Purification and Properties of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

From the mycelia of Neurospora crassa (wild type No. 6068) multiple forms of a nuclease which had very close isoelectric points (pI = 9.6 (peak I), 9.4 (peak II)) were isolated by ampholine electrofocusing column chromatography (pH 8.5 ~ 10). The nuclease was about 300-fold purified from the crude extract. The two fractions of Peak I, II were indistinguishable in their enzymatic properties and were considered as manifestation of the same enzyme with minor physicochemical differences. The molecular weight was around 41,000 as estimated by the gel filtration method. The enzyme could hydrolyze both DNA and RNA in the order of heat-denatured DNA > native DNA DNA ≧ RNA. RNA competitively inhibited DNA degradation with this enzyme. The enzyme was therefore regarded as a nuclease. The pH optimum was around pH 6.5 toward native DNA, pH 6.7 toward heat-denatured DNA and pH 7.9 toward RNA. The temperature optimum was around 40°C toward these substrates and most of the activities were lost by heating at 55°C for 15 min. The enzyme required Mg²⁺ for action toward heat-denatured DNA and Mg²⁺, Mn²⁺ or Co²⁺ toward native DNA. In the presence of EDTA, the activities toward both types of DNA were lost and recovered by addition of the respective activating metallic ions. p-CMB inhibited this nuclease, but β-mercapto-ethanol and glutathione had no effect. Polyamìnes showed no activation of the nuclease for DNA degradation.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

Folic acid effects on glycoprotein-galactosyltransferase: a re-assessment

Prostaglandin Δ¹³-reductase was purified extensively by ammonium sulfate fractionation, and DEAE-Sephadex-, hydroxylapatite-, and phosphocellulose chromatography. Enzyme activity was followed by radioimmunoassay with the use of an antiserum directed toward 15-keto-prostaglandin F_2α and antibodies directed toward 13, 14 dihydro-15-keto-prostaglandin F_2α. The purified enzyme used NADPH as a cofactor much more effectively than NADH. It specifically reduced 15-keto-prostaglandins but not 15-hydroxy-prostaglandins. The enzyme was inhibited by p-chloromercuribenzoate. It had a relatively broad pH optimum (pH 7.4 to pH 8.5), and has a molecular weight estimated to be 70,000 to 80,000.     

Purification and Some Properties of α-Galactosidase from Candida guilliermondii H-404

Candida guilliermondii H-404, isolated from soil, produced thermostable α-galactosidase, but small amounts of other glycosidases (such as β-galactosidase, α-glucosidase, and β-glucosidase). The enzyme was separated into two fractions by DEAE-Toyopearl 650M chromatography, and the two enzymes were designated galactosidase I and II. These two enzymes had the same molecular weight (270,000 by gel filtration, 64,000 by SDS-PAGE). The isoelectric points of α-galactosidase I and II were 6.16 and 6.21, respectively. These two enzymes were different from each other in pH stability, temperature stability, and effects of Fe^{2 +} and Cu^{2 +} ion on α-galactosidase activity. The enzyme had stronger transfer activity and wider acceptor specificity than α-galactosidases which have been reported.     

Synthesis of 1,2,3,4-Tetrahydro-1-oxo-pyrido[l ,2-a]benzimidazole

In Euglena gracilis arginine deiminase was located in the mitochondrial matrix. The highly purified enzyme required Co²⁺ for the enzyme reaction with the Km value of 0.23 mM, and its optimum pH was 9.7 to 10.3. The molecular weight of the native enzyme protein was 87,000 by gel filtration, and SDS-acrylamide gel electrophoresis showed that the enzyme consisted of two identical subunits with a molecular weight of 48,000. Euglena arginine deiminase was inhibited by sulfhydryl inhibitors, indicating that a sulfhydryl group is involved in the active center of the enzyme. It exhibited negative cooperativity in binding with arginine. l-α-amino-β-guanidino-propionate, d-arginine, and l-homoarginine strongly inhibited the enzyme while β-guanidinopro-pionate, γ-guanidinobutyrate, and guanidinosuccinate did not. Considerable inhibition was also observed with citrulline and ornithine. We discuss the effects of the unique properties of the Euglena arginine deiminase on the regulation of arginine metabolism in this protozoon.     

