Purification of Inulin Fructotransferase (DFA I-producing) from Arthrobacter MCI2493 and Production of DFA I from Inulin by the Enzyme

An extracellular enzyme that produces di-d-fructofuranose-2′, 1;2, 1′ dianhydride from inulin was purified from the culture broth cf Arthrobacter sp. MCI2493. The molecular weight of the enzyme was 40,000 by gel filtration and SDS polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 6.0 and 50°C. Using this purified enzyme, 100g/liter inulin was converted into 60 g/liter of DFA I, nystose, and 1-f-fructofuranosyl-nystose after incubation for 30 h.     

Purification and properties of milk-clotting enzyme from Bacillus subtilis K-26

A milk-clotting enzyme from Bacillus subtilis K-26 was purified by gel filtration and ion-exchange chromatography resulting in a 24-fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugel analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a K_m of 2.77mg/ml for κ-casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk-clotting activity was obtained at 60°C, but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy-metal ions (Hg²⁺, Pb²⁺) inactivated the enzyme.     

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.     

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 Some Properties of An Aldehyde Oxidase from Streptomyces Rimosus ATCC10970

Summary An aldehyde oxidase was purified from a cell-free extract of Streptomyces rimosus ATCC10970 to an electrophoretically homogeneous state. The molecular mass of the native enzyme was estimated to be 150 kDa by a gel filtration. SDS-polyacryamide gel electrophoresis showed that the enzyme consisted of three non-identical subunits with molecular masses of 79, 39 and 23 kDa. The absorption spectrum revealed a distinctive feature as an enzyme belonging to the xanthine oxidase family with maxima at 277, 325, 365, 415, 450, 480, and 550 nm. A variety of aliphatic and aromatic aldehydes were oxidized, but nitrogen-containing heterocyclic compounds were not. Among the substrates tested, n-heptanal was most rapidly acted on. Its optimum pH and temperature were pH 7.0 and 30 °C, respectively.     

Purification and Properties of Cyclodextrin Glucanotransferase from Brevibacterium sp. No. 9605

Cyclodextrin glucanotransferase (EC 2.4.1.19) from Brevibacterium sp. No. 9605 was purified to homogeneity by chromatography on butyl-Toyopearl 650M, γ-cyclodextrin-Sepharose 4B, and Toyopearl HW-55S. The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the purified enzyme was 2.8. The optimum pH and temperature were pH 10 and 45°C, respectively. The enzyme was stable at the range of pH 6–8 and at temperatures 50°C or less in the presence of CaCl₂. The enzyme produced mainly γ-cyclodextrin from starch in the initial stage of reaction, but later, the proportion of β-cyclodextrin was increased.     

Purification and characterization of the amylase of B. subtilis NRRL B3411

The amylase of Bacillus subtilis NRRL B3411 has been purified and partially characterized. The specific activity can be increased from 300,000 units/g to 6,000,000 units/g with a 60% recovery of total units. The purified material consists of one major and one trace anodic component as determined by disc gel electrophoresis. The molecular weight was 48,000 as determined by bio-gel filtration; the molecular weight was 44,900 ± 2400 as determined by sedimentation equilibrium methods. This purified enzyme is stable at, 70°C in the presence of 0.01 M Ca⁺⁺ and 0.1 M NaCl over a broad pH range from 5.5–9.5. The pH activity profile indicates optimum activity at pH 6.0. This amylase exhibits maximum activity at 60°C. The enzyme is a liquefying α-amylase as determined by analysis of hydrolysis products and immunological studies.     

Induction of an Increase in Nerve Growth Factor Content in the Cell-conditioned Medium of Mouse L-M Cells by Basic Peptides

Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80–82% at 30–40°C. The activity was inhibited by Cu²⁺ , Hg²⁺, Zn²⁺, and Tris.     

Production of Pentameric Hybrid Immunoglobulins Consisting of IgA and IgM

The amylomaltase from Escherichia coli IFO 3806 was purified to homogeneity seen by SDS- polyacrylamide gel electrophoresis after DEAE-Sephadex, Ultrogel AcA 44, hydroxylapatite, and 1,6- hexane-diamine-Sepharose 4B column chromatographies. The molecular weight of the purified enzyme was 93,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5 and at 35°C, and stable up to 45°C at pH 7.0 and from pH 6.0 —7.3 at 40°C on 30min incubation. The enzyme acted on maltotetraitol, maltopentaitol, and maltosylsucrose besides maltooligosaccharides, but did not act on maltitol, maltotriitol, glucosylsucrose, isomaltose, panose, isopanose, or isomaltosyl- maltose. This enzyme did not catalyze hydrolytic action on maltotetraitol, maltopentaitol, or maltosylsucrose.     

