Characterisation of 2,5-diketo-D-gluconic acid reductase from Corynebacterium sp.

Summary 2,5-diketo-D-gluconic acid reductase, that converts 2,5-diketo-D-gluconic acid into 2-keto-L-gulonic acid (the direct precursor of vitamin C) was extracted and purified from Corynebacterium sp.. The enzyme was characterised in terms of kinetic parameters, molecular weight and isoelectric point. Enzyme stability at different operating temperatures was investigated, as well.     

Purification and characterization of 2,5-diketo-D-gluconate reductase from Corynebacterium sp

2,5-Diketo-D-gluconate reductase, a novel enzyme that catalyzes the stereospecific NADPH-dependent reduction of 2,5-diketo-D-gluconate to 2-keto-L-gulonate, has been purified to homogeneity by sequential anion exchange, Cibacron blue F3GA affinity, and gel permeation chromatography from Corynebacterium sp. ATCC 31090. Molecular weight of the native form, determined by gel permeation chromatography, is 35,000 +/- 2,000. The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 34,000; thus, the enzyme is active as a monomer. A pI value of 4.4 is measured for the enzyme. Amino- and carboxyl-terminal sequences are consistent with that predicted by the DNA sequence of the reductase gene. At 25 degrees C, pH 6.4, the turnover number is 500 min-1, and the apparent Km values for 2,5-diketo-D-gluconate and NADPH are 26 mM and 10 microM, respectively. The enzyme is specific for NADPH, but the sugar binding site will also accept 5-keto-D-fructose and dihydroxyacetone as substrates. The enzyme is active over a broad pH range (pH 5-8) for the reduction of 2,5-diketo-D-gluconate; a sharp optimum at pH 9.2 is observed for the oxidation of 2-keto-L-gulonate. A Keq value of 5.6 X 10(-13) M indicates that reduction of substrate by NADPH is highly preferred. An activation energy of 12.3 kcal mol-1 is measured. Enzyme turnover is slow relative to dehydration of the gem-diol at C-5 of the substrate.     

PAI—2及其突变体的生物化学性质

为研究和比较纤深酶原激活剂抑制物２型（ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｏｒ ｉｎｈｉｂｉｔｏｒ ｔｙｐｅ ２,ＰＡＩ－２）及其突变体的生物化学性质,用ＰＣＲ、体外定点突变技术构建ＰＡＩ－２突变体ＰＡＩ－２ＣＤ和ＰＡＩ－２ＱｃＤＮＡ,将突变体ｃＤＮＡ与原核表达载体重组并转化怕杆菌。经诱导表达,ＳＤＳ－ＰＡＧＥ证实表达出相应蛋白质,均占全菌总蛋白的１４％。Ｗｅｔｅｒｎ印迹、溶圈抑制及纤维蛋白翻转板     

Biochemical Aspects of Fumaric Acid Accumulation by Rhizopus arrhizus

The accumulation and excretion of fumaric acid, and to a lesser extent malic and succinic acids, by Rhizopus arrhizus occurs under aerobic conditions in a high-glucose medium containing a limiting amount of nitrogen and a neutralizing agent (CaCO₃). An overall four-carbon dicarboxylic acid molar yield of up to 145% (moles of acid produced per mole of glucose utilized) is obtained after incubation for 4 to 5 days. Evidence is presented that fumarate is synthesized from pyruvate via a carboxylation reaction yielding oxaloacetate, which is then converted to malate and further on to fumarate via the reductive reactions of the tricarboxylic acid cycle. The possible formation of fumarate from the normal (oxidative) operation of the tricarboxylic acid cycle was not excluded by the data. Yield, ¹³C nuclear magnetic resonance, and enzymatic activity studies were carried out in a strain of R. arrhizus which produces high levels of fumarate from glucose and carbonate. The observed high fumarate molar yield (greater than 100%) can therefore be explained in terms of the carboxylation of pyruvate and the operation of the reductive reactions of the tricarboxylic acid cycle under aerobic conditions.     

Purification and properties of two 2,5-diketo-d-gluconate reductases from a mutant strain derived from Corynebacterium sp.

