Butyl Toyopearl Research Articles

Free flavins accelerate release of ferrous iron from iron storage proteins by both free flavin-dependent and -independent ferric reductases in Escherichia coli.

Ferredoxin NADP+ oxidoreductase (Fpr) and oxygen-insensitive NAD(P)H nitroreductase (NfnB) are purified from Escherichia coli JM109 (E. coli JM109) as a predominant free flavin-independent ferric reductase. In the present study, we prepared natural iron storage proteins, E. coli ferritin A (FtnA) and bacterioferritin (Bfr), to show the effective ferrous iron release from these proteins by Fpr and NfnB in the presence of free flavins. Fpr and NfnB showed flavin reductase activity for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and riboflavin, and their ferrous iron release activities were positively associated with the catalytic efficiencies (kcat/Km) for individual flavins. The ferrous iron release activity of E. coli cell-free extracts was affected by flavin reductase activity of the extracts. The Butyl TOYOPEARL column chromatography of the extracts, on the basis of NAD(P)H-dependent flavin reductase activity, resulted in the separation of six active fractions containing Fpr, NfnB, NAD(P)H-quinone oxidoreductase (QOR), flavin reductase (Fre) or alkyl hydroperoxide reductase subunit F (AhpF) as major components. Like Fpr and NfnB, recombinant QOR, Fre, and AhpF showed flavin reductase activity and ferrous iron release activity in the presence of free flavins, indicating an association of flavin reductase activity with ferrous iron releasing activity. Taken together, both free flavin-dependent and free flavin-independent ferric reductases in E. coli require free flavins to mediate an electron transfer from NAD(P)H to ferric iron in the iron storage proteins for the effective ferrous iron release.

Proteolysis of α-amylase from Saccharomycopsis fibuligera: characterization of digestion products

α-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme. However, it was found that the Km value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain, while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A.

Purification, Characterization and Cloning of a Chymotrypsin Inhibitor (CI-9) from the Hemolymph of the Silkworm, Bombyx mori

Hemolymph chymotrypsin inhibitor 9 (CI-9) from the hemolymph of the silkworm, Bombyx mori, was purified by ammonium sulfate precipitation, Butyl Toyopearl hydrophobic chromatography, gel filtration through Sephadex C-50 and chymotrypsin-sepharose 4B affinity chromatography. Checked by Native PAGE and SDS-PAGE in combination with silver staining, the final preparation appeared homogeneous. In tricine SDS-PAGE, CI-9 displayed a molecular weight of 7.5 kD, which was determined to be 7167 Da with the Voyager TOFMass analyser. The pI value for CI-9, revealed by 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), was 4.3. CI-9 exhibited inhibitory activity at a temperature as high as 100 degrees C and a stability against a wide range of pH (1-12). In N-terminal amino-acid analysis of CI-9, 40 amino acid residues were obtained. The C-terminal 22 amino acid residues were deduced by subsequently cloned cDNA and genomic fragments. MW and pI of CI-9 were predicted to be 7170.98 Da and 4.61, respectively, on the website. Its low molecular weight, high stability, conserved active site and Kunitz domain showed that CI-9 is a Kunitz-type CI. The difference of sequence and pI between CI-9 and other Kunitz type CIs indicated that it is a novel chymotrypsin inhibitor.

Crystal Structure of a Novel FAD-, FMN-, and ATP-containing l-Proline Dehydrogenase Complex from Pyrococcus horikoshii

Two novel types of dye-linked L-proline dehydrogenase complex (PDH1 and PDH2) were found in a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Here we report the first crystal structure of PDH1, which is a heterooctameric complex (alphabeta)4 containing three different cofactors: FAD, FMN, and ATP. The structure was determined by x-ray crystallography to a resolution of 2.86 angstroms. The structure of the beta subunit, which is an L-proline dehydrogenase catalytic component containing FAD as a cofactor, was similar to that of monomeric sarcosine oxidase. On the other hand, the alpha subunit possessed a unique structure composed of a classical dinucleotide fold domain with ATP, a central domain, an N-terminal domain, and a Cys-clustered domain. Serving as a third cofactor, FMN was located at the interface between the alpha and beta subunits in a novel configuration. The observed structure suggests that FAD and FMN are incorporated into an electron transfer system, with electrons passing from the former to the latter. The function of ATP is unknown, but it may play a regulatory role. Although the structure of the alpha subunit differs from that of the beta subunit, except for the presence of an analogous dinucleotide domain with a different cofactor, the structural characteristics of PDH1 suggest that each represents a divergent enzyme that arose from a common ancestral flavoenzyme and that they eventually formed a complex to gain a new function. The structural characteristics described here reveal the PDH1 complex to be a unique diflavin dehydrogenase containing a novel electron transfer system.

