定向进化提高来源于Arthrobacter ramosus的MTHase的热稳定性

麦芽寡糖基海藻糖水解酶(mahosyhrehalose hydrolase,MTHase)是以淀粉或麦芽糊精为底物制备海藻糖的关键酶之一.来源于Arthrobacter ramosus的MTHase,表达量好,比活高,但热稳定性差,限制了其工业化应用.采用定向进化技术,筛选得到L137M和A216T两个突变体,在60℃...     

Catalytic mechanism of inulinase from Arthrobacter sp. S37

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k_cat/K_m) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k_cat, but not due to variations in K_m, consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK_a of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK_a. Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme.     

Gene Analysis of Trehalose-producing Enzymes from Hyperthermophilic Archaea in Sulfolobales

The genes encoding new trehalose-producing enzymes from S. acidocaldarius ATCC33909 were cloned to analyze the distribution of these genes in Sulfolobales. Comparison of the amino acid sequences with S. solfataricus KM1 showed approximately 50% similarity. Southern analysis suggests that homologues of the trehalose-producing enzyme genes exist widely in Sulfolobales and strains in Sulfolobales were classified into three kinds of genotypes.     

L-Phase variants ofAgromyces ramosus

Agromyces ramosus, which is a numerically prevalent bacterium in soil, was easily induced into the L-phase by growing it on agar media containing low levels of penicillin or glycine. The L-forms were stable after initial contact with the inducing agent and could not be reverted to the bacterial form by any of the procedures tried. These results are discussed in relation to a possible natural occurrence of L-forms of this bacterium in soil.     

Study and immunoanalytical applications for peroxidase from Arthromyces ramosus

Some biochemical and catalytic properties of peroxidase from Arthromyces ramosus (EC 1.11.7.1) in chemiluminescent reaction of luminol oxidation by hydrogen peroxide were investigated. The second order rate constants were determined by the stopped-flow technique. Optimal conditions to quantity the enzyme were found, the detection limit being 5.10(-13) M. The peroxidase was used as a marker in the human IgG immunoassay.     

New Bacterial Polysaccharide from Arthrobacter

A bacterial strain (NRRL B-1973) isolated from soil at Guatemala City and tentatively identified as an Arthrobacter species produced a polysaccharide with unusual properties. Conditions were studied for the production of this microbial gum in shaken flasks and 20-liter fermentors. Suitable nutrients for optimal polysaccharide production included 3% glucose, 0.3% enzyme-hydrolyzed casein, magnesium sulfate, manganese sulfate, and potassium phosphate buffer (pH 7.0). Polysaccharide yields ranged from 40 to 45%, based on initial dextrose in the medium in 3- or 4-day fermentations. The gum was readily recovered from culture fluid by alcohol precipitation in the presence of an electrolyte. The Arthrobacter gum exhibited characteristics unique for a polyelectrolyte. Viscosity of solutions was not decreased by heating in the presence of salt, and the gum withstood a temperature of 121 C for 30 min. At polysaccharide levels above 0.75%, gels were formed when solutions were autoclaved with KCl. There was no significant change in viscosity over a pH range of 5.0 to 10.0.     

Production,isolation and characterization of extracellular protease of an alkaliphilic strain of Arthrobacter ramosus,MCM B-351 isolated from the alkaline lake of Lonar,India

An extracellular protease was produced by Arthrobacter ramosus isolated from the alkaline lake of Lonar, Buldhana District of Maharashtra, India when grown on a synthetic medium of pH 10 containing casein. The optimum conditions for production were 3.0% initial casein concentration, 2% inoculum of 1 × 10⁸ cells/ml, pH 9.0, temperature 30 °C and shaken culture conditions. The protease was purified by ammonium sulphate precipitation followed by Sephadex G-100 chromatography. Two proteases viz. Arthro I and Arthro II, having molecular weights 21 and 11.4 kDa respectively were isolated. The Arthro II fraction had K _m 395 g/ml and V _max 10.55 g/min for azocasein. The maximum activity of enzyme was at 55 °C and pH 8. It was thermostable (up to 80 °C), alkali stable (pH 12) and stable in commercial detergent. The enzyme may contain a thiol group at the active site.     

