盐碱土壤PAHs 降解菌的筛选鉴定及其降解特性

采用富集培养的方法,从天津大港油田PAHs污染盐碱化土壤中分离出一株能以菲、芘为唯一碳源和能源的优势菌TJB5。经形态观察和16S rDNA序列分析结果表明,该菌株为成团泛菌(Pantoea agglomerans)。采用液体培养的方法,研究了pH、盐度、菲芘的初始浓度对TJB5菌株降解菲芘效果的影响,确定了最佳降解条件。结果表明,该菌对菲、芘的降解具有较广泛的pH、盐度范围和良好的降解效果。在菲、芘浓度分别为50 mg/L、pH 6.8-9.5、盐度2%-3%、温度30°C条件下,接种15 d后菲降解率在93.3%以上,芘降解率在20%以上。     

不同植物源的黄烷酮-3-羟化酶的催化特性

[背景]黄烷酮-3-羟化酶(flavanone-3-hydroxylase,F3H)是黄酮类化合物代谢途径中的关键酶之一,不同植物来源的F3H催化特性可能存在差异,并对黄酮类化合物的生物合成产生重要影响.[目的]比较分析不同植物源F3H的酶学性质、异源催化能力差异,为今后在黄酮类化合物代谢工程中F3H的选用提供参考.[...     

多氯联苯降解菌的筛选、鉴定及其降解特性研究

以联苯为唯一碳源和能源从河海交汇处筛选、分离得到一株多氯联苯降解菌,研究其多氯联苯降解特性。以联苯(BPH)和4-一氯联苯(PCB3)为底物,探究假单胞菌属(Pseudomonas sp.)P-6-5的生长情况及降解能力。降解菌最适生长pH为7,盐度为35 g/L。以BPH和PCB3为诱导剂,均能促进降解菌的生长。P-6-5对10-100 mg/L的PCB3存在不同程度的转化能力,对浓度为10 mg/L的PCB3降解率达95.3%,最大降解速率1.9 mg/(L·h)。P-6-5对mix13(13种多氯联苯的同系物)中的四氯及四氯以下多氯联苯均有降解能力。结合产物分析,推测降解菌可能具有矿化PCB3的能力。菌株P-6-5具有海水菌的特点,表现了宽广的底物利用范围,是一株高效PCBs降解菌,对环境中PCBs的生物修复具有重要意义。     

重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷

利用重组大肠杆菌Escherichia coli Rosetta(DE3)/pET-SPase发酵生产蔗糖磷酸化酶(EC 2.4.1.7,Sucrose phosphorylase,SPase)。收集的菌体经高压破碎后离心得到粗酶液,通过镍NTA亲和层析、超滤除盐后得到电泳纯的SPase,纯化后的SPase的比酶活是原来的2.1倍,酶活回收率达到82.7%。经SDS-PAGE电泳测定,重组SPase的分子量约为59 kDa。该酶在不高于37℃,pH 6.0~6.7的条件下比较稳定,最适催化温度与最适催化pH分别为37℃,pH 6.7,该酶对蔗糖的米氏常数(Km)为7.3 mmol/L,最大反应速率(Vmax)为0.2μmol/(min.mg)。此外文中还以蔗糖和氢醌为底物,利用重组SPase催化合成α-熊果苷。其最佳反应条件为:20%蔗糖,200 U/mL的酶液,1.6%氢醌,pH 6.0~6.5,25℃,反应21 h。α-熊果苷的摩尔产率为78.3%,α-熊果苷的产量为31 g/L。     

