Purification and properties of a heat-stable inulin fructotransferase from Arthrobacter ureafaciens

An inulin fructotransferase producing difructose dianhydride I (EC 2.4.1.200) was purified from Arthrobacter ureafaciens A51-1. It had maximum activity at pH 5.5 and 45 °C, and was stable up to 80 °C. This is the highest thermal stability for this enzyme reported to date. The molecular mass was estimated to be 38000 by SDS-PAGE, and 61000 by gel filtration. It was therefore estimated to be a dimer.     

Purification and properties of neuraminidase isozymes in Arthrobacter ureafaciens mutant

An Arthrobacter ureafaciens mutant (M1057) capable of producing neuraminidase constitutively was isolated by NTG mutagenesis from A. ureafaciens KMS 3663. Four molecular species (L, M1, M2, and S) of neuraminidase isozymes were homogeneously purified from the mutant and parent strains by means of DEAE-cellulose, affinity chromatography, ammonium sulfate precipitation, chromatofocusing, and Ultrogel AcA44 gel filtration. The molecular weights of L, M1, M2, and S isozymes were shown to be approximately 88,000, 66,000, 66,000, and 52,000, respectively. The optimal pHs and Km values of these isozymes for N-acetylneuraminosyl-alpha,(2-6)-lactose were 4.5-5.5 and 0.6-0.8 mM. Neuraminidase L, M1, M2, and S were able to hydrolyze oligosaccharides, glycoproteins and gangliosides containing alpha,(2-3)-, alpha,(2-6)-, and alpha,(2-8)-linked N-acetylneuraminic acid. Among these isozymes isolated, isozyme S was most active on colominic acid.     

Action of levan fructotransferase of Arthrobacter ureafaciens on levanoligosaccharides

Levan fructotransferase of the bacterium Arthrobacter ureafaciens, which produces di-D-fructose 2,6':6,2' dianhydride (difructose anhydride IV) from levan by an intramolecular transfructosylation reaction, was purified to give a single protein band of pI 4.5-4.7 on isoelectric focusing. It had a molecular weight of 128,000 on gel-filtration on Sephadex G-200 and 60,000 on SDS-polyacrylamide disc gel-electrophoresis, suggesting that the enzyme is composed of two identical subunits. The shortest levanoligosaccharide chain required for the difructose anhydride IV formation was determined to be tetraose. TLC of the enzymic digest of a modified levanhexaose derived from levanhexaose by the reduction of the reducing end to an alditol residue with sodium borohydride gave the difructose anhydride IV spot, suggesting that the enzyme attacks the modified levanhexaose molecule from the direction of the non-reducing fructose end. The enzymic digests of levantetraose, -pentaose, and -hexaose as the substrate gave, in addition to the difructose anhydride IV spot, spots of oligofructans of lower mobility than the original substrate on TLC. From the digest of levantetraose, a hexaoligofructan and a smaller amount of a pentaoligofructan but no fructose were separated, indicating enzymic intermolecular levanbiosyl and fructosyl transfer reactions.     

Synthesis of Sialyllactose from N-Acetylneuraminic Acid and Lactose by a Neuraminidase from Arthrobacter ureafaciens

Two sialyllactose isomers, NeuAcα2→6Galβ1→4Glc and Galβ1→4(NeuAcα2→6)Glc, were prepared by incubation of a concentrated solution of N-acetylneuraminic acid and lactose in the presence of a neuraminidase from Arthrobacter ureafaciens. Each sialyllactose was isolated by a combination of ion-exchange chromatography and high performance liquid chromatography. The structure of each sialyllactose was identified by mass spectrometry, nuclear magnetic resonance spectrometry, and enzymatic analysis.     

Cystite formation of Arthrobacter ureafaciens NRIC 0157(T)

The cystite formation of Arthrobacter ureafaciens NRIC 0157(T) was studied by the use of a newly designed CT medium composed of 2.0% D-glucose, 0.28% NH(4)H(2)PO(4), 0.136% K(2)HPO(4) 0.136% KH(2)PO(4), 0.0005% MgSO(4).7H(2)O, and 0.0007% CaCl(2).2H(2)O. Cystites are drumstick-shaped and oval cells and are larger than vegetative cells. Cystites are Gram-negative, whereas vegetative cells are Gram-positive by the KOH reaction. The concentration and ratio of K(+) and Mg(2+) in CT medium mainly affected the cystite formation. Cystites are considered aberrant forms produced by nutritional imbalance.     

Action of Arthrobacter ureafaciens inulinase II on several oligofructans and bacterial levans.

