产顺式环氧琥珀酸水解酶的博德特氏菌BK-52的筛选、鉴定及其产酶条件优化

[目的]产顺式环氧琥珀酸水解酶(CESH)新型菌株的筛选、鉴定及其产酶条件优化.[方法]通过电镜、Biolog GN、(G C)含量和16S rDNA序列研究,对筛选菌株进行分类鉴定.通过红外光谱、核磁共振氢谱和碳谱、质谱以及旋光度实验,鉴定纯化产物的结构并优化CESH产酶条件.[结果]本文筛选出一株产CESH的新型菌株,该菌株可将顺式琥珀酸盐转化为D(-)-酒石酸,属于博德特氏菌属,并将其命名为博德特氏菌BK-52.最佳发酵条件为:最佳温度30℃,最佳pH7.0,最佳发酵时间36 h,最佳碳源蔗糖,最佳无机氮源硫酸铵,最佳酶诱导剂顺式环氧琥珀酸二钠.在此最佳条件下,CESH酶活最高达764 U/g生物量.[结论]本文筛选的新菌株博德特氏菌BK-52为D(一)一酒石酸的生产提供了一种新的方法.     

Two glucosylated abscisic acid derivates from avocado seeds (Persea americana Mill. Lauraceae cv. Hass)

Phytochemical investigation of avocado seed material (Persea americana Mill., Lauraceae) resulted in the isolation of two glucosylated abscisic acid derivates. One of these was not known as a natural product and can be regarded as a potential 'missing link' in abscisic acid metabolism in plants. After fractionation by high-speed countercurrent chromatography, and multiple steps of column chromatography, structures were elucidated by 1D-, 2D-NMR, electrospray-MS to be the novel beta-d-glucoside of (1'S,6'R)-8'-hydroxyabscisic acid, and (1'R,3'R,5'R,8'S)-epi-dihydrophaseic acid beta-d-glucoside. Absolute configuration was determined by circulardichroism, optical rotation, and by NOE experiments.     

Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production

The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)).     

3-羟基丙酸高产菌株的筛选及诱变选育

【目的】3-羟基丙酸是一种重要的化学平台化合物,期望得到一株能够高产3-羟基丙酸的菌株。【方法】从土壤及粪便筛选并对得到的菌株进行鉴定和复合诱变。【结果】得到了一株能够利用丙酸发酵生产3-羟基丙酸的酵母Y-11,经生理生化鉴定及18S rDNA序列分析确定其为Candida sp.(假丝酵母)。以Y-11为出发菌株,经紫外-亚硝基胍-60Coγ复合诱变得到了突变性状稳定且可遗传的高产菌株5-13B,其3-羟基丙酸的产量为11.78 g/L,是出发菌株的2.46倍。【结论】对出发菌株和突变株的发酵特性进行了比较,结果表明突变株的3-羟基丙酸产量、对底物丙酸的转化率、产物3-羟基丙酸的积累性能及丙酸的耐受性均优于出发菌株。     

Presence of fucosamine in teichuronic acid of the alkalophilic Bacillus strain C-125.

Cell walls of the alkalophilic Bacillus strain C-125 are composed of gamma-peptidoglycan, teichuronic acid and a polymer of glucuronate and glutamate. An amino sugar that was a main component of the teichuronic acid did not correspond to any of the commercially available hexosamines. The amino sugar was purified into crystalline form from the hydrolysate of the teichuronic acid by ion-exchange chromatography and then partition chromatography on a cellulose column. The amino sugar was identified as D-fucosamine (2-amino-2,6-dideoxy-D-galactose) by 400 MHz n.m.r. spectrometric analysis, measurement of optical rotation and elemental analysis.     

E-2(S)-amino-3-methyl-3-pentenoic acid from coniogramme intermedia

A new amino acid, E-2(S)-amino-3-methyl-3-pentenoic acid was isolated from Coniogramme intermedia. The structure was elucidated by elementary analysis, optical rotation, catalytic hydrogenation, ¹H and ¹³C NMR spectra.     