低温产纤维素酶菌株的筛选、鉴定及纤维素酶学性质

[目的]筛选一株低温产纤维素酶菌株并进行鉴定,初步探索其酶学性质,为微生物肥料生产筛选菌种资源.[方法]常温条件下,采用CMC-刚果红染色法初筛纤维素降解菌株.采用低温条件诱导的方法,筛选耐低温且产纤维素酶能力最强的菌株,经形态学、生理生化特征试验、ITS序列等方面分析系统分类地位.单因素试验确定温度、pH及金属离子对纤维素酶活力的影响.[结果]从秸秆还田土壤中分离出一株在13℃低温环境下高效分解纤维素的真菌M11,鉴定M11为草酸青霉(Penicillium oxalicum).发酵试验表明:以玉米秸秆粉为唯一碳氮源,13℃、200 r/min摇床发酵培养9d时,纤维素酶活力最高为33.08 U/mL.对其酶学性质初步研究表明:该酶最适pH为5.0,最适反应温度为20℃,在5℃-20℃间酶活力仍能保持在90％以上.[结论]Penicillium oxalicum M11是一株高效的纤维素降解菌株,在低温条件下可分泌纤维素酶且活性显著,具有潜在的开发价值.     

恶臭假单胞菌SJTE-1中转化17β-雌二醇的3-酰基-ACP还原酶的鉴定与功能研究

[目的]假单胞菌SJTE-1可高效转化17β-雌二醇,但是催化该转化的酶尚不清楚。本文鉴定了该菌株的一个新的3-酮酰基-ACP还原酶（ANI01589.1）,并对其进行了功能研究。[方法]首先,我们克隆了该3-酰基-ACP还原酶的编码基因,在大肠杆菌BL21（DE3）菌株中进行了异源表达;利用金属离子亲和层析法,纯化获得了重组蛋白。体外检测了重组蛋白的活性与酶学性质,并利用高效液相色谱法（HPLC）测定了该酶的催化产物。[结果]3-酮酰基-ACP还原酶可被17β-雌二醇诱导表达,重组蛋白纯化量可达19.6 mg/L。蛋白序列比对结果表明,该蛋白包含短链脱氢酶/还原酶（SDR）的2个共有区域和多个保守残基。该酶以NAD⁺为辅助因子,将17β-雌二醇转化为雌酮;其K_m值为0.071 mmol/L,k_cat值为2.4±0.06/s^-1,5 min内可转化超过95.8%的雌二醇。该酶的最佳反应温度为42℃,最佳pH为8.0。不同二价离子对该酶的活性影响不同,Mg²⁺和Mn²⁺可增强其酶活性。[结论]这一假单胞菌SJTE-1来源的3-酮酰基-ACP还原酶可高效催化17β-雌二醇的转化,该酶可能在该菌株的雌激素代谢过程中起到重要作用。     

白蚁肠道元基因组来源高温β-葡萄糖苷酶Bgl17的酶学性质

【目的】对短角球白蚁(Globitermes brachycerastes)肠道元基因组文库中筛选得到的一个新型β-葡萄糖苷酶编码基因bgl17进行酶学性质研究。【方法】通过克隆与异源表达得到纯的Bgl17酶蛋白,根据Bgl17对底物的水解活性测定其稳定性及动力学参数,利用薄层层析确定其水解产物。【结果】该酶属于糖基水解酶第一家族(GHF1),对其特异性底物4-硝基苯基-β-D-吡喃葡萄糖苷(pNPGlc)的最适反应温度为70 °C,最适pH为5.0。在最适反应条件下,该酶以pNPGlc为底物比活力为115.69 U/mg,以水杨苷为底物比活力为297.39 U/mg。以pNPGlc为底物时,其动力学参数Km值和Vmax分别为0.81 mmol/L和227.27 μmol/(mL·min)。在稳定性方面,该酶在50 °C处理1 h仍可保持50%的活性,在pH 5.0和6.0条件下,该酶的半衰期为1 h。【结论】该酶在较高的温度下具有较高的活性,且对水杨苷水解活性高,这点不同于已知的β-葡萄糖苷酶,推测其更有利于木质纤维素复杂结构的降解;该酶的最适温度远高于白蚁生存环境温度,可为研究白蚁降解纤维素的机理提供参考。     