Theczc operon ofAlcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals

Summary The plasmid-borneczc operon ensures for resistance to Cd²⁺, Zn²⁺ and Co²⁺ ions through a tricomponent export pathway and is associated to various conjugative plasmids ofA. eutrophus strains isolated from metal-contaminated industrial areas. Theczc region of pMOL30 was reassessed especially for the segments located upstream and downstream the structural genesczc CBA. In cultures grown with high concentrations of heavy metals,czc-mediated efflux of cations is followed by a process of metal bioprecipitation. These observations led to the development of bioreactors designed for the removal of heavy metals from polluted effluents.     

Cloning, sequencing and characterisation of a downstream region of ksdDI operon of Arthrobacter simplex.

An 8-kb region downstream of the ketosteroid dehydrogenase (ksdD)-ketosteroid isomerase (ksdI) genes of Arthrobacter simplex was cloned. The nucleotide sequence of the first 3-kb segment downstream of ksdD-ksdI operon was determined. Three open reading frames (ORFs) preceded by Shine-Dalgarno (SD) sequences have been found. Homology search revealed that the putative product encoded by ORF3 has high level of similarity with the 3-oxosteroid dehydrogenases from A. simplex and P. testosteroni (90% identity in their putative active sites). The role of ORF3 product as a FAD-containing dehydrogenase in steroid degradation pathway is discussed.     

Analysis of bgl operon structure and characterization of beta-glucosidase from Pectobacterium carotovorum subsp. carotovorum LY34

A putative bgl operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated. Sequence analysis of the 5,557 bp cloned DNA fragment (accession no. AY542524) showed three open reading frames (bglT, bglP, and bglB) predicted to encode 287, 633, and 468 amino acid proteins respectively. BglT and BglP ORFs show high similarity to that of the Pectobacterium chrysanthemi ArbG antiterminator and ArbF permease respectively. Also, the latter contains most residues important for phosphotransferase activity. The amino acid sequence of BglB showed high similarity to various beta-glucosidases and is a member of glycosyl hydrolase family 1. The purified BglB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. The molecular weight of the enzyme was estimated to be 53,000 Da by SDS-PAGE. The purified beta-glucosidase exhibited maximal activity at pH 7.0 and 40 degrees C, and its activity was enhanced in the presence of Mg(2+). Two glutamate residues (Glu(173) and Glu(362)) were found to be essential for enzyme activity.     

Crystallization and preliminary X-ray diffraction studies of peroxidase from a fungus Arthromyces ramosus

Peroxidase (donor: H₂O₂ oxi-doreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P4₂2₁2, with unit cell dimensions of a = b = 74.5 Å, c = 117.6 Å. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 Å resolution and are stable to X-rays. © 1993 Wiley-Liss, Inc.     

Cloning and nucleotide sequence of a gene encoding a glycogen debranching enzyme in the trehalose operon from Arthrobacter sp. Q36

A gene located just upstream of the treYZ operon was isolated from Arthrobacter sp. strain Q36. The gene, designated treX, encoded an 823-amino acid protein. The amino acid sequence of the protein had 50% identity with the TreX protein (isoamylase) from Sulfolobus acidocaldarius ATCC 33909 which has a treZXY operon on the genome. We suggest that Arthrobacter treX is an isoamylase gene, and that it is a component of a treXYZ operon.     

L-serine dehydratase from Arthrobacter globiformis.

