DEAE-Toyopearl 650M Research Articles

Purification and Properties of Two Different Azoreductases from a Yeast Candida curvata AN723.

Two azoreductases from Candida curvata AN723 have been purified and characterized. The yeast cells cultured in a GYP medium were disruped with French press. From the acetone fraction of the cell free extract, two different azoreductases (NADPH-dependent azoreductase 1 and NADH-dependent azoreductasese 2) were separated by DEAE-Toyopearl 650M chromatography followed by Toyopearl HW50F chromatography. Azoreductase 1 was further purified by ion exchange chromatography using TSK gel DEAE 5PW. Azoreductase 2 was further purified by affinity chromatography using AF-Blue Toyopearl 650 and chromatofocusing using Toyopearl HW50F and Rotofor system. Each final preparation was homogenous on polyacrylamide gel disc electrophoresis. Azoreductase 1 and 2 were purified 295- and 240-fold, respectively. The molecular mass of azoreductase 1 and 2 were calculated to be about 47,000 daltons and 56,000 daltons, respectively. The isoelectric points of azoreductase 1 and 2 were 4.5 and 4.9, respectively. Azoreductase 1 and 2 had the same optimum pH 5.5-6.0, while they were stable between pH 4.0 and 6.5, and between pH 5.0 and 6.0, respectively. The optimum temperature of azoreductase 1 was 50°C, and that of azoreductase 2 was 40°C, while the former was stable below 75°C, and the latter below 50°C. Azoreductase 1 and 2 had low substrate specificity. Spectrum of these two purified enzymes showed no characteristic absorption band of flavine in the visible range of wavelengths.

Metabolism of DFA III byArthrobactersp. H65-7: Purification and Properties of a DFA III Hydrolysis Enzyme (DFA IIIase)

Assimilation of DFA III by Arthrobacter sp. H65-7 was found to consist of two sequential enzyme steps, hydrolysis of DFA III to inulobiose (1-O-β-d-fructofuranosyl-d-fructopyranose) and inulobiose to fructose, that is, α-(2 → 3′) and β-(2′ → 1) fructosidic linkages were split separately. The enzyme catalyzing the first step, named DFA III hydrolysis enzyme (DFA IIIase), has been purified from the cell-free extracts of Arthrobacter sp. H65-7 to an electrophoretically pure state by heat-treatment, ammonium sulfate fractionation, and chromatographies on DEAE-Toyopearl 650M, Butyl-Sepharose 4B and TSKgel G3000SWxl, and Biophoresis III. The molecular weight of the purified enzyme was estimated to be 125,000 by gel filtration, and 61,000 by SDS–polyacrylamide gel electrophoresis. The enzyme showed the highest activity at pH 6.0 and 45°C, and was stable from pH 4.5 to 10.0 and up to 60°C. The Km of this enzyme for DFA III was 12.5 mm. The enzyme also catalyzed the reverse reaction, inulobiose to DFA III.

Bacterial Expression, Purification, and Characterization of Akazara Scallop Troponin C

To investigate structure-function relationship for akazara scallop troponin C (TnC), we constructed a bacterial expression system using a cDNA clone which we isolated previously (Ojima, T., Tanaka, H., and Nishita, K. Arch. Biochem. Biophys., 311, 272-276 (1994)). The cDNA for akazara scallop TnC was subcloned into the expression plasmid pET-16b and expressed in Escherichia coli BL21 (DE3) by the induction with isopropyl-β-D (-) -thiogalactopyranoside. Amount of the expressed TnC increased up to approximately 10% of total protein during 4h incubation. The TnC was thenextracted from the bacterial pellet and purified by subsequent column chromatographies on DEAE-Toyopearl 650M and Phenyl-Sepharose CL-4 B. Although the expressed TnC possessed an extra moiety of 6 amino acids at N-terminus, which was derived from adaptor DNA of λgt11 and 5'-untranslated region of the cDNA clone, it showed basically the same properties as those of native TnC with respect to the Ca2+-induced difference UV-absorption spectrum and Ca2+-regulatory ability when reconstitutedwith native tropo-nin T and troponin I.

