Enzymatic preparation of pyruvate by a whole-cell biocatalyst coexpressing l-lactate oxidase and catalase

Enzymatic oxidative dehydrogenation of biomass-derived l-lactate in the presence of O₂ is regarded as a green alternative for pyruvate production, but the process also results in H₂O₂ accumulation. To remove the effect of H₂O₂ on pyruvate production, various potential catalases from different species were screened for their efficiency in H₂O₂ degradation. Then, an in vitro cascade biocatalysis was designed using lactate oxidase from Aerococcus viridans (AvLOX) and catalase from Ureibacillus thermosphaericus (UtCAT). The in situ removal of H₂O₂ by UtCAT significantly improved the yield and productivity of pyruvate. To simplify the technological processes and reduce production costs, a whole cell biocatalyst without the addition of catalase for the production of pyruvate was established by coexpressing AvLOX and UtCAT in Escherichia coli. By applying suitable coexpression strategies and changing the ribosome binding site (RBS) strengths, the optimal ratio of AvLOX:UtCAT(12.6:4127.3) in E. coli-AvLOX-_(rbs3)UtCAT was finally achieved. Under the optimized transformation conditions, pyruvate was produced at a titer of 59.9 g/L and a yield of 90.8% in a substrate fed-batch process, promising an alternative route for the green production of pyruvate.     

<Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> as a novel biocatalyst for pyruvate production from<Emphasis Type="SmallCaps"> DL</Emphasis>-lactate

Pseudomonas stutzeri SDM oxidized dl-lactic acid (25.5 g l^-1) into pyruvic acid (22.6 g l^-1) over 24 h. Both NAD⁺-independent d-lactate dehydrogenase and NAD⁺-independent l-lactate dehydrogenase were found for the first time in the bioconversion of lactate to pyruvate based on the enzyme activity assay and proteomic analysis. Jianrong Hao and Cuiqing Ma contributed equally to this work     

Biodegradation and detoxification of nicotine in tobacco solid waste by a Pseudomonas sp.

A Pseudomonas sp. grew with nicotine optimally 3 g l^–1 and at 30 °C and pH 7. Nicotine was fully degraded within 10 h. The resting cells degraded nicotine in tobacco solid waste completely within 6 h in 0.02 m sodium phosphate buffer (pH 7) at maximally 56 mg nicotine h^–1 g dry cell^–1.     

铜绿假单胞菌对青霉素类抗生素异质性耐药研究

【背景】铜绿假单胞菌是临床上常见的条件致病菌,其异质性耐药的发生常导致临床治疗失败。【目的】研究铜绿假单胞菌对青霉素类抗生素的异质性耐药情况,为相关临床感染治疗提供一定的依据。【方法】收集临床分离的50株铜绿假单胞菌,采用纸片扩散法(diskdiffusion method)即Kirby-Bauer (K-B)法、菌落谱型分析(population analysis profile,PAP)法、生长实验以及传代稳定性实验探究铜绿假单胞菌的异质性耐药特征。【结果】K-B法初筛得到铜绿假单胞菌对哌拉西林(piperacillin,PIP)、哌拉西林/他唑巴坦(piperacillin/tazobactam,TZP)和替卡西林/克拉维酸(ticarcillin/clavulanic acid,TIM)的异质性耐药率分别为52%、52%和54%。PAP实验确认后有13株异质性耐药菌,其检出率占总实验菌株的26%。随机选取8株异质性耐药菌株,其耐药亚群的发生频率为7.3×10-7-1.2×10-5。通过无抗生素压力的生长实验发现,异质性耐药菌株PAS92、PAS57与其各自的3株最高PIP浓度平...     

Cloning, expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp. TS1138

L-cysteine desulfhydrase (CD) plays an important role in L-cysteine decomposition. To identify the CD gene in Pseudomonas sp. TS1138 and investigate its effect on the L-cysteine biosynthetic pathway, the CD gene was cloned from Pseudomonas sp. TS1138 by polymerase chain reaction (PCR) method. The nucleotide sequence of CD gene was determined to be 1,215 bp, and its homology with other sequences encoding CD was analyzed. Then the CD gene was subcloned into pET-21a(+) vector and expressed in Escherichia coli (E. coli) by isopropyl-β-D-thiogalactopyranoside (IPTG) inducement. The recombinant CD was purified by Ni-NTA His-Bind resin, and its activity was identified by the CD activity staining. The enzymatic properties of the recombinant CD were characterized and its critical role involved in the L-cysteine biosynthetic pathway was also discussed. __________ Translated from Microbiology, 2006, 33(4): 21–26 [译自: 微生物学通报]     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined –16 to –25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