1. L-Serine dehydratase (EC 4.2.1.13) was purified 970-fold from glycine-grown Arthrobacter globiformis to a final specific activity of 660micronmol of pyruvate formed/min per mg of protein. 2. The enzyme is specific for L-serine; D-serine, L-threonine and L-cysteine are not attacked. 3. The time-course of pyruvate formation by the purified enzyme, in common with enzyme in crude extracts and throughout the purification, is non-linear. The reaction rate increases progressively for several minutes before becoming constant. The enzyme is activated by preincubation with L-serine and a linear time-course is then obtained. 4. The substrate-saturation curve for L-serine is sigmoid. The value of [S]0.5 varies with protein concentration, from 6.5mM at 23microng/ml to 20mM at 0.23microng/ml. The Hill coefficient remains constant at 2.9.5 The enzyme shows a non-specific requirement for a univalent or bivalent cation. Half-maximal activity is produced by 1.0mM-MgCl2 or by 22.5mM-KCl. 6. L-Cysteine and D-serine act as competitive inhibitors of L-serine dehydratase, with Ki values of 1.2 and 4.9mM respectively. L-Cysteine, at higher concentrations, also causes a slowly developing irreversible inhibition of the enzyme. 7. Inhibition by HgCl2 (5micronM)can be partially reversed in its initial phase by 1mM-L-cysteine, but after 10 min it becomes irreversible. 8. In contrast with the situation in all cell-free preparations, toluene-treated cells of A. globiformis form pyruvate from L-serine at a constant rate from the initiation of the reaction, show a hyperbolic substrate-saturation curve with an apparent Km of 7mM and do not require a cation for activity.     

细菌黄嘌呤氧化酶的稳定性

研究pH、温度、金属离子和一些添加剂对黄嘌呤氧化酶稳定性的影响。结果表明：黄嘌呤氧化酶在pH4．5～7．5的范围内较稳定;反应最适温度为37℃。在常温25～35℃该酶比较稳定,经45℃处理2h．可保持50％左右,不同种类、不同浓度的金属离子对黄嘌呤氧化酶活性表现出程度不同的激活或抑制作用;添加谷氨酸和天门冬氨酸,能有效提高黄嘌呤氧化酶的存放稳定性.     

Isopullulanase from Arthrobacter globiformis T6

The physico-chemical properties of the purified glucose isomerases [d-xylose ketol isomerase, EC 5.3.1.5] of Streptomyces olivochromogenes and Bacillus stearothennophilus were examined. The molecular size and shape of both enzymes were similar. The molecular weights, sedimentation coefficients, partial specific volumes, diffusion constants and Stokes’ radii of the Streptomyces and Bacillus enzymes were determined to be 120,000 and 130,000, 7.55 S and 9.35 S, 0.725 and 0.736 ml/g, 5.87 × 10^-7 and 6.82 × 10^-7 cm²/sec, and 51 and 53 Å, respectively. The Streptomyces glucose isomerase was found to consist of two subunits, each having a molecular weight of 56,000. Large differences were found in the amino acid compositions of these two enzymes, especially in their serine, proline, tyrosine, lysine and arginine contents. The enzymatic properties of both these purified glucose isomerases were also examined, and it was seen that they both displayed activity on d-xylose, d-xylulose, d-glucose, d-fructose, d-arabinose and d-ribose. The smaller Km values and the larger molecular activities for d-xylose and d-xyluIose indicated that both enzymes are essentially d-xylose isomerases. The optimum temperature was 80°C for both enzymes. The optimum pH was 8 to 10 for the Streptomyces enzymes and 7.5 to 8.0 for the Bacillus enzyme. The Bacillus enzyme was more thermostable than the Streptomyces enzyme, but required cobalt ions in addition to magnesium ions for the full expression of its activity.     

Isolation of Arthrobacter Bacteriophage from Soil

Soil was percolated with water and various nutrient solutions, and then the percolates were analyzed for bacteriophages which produced plaques on various Arthrobacter strains. The water percolates did not contain detectable phage. In contrast, phages for A. globiformis strains ATCC 8010 and 4336, and for several recent Arthrobacter species soil isolates, were easily detected in nutrient broth, soil extract, and cation-complete medium percolates. These percolates did not contain phage that produced plaques on A. oxydans and a recent Arthrobacter species soil isolate. Percolation with a selective nicotine-salts solution was required for demonstrating phage for these bacteria. None of the percolates contained phage for five additional named Arthrobacter species. In addition, phages were not detected for A. crystallopoietes in a 2-hydroxypyridine percolate of soil. Based on their lytic spectra, the phage isolates from this soil were relatively host specific.