Two 2,5-diketo-d-gluconate reductases, I and II, were purified respectively 918-fold and 28-fold from a mutant strain derived from Corynebacterium sp. SHS 0007. The enzymes appeared to be homogeneous on polyacrylamide gel electrophoresis. Both reductases converted 2,5-diketo-d-gluconate to 2-keto-l-gulonate in the presence of NADPH and seemed to be active only for reduction. The molecular weights of reductases I and II were estimated to be 29,000 and 34,000, respectively; and both were monomeric. Their isoelectric points were respectively pH 4.3 and pH 4.1. The optimum pH was 6.0 to 7.0 for reductase I, and 6.0 to 7.5 for reductase II. The K_m values (pH 7.0, 30°C) of reductase I for 2,5-diketo-d-gluconate and for NADPH were 1.8 mM and 12 μM, respectively; and the corresponding values of reductase II were 13.5 mM and 13 μM. Both reductases converted 5-keto-d-fructose to l-sorbose in the presence of NADPH.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Starch Accumulation

The investigation dealt with starch accumulation in four species of Cuscuta (Cuscuta campestris, C. indecora, C. planiflora and C. reflexa), a leafy mistletoe (Dendrophthoe falcata) and a chlorophyll-lacking root parasite (Orobanche aegyptiaca). The highest content of starch occurred In O. aegyptiaca, with a maximum of 45 per cent of dry weight Starch in Cuscuta filaments and mistletoe leaves showed a maximum of about 10 per cent of dry weight. The starch content varied along the length of the Cuscuta vine, with a maximum in the apical region. Orobanche had a higher starch content when it was still submerged than it was fully developed. Cuscuta vines did not show any marked diurnal alteration in the starch content. The content of ethanol-soluble carbohydrate was only a tenth of the starch in Orobanche, but was relatively higher in the other parasites. the neutral sugars in Cuscuta filaments were sucrose and glucose, whereas fructose was also present in mistletoe and Orobanche. Raffinose and stachyose were absent or present only ill traces in parasite tissue. Starch granules from Cuscuta and Orobanche bad ADPG/UDPG-starch synthetase activity and homogenates starch phosphorylase activity. The former enzyme appeared to be responsible for synthesis of starch and the latter for utilization. The four different species of Cuscnta, growing on alfalfa, had more or less the same activity of starch synthetase and also of phosphorylase activity. Hosts infected by Cuscuta had significantly less starch per plant than the controls. A characteristic feature of invasion by Cuscuta and Orobanche was increased phosphorylase activity in the host tissues. The protein content of the tissues of Cuscuta and Orobanche was of a lower level than that of the host shoot system or foliage, indicating that the parasite differed from the host in having a higher carbon (of starch) to nitrogen (of protein) ratio.     

Synthesis of 2-keto-L-gulonic acid from gluconic acid by co-immobilized Gluconobacter oxydans and Corynebacterium sp.

2-Keto-L-gulonic acid was produced from gluconic acid using co-immobilized cells of Gluconobacter oxydans and Corynebacterium sp. with 2,5-diketo-D-gluconic acid. Gluconobacter oxydans and Corynebacterium sp. were entrapped together with polyvinylalcohol and alginate. 50 g/l glucose, 50 g/l gluconic acid, and the mixture of equal volume of 50 g/l glucose and 50 g/l gluconic acid were used as substrates. When the ratio of two cells was 1 to 1 with 100 mg cells/ml, the conversion of 2-KLG from gluconic acid was 38% (g/g). © Rapid Science Ltd. 1998     

Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of Acinetobacter sp. Strain ADP1

Hydroxycinnamates, aromatic compounds that play diverse roles in plants, are dissimilated by enzymes encoded by the hca genes in the nutritionally versatile, naturally transformable bacterium Acinetobacter sp. strain ADP1. A key step in the hca-encoded pathway is activation of the natural substrates caffeate, p-coumarate, and ferulate by an acyl:coenzyme A (acyl:CoA) ligase encoded by hcaC. As described in this paper, Acinetobacter cells with a knockout of the next enzyme in the pathway, hydroxycinnamoyl-CoA hydratase/lyase (HcaA), are extremely sensitive to the presence of the three natural hydroxycinnamate substrates; Escherichia coli cells carrying a subclone with the hcaC gene are hydroxycinnamate sensitive as well. When the hcaA mutation was combined with a mutation in the repressor HcaR, exposure of the doubly mutated Acinetobacter cells to caffeate, p-coumarate, or ferulate at 10⁻⁶ M totally inhibited the growth of cells. The toxicity of p-coumarate and ferulate to a ΔhcaA strain was found to be a bacteriostatic effect. Although not toxic to wild-type cells initially, the diphenolic caffeate was itself converted to a toxin over time in the absence of cells; the converted toxin was bactericidal. In an Acinetobacter strain blocked in hcaA, a secondary mutation in the ligase (HcaC) suppresses the toxic effect. Analysis of suppression due to the mutation of hcaC led to the development of a positive-selection strategy that targets mutations blocking HcaC. An hcaC mutation from one isolate was characterized and was found to result in the substitution of an amino acid that is conserved in a functionally characterized homolog of HcaC.     

