity were around 4.0 and 5.5 when PPi and ATP were used as phosphoryl donors, respectively. The Km for AsA was 147 mm. The enzyme activity was inhibited by Cu²⁺, but not by sulfhydryl reagents.

The enzyme simultaneously had phosphatase activity at weakly acidic or neutral pH and the Km for p-NPP in the phosphatase activity was 0.38 mm. The enzyme was tentatively named “ascorbic acid phosphorylating enzyme.”     

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 and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

New Nematicidal Metabolites from a Fungus,Irpex lacteus

N-Benzoyl-l-alanine amidohydrolase was purified from a cell-free extract of Corynebacterium equi H-7 which was grown in a medium containing hippuric acid as the sole carbon source. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The molecular weight was 230,000 and the enzyme consisted of six subunits, identical in molecular weight (approximately 40,000). The isoelectric point of the enzyme was pH 4.6. The optimum pH of the enzyme reaction was 8.0 and the enzyme was stable from pH 7.0 to 8.0. The enzyme hydrolyzed N-benzoyl-l-alanine, N-benzoylglycine, and N-benzoyl-l-aminobutyric acid. The Km values for these substrates were 4.3 mm, 6.7 mm, and 4.3 mm, respectively. The enzyme was activated by Co²⁺.     

Crystallization and Characterization of Agmatine Oxidase from Penicillium chrysogenum

Agmatine oxidase was purified and crystallized with an overall yield of about 30% from a mycelial extract of Penicillium chrysogenum by a procedure involving ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, 1,8-diaminooctane-Sepharose 4B and Sephadex G-200 column chromatographies. The purified enzyme was homogeneous on disc gel electrophoresis and the pink crystals appeared as a hexagonal board on addition of solid ammonium sulfate. The molecular weight of the native monomer form was determined to be 160,000 by gel filtration, and it was composed of two identical subunits. The prosthetic group was identified as copper and its content was determined to be 2 mol per mol of the enzyme. The enzyme was inhibited by hydroxylamine, hydrazine, phenylhydrazine, semicarbazide, KCN, PCMB, Ag⁺, Hg²⁺ and Cu²⁺. The apparent Km values for agmatine, histamine and putrescine were calculated to be 2.51 × 10^?4m, 4.25 × 10^?4m and 1.64 × 10^?2m, respectively.     

A Highly N-Glycosylated Chitin Deacetylase Derived from a Novel Strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2–4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1–10 mM Co²⁺ and strongly inhibited by 10 mM Cu²⁺. It required at least two GlcNAc residues for catalysis. When (GlcNAc)₆ was used as substrate, K _m and V _max were determined to be 1.1 mM and 54.6 μmol min^?1 respectively.     

Pisiferdiol,a Novel Phenolic Diterpenoid from Chamaecyparis pisifera Endl

Isocitrate lyase was purified from the purple nonsulfur bacterium Rhodopseudomonas sp. No. 7. The purified enzyme was electrophoretically homogeneous. The molecular weights of the native enzyme and its subunit were estimated to be approximate 250,000 and 62,000 by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively. The optimum pH for its activity was 6.5. The optimum temperature was 45°C. The Km for dl-isocitrate was 0.136 mm in potassium phosphate buffer (pH 6.0). Mg²⁺ was required for full activity of the enzyme as a non-essential activator. The enzyme activity was inhibited by SH-blocking reagents. Non-competitive inhibitory effects on the enzyme were examined with malate and succinate. The Ki for malate and succinate were 2.7 and 0.24 mm, respectively.     

Purification and Properties of Pantothenate Kinase from Brevibacterium ammoniagenes IFO 12071

Pantothenate kinase (ATP: pantothenate 4′-phosphotransferase, EC 2.7.1.33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45,000. The enzyme catalyzed the formation of pantothenic acid 4′-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^?5 m for pantothenate, 3.5×10^?5 m for ATP, and 10^?3 m for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.     

A New Simple Method for Isolating Multistress-Tolerant Semidominant Mutants of Saccharomyces cerevisiae by One-Step Selection under Lethal Hydrogen Peroxide Stress Condition

A tetrathionate-decomposing enzyme that catalyzes the decomposition of tetrathionate into thiosulfate and sulfate was purified to homogeneity from tetrathionate-grown Thiobacillus thiooxidans. The enzyme had an apparent molecular weight of 104,000, and was composed of two identical subunits (MW = 58,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point at 9.6 and was most active at pH 3.0–3.5 and 40°C. Enzyme activity was increased approximately 100-fold in the presence of 400 mm sulfate ion. The Michaelis constant of this enzyme for tetrathionate in the presence of 20, 50, and 200 mm of sulfate ion was 2.4 mm. Mercuric and ferric ions completely inhibited the enzyme activity at 1 mm. Though cupric ion up to 0.01 mm markedly stimulated the activity in the presence of 20 mm sulfate ion, a higher concentration (1 mm) rather strongly inhibited the activity. Ethylenediaminetetraacetic acid (EDTA) strongly inhibited the activity, but this inhibiton was completely restored by cupric ion.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Effect of Substrate Concentration on Plastein Productivity and Some Rheological Properties of the Products

A bacterial arginase was purified to homogeneity from a strain of Bacillus brevis. The native enzyme, with an estimated MW of 143,000, migrated on SDS-PAGE as a single polypeptide of estimated MW of 33,000. The enzyme, highly specific to l-arginine, showed the maximum activity at pH 11.0 in the presence of Mn²⁺ ions and the pI was 4.8 by isoelectric focusing. The enzyme activity was increased significantly by the addition of Mn²⁺, Ni²⁺, or Co²⁺ ions, and inhibited potently by chemicals such as HgCl₂, N-bromosuccinimide, or glutathione. The K_ms for l-arginine and l-canavanine were 0.69 and 22.2 mm, respectively. The enzyme was inhibited competitively by γ-guanidinobutyric acid, and non-competitively by l-lysine, l-ornithine, creatine, blasticidin S, and edeine B₁ Analysis of the N-terminal amino acid sequence of the purified bacterial enzyme found 33–36% homologies with the Agrobacterium, yeast, rat, and human enzymes.     