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.     

Constituents of the Essential Oil of Chrysanthemum japonense var. debile

One hundred fifty strains of actinomycetes were isolated from soils on plate cultures containing beet arabinan as the sole carbon source. About one-third of the culture fluids were found to have arabinosidase activity. A wild-type strain, Streptomyces sp. No. 17-1, was selected as the best producer of arabinosidase. The highest enzymatic activity was obtained in the culture fluid when the initial pH was adjusted to 9.0. An α-l-arabinofuranosidase was highly purified from the culture filtrate of No. 17-1 by combining column chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and isoelectric focusing. The molecular weight of the purified enzyme was estimated to be about 92, 000, and its isoelectric point was pH 4.4. The enzymatic activity was maximum at pH 6.0 and was completely inhibited by Hg²⁺. The apparent Km value of the enzyme for p-nitrophenyl-α-l-arabinofuranoside was determined to be 3.6 mM.     

An Arylamidase from Flavobacterium meningosepticum

An arylamidase was purified from Flavobacterium meningosepticum by a series of chromatographies on CM-cellulose, DEAE-Sephadex A-50 and Sephadex G-150. The purified enzyme appeared homogeneous on SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be more than 500,000 dalton by using a column of Sepharose 4B and to be 62,000 when checked by SDS-gel electrophoresis. The enzyme was most active at pH 7.5 toward Leu-β-naphthylamide (Leu-β-NA). It catalyzed the hydrolysis of not only various amino acid-β-naphthylamides but also some peptides, but the hydrolysis rate of the latter substrates was quite low. Cys-di-β-naphthylamide was split by this enzyme at an optimal pH of 6.2. Incubation of oxytocin with the enzyme resulted in a decrease in the biological activity, indicating that this arylamidase possesses an oxytocinase (cystyl aminopeptidase)-like activity.     

Isolation and Characterization of Nα-Benzyloxycarbonyl Amino Acid Urethane Hydrolase II from Lactobacillus fermenti 36 ATCC 9338

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of N^α-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co²⁺, is essential for its activity.     

Purification and Some Properties of Metal Proteinases from Lentinus edodes

To investigate the function of proteinases in the fruiting of Basidiomycetes, we purified the neutral proteinase in vegetative mycelium of Lentinus edodes. About 1.6 mg of purified enzyme was obtained from 1.5 kg of mycelium. The purified enzyme was confirmed to be monodispersive on disc electrophoresis.

The neutral proteinase was most active around pH 7.5 toward hemoglobin and 7.0 toward casein and was extremely labile with temperature. The enzyme was strongly inhibited by EDTA or Talopeptin (MK-I). The molecular weight and isoelectric point of the enzyme were 45,000 and pH 5.3, respectively. The enzyme contained no methionine residues. The enzyme hydrolyzed the bonds involving hydrophobic or bulky amino acid residues of oxidized insulin B-chain such as His-Leu (10–11 and 5–6), Leu (17)-Val (18) and Ala (14)-Leu (15).

These characteristics are compared with those of the metal proteinase in the fruit-body of the same fungus, which was purified and characterized at the same time as in vegetative mycelium. We also compare it with proteinases from other microbes.     

Purification and Some Properties of Saccharomyces logos α-Glucosidase

α-Glucosidase was purified from Saccharomyces logos by precipitation with ethanol, and chromatographies on Sephadex G–200, DEAE-Sephadex, DEAE-ceiluiose and Duolite A–2. The purified α-glucosidase was homogeneous on ultracentrifugation and zone electrophoresis using cellulose acetate membrane. The sedimentation coefficient was calculated to be 9.6 S. The molecular weight was estimated to be approximately 2.7 × 10⁵ by gel-filtration technique.

