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.     

Purification and characterization of secreted acid phosphatase under phosphate-deficient condition inPholiota nameko

Activity of acid phosphatase secreted by mycelia ofPholiota nameko on cultivation for 30d in Pi-depleted medium was 88-fold higher than the corresponding activity in the Pi-supplied medium. One isozyme of the secreted acid phosphatases was purified from the culture filtrate of Pi-depleted medium by ammonium sulfate fractionation and cation exchange chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed change chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed that the native molecule had a molecular weight of 117,000. The molecular weight on gel electrophoresis with SDS was 52,000, indicating that the native form of the enzyme was a homodimer. The optimum pH and temperature of the enzyme were, 5.5 and 45°C, respectively, and the isoelectric point of the enzyme was pH 6.9. Adsorption on Con A-Sepharose and periodic-Schiff stain suggested that the enzyme is a glycoprotein. The enzyme hydrolyzed a wide variety of phosphate esters, nucleoside phosphates, sugar phosphates, and phosphorylated amino acids. Cu²⁺, Fe²⁺, Hg²⁺, iodoacetate, molybdate, tartaric acid, and SDS inhibited the enzyme activity. Fe³⁺ (1 mM), Triton X-100, methanol, and ethanol activated it. Fifteen residues of the N-terminal amino acid sequence were determined.     

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 a Dehyrodicaffeic Acid Dilactone-forming Enzyme from a Mushroom,Inonotus sp. K-1410

A dehydrodicaffeic acid dilactone-forming enzyme was purified from the mycelia of a mushroom, Inonotus sp. K-1410 by calcium acetate treatment, ammonium sulfate precipitation and column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and caffeic acid-bound AH-Sepharose 4B. The enzyme was purified about 1200-fold from a crude extract and shown to be almost completely homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 39,000. The optimal pH for the enzymic conversion of caffeic acid to dehydrodicaffeic acid dilactone is around 6.0. The enzyme is stable up to 60°C and preincubation of the enzyme at 40°C for 10 min gives 1.5-fold activation compared with preincubation at 0°C. The optimal temperature for the enzyme reaction is 40°C.     

Purification and Some Properties of β-Mannanase from Penicillium purpurogenum

A β-mannanase was purified from the culture filtrate of Penicillium purpurogenum No. 618 by 1st and 2nd DEAE-cellulose column chromatographies, and subsequent Ultro-gel chromatography. The final preparation thus obtained showed a single band on polyacrylamide disc-gel and SDS-polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were determined to be 57,000 and pH 4.1 by SDS-polyacrylamide gel electrophoresis and isoelectric focusing, respectively. The purified mannanase contained the following amino acids: glycine > serine >glutamic acid > alanine > aspartic acid. The mannanase exhibited maximum activity at pH 5 and 70°C, and was stable in the pH range of 4.5 to 8 and at temperatures up to 65°C. The enzyme activity was not affected considerably by either metal compounds or ethyl- enediaminetetraacetic acid. Copra galactomannan (Gal: Man =1 :14) was finally hydrolyzed to galactose, mannose and β-1,4-mannobiose through the sequential actions of the purified mannanase and the α-galactosidase purified from the same strain.     

Studies on Tannin Acyl Hydrolase of Microorganisms

Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.

The molecular weight of the enzyme estimated by gel filtration method was about 200,000.

The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.

Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water.     

Purification and Some Properties of Active β-Amylase in Rice

An active β-amylase was purified from germinated rice seeds by precipitation with ammonium sulfate, acid treatment, chromatographies on DEAE-cellulose and DEAE-Sephadex A-50, and gel filiations on Sephadex G-75. The purified enzyme was homogeneous in disc electrophoretic analysis.

The molecular weight was estimated to be approximately 53,000 by thin-layer gel filtration and polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 5.0 by disc electrofocusing.

The optimum pH was found to be in the pH range of 5.5 to 6.5. The Km value for soluble starch was 3 mg/ml. The enzyme was inhibited by sulfhydryl reagents or heavy metal ions.

The active β-amylase was oxidatively dimerized by treatment with 0.3 m ferricyanide in 3 m urea. The dimerized enzyme was thought to be one of inert β-amylases in ungerminated rice seeds.     

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.     

Ester Formation by Alcohol Acetyltransferase from Brewers’ Yeast

Alcohol acetyltransferase responsible for the formation of acetate esters during beer fermentation was found to be localized at the cell membrane of brewers’ yeast. This cell membrane-bound enzyme was purified 120-fold by solubilization with Triton X-100, gel filtration on a Sepharose 6B column and chromatography on a DEAE-Sephadex A-50 column. The enzyme was most active at 30°C at pH 7 ? 8. It was least active against C₃ alcohol among C₁ ? C₆ alcohols, and slightly more active against straight-chain alcohols than against branched-chain alcohols with the same carbon number. The enzyme was strongly inhibited by unsaturated fatty acids, heavy metal ions and sulfhydryl reagents.     