金黄喇叭菌中一个新的炔酸化合物

从担子菌金黄喇叭菌(CratereUus aureus)子实体中分离到3个炔酸类化合物,其中-个为新化合物,其化学结构通过波谱学方法和量子化学计算鉴定为(8E,10R,14Z)-10-羟基-8,14-十八碳二烯-12-炔酸(1).     

Production of D-(--)-3-hydroxyalkanoic acid by recombinant Escherichia coli

Pathways for extracellular production of chiral D-(-)-3-hydroxybutyric acid (3HB) and D-(-)-3-hydroxyalkanoic acid (mcl-3HA) were constructed by co-expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB) and 3-hydroxyacyl-ACP CoA transacylase (phaG), respectively, in Escherichia coli strain DH5alpha. The effect of acrylic acid and glucose on production of both 3HB and mcl-3HA was investigated. It was found that the addition of acrylic acid significantly increased production of 3HB and mcl-3HA consisting of 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid in a ratio of 1:3 from 199 mg x l(-1) to 661 mg x l(-1) and from 27 mg x l(-1) to 135 mg x l(-1), respectively, in shake flask studies when glucose was present in the medium at the very beginning of fermentation. The timing of glucose addition had no effect on 3HB production. In contrast, mcl-3HA production was affected by glucose addition, an mcl-3HA concentration of 193 mg x l(-1) was obtained when glucose was added to the culture at 12 h. A more than seven-fold increase was obtained when compared with that in medium containing glucose at the beginning of fermentation. However, a decrease in production of 3HB and mcl-3HA was found when glucose was added at 12 h to the culture containing acrylic acid. The repressive effect of acrylic acid on acetic acid production was also evaluated and discussed.     

Chromatographic determination of optical configuration of 3-hydroxy fatty acids composing microbial surfactants

Abstract The chromatographic determination of the optical configuration of 3-hydroxy fatty acids of microbial surfactants was achieved in chiral high pressure liquid chromatography (HPLC) by injecting 3,5-dinitroaniline-derivatives of crude hydrolysates (less than 1 mg). Serrawettin W2, a surface-active cyclodepsipeptide of Serratia marcescens , was shown to contain d -3-hydroxydecanoic acid. Rubiwettin R1 and RG1, surface active glycolipid and linked fatty acids of Serratia rubidaea , were shown to contain d -3-hydroxytetradecanoic acid and d -3-hydroxydecanoic acid. The new method does not require purified sample or authentic optical isomers, and could be useful in the structural analysis of microbial lipids.     

耐温性L-谷氨酸发酵菌种的选育

应用基因组改组技术提高,L-谷氨酸生产菌在高温发酵条件下的谷氨酸产量。以天津短杆菌T6—13变异株SW07-1为原始亲株,分别经紫外线（UV）-硫酸二乙酯（DES）和X射线诱变,获得5株耐温性能略有提高的突变菌株。经2轮基因组改组,获得耐高温（能在44℃生长）的L-谷氨酸菌株F2-50。F2—50在38℃下,摇瓶发酵40h,发酵液中L-谷氨酸浓度比原始出发菌株提高了近41％,在41℃高温下,摇瓶发酵40h,L-谷氨酸浓度比原始出发菌株提高了近2倍。     

Antigenic polysaccharides of bacteria. 37. Structure of the polysaccharide chain of Pseudomonas syringae pv.tabaci (serogroup VII) lipopolysaccharide]

The structure of the O-specific polysaccharide chain of Pseudomonas syringae pv. tabaci strain 223 (serogroup VII) lipopolysaccharide was established on the basis of one- and two-dimensional 1H NMR analysis, 13C NMR analysis and calculation of optical rotation. The structure determined by the non-destructive way was confirmed by acid hydrolysis and methylation. (Sequence: see text). O-Antigen of the strain studied is similar in structure and serological properties to O-antigens of Pseudomonas syringae strains belonging to serogroup I.     

Production of colominic acid by Pasteurella haemolytica serotype A2 organisms

Using chemical analysis and ¹³C-nuclear magnetic resonance (NMR) spectroscopy, capsular polysaccharide purified from culture supernatants of a strain of Pasteurella haemolytica serotype A2 was shown to consist of a (2 → 8)-α-linked polymer of N-acetylneuraminic acid. This is identical to the capsular polysaccharides of Neisseria meningitidis group B and Escherichia coli K1, and is known as colominic acid. Polymer isolated from a second strain was contaminated with α-1,4-linked dextran. The known poor immunogenicity of these two polymers explains the failure by others to produce effective extract vaccines for this important ovine pathogen.     