1. Arthrobacter ureafaciens inulinase II which converts inulin to di-D-fructofuranose 1,2' : 2,3' dianhydride (difructose anhydride III) leaving a small amount of oligosaccharides, was investigated in order to characterize its mode of action. 2. After the enzymatic reaction on the glucose-terminated inulin molecules had been completed, the oligosaccharides left in the enzyme digest were isolated, and identified to be the fructose-glucose oligosaccharides; O-beta-D-fructofuranosyl-(2 leads to 1)-O-beta-D-fructofuranosyl alpha-D-glucopyranoside (1-kestose), O-beta-D-fructofuranosyl-[(2 leads to 1)-O-beta-D-fructofuranosyl]2 alpha-D-glucopyranoside and O-beta-D-fructofuranosyl-[(2 leads to 1)-O-beta-D-fructofuranosyl]3 alpha-D-glucopyranoside. The difructose anhydride formation from the three fructose-glucose oligosaccharides in the separate reaction system with an increased substrate concentration was observed only with the latter two substrates, but not with the first one. 3. The difructose anhydride formation with several (2 leads to 1)-beta-linked fructose oligosaccharides and bacterial (2 leads to 6)-beta-fructans was examined. The (2 leads to 1)-beta-linked fructose oligosaccharides were effective as substrates for the enzyme with the exception of inulobiose, but the (2 leads to 6)-beta-fructans remained unaffected. 4. It was concluded that the enzyme attacks (2 leads to 1)-beta-linked fructan molecules from the nonreducing fructose ends and requires the presence of at least two adjacent (2 leads to 1)-beta-fructofuranosyl linkages.     

Characteristics of levan fructotransferase from Arthrobacter ureafaciens K2032 and difructose anhydride IV formation from levan

A microorganism producing levan fructotransferase was isolated from sugar-disclosed soil and it was identified as Arthrobacter ureafaciens. The major product from levan by enzyme reaction was identified as di-D-fructofuranose 2,6':6,2' dianhydride by mass spectrometry, nuclear magnetic resonance, and chemical analyses. Small amounts of several oligosaccharides and free fructose were also formed by enzyme reaction. An extracellular enzyme that produces di-D-fructofuranose 2,6':6,2' dianhydride from levan was purified from the culture broth of A. ureafaciens K2032. The enzyme had optimum activity around pH 5.8 and 45 degrees C and had a dimeric form in solution. The N-terminal amino acid residues of the purified enzyme were SAPGSLRAVYHMTPPSGXLXDPQ. The enzyme has narrow substrate range and converts the levan to di-D-fructofuranose 2,6':6,2' dianhydride with around 62.5% conversion yield.     

Intermolecular fructosyl and levanbiosyl transfers by levan fructotransferase of Arthrobacter ureafaciens

A purified levan fructotransferase preparation from the culture of the bacterium Arthrobacter ureafaciens, which produces di-D-fructose 2,6':6,2' dianhydride (difructose anhydride IV) from levan by an intramolecular levan fructosyl transfer (ILFT) reaction, was found to produce a trioligofructan and a tetraoligofructan from levan in the presence of levanbiose, indicating the intermolecular fructosyl and levanbiosyl transfer (LFT and LBT) reactions. The tri- and tetraoligofructans were identified to be levantriose and -tetraose respectively. Increase in the levanbiose concentration brought about increased production of both oligofructans with decreased formation of difructose anhydride IV, supporting the previous theory proposed by Tanaka et al. (1983) that the ILFT, LFT, and LBT reactions are catalyzed by the same enzyme. In addition, there existed a roughly stoichiometric relationship between the increase and decrease in the productions of these oligofructans, and the LBT reaction was found to occur more intensively than the LFT reaction. Acceptor specificity of the LFT and LBT reactions was studied using fifteen sugars including mono-, di-, and trisaccharides. The enzyme showed both of the reactions only with levanbiose, -triose, and kestose, indicating that the exposed non-reducing levanbiosyl residue was essential for the acceptor and suggesting the existence of a levanbiosyl acceptor site on the enzyme molecule.     

Action of Levan Fructotransferase of Arthrobacter ureafaciens on a Mixture of Branched Levanpentasaccharides

Two coryneform bacteria, Arthrobacter globiformis IFO 12137 (ATCC 8010) and Brevibacterium helvolum IFO 12073, which have the arginine oxygenase pathway, could utilize L-ornithine, L-citrulline, and D-arginine. The cells of the bacteria grown on these amino acids contained high levels of guanidinobutyrase and induced levels of the enzymes of the preceding steps of the pathway. 4-Guanidinobutyrate induced guanidinobutyrase but failed to induce the other enzymes, indicating that it was the direct inducer of guanidinobutyrase. These amino acids and L-arginine also induced L-arginine: 2-ketoglutarate aminotransferase. 4-Aminobutyrate was formed on incubation of L-citrulline with L-citrulline-grown cells of A. globiformis in the presence of gabaculine; its amount was about 50% of the L-citrulline degraded. The L-arginine-grown cells produced 4-aminobutyrate and urea from L-arginine in the presence of aminooxyacetate or gabaculine; the amount of 4-aminobutyratewas 80% or more of that of the L-arginine degraded. When the oxygenase pathway was blocked with thioglycolate, the degradation of L-arginine and the formation of urea and 4-aminobutyrate were greatly suppressed. These results indicate that these amino acids are degraded via the arginine oxygenase and the arginine aminotransferase pathways and the major route is the former. Agmatine was degraded in these bacteria and induced agmatine deiminase, carbamoylputrescine hydrolase, putrescine oxidase, and aminobutyraldehyde dehydrogenase. None of the enzymes was induced by L-arginine.     