Mineralization of 2-chloro- and 2,5-dichlorobiphenyl by Pseudomonas sp. strain UCR2

Pseudomonas sp. strain UCR2 was isolated from a multi-chemostat mating experiment between a chlorobenzoate-degrader, Pseudomonas aeruginosa strain JB2, and a chlorobiphenyl-degrader, Arthrobacter sp. strain B1Barc. Strain UCR2 differed from either of the parental organisms in that it grew on both 2-chloro- and 2,5-dichlorobiphenyl with concomitant release of chloride. Phenotypic typing by the Biolog system indicated that strain UCR2 shared greater similarity with strain JB2 (88%) than strain B1Barc (3%). In DNA:DNA hybridization experiments, genomic DNA from strain UCR2 hybridized with both strain JB2 and strain B1Barc, with the former pairing yielding a much stronger signal than the latter. In contrast, no hybridization whatsoever was observed when the parental organisms strains JB2 and B1Barc were probed against each other.     

Production of Polyalcohol by a Corynebacterium sp.

A gluconate-utilizing strain of Corynebacterium was found to be capable of utilizing aldopentoses and producing corresponding pentitols when pentoses were added to the medium containing gluconate as a carbon source during the cultivation of the organism.

Pentitols produced from d-xylose, l-arabinose, and d-ribose were isolated from the cultured medium and identified as xylitol, l-arabitol, and ribitol, respectively.

The pentitol production was significantly influenced by the concentration of gluconate in the initial medium and that of pentose added to the medium during the cultivation.

The amount of xylitol, l-arabitol, and ribitol reached 69 mg/ml, 60 mg/ml, and 32 mg/ml, respectively, after 14 days of incubation when pentoses were added to the medium containing 9.6% potassium gluconate to give a final concentration of 150 mg/ml.     

Oxidation of acyclic terpenoids by Corynebacterium sp.

Squalene analogs such as lycopersene, geranylfarnesyl, digeranyl, and 2-hydroxy-2,3-dihydrosqualene and terpene alcohol derivatives such as farnesyl benzyl ether, farnesyl pivalate, geranylgeranyl pivalate, geranyl pivalate, and geranyl benzyl ether were oxidized by Corynebacterium sp. strain SY-79, which was isolated from soil by using squalene as a carbon source. Lycopersene and geranylfarnesyl gave no major product. Digeranyl, geranyl benzyl ether, and geranyl pivalate gave terminal oxidation products, and 2-hydroxy-2,3-dihydrosqualene, farnesyl benzyl ether, farnesyl pivalate, and geranylgeranyl pivalate were degraded to give lower molecular carboxylic acids. Strain SY-79 showed promising oxidative activities toward acyclic terpenes, although the metabolites obtained were variable, depending upon the structure of the substrate.     

Purification and characterization of membrane-bound endoglycoceramidase from Corynebacterium sp.

Endoglycoceramidase catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. We found that a bacterial strain Corynebacterium sp., isolated from soil, produced endoglycoceramidase both intracellularly and extracellularly. The intracellular enzyme bound to the cell membrane was solubilized with 1% Triton X-100 and purified to homogeneity about 170-fold with 60% recovery. The molecular mass of the enzyme was approximately 65 kDa. The enzyme is most active at pH 5.5-6.5 and stable at pH 3.5-8.0. Various neutral and acidic glycosphingolipids were hydrolyzed by the enzyme in the presence of 0.1% Triton X-100. Ganglio- and lacto-type glycosphingolipids were readily hydrolyzed, but globo-type glycosphingolipids were hydrolyzed slowly.     

Production of l-Glutamic Acid from Hydrocarbon by Penicillin-resistant Mutants of Corynebacterium hydrocarboclastus

Penicillin-resistant mutants were derived from Corynebacterium hydrocarboclastus R-7. One of them produced 84 g/liter of l-glutamic acid from hydrocarbon, though its parent strain produced 26 g/liter.

The penicillin-resistant mutant had stronger activities of substrate consumption and oxygen absorption than the parent strain, and this was one of the reasons for the accumulation of a larger quantity of l-glutamic acid.

The interacellular content of phosphatidyl inositol mannoside (P.I.M) was related to the glutamate productivity, and the higher glutamate productivity of the penicillin-resistant mutant was supposed to be related to the remarkable diminution in the content of P.I.M.     

