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.     

Studies on Glucose-Isomerizing Enzyme of Bacteria

d-Glucose-isomerizing enzyme from Escherichia intermedia HN-500, which converts d-glucose to d-fructose in the presence of arsenate, was purified by treating with manganous sulfate, rivanol, and DEAE-Sephadex column chromatography. About 180-fold purified enzyme preparation was obtained by the above procedures. The purified preparation was free from the activities of d-glucose-, d-galactose-, glucose-6-phosphate-, mannitol-, and sorbitol-dehydrogenases and was homogeneous on polyacrylamide gel in zone electrophoresis. Optima of pH and temperature for the enzyme were found to be pH 7.0 and 50°C, respectively. The enzyme was completely inactivated by heating at 60°C for ten minutes and stable in the pH range of 7.0~9.0 at 30°C. Activation energy for the isomerizing enzyme was calculated to be 15,300 calories per mole degree from Arrhenius' equation. Either in the absence or presecne of arsenate, d-mannose, d-xylose, d-mannitol and d-sorbitol could not be isomerized by the purified enzyme at all, but the present enzyme isomerized exclusively glucose-6-phosphate and fructose-6-phosphate in the absence of arsenate.     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

Purification and Some Properties of Chaetomium trilaterale β-Xylosidase

A β-xyloside hydrolytic enzyme of the fungus Chaetomium trilaterale was further purified by a modification of Kawaminami’s procedure (DEAE-Sephadex A-25 and Sephadex G-75 column chromatography), followed by isoelectric focusing. The purified preparation was homogeneous by polyacrylamide disc gel electrophoreses at pH 4.3 and pH 8.3. The purified enzyme hydrolyzed β-d-glucopyranosides as well as β-d-xylopyranosides, and the ratio of β-glucosidase activity against β-xylosidase activity increased about 3 fold during the purification steps. The molecular weight of this preparation was estimated to be about 240,000 by Sephadex G-200 gel filtration and 118,000 by SDS-polyacrylamide slab gel electrophoresis. The isoelectric point was 4.86 and the amino acid composition was also determined.

The optimum pH was at 4.2 for phenyl β-d-glucoside and around 4.5 for phenyl β-d-xyloside. The β-xylosidase activity was relatively stable but β-glucosidase activity was rapidly inactivated, at the alkaline pH range above 11. The heating of the preparation at 60°C didn’t show a parallel inactivation of the two activities. N-Bromosuccinimide strongly inactivated both enzyme activities. Nojirimycin and glucono-l,5-lactone showed a stronger inhibition on β-xylosidase activity than on β-glucosidase activity. The maximal velocities decreased in the order; phenyl β-d-glucoside > cellobiose > phenyl β-d-xyloside > xylobiose; the value with phenyl β-d-glucoside was about 28-fold higher than that with phenyl β-d-xyloside.     

Metabolism of Aromatic Amino Acid in Microorganisms

Phenylalanine ammonia-lyase, which catalyzes the conversion of l-phenylalanine to trans-cinnamic acid and ammonia, has been partially purified from the cells of Rhodotorula. Some of the properties of this phenylalanine ammoyia-lyase were investigated. The enzyme was stable in phosphate buffer of pH over the range of 6.0 to 7.0 On heating, the enzyme was stable up to 50°C, but above 60°C, it was destroyed. The enzyme activity was strongly inhibited by p-chloromercuribenzoate at 10^?5 m and almost recovered by the addition of glutathione or mercaptoethanol at 10^?3 m. The present enzyme preparation of Rhodotorula also catalyzed the deamination of l-tyrosine to trans-p-coumaric acid. trans-p-Coumaric acid was isolated from the reaction mixture and identified by its absorption spectra. The rates of deamination showed optima at pH 9.0 and 9.5 for l-phenylalanine and l-tyrosine, respectively.     