Purification and properties of mutanase from Bacillus circulans

Bacterial strain HU-M1 was isolated as a mutan-degrading microbe from soil and identified as Bacillus circulans. From an HU-M1 culture supernatant mutanase was purified 260-fold by ammonium sulfate fractionation and DEAE-Toyopearl 650M (twice), Toyopearl HW55-F, and Butyl-Toyopearl 650M column chromatography with an activity recovery of 10%. The M r of the mutanae is 160,000 (monomer). The optimal pH and temperature for the mutanase are pH 6.9 and 50°C for a 10-min reaction. The mutanase is stable at up to 40°C for 60-min and at pH 6.0 to 11.0 at 4°C for 48 h, and logarithmically inactivated at 54°C with a half life of 2.6 min. Mutant hydrolysates produced by the enzyme had di-, tri-, and tetramer of glucose and the final hydrolysis was 38%.

Cloning and expression of an intracellular alkaline protease gene from alkalophilic Thermoactinomyces sp. HS682.

An intracellular alkaline serine protease gene of alkalophilic Thermoactinomyces sp. HS682 was cloned and expressed in Escherichia coli. Sequence analysis showed a putative promoter region, a putative transcriptional termination signal, and an open reading frame of 963 bases, coding for a polypeptide of 321 amino acids. The protease expressed in E. coli was purified by DEAE-Toyopearl 650M and Sephadex G-75 chromatography. The N-terminal sequence (30 amino acids) of the purified protein was coincident with Asp16-Val45 of the deduced amino acid sequence of the ORF. Fifteen amino acids in the N-terminal region were removed during the purification procedures. The deduced amino acid sequence showed high similarity with microbial intracellular serine proteases. The molecular mass of this enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme was stable at pH 6.0-12.0 and below 60 degrees C in the presence of Ca2+. The temperature and pH optima of the enzyme were 65 degrees C and pH 11.0, respectively. The enzyme was inhibited by DFP and PMSF, but not by MIA and EDTA.

Isolation of an l-rhamnose isomerase-constitutive mutant of Pseudomonas sp. strain LL172: Purification and characterization of the enzyme

A soil bacterium, strain 172a, which inductively produced l-rhamnose isomerase ( l-rhamnose ketolisomerase, EC 5.3.1.14) and could not grow on l-lyxose acquired the ability to grow on l-lyxose following cultivation in a mineral salts medium containing l-lyxose as the sole carbon source for about 4 d. This l-lyxose-utilizing mutant (LL172) was isolated and found to produce l-rhamnose isomerase constitutively. Based on various bacteriological characteristics, the parent strain was identified as Pseudomonas sp. and the mutant was confirmed to be derived from the parent strain. l-Rhamnose isomerase was purified from extracts of Pseudomonas sp. LL172 by polyethylene glycol precipitation, anion exchange chromatography on DEAE-Toyopearl 650M and gel filtration on Sephadex G-150. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The apparent molecular weight of the enzyme was estimated to be 150,000 by gel filtration on Sephadex G-150. Based on sodium dodecyl sulfate (SDS) gel electrophoresis, the enzyme is most likely composed of 4 identical subunits of molecular weight of approximately 42,000. The enzyme was optimally active at pH 9.0 and was stable in the pH range of 5.0–11.0. The optimum temperature for activity was 60°C (10 min, pH 9.0) and the enzyme was stable up to 60°C (10 min, pH 9.0). The isoelectric point of the enzyme was estimated to be 5.1. The substrate specificity of the enzyme was broad and the enzyme required manganese ions for maximum activity. The K m for l-rhamnose isomerase was 55 mM and the V max was 182.6 U/mg. The equilibrium ratio between l-rhamnose and l-rhamnulose was 55:45.

Production of d-tagatose 3-epimerase of Pseudomonas cichorii ST-24 using recombinant Escherichia coli

The d-tagatose 3-epimerase ( d-TE) gene of Pseudomonas cichorii ST-24 was expressed in Escherichia coli under the control of the trc promoter. The d-TE production level was highest in E. coli JM105 as a host strain and in NZC medium as a culture medium. Production of d-TE by E. coli JM105 was about 100-fold higher than that of d-TE by P. cichorii ST-24, and the enzyme constituted ∼5% of the total protein. d-TE from E. coli JM105 was purified by polyethylene glycol precipitation, DEAE-Toyopearl 650M column chromatography, and Sephadex G-150 column chromatography. The overall purification procedure resulted in 16.7-fold purification with 18.2% recovery. The molecular weight of the purified d-TE was estimated to be 33.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and agreed with that of the d-TE from P. cichorii ST-24. d-TE produced by E. coli JM105 had generally the same enzymological properties, i.e., the optimum pH and temperature, pH and thermal stabilities, and K m value, as that from Pseudomonas sp. ST-24, but the V max value of d-TE from E. coli was 6 times higher than that from Pseudomonas sp. ST-24. The N-terminal amino acid sequence of the recombinant d-TE agreed with that of the d-TE from P. cichorii ST-24.