Purification and Properties of Pyranose Oxidase from Coriolus versicolor

Coriolus versicolor KY2912 grown on a medium containing glucose, sucrose or glycerol produced pyranose oxidase. Pyranose oxidase (glucose-2-oxidase) was purified by HPA-75 chromatography, Sepharose 4B and Sephadex G-100 gel filtration, and hydroxyapatite chromatography. The purified enzyme preparation showed a single protein band on acrylamide gel electrophoresis. The highest activity was obtained when D-glucose was employed as substrate and molecular oxygen as electron acceptor. The enzyme was most active at pH 6.2 and 50°C, stable in the pH region between 5.0 and 7.4, and the activity was completely lost above 70°C. The activity was inhibited by Ag⁺ , Cu²⁺ and PCMB. The enzyme contained FAD covalently bound to the polypeptide chain. The enzyme consisted of identical subunits with a molecular weight of 68,000, and showed a total molecular weight of 220,000.     

Purification and Properties of an Asymmetric Reduction Enzyme of 2-Methyl-3-oxobutyrate in Baker’s Yeast

The benzyl 2-methyl-3-hydroxybutyrate dehydrogenase was purified from the cells of baker’s yeast by streptomycin treatment, Sephadex G-50 gel filtration, SP-Sephadex C-50 chromatography, and Toyopearl HW-60F gel filtration. The purified enzyme preparation was homogeneous and the molecular weight was about 31,000 to 32,000. The enzyme was NADPH-dependent and its maximum activity was at pH 7.0 and 45°C. It was stable between pH 6 and 9. The Km values at pH 7.0 were 0.42 mM for benzyl 2-methyl-3-oxobutyrate (1) and 4.2 mM for α-methyl β-hydroxy ester [syn-(2) and anti-(3)]. This enzyme reduced only benzyl 2-methyl-3-oxobutyrate (1) but had no effect on other synthetic substrates.

The reduced products [syn-(2) and anti(3)] produced by the purified enzyme were identified by 400 MHz NMR.     

Purification and Properties of Alcohol Oxidase from Candida methanosorbosa M-2003

Alcohol oxidase from Candida methanosorbosa was purified about sixfold with a yield of 37.6% from the culture broth of Candida methanosorbosa M-2003. The enzyme preparation was homogeneous on slab gel electrophoresis. The purified enzyme had an optimal pH from 6.0 to 9.0 and was stable in the range 6.0–8.5. Its optimal temperature of reaction was 50°C, and it was stable below 50°C. In the presence of NaN₃, the enzyme retained its initial activity at 30°C for 35 days, indicating stability for a long term, so far. The isoelectric point was pH 4.3. Its molecular weight was 620,000 by gel filtration chromatography and 80,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. These results indicate that the enzyme consists of 8 subunits. Received: 1 October 1996 / Accepted: 12 December 1996     

Some Properties of Transglycosylation Activity of Sesame β-Glucosidase

The transglycosylation reaction of partially purified β-glucosidase from sesame seeds with cellobiose is described. Sesame β –glucosidase was partially purified by ammonium sulfate fractionation and gel filtration. The molecular weight of the enzyme was 200,000 by gel filtration. Sesame β-glucosidase showed strong transfer activity to synthesize the trisaccharide from cellobiose. The optimum pH and temperature of the transglycosylation reaction were pH 4.0 and 60°C.     

Purification and Properties of Extracellular Acid Phosphatase from Zizania latifolia-pavasite,stilago esculenta

An extracellular acid phosphatase from Ustilago esculenta was purified to homogeneity on the basis of polyacrylamide gel electrophoresis. It was a glycoprotein with an isoelectric point of 4.7. The molecular weight of the enzyme was estimated to be about 343,000 by gel filtration on Sephadex G-200, whereas on SDS-polyacrylamide gel electrophoresis, the enzyme gave a single protein band with a molecular weight of 116,000. This result suggests that the enzyme consists of three identical subunits. The enzyme showed an optimum activity at pH 4.5, retained 90% of its activity for 10 min at 55°C and had a Km value of 0.25 mm for p-nitrophenylphosphate. No definite substrate specificity of the enzyme was observed.     