Purification and characterization of alpha-L-arabinopyranosidase and alpha-L-arabinofuranosidase from Bifidobacterium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and Rc.

Two arabinosidases, alpha-L-arabinopyranosidase (no EC number) and alpha-L-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. alpha-L-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 micro mol/min/mg. alpha-L-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 micro mol/min/mg. The molecular mass of alpha-L-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that of alpha-L-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. alpha-L-Arabinopyranosidase and alpha-L-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40 degrees C and pH 4.5 and 45 degrees C, respectively. Both purified enzymes were potently inhibited by Cu(2+) and p-chlormercuryphenylsulfonic acid. alpha-L-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinopyranoside, followed by ginsenoside Rb2. alpha-L-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-beta-galactopyranoside or p-nitrophenyl-beta-D-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified alpha-L-arabinosidases. This is the first reported purification of alpha-L-arabinopyranosidase from an anaerobic Bifidobacterium sp.

Purification and characterization of a heat stable inulin fructotransferase (DFA I-producing) from Arthrobacter pascens a62-1

An inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter pascens a62-1 was purified and the properties of the enzyme were investigated. The enzyme was purified from culture supernatant of the microorganism 58.5 fold with a yield of 8.32% using Super Q Toyopearl chromatography and butyl Toyopearl chromatography. It showed maximum activity at pH 5.5 and 45 °C and was stable up to 75 °C. This heat stability was highest in the inulin fructotransferases (DFA I-producing) reported until now. The molecular mass of the enzyme was estimated to be 37,000 by SDS-PAGE and 60,000 by gel filtration, and was considered to be a dimer. The N-terminal amino acid sequence (20 amino acid residues) was determined as Ala-Asn-Thr-Val-Tyr-Asp-Val-Thr-Thr-Trp-Ser-Gly-Ala-Thr-Ile-Ser-Pro-Tyr-Val-Asp.

Purification and properties of a heat stable inulin fructotransferase (DFA III-producing) from Arthrobacter pascens T13-2

An inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter pascens T13-2 was purified and the properties of the enzyme were investigated. The enzyme was purified from a culture supernatant of the microorganism 18.5-fold with a yield of 13.1% by Super Q Toyopearl chromatographies and a butyl Toyopearl chromatography. It gave a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the enzyme was estimated to be 44 000 by SDS-PAGE and 79 000 by gel filtration and was therefore considered to be a dimer. The N-terminal amino acid sequence was determined as Ala-Gln-Asp-Ala-Lys-Ala-Gly-Pro-Phe-Asn-Ser-Pro-Asn-Thr-Tyr-Asp-Val-Thr. The enzyme showed maximum activity at pH 5.5–6.0. The optimum temperature for the enzyme activity was 50 °C. The enzyme was stable up to 75 °C. The enzyme activity was inhibited strongly by Hg 2+, and inhibited slightly by Fe 3+, and Zn 2+. An immobilized enzyme was prepared using Chitopearl BCW3510 as a carrier. The immobilized enzyme was able to use eight times without a significant loss of the enzyme activity.

Tributyrin specifically induces a lipase with a preference for the sn-2 position of triglyceride in Geotrichum sp. FO401B.

The extracellular Lipases A and C produced by Geotrichum sp. FO401B have a preference for the sn-1,3 and sn-2 positions of triglyceride, respectively. Total production of these lipases was increased by plant oils and tributyrin. Butyl Toyopearl column chromatography demonstrated that only Lipase C was produced in the presence of tributyrin. Lipase C hydrolysed natural fats except sardine oil preferentially at the sn-2 position, but it showed little stereoselectivity for triolein.

Purification and Characterization of Feruloyl Esterase from Aspergillus awamori.

A. awamori feruloly esterases, termed FAE-a and FAE-b, were purified to homogeneity by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, DEAE-Toyopearl 650 column chromatography, hydrophobic chromatography on Butyl Toyopearl 650S, and gel filtration on Toyopearl HW55. The molecular weights (Mr) of FAE-a and FAE-b were estimated to be 31 KDa and 29 KDa by SDS-PAGE; these isoelectric points were 3.9 and below 3.4, respectively. Both enzymes were optimally active at 45-50°C and pH 5.0-5.5. FAE-a was stable up to 37°C, and FAE-b was stable up to 50°C. Both enzymes hydrolyzed predominantly the feruloylated carbohydrate ester prepared from pineapple stem residue rather than methyl esters of ferulic acid.