Purification and properties of two chitinases from Vibrio alginolyticus H-8

Chitinases C1 and C3 from Vibrio alginolyticus H-8 were purified by column chromatography on DEAE-Toyopearl 650M and Superdex 200HR. The molecular weights were estimated by SDS-gel electrophoresis (SDS-PAGE) to be 81,000 Da and 68,000 Da for C1 and C3, respectively. The pIs for C1 and C3 were 3.9 and 3.6, respectively. The activities of both enzymes were inhibited by Ag + and Hg 2+.

Purification and some properties of phospholipase B from Schizosaccharomyces pombe.

Phospholipase B from Schizosaccharomyces pombe was purified by ammonium sulfate fractionation and chromatographed on phenyl-Sepharose CL-4B, DEAE-Toyopearl 650M, and TSK gel G4000SW columns. The purified enzyme was a glycoprotein with molecular weight of approximately 300,000 and 100,000-150,000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point was pH 4.7. The optimum pH of the enzyme was 2.5 and no activity was detected at neutral and alkaline pHs. The enzyme was not heat-stable. Enzyme activity was slightly stimulated by divalent ions except Fe2+ and 0.1% sodium deoxycholate, and inhibited by Fe2+, Fe3+, 0.1% sodium dodecyl sulfate, and 0.01% cetyltrimethylammonium bromide. The enzyme hydrolyzed mono- and diacylphospholipids, and phosphatidylinositol was hydrolyzed most preferentially. Triglyceride was not hydrolyzed. The enzyme also had acyltransferase activity on lysophosphatidylcholine, forming the corresponding diacylphosphatidylcholine.

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases produced by Agrobacterium sp. K-03

A poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Agrobacterium sp. strain K-03, isolated from soil, secreted two PHB depolymerases into the culture fluid when it was cultivated on PHB, 3-hydroxybutyrate (3HB), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 43 mol% 3-hydroxyvalerate (P(3HB-co-43%3HV)) as the sole source of carbon. The two extracellular PHB depolymerases, designated E1 and E2, were purified from the culture fluid using DEAE-Toyopearl 650M column chromatography followed by gel filtration in Sephadex G-75 column chromatography. E1 and E2 are basic proteins having molecular weights of 46 and 44 kDa, and isoelectric points of 9.0 and 8.9, respectively. The optimal pHs of E1 and E2 for degradation of PHB are 8.1 and 7.9, respectively, and both of them have optimal PHB degradation temperatures of 45°C. Their enzymatic activities were considerably inhibited by dithiothreitol, phenylmethanesulfonyl fluoride, and Tween 20. The K m values of E1 and E2 for PHB were 17.8 μg/ml and 70.5 μg/ml, respectively. At pH 8.0 and 30°C, both enzymes completely degraded PHB, and they degraded 94% of P(3HB-co-43%3HV). The methyl ester of 3HB dimer was degraded by each enzyme to form the free dimer and the free monomer of 3HB.

Purification of membrane-bound ATPase of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 and characterization as a F0F1-type enzyme.

The ATPase bound to the inner membrane of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 (Vibrio ABE-1) was extracted with Triton X-100 and purified by fractionation with polyethylene glycol, sucrose density gradient centrifugation, and DEAE-Toyopearl 650M column chromatography. The molecular masses of subunits constituting the purified ATPase were estimated as 54, 49, 33.5, 27, 23.5, 18.5, and 15 kDa by SDS-PAGE. The composition and molecular masses of the subunits of the purified ATPase were similar to those of Escherichia coli F0F1-ATPase (EF0F1). The 54-, 49-, and 18.5-kDa polypeptides of the Vibrio ABE-1 ATPase strongly cross-reacted with the antibodies against the EF0F1 alpha, beta, and b subunits, respectively. However, the Vibrio ABE-1 ATPase contained no cross-reactive polypeptide with the antibodies against A and B subunits of V-type H(+)-ATPase from mung bean tonoplasts. The ATPase activity showed two pH optimum peaks at pH 5.3 and 8.0 and was strongly inhibited by N,N'-dicyclohexyl carbodiimide (DCCD) and NaN3. It hydrolyzed ATP, GTP, and ITP at similar rates. These properties confirm that the purified ATPase is a F0F1-type. The optimum temperature for the ATP-hydrolyzing activity of the enzyme was observed at 50 degrees C, but the DCCD-sensitivity of the enzyme was markedly decreased above 30 degrees C, suggesting that the F1-moiety is released from the enzyme complex at high temperatures. This characteristic is compatible with the psychrophilic nature of Vibrio ABE-1.