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.     

α-Galactosidase from Alfalfa Seeds (Medicago sativa L.)

An α-galactosidase from alfalfa seeds was purified 140-fold by ammonium sulfate fractionation, and column chromatography on Sephadex G-100, DEAE- and CM-Sephadex. Polyacrylamide-gel electrophoresis of the purified enzyme showed a single protein band. The molecular weight was estimated to be approximately 57,000 by gel-filtration. The purified enzyme hydrolyzed p-nitrophenyl α-d-galactoside more rapidly than raffinose. The maximal enzyme activities were obtained at pH 4.0 and 5.5 for p-nitrophenyl α-d-galactoside and at 4.5 for raffinose. The enzyme was shown to be inhibited by Hg²⁺ and Ag⁺ ions, and d-galactose.     

Synthesis and Degradation of 1-Aminocyclopropane-1-carboxylic Acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M _r 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the K _m for S-adenosyl-L-methionine of 1.74 mM and k _cat of 0.56 s^-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M _r 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a K _m for ACC of 4.8 mM and k _cat of 3.52 s^-1. The presence of 7 mM Cu²⁺ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

Characterization of Endo β-1,3-Glucanase IV from Flavobacterium dormitator var. glucanolyticae FA-5

Endo β-1,3-glucanase IV (E.C. 3.2.1.6, endo-1,3(4)-β-d-glucanase) from Flav. dormitator var. glucanolyticae FA-5 was shown to be a glycoprotein by gel filtration and sodium dodecyl sulfate gel electrophoresis. The carbohydrate moiety was composed of 17 hexose units. The enzyme had an apparent molecular weight of 3.3 x 10⁴, determined by gel filtration, sodium dodecyl sulfate gel electrophoresis and ultracentrifugation. The enzyme showed maximum reactivity at pH 6.0 and 6.5 for living yeast cells and laminaran, respectively. The enzyme predominantly released laminaripen-taose from a variety of linear β-1,3-glucans and showed transglucanosylation activity. The amino acid composition of the enzyme and some of its physicochemical and enzymatic properties are described.     

Purification and some Properties of β-Xylosidase from Trichoderma viride

β-Xylosidase was purified 25 fold from a culture filtrate by ammonium sulfate fractionation, DEAE-Sephadex chromatography, column electrophoresis, gel filtration on Biogel P-100, and isoelectric focusing. The purified β-xylosidase was found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis and on disc electrophoresis. A molecular weight of 101,000 was estimated by chromatography on Sephadex G-200, and 102,000 was obtained by SDS polyacrylamide gel electrophoresis. The purified p-xylosidase had an isoelectric point at pH 4.45, and contained 4.5% carbohydrate residue. The optimum activity for the enzyme was found to be at pH 4.5 and 55°C. The enzyme activity was inhibited by Hg^{2 +}, and N-bromosuccinimide at a concentration of 1 x 10^?3 m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o—13.0 sec”¹), p-nitrophenyl β-d-xyloside (k_o=2l.3 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 22.2 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 20.0 sec^?1), p-methylphenyl β-d-xyloside (k_o~9.0 sec^?1), o-methylphenyl β-d-xyloside (k_o= 10.7 sec^?1), p-methoxyphenyl β-d-xyloside (k_o=10.3 sec^?1), o-methoxyphenyl β-d-xyloside (&;_o=10.9 sec^?1), xylobiose (k_o = 36A sec^?1), xylotriose (k_o = 34.5 sec^?1), xylotetraose (k_o~HA sec^?1), and xylopentaose (k_o= 13.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of configuration. The purified p-xylosidase was practically free of α-xylosidase and β-glucosidase activities.     

Purification and Properties of a Novel Enzyme,Maltooligosyl Trehalose Synthase,from Arthrobacter sp. Q36

Arthrobacter sp. Q36 produces a novel enzyme, maltooligosyl trehalose synthase, which catalyzes the conversion of maltooligosaccharide into the non-reducing saccharide, maltooligosyl trehalose (α-maltooligosyl α-D-glucoside) by intramolecular transglycosylation. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on Sepabeads FP-DA13, DEAE-Sephadex A-50, Ultrogel AcA44, and Butyl-Toyopearl 650M. The enzyme was specific for maltooligosaccharides except maltose, and catalyzed the conversion to form maltooligosyl trehalose. The K_m of the enzyme for maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were 22.9mM, 8.7mM, 1.4mM, and 0.9mM, respectively. The enzyme had a molecular mass of 81,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal and C-terminal amino acids of the enzyme were methionine and serine, respectively. The enzyme showed the highest activity at pH 7.0 and 40°C, and was stable from pH 6.0 to 9.5 and up to 40°C. The enzyme activity was inhibited by Hg²⁺ and Cu2⁺.