对硝基苯酚降解菌Pseudomonas sp. PDS-7的降解特性及 其降解相关基因的克隆

[目的]本研究的目的是分离对硝基苯酚(PNP)降解菌,研究其对PNP的降解特性;克隆其降解相关基因,并进行表达.[方法]本研究通过富集培养法和系列稀释平板涂布法分离PNP降解菌株;采用形态观察、生理生化特征测定和16S rDNA分析对菌株进行初步鉴定;通过摇瓶试验研究菌株降解特性;利用SEFA-PCR技术克隆降解相关基因,并亚克隆到表达载体pET29a中,构建重组表达质粒pETpnpC,再转入受体菌E.coli BL21(DE3)中进行诱导表达;通过分光光度法测定表达产物的酶活力.[结果]分离到一株PNP降解菌PDS-7,将该菌株鉴定为假单胞菌属(Pseudomonassp.);该菌株能够以PNP作为唯一碳源、氮源和能源生长,菌株对PNP的最高耐受浓度为80 mg/L,最适降解温度为30℃,偏碱性条件有利于菌株对PNP的降解;克隆了PNP降解过程中的偏苯三酚1,2-双加氧酶基因pnpC及马来酰醋酸还原酶基因pnpD(GenBank登陆号EU233791);将pnpC在E.coli BL21(DE3)菌株进行了诱导表达,表达产物对偏苯三酚和邻苯二酚均有邻位开环活性,比活力分别为0.45 U/mg protein和0.37 U/mg protein,表明偏苯三酚1,2-双加氧酶基因pnpC得到了活性表达.[结论]分离鉴定了一株PNP降解菌Pseudomonas sp.PDS-7,研究了该菌株的降解特性,克隆和表达了降解相关基因.     

拟南芥肌醇半乳糖苷合成酶与棉子糖合成酶的体外催化活性比较

[目的]基因克隆及原核表达纯化后比较拟南芥的2个肌醇半乳糖苷合成酶及2个棉子糖合成酶的体外催化活性,为微生物法或酶法合成棉子糖尊定基础。[方法]RT-PCR克隆拟南芥的肌醇半乳糖苷合成酶(GolS1及GolS3)与棉子糖合成酶(RafS1及RafS5)的基因,分别构建原核表达菌株,诱导表达纯化获得酶,电泳检测及蛋白定量后进行体外酶催化反应,HPLC分析产物。[结果]克隆到GolS1与GolS3及RafS1与RafS5的基因,原核表纯化获得纯酶,以反应体系中目标产物生成速率衡量,GolS1与GolS3催化速率分别为0.51和0.28mmol/(mg·min),RafS1与RafS5的催化速率分别为0.45和0.21mmol/(mg·min)。[结论]拟南芥的肌醇半乳糖苷合成酶(GolS1及GolS3)与棉子糖合成酶(RafS1及RafS5)基因经异源表达后具有良好酶活,其中GolS1酶活是GolS3的1.82倍,RafS1酶活是RafS5的2.14倍。     

右旋糖酐蔗糖酶工程菌株的构建及其培养条件的研究

[目的]右旋糖酐蔗糖酶是一种以蔗糖为底物,催化转移D-葡萄糖基生成α-葡聚糖或低聚糖的葡萄糖基转移酶.[方法]利用PCR扩增技术,将已获得的右旋糖酐蔗糖酶基因dexYG亚克隆到表达载体PET28a( )上,转化E.coli BL21(DE3),经过卡那霉素抗性筛选和酶切验证后,得到右旋糖酐蔗糖酶工程菌株BL21(DE3)/pET28-dexYG.[结果]经IPTG诱导该基因在E.coli BL21(DE3)中能有效表达,在诱导过程中菌体生长受到抑制.通过对培养时间、IPTG浓度、培养温度、菌浓(OD600)和pH值等产酶因素的优化考察,得到最佳培养条件为:培养时间5h、IPTG浓度0.5mmol/L、25℃、OD600值1.0和pH6.0.酶活力由最初的5.39U/mL提高到35.62U/mL,其中pH值对产酶活力影响最大,在pH6.0时的最高产酶活力是LB原始pH条件下最高酶活的3.5倍,并且pH值也是导致在诱导后期酶活迅速下降的主要原因之一.[结论]酶的表达和酶活的研究结果表明,构建的工程菌株能够异源高效表达右旋糖酐蔗糖酶,并且表现出较高的酶活力.     