Partial Purification and Characterization of Polyphenol Oxidase Isozymes in Banana Bud

Polyphenol oxidase was extracted from banana buds in the presences of Triton X-100, isoascorbate, and Polyclar AT, and two isozymes I and II have been separated and partially purified by chromatographies on Butyl Toyopearl 650 and DEAE-cellulose. I and II had different mobility in polyacrylamide gel electrophoresis with optimum pHs of 6.8 and 5.5, respectively. Both enzymes showed the apparent K_m values of 0.5 mM for dopamine with substrate inhibitions at its higher concentrations. I and II were inhibited competitively by NaCI with the K_i values of 140 mM and 40 mM, respectively. I and II have a high heat stability, and 88 and 95% of the initial activities were retained after 1-hr incubation at 70°C, respectively.     

Partial Purification and Some Properties of Extracellular Asparaginase from Candida utilis

Extracellular asparaginase from Candida utilis was partially purified by precipitation with acetone and by column chromatography on DEAE Sephadex A-50 and Sephadex G-200. The specific activity of the enzyme preparation was 3900 units per mg of protein. Candida asparaginase characteristically had deaminating activity for d-asparagine as well as for l-asparagine. But this enzyme was not able to hydrolyzed l- or d-glutamine. SH inhibitor, chelating agents and metal ions did not show any inhibition or activation of l-asparaginase activity. Optimum pH was about 6 for both l- and d-asparagine. This asparaginase was stable between pH 4 and pH 10 in heating for 10 min at 50°C.     

Cyclic (1→2)-β-d-Glucan-hydrolyzing Enzymes from Acremonium sp. 15 Purification and Some Properties of Endo-( 1 → 2)-α-d-glucanase and β-d-Glucosidase

Acremonium sp. 15 a fungus isolated from soil, produces an extracellular enzyme system degrading cyclic (1→2)-β-d-glucan. This enzyme was found to be a mixture of endo-(1→2)-β-d-glucanase and β-d-glucosidase. The (1→2)-β-d-glucanase was purified to homogeneity shown by disc-electrophoresis after SP-Sephadex column chromatography, Sephadex G-75 gel filtration, and rechromatography on SP-Sephadex. The molecular weight of the enzyme was 3.6 × 10⁴ by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was pH 9.6. The enzyme was most active at pH 4.0—4.5, and stable up to 40°C in 20 mm acetate buffer (pH 5.0) for 2 hr of incubation. This enzyme hydrolyzed only (l→2)-β-d-glucan and did not hydrolyze laminaran, curdlan, or CM-cellulose. The hydrolysis products from cyclic (1→2)-β-d-glucan were mainly sophorose.

The β-d-glucosidase was purified about 4000-fold. The rate of hydrolysis of the substrates by this β-d-glucosidase decreased in the following order: β-nitrophenyl-β-d-glucoside, sophorose, phenyl-β-d-glucoside, laminaribiose, and salicin. This enzyme has strong transfer action even at the low concentration of 0.75 mm substrate.     

Purification and Characterization of an Intracellular α-D-Xylosidase II from Aspergillus flavus MO-5

α-D-Xylosidase II activity from Aspergillus flavus MO-5 was increased roughly 5- to 10-fold by use of xylose instead of methyl α-D-xylopyranoside (α-MX) as a carbon source.

The enzyme was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SWXL. The purified enzyme hydrolyzed isoprimeverose [α-D-xylopyranosyl-(1→6)-D-glucopyranose] and p-nitrophenyl α-D-xylopyranoside (α-p-NPX), but not α-MX or xyloglucan oligosaccharide. The apparent K_m and V_max of the enzyme for α-p-NPX and isoprimeverose were 0.97 mM and 28.0 µmol/min/mg protein, and 47.62 mM and 2.0 µmol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 67,000 by SDS-polyacrylamide gel electrophoresis and 180,000 by gel filtration chromatography (TSK-gel G3000SWXL).

The enzyme showed the highest activity at pH 6.0 and 40°C, and was stable in the pH range from 6.0 to 7.0 and at the temperatures up to 40°C. The activity was inhibited by Cu²⁺, Zn²⁺, Hg²⁺, p-CMB, SDS, Fe³⁺, and N-ethylmaleimide.