Purification and properties of aromatic l-amino acid decarboxylase from liver of skipjack tuna (Katsuwonus pelamis, Teleostei: Scombridae)

Aromatic l-amino acid decarboxylase (AADC; EC 4.1.1.28) was extracted from skipjack (Katsuwonus pelamis) liver with potassium phosphate buffer solution. This enzyme was purified by ammonium sulfate fractionation, sepharose CL-6B, QAE-Toyopearl 550C, DEAE-Toyopearl 650M, Bio-gel HT, and Toyopearl HW-55F chromatography. The molecular weight of the enzyme was estimated to be about 110,000 by gel filtration on Sepharose CL-6B. The enzyme was inhibited by Co2+, Cu2+ and Zn2+ ions, but activated by K+, Li+, Na+, Ba2+, and Mn2+ ions.

Purification and Characterization of Chitin Deacetylase from Absidia coerulea

Chitin deacetylase, which releases the acetyl groups of glycol chitin was purified from a fungus, Absidia coerulea, and characterized. The enzyme was purified 516-fold to homogeneity by means of 65-80% ammonium sulfate precipitation followed by chromatography on Butyl Toyoperal-650M, Gigapite (hydroxyapatite), and DEAE Toyopearl-650M. It had an apparent molecular weight of 75 kDa both on sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration chromatography, indicating that the enzyme exists as a monomer. The amino-terminal sequence was determined to be Gly-Glu-Tyr-Trp-Gln-Ser-Phe-. The enzyme is active on chitooligosaccharides with more than two N-acetylglucosamine residues (chitobiose) and is able to convert the nascent chitin synthesized by chitin synthase to chitosan in vitro. When O-hydroxyethylated chitin (glycol chitin) was used as a substrate, the optimum pH for enzyme activity was 5.0 and the optimum temperature was 50 degrees C. The enzyme was heat-stable and strongly inhibited by Fe3+. Furthermore, chitin deacetylase was demonstrated to be localized near the inner face of the cell wall (periplasmic space) in the mycelia by using immunoelectron microscopy.

Chemoattraction of male gametes by a pheromone produced by female gametes of Chlamydomonas.

In isogamous species of Chlamydomonas, such as Chlamydomonas reinhardtii and Chlamydomonas eugametos, the sexual process involves the use of flagella agglutinins by which the gametes of compatible strains adhere through chance encounter and ultimately pair and fuse to form zygotes. In a newly described heterogamous species, Chlamydomonas allensworthii, the sexual process is initiated by the chemoattraction of small sperm to a sexually competent female gamete, which continues to secrete the pheromone until it has fused with one of the sperm so attracted. From bacteria-free female strains of C. allensworthii, the chemoat-tractant has been isolated and identified as a pentosylated hydroquinone (Mr = 532) whose spectral, chemical, and physical properties are in accord with the structure of a 2,3-dimethyl-5-(triprenylcarboxymethyl)-1,4-benzohydroquinone-1-(beta-xyloside). A rapid bioassay of the pheromone uses DEAE-Toyopearl 650M beads to which the pheromone adsorbs. When such activated beads are placed in a suspension of sperm, they act as surrogate females and attract the small motile sperm. The purified pheromone shows activity at a concentration as low as 1 pM.

Purification and Characterization of Protein Disulfide Isomerase from Soybean

Protein disulfide isomerase (PDI), which catalyses the folding of newly synthesized or denatured proteins through correct disulfide formation, was purified from soybean (Glycine max). The enzyme was purified 12,000-fold over crude extracts to apparent homogeneity in six purification steps: 60-70% ammonium sulfate fractionation, and chromatography on DEAE Toyopearl 650M, Q-Sepharose Fast Flow, Hiload Superdex 200 pg, Phenyl Sepharose HP, and TSK G-3000 SW. The native enzyme had a molecular weight of 120 kDa on gel filtration. Subunit molecular weight was estimated as 63 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, thus indicating the enzyme to be comprised of two identical subunits. The enzyme pH optimum was 8.0 with reactivation of scrambled RNase, and the pI 7.65. The N-terminal amino acid sequence of soybean PDI was homologous to that of mature alfalfa as deduced from the cDNA sequence. Two identical active site sequences, APWCGHCK, were obtained from different proteolytic peptide fragments of soybean PDI. Soybean PDI facilitated reactivation not only of scrambled RNase, but denatured and reduced lysozyme and the Bowman Birk soybean trypsin inhibitor as well. This is the first report to appear on the the purification, characterization and amino acid sequence analysis of the active site of a plant PDI.