一株烟酸羟基化转化菌株的筛选和鉴定

从南京地区的土壤中筛选到一株高效转化烟酸为 6_羟基烟酸的菌株NA_1。形态及生理生化特征测定结果表明 ,NA_1菌株与假单胞菌属 (Pseudomonas)中的恶臭假单胞菌 (P .putida)种的特征基本一致。测定了该菌株的16SrDNA序列并根据 16SrDNA构建了系统发育树 ;在系统发育树中 ,NA_1菌株与恶臭假单胞菌形成一个类群 ,序列同源性为 99%。因此将NA_1菌株鉴定为恶臭假单胞菌     

Model systems for the immunolocalisation of cis,trans abscisic acid in plant tissues

To explore the feasibility of immunolocalisation of endogenous abscisic acid (ABA), model systems were developed for testing quantitatively the sensitivity of the second antibody peroxidase/antiperoxidse (PAP) method for immunolocalisation of ABA on plant tissues. Exogenous (±)ABA was fixed to carrot sections on glass slides or to homogenised pea cotyledon material on microtitre plates, either directly by carbodiimide fixation or by glutaraldehyde fixation of ABA-protein conjugates linked through the C1 carboxyl by 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride (EDC). Backgrounds were decreased by including 0.1% normal goat serum in the incubations, by including 0.1% Triton X-100 as a wetter, by including glycine in the rinses after EDC fixation and by using low-pH rinses after incubation with the primary antibody. Serum antibodies recognising the peptide bond between the protein and abscisic acid were removed by preincubating the serum with acetic acid conjugated to protein. Positives were only accepted when they could be eliminated by adding an excess of ABA-protein conjugate in the primary antiserum. By using a soluble peroxidase reaction product to facilitate quantitation, the limit of reliable exogenous ABA detection was found to be only of the order of 1 pmol. For the histochemical immunolocalisation of endogenous ABA, better antisera and lower backgrounds will be required.The efficiency of fixation of exogenous ABA was determined using [³H] or [¹⁴C]ABA. When aqueous EDC or di-isopropyl carbodiimide (IPC) were used the fixation efficiency was low (up to 5%), but much higher efficiencies (up to 80%) were obtained using IPC vapour with freeze-dried material. Similarly efficient fixation of endogenous ABA in pea cotyledon material, as determined by gas chromatography-mass spectrometry analysis, was obtained using the same technique. The PAP method failed to detect fixed endogenous ABA in pea cotyledons, even though the total tissue amounts present exceeded 1 pmol, evidence that not enough of the ABA was accessible to the antibody.Abbreviations ABA abscisic acid - ACE-ALP acetic acid-alkaline phosphatase - EDC 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride - GC-MS gas chromatographymass spectrometry - IgG Immunoglobulin G - HSA humanserum albumin - IPC dinsopropyl carbodiimide - LINK goat anti-rabbit IgG - OD optical density - PAP peroxidase/rabbit antiperoxidase complex     

Increased urinary excretion of free N-acetylneuraminic acid in thirteen patients with Salla disease

Thirteen severely retarded patients with Salla disease, a new type of lysosomal storage disorder, have been studied biochemically. All patients excreted approximately ten times more free sialic acid than normal individuals. The isolated sialic acid was characterized by paper chromatography, thin-layer chromatography, optical rotation, 13C and 1H nuclear magnetic resonance spectroscopy, and mass spectrometry of its permethylated derivative. The results clearly indicated that the excreted sialic acid was identical to N-acetylneuraminic acid. The main sialylated trisaccharide present in the urine of the patients was identified as 3'-sialyllactose by sugar and methylation analysis. The excreted amounts were found to be within normal range.     

Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid

AIMS: The aim of the present study was to isolate and characterize a bacterium, strain EN3, capable of using diesel oil as a major carbon and energy source, and to analyse the enhancement of diesel oil degradation by this organism using synthetic mycolic acid (2-hexyl-3-hydroxyldecanoic acid). METHOD AND RESULTS: An actinomycete with the ability to degrade diesel oil was isolated from oil contaminated soil and characterized. The strain had phenotypic properties consistent with its classification in the genus Rhodococcus showing a 16S rRNA gene similarity of 99.7% with Rhodococcus baikonurensis DSM 44587(T). The ability of the characterized strain to degrade diesel oil at various concentrations (1000, 5000, 10 000 and 20 000 mg l(-1)) was determined. The effect of synthetic mycolic acid on the biodegradation of diesel oil was investigated at the 20 000 mg l(-1) concentration; the surfactant was added to the flask cultures at three different concentrations (10, 50 and 100 mg l(-1)) and degradation followed over 7 days. Enhanced degradation was found at all three concentrations of the surfactant. In addition, the enhancement of diesel oil degradation by other surfactants was observed. CONCLUSIONS: The synthetic mycolic acid has potential for the remediation of petroleum-contaminated sites from both an economic and applied perspective as it can stimulate biodegradation at low concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the synthesized mycolic acid can be used for potential applications in the bioremediation industries, for example, in oil spill clean-up, diesel fuel remediation and biostimulation.     

Structural studies on the core oligosaccharide of Phenylobacterium immobile strain K2 lipopolysaccharide. Chemical synthesis of 3-hydroxy-5c-dodecenoic acid

The fatty acid composition of the bound lipids of 17 strains from Phenylobacterium immobile was investigated. Ester-linked 3-hydroxy-5c-dodecenoic acid was found to be the major substituent in the lipopolysaccharide. Therefore the occurrence of this unusual acid can be taken as a useful marker for the determination of P. immobile by simple fatty acid analysis. Lipid A backbone was found to consist of 2,3-diamino-2,3-dideoxy-D-glucose, which up to this time has only been detected in group I of purple nonsulfur bacteria and their non-phototrophic relatives. A convenient chemical synthesis for 3-hydroxy-5c-dodecenoic acid is described. The polysaccharide region of P. immobile strain K2 lipopolysaccharide was investigated. The methods used included gel filtration procedures, methylation analysis, periodate oxidation, Smith degradation, oxidation with chromium trioxide and enzymic degradations of the isolated oligosaccharide. We found that strain K2 has a rough-type lipopolysaccharide, devoid of an O-specific side chain. For the core oligosaccharide the following structure is proposed: (Formula: see text) beta-D-Glcp(1-3)-L-alpha-D-Hep rho(1-3)-L-alpha-D-Hep rho(1-3-L-alpha-D-Hep rho(1-4/5)dOclA.     

Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae

A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens. Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride. DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position. Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24. The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.     

Production of D-lactic acid from 1,2-propanediol byPseudomonas sp. strain TB-135

Summary A newly isolated bacterium, strain TB-135, produces solely D-lactic acid from 1,2-propanediol (1,2-PD). From taxonomical studies, the strain was concluded to belong to the genusPseudomonas. The optimal conditions for the acid production were found to be at 4% 1,2-PD and 0.2% yeast extract, when the strain produced 21mg/ml of the acid of a high optical purity (over 99% e.e.) after 4days of cultivation. Since it was considered that the acid was produced from (R)-(–)-1,2-PD, the molar yield was estimated to be 86%.     

Structure of the type 5 capsular polysaccharide of Staphylococcus aureus

The Staphylococcus aureus type 5 capsular polysaccharide is composed of 2-acetamido-2-deoxy-L-fucose (1 part), 2-acetamido-2-deoxy-D-fucose (1 part), and 2-acetamido-2-deoxy-D-mannuronic acid (1 part). On the basis of methylation analysis, optical rotation, high-field one- and two-dimensional 1H- and 13C-n.m.r. experiments, and selective cleavage with 70% aqueous hydrogen fluoride, the polysaccharide was found to be a partially O-acetylated (50%) polymer of the repeating trisaccharide unit, [----4)-3-O-Ac-beta-D-ManpNAcA-(1----4)-a-L-FucpNAc-(1----3) -beta-D-FucpNAc-(1----]n.