L-Tyrosine production by Polyauxotrophic Mutants of Corynebacterium glutamicum

Polyauxotrophic mutants of Corynebacterium glutamicum which have additional requirements to L-phenylalanine were derived from L-tyrosine producing strains of phenylalanine auxotrophs, C. glutamicum KY 9189 and C. glutamicum KY 10233, and screened for L-tyrosine production. The increase of L-tyrosine production was noted in many auxotrophic mutants derived from both strains. Especially some double auxotrophs which require phenylalanine and purine, phenylalanine and histidine, or phenylalanine and cysteine produced significantly higher amounts of L-tyrosine compared to the parents, A phenylalanine and purine double auxotrophic strain LM–96 produced L-tyrosine at a concentration of 15.1 mg per ml in the medium containing 20% sucrose. L-Tyrosine production by the strain decreased at high concentrations of L-phenylalanine.     

l-Isoleucine Production by Analog-resistant Mutants Derived from Threonine-producing Strain of Corynebacterium glutamicum

A thiaisoleucine-resistant mutant, ASAT–372, derived from a threonine producer of Corynebacterium glutamicum, KY 10501, produced 5 mg/ml each of l-isoleucine and l-threonine. l-Isoleucine productivity of ASAT–372 was improved stepwise, with concurrent decrease in threonine production, by successively endowing it with resistivity to such substances as ethionine, 4-azaleucine and α-aminobutyric acid. The mutant strain finally selected, RAM–83, produced 9.7 mg/ml of l-isoleucine with a medium containing 10% (as sugar) molasses.

l-Isoleucine production was significantly affected by the concentration of ammonium sulfate in the fermentation medium. At 4% ammonium sulfate l-isoleucine production was enhanced whereas l-threonine production was suppressed. At 2% ammonium sulfate l-threonine production was stimulated while l-isoleucine production decreased.     

L-Tyrosine Production by Analog-resistant Mutants Derived from a Phenylalanine Auxotroph of Corynebacterium glutamicum

Mutants resistant to various phenylalanine- or tyrosine-analogs were isolated from a phenylalanine auxotroph of Corynebacterium glutamicum KY 10233 by treatment with N- methyl-N′-nitro-N-nitrose guanidine (NTG) and screened for L-tyrosine production. A mutant, 98–Tx–71, which is resistant to 3-aminotyrosine, p-aminophenylalanine, p-fluoro-phenylalanine, and tyrosine hydroxamate was found to produce L-tyrosine at a concentration of 13.5 mg/ml in the cane molasses medium containing 10% of sugar calculated as glucose. A tyrosine-sensitive mutant, pr–20 which was derived from 98–Tx–71 produced L-tyrosine at a concentration of 17.6 mg/ml. L-Tyrosine formation in the strain pr–20 was found to be still inhibited by L-phenylalanine though it was not inhibited by L-tyrosine. The L-tyrosine formation in the mutant was repressed neither by L-phenylalanine nor by L-tyrosine.     

Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1.

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS2, FeS2, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.     

Corynebacterium uberis sp. nov. frequently isolated from bovine mastitis

Several strains belonging to the genus Corynebacterium, but not to any described species of the genus were isolated from bovine mastitic milk samples over the past five years in the diagnostic unit of the University of Bern. Six of these strains (18M0132^T, 17M2518, 18M0913, 19M0083, 20M1046 and 20M1090) that were phenotypically similar were further characterized genotypically. Gram-positive coryneform rods were catalase positive, facultative anaerobe and CAMP-test negative. Whole genome sequencing and subsequent phylogenetic analysis revealed their genome size to be 2.53 Mb and their G + C content to be between 65.4 and 65.5 mol%. Digital DNA-DNA hybridisation (dDDH) showed the highest similarity of only less than 20% with Corynebacterium mastitidis and Corynebacterium frankenforstense, which indicated that the isolates belong to an undescribed Corynebacterium species. This was confirmed by studying the average nucleotide identity (ANI) where the accepted species boundary is around 95% and which ranged between 70.3% and 74.9% with the most closely related species C. mastitidis. We established MALDI-TOF fingerprints of the species, which allows a clear separation from related species and can be used by other laboratories for diagnostic purposes.Based on our analyses we conclude that the selected strains belong to a previously undescribed species and propose the name Corynebacterium uberis sp. nov. The proposed type strain is 18M0132^T (=DSM 111922^T, = CCOS 1972^T).