The optimum pH was found to be in the range of 4.6~5.0, and the optimum temperature was 40°C. The enzyme exhibited higher hydrolytic activity toward maltose rather than toward phenyl-α-glucoside and turanose, and no activity toward sucrose.

The enzyme was a glycoprotein containing carbohydrate of about 50%.     

Effects of Ionic Strength on Thermostability of Lactoperoxidase

An extracellular enzyme that produces di-D-fructofuranose 2′,1;2,1′-dianhydride (difructose anhydride I= DFA I) from inulin was purified from the culture broth of Streptomyces sp. MCI-2524. The purification enhanced the specific activity 7-fold with an overall yield of 17%. The purified enzyme, when electrophoresed on a SDS polyacrylamide gel, gave a single band corresponding to a molecular weight of 36 kDa. Gel filtration chromatography gave a single peak that eluted at a position corresponding to 70 kDa. The enzyme was active from pH 3.0 to pH 9.0, and at temperatures up to 65°C. Maximal activity was observed at pH 6.0, at 55°C. The enzyme was inhibited by Cu²⁺.     

Aminopeptidases in the Acidic Fraction of the Yeast Autolysate

Two aminopeptidases, I and II, were found in the acidic fraction of the yeast autolysate, adsorbed on DEAE-cellose and DEAE-Sephadex A&50. Aminopeptidase I was purified as a single protein with a molecular weight of 200,000. The enzyme required Zn for its activity and hydrolyzed dipeptides, and a polypeptide (glucagon). It also hydrolyzed amides, naphthylamides and the p-nitroanilide of amino acids. The enzyme was strongly inhibited by sulfhydryl reagents. Aminopeptidase II seemed also to be a metal enzyme with a molecular weight of 34,000. The enzyme hydrolyzed the dipeptide and tetrapeptide but not leucine-p-nitroanilide.     

The presence of two serine racemases in Streptomyces garyphalus,a d-cycloserine producer

Two serine racemases (I and II) were isolated from Streptomyces garyphalus. Serine racemase I (molecular weight 93,000) was purified to a single band in an analytical electrofocusing system. Serine racemase II (molecular weight 73,000) was partially purified. Both enzymes used pyridoxal-5-phosphate as cofactor. Besides serine the enzymes utilized alanine as substrate but no other amino acid tested. The K _m values of l-alanine and l-serine for enzyme I were 111 mM and 35 mM respectively. Enzyme I was not inhibited by d-cycloserine but by hydroxylamine. Both substances inhibited enzyme II. The serine racemases may be involved in the biosynthesis of d-cycloserine in S. garyphalus.     

Purification and characterisation of a novel alkalophilic α-D-mannosidase from Pseudomonas fluorescens

An extracellular alkaline α-D-mannosidase in the cell culture of a marine bacterium Pseudomonas fluorescens JK-02 was purified to homogeneity with a 30.7-fold by ammonium sulphate fractionation, anion-exchange chromatography and gel-filtration chromatography. The molecular weight of the purified enzyme was estimated to be 50.5 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme were 8.5 and 30°C. The K_m and V_max values of the purified enzyme towards p-nitrophenyl-α-D-mannopyranoside were determined to be 77 µM and 0.23 µM min^?1mg^?1 of protein, respectively. The α-D-mannosidase showed higher substrate specificity to α-1,3-mannobiose than other isomeric substrates such as α-1,2- and α-1,6-mannobiose. In addition, molecular characterisation of this enzyme reveals that it belongs to a class II α-mannosidase from the glycosyl hydrolase family 38. To the best of our knowledge, this is the first report on the alkalophilic α-1,3 D-mannosidase of Pseudomonas species, which has selective algal-lytic activity against Alexandrium tamarense, Akashiwo sanguine, Gymnodinium catenatum, Gymnodinium mikimotoi and Prorocentrum dentatum.     

Isolation and Characterization of a Novel Endo-β-galactofuranosidase from Bacillus sp.