Purification and Characterization ofPenicillium roquefortiIAM 7268 Lipase

An extracellular lipase from Penicillium roqueforti IAM 7268 was purified by a procedure involving ethanol precipitation, ammonium sulfate precipitation, and DEAE-Toyopearl 650M, Phenyl-Toyopearl 650M, and Toyopearl HW-60 column chromatographies. The purified lipase was homogeneous with 25 kDa of molecular mass by SDS–polyacrylamide gel electrophoresis, and had high specificities toward short-chain fatty acid esters.

Purification and characterization of a new lipase from Fusarium sp. YM-30.

The extracellular lipase from Fusarium sp. YM-30 was purified by a procedure involving ultrafiltration, ammonium sulfate precipitation, and DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Butyl-Toyopearl 650M column chromatographies. The purified lipase was homogeneous with 12kDa of molecular mass by SDS-PAGE, and had high specificities for mono- and diacylglycerols, but low toward triacylglycerols. The enzyme had maximum activity at pH 7.0 to 8.0 and 37 degrees C, and hydrolyzed digalactosyl diglyceride too.

Production and release of polyphosphate by a genetically engineered strain of Escherichia coli

A recombinant strain of Escherichia coli MV1184, which contains plasmid-borne genes encoding the phosphate-specific transport (Pst) system and polyphosphate (polyP) kinase, accumulated high levels of Pi and released polyP into the medium. PolyP could be separated from the culture supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution 31P nuclear magnetic resonance spectroscopy. Once E. coli recombinants accumulated high levels of polyP, they released polyP concomitantly with Pi uptake. PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a result of cell lysis. PolyP release ceased when Pi became depleted in the medium and resumed upon addition of Pi to the medium. When Pi uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), no polyP release was observed. Furthermore, neither Pi uptake nor polyP release occurred when cells were incubated at 4 degrees C. These findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concentration in E. coli recombinants. High-resolution 31P nuclear magnetic resonance spectroscopy also detected a surface pool of polyP in intact cells of the E. coli recombinant. The polyP resonance increased when cells were treated with EDTA and broadened upon the addition of a shift reagent, praseodymium. Although the mechanism of surface polyP accumulation is unclear, surface polyP seems to serve as the source for polyP release.

Purification and some properties of cycloinulo-oligosaccharide fructanotransferase from Bacillus circulans OKUMZ 31B

Cycloinulo-oligosaccharide fructanotransferase was purified from the cultured medium of Bacillus circulans OKUMZ 31B, to electrophoretic homogeneity, by anion-exchange column chromatography on DEAE-Toyopearl 650M, hydrophobic column chromatography on Butyl-Toyopearl 650M, gel-filtration column chromatography on Sephacryl S-2000HR and anion-exchenge column chromatography on SuperQ-Toyopearl 650M. The enzyme has a molecular weight of 132 000 and a pI of 4.1. The enzyme was most active at pH 7.5 and 40°C, and was stable at pH 6.0–9.0 and below 40°C. The enzyme catalyses the conversion of inulin into cycloinulohexaose and cycloinuloheptaose in the ratio of ca. 4:1, and a small amount of cycloinulo-octaose. The enzyme has an isoform which may be a proteolyticaly modified species of the CFTase because of its reduced molecular weight, 126 000.

Adsorption and separation of proteins on composite anion exchangers with poly(N-diethylaminoethylacrylamide) bonded phases

Composite anion exchangers for chromatography of proteins were prepared by chemical adsorption of poly( p-nitrophenyl acrylate) on γ-aminopropylsilicas followed by coupling of the esters to 2-diethylaminoethylamine. Separation of standard proteins (bovine serum albumin and ovalbumin) on these anion exchangers established their enhanced selectivity and milder desorption conditions as compared with polyethyleneiminesilicas and DEAE-Toyopearl 650M. Frontal analysis was used to evaluate the maximum binding capacity for ovalbumin adsorption on the new packing, which was found to be 13 mg/ml and so nearly half those observed with polyethyleneiminesilicas of comparable pore diameter. The possible role of the excluded volume of the attached polymer is discussed with respect to the adsorptivity of the composite ion exchangers.