Biodegradation of 4-chlorophenol via a hydroquinone pathway by Arthrobacter ureafaciens CPR706

Abstract A newly isolated Arthrobacter ureafaciens , strain CPR706, could degrade 4-chlorophenol via a new pathway, in which the chloro-substituent was eliminated in the first step and hydroquinone was produced as a transient intermediate. Strain CPR 706 exhibited much higher substrate tolerance and degradation rate than other strains that degraded 4-chlorophenol by the hydroxylation at the second carbon position to form chlorocatechol. Strain CPR706 could also degrade other para -substituted phenols (4-nitro-, 4-bromo-, 4-iodo-, and 4-fluoro-phenol) via the hyroquinone pathway.     

Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790.

Arthrobacter sp. strain ATCC 33790 was grown with pentachlorophenol (PCP) as the sole source of carbon and energy. Crude extracts, which were prepared by disruption of the bacteria with a French pressure cell, showed no dehalogenating activity with PCP as the substrate. After sucrose density ultracentrifugation of the crude extract at 145,000 x g, various layers were found in the gradient. One yellow layer showed enzymatic conversion of PCP. One chloride ion was released per molecule of PCP. The product of the enzymatic conversion was tetrachlorohydroquinone. NADPH and oxygen were essential for this reaction. EDTA stimulated the enzymatic activity by 67%. The optimum pH for the enzyme activity was 7.5, and the temperature optimum was 25 degrees C. Enzymatic activity was also detected with 2,4,5-trichlorophenol, 2,3,4-trichlorophenol, 2,4,6-trichlorophenol, and 2,3,4,5-tetrachlorophenol as substrates, whereas 3,4,5-trichlorophenol, 2,4-dichlorophenol, 3,4-dichlorophenol, and 4-chlorophenol did not serve as substrates.     

Enzymic hydrolysis of di-D-fructofuranose 1, 2'; 2, 3' dianhydride with Arthrobacter ureafaciens.

Enzymic hydrolysis of di-D-fructofuranose 1, 2'; 2, 3' dianhydride with the bacteria Arthrobacter ureafaciens was studied to elucidate its mechanism. Hydrolysis of the difructose dianhydride to D-fructose, which did not occur with yeast invertase [EC 3.2.1.26], was found to occur on incubation with an enzyme preparation from an autolysate of the above bacteria. However, incubation with enzyme which had been treated at 60 degrees for 30 min yielded an intermediate hydrolysis product. The product isolated was found to be inulobiose and to be hydrolyzed to D-fructose by the original enzyme, as well as by yeast invertase. It was thus shown that the hydrolysis of the difructose dianhydride to D-fructose with the crude enzyme took place not in a single step but in two separate steps at 2, 3' and 1, 2' linkages. It was not determined whether the entire process is mediated by one and the same beta-fructofuranosidase or by different enzymes.     

Enzymatic Characterization of an Amine Oxidase from Arthrobacter sp. Used to Measure Phosphatidylethanolamine

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS–PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu²⁺, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.     

Enzymatic characterization of an amine oxidase from Arthrobacter sp. used to measure phosphatidylethanolamine

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS-PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu2+, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).     

Enzymatic dehalogenation of 4-chlorobenzoate by extracts from Arthrobacter sp. SU DSM 20407

In extracts from Arthrobacter sp. SU DSM 20407 an enzyme was detectable, that converted 4-chlorobenzoate into 4-hydroxybenzoate. This conversion was also observed when no oxygen was present in the reaction mixture. Boiling for 5 min destroyed the enzyme activity. 4-Bromo- and 4-iodobenzoate were substrates for the enzyme too, but not 4-fluorobenzoate, 4-chlorophenylacetate and 4-chlorocinnamic acid. The enzyme showed optimum activity at 16 degrees C and at pH 7-7.5. The specific activity in the extracts varied between 0.5 and 5 mU/mg of protein. Zn2+ and Cu2+ inhibited the enzyme, while H2O2 slightly activated. In contrast to all other 4-chlorobenzoate dehalogenases described before the enzyme was not inhibited by EDTA, nor was it activated by Mn2+. Other divalent ions also had no effect. The molecular mass of the enzyme was 45,000 +/- 5,000 Da as judged by gel-filtration.