Crystallization and Some Properties of Alkaline Proteinase from Aspergillus sulphureus

An alkaline proteinase of Aspergillus sulphureus (Fresenius) Thorn et Church has been purified in good yields from wheat bran culture by fractionation with ammonium sulfate, treatment with acrynol, and DEAE-Sephadex A-50 column chromatography. The crystalline preparation was homogeneous on sedimentation analysis and polyacrylamide gel zone electrophoresis. The molecular weight was calculated to be 23,000 by gel filtration. The amino acid composition of the enzyme was determined. The enzyme did not precipitate with acrynol. Optimum pH for the hydrolysis of casein was 7 to 10 at 35°G for 15 min. Optimum temperature was 50°C at pH 7 for 10 min. The enzyme was highly stable at the range of pH 6 to 11 at 5°C, whereas relatively stable at pH 6 to 7 at 35°C. Metalic salts tested did not affect activity. Chelating agents, sulfhydryl reagents, TPCK, and oxidizing or reducing reagents tested, except iodine, had no effect on the activity. Diisopro-pylfluorophosphate and N-bromosuccinimide almost completely inactivated the proteinase.     

Purification and Some Properties of ATP: Nucleotide Pyrophospho-transferase of Streptomyces adephospholyticus

ATP: nucleotide pyrophosphotransferase was purified from culture filtrate of Streptomyces adephospholyticus A–4668 about 13,000 fold by the method including ammonium sulfate fractionation, Amberlite IRC–50 treatment and column chromatography with DEAE-cellulose, DEAE-Sephadex A–25, SP-Sephadex C–25 and Sephadex G–75. The purified enzyme was homogenous on disk gel electrophoresis and ultracentrifugation and the specific activity was 915 units per mg protein, The molecular weight was determined as 28,000 by gel filtration on Sephadex G–75. The enzyme was found to be stable in the pH range of 5.5 to 10.5. More than 80% of the activity was remained after heating at 60°C for 30 min. The enzyme exhibited maximum activity at 50°C.     

Purification and Properties of Mycodextranase from Streptomyces sp. J-13-3

An enzyme hydrolyzing nigeran (alternating α-l,3-and α-l,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-I00. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS–PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50°C. The enzyme activity was inhibited significantly by Hg⁺, Hg²⁺, and p-chloromercuribenzoic acid. The K_m (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the α-l,4-glucosidic linkages in nigeran.     

Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum.

A β-glucosidase was isolated from $\text{Clostridium thermocellum}$; the enzyme was localized in the periplasmic space.It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing.The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity.Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.     

Purification and characterization of novel cutinase from nasturtium (Tropaeolum majus) pollen.

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C₂ to C₁₈ fatty acids with similar K_m and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Purification and Properties of Three Types of Xylanases Induced by Methyl β-Xyloside from Streptomyces sp.

When mycelia of Streptomyces sp. No. 3137 were cultivated in a medium containing methyl β-xyloside, xylanases (EC 3.2.1.8) were inductively produced into the medium. Three types of enzyme from the culture filtrate have been purified by ultrafiltration with DIAFLO UM-10, chromatography on DEAE-Sephadex A-25, gel filtration on Bio Gel P-100, and isoelectric focusing with Servalyt 6~8 or 9~11. The three purified enzymes, tentatively named X-I, X-II-A, and X-II-B, were homogeneous by Polyacrylamide gel electrophoresis at pH 4.3. The molecular weight of X-I was about 50,000 by SDS-polyacrylamide gel electrophoresis or gel filtration on Bio Gel P-100. The molecular weight of X-II-A and X-II-B were both approximately 25,000 by SDS-polyacrylamide gel electrophoresis and that of X-II-B was 25,680 by the sedimentation-equilibrium method. X-I had an isoelectric point at 7.10, and X-II-A and X-II-B had different isoelectric points, 10.06 and 10.26, respectively. The three enzymes were optimally active at 60~65°C and stable to 55°C. The optimal pH of X-I, X-II-A, and X-II-B were pH 5.5~6.5, 5.0~6.0, and 5.0~6.0, respectively. The ranges of two X-I’s pH stability (pH 1.5 ~ 11.5) were wider than that of X-I’s (pH 3.0 ~ 10.5). These purified preparations hydrolyzed xylotriose, xylotetraose, and xylan but not xylobiose, cellobiose, maltose, carboxymethyl cellulose, or soluble starch. Their actions were inhibited by Hg²⁺ and Fe³⁺ ions, sodium dodecyl sulfate, and N-bromosuccinimide.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

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 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.     

Purification and partial characterization of an acid phosphatase from the body wall of sea cucumber Stichopus japonicus

A high molecular weight (HMW) acid phosphatase from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of anion exchange chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC). The enzyme was purified 19.3-fold with a total yield of 1.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 147.9 kDa. The enzyme displayed maximum activity at pH 4.0 and 50 °C with p-nitrophenyl phosphate as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg²⁺, whereas inhibited strongly by Cu²⁺ and Zn²⁺. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The amino acid sequences of three segments of the purified enzyme were analyzed by mass spectroscopy, which did not have any homology with previously described acid phosphatase.