Purification and characterization of glycyrrhetic acid mono-glucuronide beta-D-glucuronidase in Eubacterium sp. GLH.

Glycyrrhetic acid mono-glucuronide (GAMG), 1-(18beta-glycyrrhet-3-yl)-beta-D-glucopyranuroic acid, was hydrolyzed to glycyrrhetic acid (GA) by GAMG beta-D-glucuronidase in Eubacterium sp. GLH from human intestinal bacteria. The enzyme had an optimum pH of 5.0 and was purified from a crude extract by Butyl Toyopearl 650 S, Toyopearl HW-55 S, Hydroxyapatite and DEAE-Toyopearl 650 M column chromatography. The purified enzyme showed a specific activity of 495 nmol/min/mg protein and a single band on Coomassie brilliant blue staining and a molecular weight of about 43 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight was 49.5 kDa, as estimated by Toyopearl HW-55 S column chromatography. Also, the enzyme seemed to have a sulfhydryl group(s) in its active site with a Km value of 77 x 10(-3) M.

Purification and characterization of galacto‐oligosaccharide‐producing β‐galactosidase from Sirobasidium magnum

Galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p-aminobenzyl 1-thio-β-d-galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β-galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β-galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The Km values for o-nitrophenyl-β-d-galactopyranoside and lactose were 14·3 and 5·5 mmol l−1, respectively, and the Vmax values for these substrates were 33·4 and 94·5 μmol min−1 mg of protein−1, respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml−1 galacto-oligosaccharide was produced from 200 mg ml−1 lactose.

Kinetic parameters of β-fructofuranosidase from Scopulariopsis brevicaulis

β-Fructofuranosidase was purified to homogeneity by a series of column chromatographies on Butyl Toyopearl 650M, DEAE-Sephadex A-50, Toyopearl HW-55F, and hydroxyapatite with a yield of 4.6% and 1,100-fold purification from the cell extract of Scopulariopsis brevicaulis N-01. The activity was optimal at 40°C and pH 6–9. The enzyme was a homologous dimer protein and the molecular weight of a subunit was 110,000. Kinetic studies were carried out using the purified β-fructofuranosidase. The K m and V max values for the formation of 1-kestose, nystose, fructosylxyloside, and difructosylxyloside from sucrose, 1-kestose, sucrose and xylose, and fructosylxyloside were respectively determined. Sucrose inhibited nystose formation from 1-kestose, and glucose inhibited the formation of 1-kestose, fructosylxyloside, and difructosylxyloside competitively. The kinetic constants for the hydrolyses of all the above oligosaccharides were determined at lower concentrations, because at higher concentrations the hydrolyses were inhibited. Taking into the consideration the kinetic studies, the preferential production of 1-kestose over nystose and of fructosylxyloside over 1-kestose and difructosylxyloside in fermentations by S. brevicaulis are discussed.

Isolation of β‐galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

β‐Galactosidase (EC 3.2.1.23) was purified from the cell wall of the fruit of Japanese pear (Pyrus serotina Rehder var. culta Rehder cv. Hosui) and characterized. Five peaks of β‐galactosidase activity, designated as Gal I to V, were separated by hydrophobic chromatography on butyl toyopearl and ion exchange chromatography on Mono S. These isolated β‐galactosidases were investigated with regard to their abilities to release monomeric galactose from the fractionated polymers of native cell wall (cyclo‐hexane‐trans‐1,2‐diamine tetraacetic acid‐, Na2CO3‐, guanidine thiocyanate‐ and KOH‐soluble fractions) and arabinogalactan (from larch wood). All the β‐galactosidase fractions were active against native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall and arabinogalactan. Gal II released much galactose only from KOH‐soluble polymers and arabinogalactan. Gal III released the most galactose. from cyclohexane‐trans‐1,2‐diamine tetraacetic acid‐, Na2CO3‐ and guanidine thiocyanate‐soluble cell wall polymers which probably contained galactosyl side chains of pectic polymers, although it did not react much to arabinogalactan. In addition, the activity of Gal Ill dramatically increased as ripening proceeded. Furthermore, Gal III was purified to homogeneity by gel filtration on TSKgel 3000SW and the size of a polypeptide was 80 kDa on SDS‐PAGE.

Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures

To obtain a monospecific antibody against NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), the enzyme was purified to homogeneity from cultured rice cells (Oryza sativa) with column chromatography using Butyl Toyopearl 650M, Sephacryl S-300, Blue Sepharose CL-6B, and Butyl Toyopearl 650S. The specific activity at the final stage of the purification was 9.8 micromoles of glutamate formed per minute per milligram of protein. The yield was 6.1% and purification was 815-fold. Analysis by denaturing gel electrophoresis revealed a single polypeptide with an apparent molecular weight of 196,000, similar to the value of 194,000 estimated for the native protein. Apparent K(m) values for l-glutamine, 2-oxoglutarate, and NADH were 811, 76, and 3.0 micromolar, respectively. Neither NADPH nor l-asparagine substituted for NADH and l-glutamine, respectively. The enzyme had its absorption maxima at 273, 373, and 440 nanometers with a shoulder at 475 nanometers, suggesting that the rice NADH-GOGAT is a flavoprotein. Monospecific antibody raised against NADH-GOGAT purified from the rice cells was obtained as the first instance for the enzyme in higher plants. Immunological analyses showed that the antibody for rice cell NADH-GOGAT reacted with only the enzyme in extracts from the cells. The anti-NADH-GOGAT antibody did not recognize the ferredoxin-GOGAT purified from rice leaves, and likewise the anti-rice leaf ferredoxin-GOGAT antibody did not react with the NADH-GOGAT purified from the cultured rice cells.

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 Km values of 0.5 mM for dopamine with substrate inhibitions at its higher concentrations. I and II were inhibited competitively by NaCI with the Ki 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.

A novel Geotrichum candidum lipase with some preference for the 2-position on a triglyceride molecule

A new component of Geotrichum candidum lipase with a unique positional specificity was isolated from culture broth together with a known major component. The purification included DEAE-Sephadex A-50 ion exchange chromatography. Sephadex G-100 gel filtration, and Butyl Toyopearl 650S hydrophobic interaction chromatography. The newly isolated component, though only a minor one, cleaved the 2-positioned ester bond nearly twice as fast as the 1(3)-positioned ester bond of a triglyceride molecule. In contrast, the major component hydrolysed all the ester bonds indiscriminately, which is consistent with the widely accepted positional specificity of the lipase from G. candidum.

Purification and Characterization of a Novel Thermostable Lipase from Bacillus sp.

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.

Purification of 5β-reductase from hepatic cytosol fraction of chicken

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5β-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5β-reduced metabolites derived from [4- 14C]testosterone. During the purification procedures, 17β-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5β-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3α- and 3β-hydroxysteroid dehydrogenases were able to be removed from 5β-reductase fraction. The final enzyme preparation was apparently homogenous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5β-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. K m of 5β-reductase for NADPH was estimated as 4.22 × 10 −6M and for testosterone, 4.60 × 10 −6M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5β-reductase were examined.

Chiral separation and determination of propranolol enantiomers in rat or mouse blood and tissue by column switching high performance liquid chromatography with ovomucoid bonded stationary phase

Resolution of propranolol (PL) enantiomers in biological samples was accomplished by column switching high performance liquid chromatography using a short precolumn and an analytical column of ovomucoid chiral phase. Plasma, whole blood or tissue homogenate sample was directly injected into the precolumn, and PL was adsorbed on Butyl Toyopearl 650-M. After column switching, the PL was backflushed and transferred to the analytical column (Ultron ES-OVM) by the eluant. Fluorometric detection was carried out at lambda ex = 297 nm and lambda em = 340 nm with a detection limit of 0.5 pmol (signal to noise ratio = 2). The recovery (98.8-103%), reproducibility (coefficient of variance less than 3%) and enantiomer resolution (separation factor 1.15) were satisfactory using as eluant 50 mM sodium dihydrogenphosphate (pH 4.6) containing 12% ethanol. The time course of elimination of PL enantiomers in rat or mouse blood and tissues was also studied.

Preparation of Lactoferrin by Hydrophobic Interaction Chromatography from Milk Acid Whey

Lactoferrin was isolated from 1.8 M ammonium sulfate whey, which had been prepared from bovine milk acid whey, by hydrophobic interaction chromatography using Butyl Toyopearl 650M and by DEAE ion exchange chromatography. The hydrophobic interaction chromatography of 1.8 M ammonium sulfate whey supernatant resulted in concentration of the whey proteins and fractionation into a fraction eluting in deionized water, a fraction eluting in .25 M acetic acid, and a fraction eluting in .2N NaOH. Lactoferrin was contained only in the fraction released by .25 M acetic acid from the matrix of Butyl Toyopearl 650M. It is estimated that approximately 80mg of lactoferrin can be obtained by this method from 1L of neutralized acid whey. The isoelectric point of lactoferrin is between pH 4.8 and pH 5.3 as determined by isoelectric focusing.