This enzyme had nothing in common with α-D-xylosidase I and four α-D-xylosidases reported already.     

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.     

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.     

Studies on the Pentose Isomerases of Lactic Acid Bacteria

Production of d-xylose and l-arabinose isomerases by lactic acid bacteria was greatly promoted by the addition of manganese ions in cultural medium. Effective concentration of the ions was 5 × 1O^-3 m. Ferrous ions were also effective for the production of d-xylose isomerase and cobaltous ions were somewhat effective for the production of l-arabinose isomerase. Zinc and cadmium ions inhibited bacterial growth. It was possible to increase the production of isomerase by changing MnSO₄ concentration to 5× 10^-3 m (0.l1 %) in place of 0.001 per cent in the normal medium.

Column chromatographic procedures for the purification of pentose isomerases were carried out. Cation and anion exchange resins were not suitable because of their low exchange capacities and instability of the enzyme at acidic pH range. But the isomerases were successfully purified by DEAE-cellulose column chromatography with high recovery (85~90%). Using a Tris buffer, KCl concentration was increased in gradient. d-Xylose isomerase was eluted at pH 7.0 at 0~0.2 m KCl, and l-arabinose isomerase at pH 8.0 at 0~0.4 m KCl. The purified isomerases, d-xylose isomerase and l-arabinose isomerase, both required manganese ions specifically for their activities.

D-Xylose isomerase and l-arabinose isomerase are different enzymes which can be separated from each other with acetone fractionation at pH 4.8~5.0, heat treatment or chromatography on a colnmn of DEAE-cellulose. In DEAE-cellulose chromatography with a linear gradient elution method, d-xylose isomerase is recovered in the first peak at pH 7.0 (Tris bnffer) with 0~0.2 m KCl, and l-arabinose isomerase is eluted in the second peak at pH 8.0 (Tris buffer) with a larger ionic strength.     

Identification of Arsenobetaine as a Major Arsenic Compound in the Ivory Shell Buccinum striatissimum

A restriction endonuclease, designated as DmaI, was purified from cell-free extracts of Deleya marina IAM 14114 by streptomycin treatment, ammonium sulfate fractionation and two steps of chromatographies on heparin-Sepharose CL-6B and Mono Q (HR 5/5, FPLC). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis and a ligation-recutting test. The relative molecular mass measurements of the purified enzyme gave 28,000 daltons by SDS-polyacrylamide gel disk electrophoresis and 56,000 daltons by gel filtration. These data indicated that the purified enzyme (56,000 daltons) has a dimeric structure composed of two 28,000-dalton subunits. The isoelectric point was 5.5. The purified enzyme worked best at 37°C in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10 mm Tris–HCl, 7 mm 2-mercaptoethanol, 7 mm MgCl₂ and 100 mm NaCl (pH 7.5). The enzyme was stable up to 55°C and between pH 7.0 and 9.0. The purified enzyme recognizes the palindromic hexanucleotide DNA sequence 5′-CAGCTG-3′, cuts between G and C and produces a flush end (isoschizomer of PvuII).     

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.     

Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.     

Multiple Forms of α-Glucosidase from Pig Duodenum

Multiple forms of neutral α-glucosidase (pH optima, 6.0~6.5) were purified from pig duodenal mucosa by a procedure including Triton X-100 treatment, fractionation with ammonium sulfate, fractionation with ethyl alcohol, DEAE-cellulose column chromatography and preparative polyacrylamide disc gel electrophoresis. All of the α-glucosidases, I_a, II_a, I_b and II_b, were found to be homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights, isoelectric points and optimum temperatures of α-glueosidases I_a and II_a were 145,000~150,000, pH 3.5~3.7 and 55°C, respectively, and both enzymes were stable up to 55°C on treatment at pH 6.0 for 15 min; whereas those of the other two α-glucosidases, I_b and II_b, were 80,000, pH 4.0~4.1 and 65°C, respectively, and both enzymes were stable up to 70°C on the same treatment. The Km values of enzyme II_a for maltose, maltotriose and amylose were 1.72mm, 0.37 mm and 1.67mg/ml, while those of enzyme II_b were 3.33 mm, 2.61 mm and 11.8 mg/ml, respectively. All enzyme hydrolyzed α-1,4-, α-1,3- and α-1,2-glucosidic linkages in substrates, but showed no activity on sucrose or isomaltose. Enzymes II_a and II_b hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside, and maltotriose was formed as the main α-glucosyltransfer product from maltose. It was revealed that two types of neutral α-glucosidases having no activity toward sucrose or isomaltose existed in pig duodenal mucosa, and that one type comprised α-glucosidase having both maltose- and amylaceous α-glucan-hydrolyzing activities and the other type heat-stable maltooligosaccharidases which hydrolyzed amylaceous α-glucan weakly.     