假单胞菌T1-3-2的抑菌特性及其对杉木的抗病促生作用

【背景】生物防治是“基于自然的解决方案”,有利于生态文明和可持续发展,开展生物防治技术研究的基础是明确菌株的生防作用和抑菌特性。【目的】探究杉木内生菌株T1-3-2的抑菌促生特性,为研制该菌株生防菌剂、防治杉木炭疽病(Cunninghamia lanceolata anthracnose)奠定基础。【方法】通过形态学特征、生理生化特性及16S rRNA基因序列分析,确定菌株T1-3-2的分类地位;通过平板对峙、菌落径向生长抑制率和平板倒扣等方法测定细菌及其挥发性气体和次级代谢产物的抑菌作用;同时,测定其促生作用和室内防效。【结果】菌株T1-3-2与桉生假单胞菌(Pseudomonas eucalypticola)亲缘性较近,属于假单胞菌属。该菌株对分属于6个属的10株靶标菌株具有较强的拮抗作用,尤其对炭疽菌属、拟盘多毛孢属、黑孢霉属和葡萄座腔菌属的6株靶标菌株抑制率高达80%以上。室内盆栽试验显示：菌株T1-3-2用Kings Medium B液体培养基的发酵菌液对杉木炭疽病的防效可达74.20%,同时能有效改善杉木幼苗的生长状况、增加生物量。【结论】菌株T1-3-2隶属于假单胞菌属,对杉木具有良好的抗病促生作用,是一株具有开发潜力的生防菌株。     

假单胞菌铁载体不产生突变体形成的进化机理

假单胞菌铁载体(Pvd)的产生是社会微生物学研究的一个重要模式系统:产生菌是合作者,而不产生菌是欺诈者。欺诈者可以不用付出Pvd合成的能量代价,而利用其他细菌产生的Pvd来获取生长所需的铁离子,因而处于竞争优势地位。合作与欺诈成为了当前解释自然界广泛存在的Pvd不产生菌进化形成的主要原理。本文将阐述荧光假单胞菌Pvd不产生菌形成的多条进化途径,并在此基础上剖析合作与欺诈原理的普遍适用性问题。     

Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate debalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalysing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4-hydroxybenzoate with concomitant release or coenzyme A.     

微生物燃料电池中假单胞菌F026的产电性能研究

基于微生物燃料电池的反应装置,从污水处理厂曝气池的污泥中通过富集,筛选和基于16S rRNA基因序列的系统发育分析等手段驯化出1株高效产电假单胞菌F026。以F026为阳极产电菌制作微生物燃料电池,考察了底物种类、温度和p H值等因素对微生物燃料电池产电性能的影响。结果表明,F026最适合在以可溶性淀粉为底物,p H为中性偏碱性,温度在30~35℃的环境下生长。在此条件下,微生物燃料电池的最高电压达到500 m V,体积功率密度达到2 W/m3。     

基于生态风险评估探究溶藻细菌2-4的抑藻特性

[目的]基于生态安全评估,探索溶藻细菌及其分泌物的抑藻效果与机制。[方法]分离获得溶藻细菌2-4 (Pseudomonassp.),通过正交试验和动力学模型研究了菌株2-4及其分泌物的抑藻特性,并通过急性毒性试验对菌株2-4及其分泌物进行生态风险评估。[结果]菌株2-4在最优条件下接种体积比(V/V)为15%时对铜绿微囊藻的4 d抑制率达92.81%,抑藻反应符合一级动力学模型(t_1/2=126 h)。菌株2-4具有抑藻多样性,首次报道了假单胞菌对斜生四链藻、蛋白核小球藻和丝藻的抑藻效果。菌株2-4分泌的抑藻活性物质分子量小于500 Da,不耐高温和强酸强碱。急性毒性试验结果显示,高于1.5%的菌液对大型溞有毒,高于2%的菌株分泌物对大型溞和稀有鮈鲫有毒,但对明亮发光杆菌无毒。较安全的使用范围内,菌株2-4 (V/V=1.5%)及其分泌物(V/V=10%)对藻华湖泊水样的叶绿素a去除率为4.83%–42.94%和30.62%–68.69%。[结论]本研究客观分析了假单胞菌2-4及其分泌物在生态安全使用范围内的实际抑藻效果,明晰了溶藻细菌生态毒性与抑藻效率的关系,为生物控藻实践提供理论参考。     

Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other gram-negative bacteria

Summary The two-step protein secretion pathway in Pseudomonas aeruginosa is dependent on the xcp genes. We investigated whether a similar secretion mechanism is present in non-pathogenic Pseudomonas spp. and in other gram-negative bacteria. The plant growth stimulating Pseudomonas strains P. putida WCS358, P. fuorescens WCS374 and Pseudomonas 1310 appeared to secrete proteins into the extracellular medium. Southern hybridization experiments showed the presence of xcp genes in these strains and also in other gram-negative bacteria, including Xanthomonas campestris. Complementation experiments showed that the xcp gene cluster of P. aeruginosa restored protein secretion in an X. campestris secretion mutant. The secretion gene cluster of X. campestris however, restored secretion capacity in P. aeruginosa mutants only to a low degree. Two heterologous proteins were not secreted by P. fuorescens and P. aeruginosa. The results suggest the presence of a similar two-step protein secretion mechanism in different gram-negative bacteria, which however, is not always functional for heterologous proteins.     

Statistical optimization of medium for the production of pyruvate oxidase by the recombinant Escherichia coli

Pyruvate oxidase (PyOD) is a very useful enzyme for clinical diagnostic applications and environmental monitor. Optimization of the fermentation medium for maximization of PyOD constitutively, production by Escherichia coli DH5α/pSMLPyOD was carried out. Response surface methodology (RSM) was used to optimize the medium constituents. A 2^6–2 fractional factorial design (first order model) was carried out to identify the significant effect of medium components towards PyOD production. Statistical analysis of results shows that yeast extract, ammonium sulfate and composite phosphate were significant factors on PyOD production. The optimized values of these three factors were obtained by RSM based on the result of a 2³ central composite rotatable design. Under these proposed optimized medium, the model predicted a PyOD activity of 610 U/L and via experimental rechecking the model, an activity of 670 U/L was attained.     

Glucose, pyruvate and lactate efflux by the perfused liver of a teleost, Clarias batrachus during aniso-osmotic exposure

Glucose, lactate and pyruvate efflux by the perfused liver of the walking catfish, Clarias batrachus was studied during aniso-osmotic exposure. During hypo-osmotic exposure (−80 mOsmol l⁻¹, maintained with NaCl), glucose, lactate and pyruvate efflux by the perfused liver significantly decreased by 55, 19 and 16%, respectively. During hyper-osmotic exposure (+80 mOsmol l⁻¹, maintained with NaCl), efflux increased by 57, 12 and 18%, respectively. Similar effects of glucose, lactate and pyruvate efflux by the perfused liver was also seen when the anisotonicity of the medium was adjusted with mannitol instead of NaCl. The decrease of glucose, lactate and pyruvate efflux during hypo-osmotic exposure was correlated with the stimulation of glycogen synthesis and the reverse was true during hyper-osmotic exposure. These observations were supported by changes in glycogen phosphorylase a (GPase a) and glycogen synthase a (GSase a) activities. During hypo-osmotic exposure (−80 mOsmol l⁻¹), the GPase a activity decreased by 1.93 fold and GSase a activity increased by 1.63 fold, while during hyper-osmotic exposure (+80 mOsmol l⁻¹), the GPase a activity increased by 1.58 fold and GSase a activity decreased by 1.95 fold. The total activity of both the enzymes were not effected by a short term exposure to aniso-osmotic conditions, suggesting that the alterations in GPase a and GSase a activity were mainly due to changes of their phosphorylation status during aniso-osmotic exposure. A direct correlation exists between glucose efflux and the hydration status of the perfused liver. These alterations of glucose metabolism are probably necessary by this walking catfish to meet the different energy demand, and also for maintenance of glucose homeostasis under osmotic stress.     

DNA sequences in chromosomes 11 and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains

Summary A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the K _m values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.A preliminary report of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology, The Hague, Netherlands. Abstract appeared in Yeast 6, S-240 (1990)     

Biological control of brown rot (Moniliana laxa Ehr.) on apricot (Prunus armeniaca L. cv. Hacıhaliloğlu) by Bacillus, Burkholdria, and Pseudomonas application under in vitro and in vivo conditions