A soil bacterium capable of growing on a polysaccharide containing β(1→6)galactofuranoside residues derived from the acidic polysaccharide of Fusarium sp. as a carbon source has been isolated. From various bacteriological characteristics, the organism was identified as a Bacillus sp. The bacterium produced β- galactofuranosidase inductively in the culture media. The most effective inducer for the β-galactofuranosidase production was a polysaccharide containing β(1→5) or β(1→6)-linked galactofuranoside residues, but gum arabic, gum guar, gum ghati, arabinogalactam, araban, and pectic acid did not induce the enzyme. The enzyme had three different molecular weight forms. The low molecular-weight form was purified by a combination of Toyopearl HW-55 and DEAE-Toyopearl 650S column chromatographies, and preparative polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 67,000 by SDS–polyacrylamide gel electrophoresis. The enzyme was most active at pH 6 and 37°C, and was stable between pH 4 to 8 at 5°C. The action of the enzyme was inhibited by the addition of Cd²⁺, Co²⁺, Hg²⁺, Zn²⁺, iodoacetic acid, and EDT A. The purified enzyme cleaved β(1→5) and β(1→6)-linked galactofuranosyl chains. Based upon the mode of liberation of galactofuranosyl residues from pyridylamino β(1→6)-linked galactofuranoside oligomers, the enzyme can be classified as an endo-β-galactofuranosidase that randomly hydrolyzes the linkage.     

Purification and Properties of Cyclodextrin Glycosyltransferase of an Alkalophilic Bacillus sp.

Extracellular cyclodextrin glycosyltransferase (α-1,4-glucan 4-glycosyltransferase, cyclizing, EC 3.2.1.19) of an alkalophilic Bacillus sp. (ATCC 21783) was purified about 74-fold and shown to be a single, homogeneous protein by disc polyacryl amide gel electrophoresis and ultracentrifugation. The molecular weight and isoelectric point were 88,000 and pH 5.4. The optimum pH for the enzyme action was 4.5-4.7. The apparent V_max and Km values for α-, β- and γ-cyclodextrin at the constant concentration of sucrose were 133.3, 23.4, 12.3 µmoles glucose/min per mg protein and 5.88, 0.39, 0.25 mm, respectively. The enzyme converted about 73% of starch, 65% of amylopectin, 45% of glycogen and 25% of amylopectin (β-limit dextrin to cyclodextrins.     

Some Properties of Two Proteinases from a Luminous Bacterium,Vibrio harveyi Strain FLN-108

Two proteinases (I and II) from a marine luminous bacterium, FLN-108, were purified to homogeneity. The molecular weights of proteinases I and II were estimated to be 49,000 and 46,000, comprising a dimer of 23,000 molecular weight subunits, respectively. These enzymes were most active at from pH 8.0 to pH 9.0 and 50°C, and stable below 45°C. These enzyme activities were inhibited by EDTA and orthophenanthrolin. Phosphoramidon inhibited the activity of proteinase II, but not that of proteinase I. Metal ions such as Cu²⁺ , Hg²⁺ , and Ni²⁺ strongly inhibited these activities. These results indicate that the proteinases I and II are metal-chelater-sensitive, alkaline proteinases.     

Heat-Moisture Treatment of Cereal Starch Observed by X-ray Diffraction

Chitinases I and II were purified from the culture supernatant of Aeromonas sp. 10S-24 by ammonium sulfate precipitation, SP-Sephadex C-50 chromatography, Sephacryl S-200 gel filtration, and chromatofocusing. Both enzymes were most active at pH 4.0 and the optimum temperature for I and II were 50°C and 60°C. Chitinase I was stable at pHs between 4 and 9 and at temperatures below 50°C and chitinase II was stable at pHs between 5 and 7 and at temperatures below 45°C. The molecular weights were estimated by 8D8 polyacrylamide gel electrophoresis to be 112,000 and 115,000 for I and II respectively, while gel filtration showed the molecular weight to be 114,000 for both types of the enzyme. The pIs for I and II were 7.9 and 8.1, respectively. The activities of both enzymes were inhibited by Ag⁺ and iodoacetic acid.     

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.