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⁺.     

Studies on d-Glucose-isomerizing Activity of d-Xylose-grown Cells from Bacillus coagulans,Strain HN-68

d-Glucose-isomerizing enzyme has been extracted in high yield from d-xylose-grown cells of Bacillus coagulans, strain HN-68, by treating with lysozyme, and purified approximately 60-fold by manganese sulfate treatment, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex column. The purified d-glucose-isomerizing enzyme was homogeneous in polyacrylamide gel electrophoresis and ultracentrifugation and was free from d-glucose-6-phosphate isomerase. Optimum pH and temperature for activity were found to be pH 7.0 and 75°C, respectively. The enzyme required specifically Co⁺⁺ with suitable concentration for maximal activity being 10^?3 m. In the presence of Co⁺⁺, enzyme activity was inhibited strongly by Cu⁺⁺, Zn⁺⁺, Ni⁺⁺, Mn⁺⁺ or Ca⁺⁺. At reaction equilibrium, the ratio of d-fructose to d-glucose was approximately 1.0. The enzyme catalyzed the isomerization of d-glucose, d-xylose and d-ribose. Apparent Michaelis constants for d-glucose and d-xylose were 9×10^?2 m and 7.7×10^?2 m, respectively.     

Purification and Properties of d-Xylose Isomerase from Alkalophilic Bacillus No. KX-6

An alkalophilic Bacillus No. KX-6 isolated from soil produced a d-xylose isomerase in alkaline media. The striking characteristic of this bacterium was its especially good growth in alkaline media. The d-xylose isomerase of this bacterium was purified by ammonium sulfate fractionation, DEAE-Sepharose ion exchange column chromatography and G-200 gel Alteration. The molecular weight and sedimentation constant were approximately 120,000 and 9.35 S, respectively. The enzyme was most active at pH 7~10 and was stable at pH 6.0 to 11.0. Enzyme activity was stimulated by cobalt ion but inhibited by Hg^{2 +}, Ag^{2 +}, and Cu^{2 +}. Substrate specificity studies showed that this enzyme was active on d-xylose, d-glucose, d-ribose, and d-arabinose. The smaller Km value and larger V_max value for d-xylose indicated that this enzyme is essentially d-xylose isomerase.     

Properties of Nucleoside Phosphorylase from Enterobacter aerogenes

The properties of uridine Phosphorylase (UPase) and purine nucleoside Phosphorylase (PNPase) at high temperature were investigated. Both enzymes were found to be distributed in a wide range of bacteria and were partially purified from Enterobacter aerogenes AJ 11125 by heat treatment, ammonium sulfate fractionation and column chromatographies onDEAE-cellulose and Sephadex G-150. The UPase was purified 109-fold, and it showed an optimum pH of 8.5 and optimum temperature of 65°C, and activity toward uridine, 2′-deoxyuridine, thymidine and uracil arabinoside but not cytidine. The Km values of UPase for uridine were 0.7 mm at 40°C and 1.8 mm at 60°C. The PNPase was purified 83-fold, and it showed an optimum pH of 6.8 and optimum temperature of 60°C, and significant activity toward purine arabinosides as well as purine ribosides. The Km values of PNPase for inosine were 0.8 mm at 40°C and 2.2 mm at 60°C.