In 2002 and 2003, a study was conducted to determine the effect of bacterial strains, Burkholdria OSU 7, Bacillus OSU 142, and Pseudomonas BA 8, on biological control of brown rot disease (Monilinia laxa Ehr.) on apricot cv. Hacıhaliloğlu in Malatya province of Turkey. Apricot orchard at full blooming stage was inoculated with conidial suspension (1 × 10⁶ spores/ml) of M. laxa Ehr. After inoculation, two apricot trees for each application were treated with each of the three biological control agents (Burkholdria gladii OSU 7, Bacillus subtilis OSU 142, and Pseudomonas putida BA 8) by spraying (1 × 10⁹ cfu/ml) on inoculated branches. Disease incidence was evaluated for untreated (control 1) and four different treatment groups including commercial disease management (control 2, positive control: 3% Bourdox in fall, 50% Cupper at pink flower, 30 g/100 l Corus at first blooming, and 300 g/100 l Captan at last blooming stage) and treatments including each of the three bacterial strains (OSU 7, OSU 142, and BA 8). The results showed that disease incidence for negative control (control 1) was 9.94, which was significantly higher than disease incidence for commercial application (2.57%) or bacterial treatments (2.82–5.00%) in the first year. In 2003, the lowest disease incidence observed in OSU 7 treatment (6.80%), while disease incidence rate for positive control and negative control were 9.45% and 28.46%, respectively. This result may suggest that OSU 7 has potential to be used as biopesticide for effective management of brown rot disease on apricot.     

微嗜酸寡养单胞菌中磷酸吡哆胺氧化酶基因pnpox生物学功能研究北大核心

【目的】研究微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila)CW117中磷酸吡哆胺氧化酶基因pnpox(phosphopyridoxamine oxidase,pnpox)在维生素B6(VB6)合成中的贡献及对黄曲霉毒素B1(aflatoxin B1,AFB1)的降解活性。【方法】采用基因插入突变方式,对菌株CW117中磷酸吡哆胺氧化酶基因pnpox进行突变,得到突变菌株。通过高效液相色谱法(high performance liquid chromatography,HPLC)检测突变株对AFB1的降解活性,以及突变株中吡哆醇和吡哆醛的合成情况,确定基因pnpox在寡养单胞菌体内VB6合成中的贡献和黄曲霉毒素降解代谢作用。【结果】成功构建了磷酸吡哆胺氧化酶基因突变子pnpox::pK19mobΩ2HMB,突变子吡哆醛的合成量较野生型菌株显著减少,吡哆醇合成量与野生型菌株无显著性差异;同时,突变子与野生型株CW117对AFB1的降解活性未发现显著性差异。【结论】菌株CW117中磷酸吡哆胺氧化酶在吡哆醛合成的过程中起着重要作用,该基因突变会导致VB6的严重缺乏,影响寡养单胞菌正常生长,但该基因对CW117降解黄曲霉毒素无显著性贡献。     

野大豆内生假单胞菌YDX26的鉴定及促生抗逆特性

[背景] 野大豆具有栽培大豆所不具有的一些优良特性,这与其特有内生菌有关,已成为当下研究热点。[目的] 对野大豆内生细菌进行分离鉴定,并从中筛选出具有促生和抗逆潜能菌株。[方法] 以组织分离法和涂布划线法进行细菌分离培养;根据形态特征、生理生化特性及基于16S rRNA基因序列系统发育分析对菌株进行菌种鉴定;通过选择性培养基或比色法等对菌株进行促生特性分析,采用水培试验分析菌株对水稻幼苗生长的影响;利用盐胁迫及聚乙二醇（Polyethylene Glycol,PEG）-6000模拟干旱胁迫探究菌株抗逆性。[结果] 分离得到一株编号为YDX26的细菌,基于菌株形态、理化特性和16S rRNA基因序列系统发育分析,将菌株YDX26初步鉴定为假单胞菌（Pseudomona s sp.）。该菌株溶磷量为46.12 mg/L,吲哚-3-乙酸（Indole-3-Acetic Acid,IAA）活性为19.97 μg/mL, 1-氨基环丙烷-1-羧酸（1-Amino-Cyclopropane-1-Carboxylic Acid,ACC）脱氨酶活性为3.95μmol/（mg·h）。菌株YDX26对水稻幼苗株高、根长、地上部干重与地下部干重均有明显促进作用,分别提高了17.34%、12.19%、5.32%和10.70%。相较于对照菌株DX22,在盐胁迫和干旱胁迫下,菌株YDX26表现出较好的耐盐和抗旱能力。[结论] 野大豆内生细菌菌株YDX26具有较好的促生、耐盐和抗旱能力,可以作为农业生产上新的菌种资源。