Glycogen Formation from Glycols and their Esters in the Livers of Chicks

Purification was conducted on polyvinyl alcohol (PVA) degrading enzyme produced and secreted into the culture medium by Pseudomonas O–3 strain. The enzyme was found to separate into several fractions by ion-exchange chromatography and gel filtration. Among these fractions, a fraction adsorbed to SP-Sephadex C–50 at pH 6.0 was purified to homogeneity by polyacrylamide gel electrophoresis. Some properties of this purified enzyme were examined. Optimum pH and temperature were 9.0 and 40°C, respectively. The enzyme was stable up to 50°C and in a pH range between 5 and 11 at 5°C. The enzyme activity was inhibited by Co²⁺, Ni²⁺, EDTA, hydroxylamine and salicylaldoxime. In substrate specificity, this enzyme oxidized several kinds of modified PVA, as well as normal PVA, but did not oxidize other synthetic polymers, such as vinylon, polyacrylamide and polyvinyl acetate. The effect of oxygen on this enzyme was examined, and without oxygen, PVA was not broken down by this enzyme. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G–100 to be approximately 26,000.     

Purification and Some Properties of Carboxylesterase from Arthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.     

Isolation and Some Properties of Acid Phosphatase-11 from Tomato Leaves

An isozyme of acid phosphatase-1, acid phosphatase-1¹, was purified from the leaves of tomato (Lycopersicon esculentum) to homogeneity and characterized. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The gel filtration analysis showed that the native molecule had a relative molecular mass of about 61 kilodaltons (kDa). The relative molecular mass of the subunit on gel electrophoresis with sodium dodecyl sulfate was about 32 kDa, indicating that the native form of the enzyme was a homodimer. It was suggested by periodic acid-Schiff staining on the gel that the enzyme was a glycoprotein. The Km for p-nitrophenylphosphate was 2.9 × 10^?3 m. The enzyme had a pH optimum of 4.5 in 0.15 m potassium acetate buffer with p-nitrophenylphosphate as a substrate. This enzyme was activated by divalent metal ions, such as Zn²⁺, Mg²⁺, and Mn²⁺. The N-terminal amino acids were sequenced after the purified enzyme was treated with pyroglutamylpeptidase. It was suggested that the N-terminal amino acid was pyroglutamate.     

Purification and characterization of an extracellular chitobiase fromTrichoderma harzianum

Chitobiase (EC 3.2.1.29), from the culture filtrate ofTrichoderma harzianum, was purified in sequential steps by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The physical and biochemical properties of the enzyme have been determined. The native enzyme has a molecular weight of 118 kDa when determined by gel filtration, and 64 kDa by SDS-PAGE. The enzyme catalyzed the hydrolysis of N,N-diacetylchitobiose andp-nitrophenyl--N-acetyl glucosamine with apparent Km of 575 µM and 235 µM, respectively. The pH optimum for the enzyme was pH 5.5, and maximum activity was obtained at 50°C. Glucosamine and N-acetylglyucosamine strongly inhibited the enzyme.     

Purification and Characterization of Poly(γ-glutamic acid) Hydrolase from a Filamentous Fungus,Myrothecium sp. TM-4222

Poly(γ-glutamic acid) (PGA) hydrolase was purified from the culture filtrate of a filamentous fungus, Myrothecium sp. TM-4222 and its general properties, especially the mode of hydrolytic action on the γ-glutamyl bond of PGA, were investigated. The purified preparation demonstrated a homogeneous band on an acidic slab gel of pH 4.3 with polyacrylamide gel electrophoresis. The enzyme showed its maximum activity at 37°C and at pH 5.0, being stable up to 40°C. The molecular mass was estimated to be 68 kDa by gel filtration. The hydrolytic action of the enzyme was specific for PGA, but not for other γ-glutamyl peptides or amides. The enzyme converted 38% of the original PGA with an average molecular mass of 500 kDa to smaller peptides, and then depolymerized these fragments to a mixture of γ-oligopeptides which consisted of only L-glutamic acid. L-Glutamic acid monomer was negligible in the reaction mixture. The remaining 62% of PGA was resistant to the enzyme action, in which D-glutamic acid was mainly detected. This study demonstrated a novel endo-type specificity of hydrolysis on PGA by the enzyme.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.