嗜麦芽寡养单胞菌PX1的降解芘特性及定殖效能

为丰富多环芳烃降解菌菌种库、降低农作物的污染风险,本研究对一株可高效降解多环芳烃(PAHs)的植物内生菌进行筛选鉴定,并初步探究其降解途径以及定殖效能。结果表明: 菌株PX1为嗜麦芽寡养单胞菌。该菌株对多环芳烃的降解具有广谱性,7 d几乎可彻底降解PAH无机盐培养基中的萘,在分别含有50.0 mg·L^-1菲、20.0 mg·L^-1芘、20.0 mg·L^-1荧蒽和10.0 mg·L^-1苯并[a]芘的培养体系中,对菲、芘、荧蒽、苯并[a]芘的降解率分别为72.6%、50.7%、31.9%和12.9%。选取芘作为PAHs模型研究菌株PX1的降解特性。酶活性试验表明,芘可诱导菌株PX1体内邻苯二甲酸双加氧酶、邻苯二酚-1,2-双加氧酶和邻苯二酚-2,3-双加氧酶的活性。在芘降解过程中检测到4,5-环氧化芘、4,5-二羟基芘、龙胆酸/原茶儿酸、水杨酸、顺-己二烯二酸/2-羟粘糠酸半醛、顺-2′-羧基苯丙酮酸、1-羟基-2-萘甲酸、水杨醛等中间产物。浸种定殖试验表明,菌株PX1可高效定殖到空心菜和小麦体内,显著促进空心菜和小麦生长,并能够将空心菜、小麦体内及其生长基质中的芘浓度分别降低29.8%～50.7%、52.4%～67.1%和8.0%～15.3%。表明菌株PX1主要通过“水杨酸途径”和“邻苯二甲酸途径”降解芘,且可以定殖到植物体内,促进植物生长。     

Purification and Characterization of a Three-Component Salicylate 1-Hydroxylase from Sphingomonas sp. Strain CHY-1

In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which so far has not been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent K_m for salicylate of 1.1 ± 0.2 μM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (K_m, 28 ± 6 μM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a K_i of 0.68 μM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

Biodegradation of phenanthrene by<Emphasis Type="Italic"> Pseudomonas</Emphasis> sp. strain PP2: novel metabolic pathway,role of biosurfactant and cell surface hydrophobicity in hydrocarbon assimilation

Pseudomonas sp. strain PP2 isolated in our laboratory efficiently metabolizes phenanthrene at 0.3% concentration as the sole source of carbon and energy. The metabolic pathways for the degradation of phenanthrene, benzoate and p-hydroxybenzoate were elucidated by identifying metabolites, biotransformation studies, oxygen uptake by whole cells on probable metabolic intermediates, and monitoring enzyme activities in cell-free extracts. The results obtained suggest that phenanthrene degradation is initiated by double hydroxylation resulting in the formation of 3,4-dihydroxyphenanthrene. The diol was finally oxidized to 2-hydroxymuconic semialdehyde. Detection of 1-hydroxy-2-naphthoic acid, alpha-naphthol, 1,2-dihydroxy naphthalene, and salicylate in the spent medium by thin layer chromatography; the presence of 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-2,3-dioxygenase activity in the extract; O(2) uptake by cells on alpha-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate and catechol; and no O(2) uptake on o-phthalate and 3,4-dihydroxybenzoate supports the novel route of metabolism of phenanthrene via 1-hydroxy-2-naphthoic acid --> [alpha-naphthol] --> 1,2-dihydroxy naphthalene --> salicylate --> catechol. The strain degrades benzoate via catechol and cis,cis-muconic acid, and p-hydroxybenzoate via 3,4-dihydroxybenzoate and 3-carboxy- cis,cis-muconic acid. Interestingly, the culture failed to grow on naphthalene. When grown on either hydrocarbon or dextrose, the culture showed good extracellular biosurfactant production. Growth-dependent changes in the cell surface hydrophobicity, and emulsification activity experiments suggest that: (1) production of biosurfactant was constitutive and growth-associated, (2) production was higher when cells were grown on phenanthrene as compared to dextrose and benzoate, (3) hydrocarbon-grown cells were more hydrophobic and showed higher affinity towards both aromatic and aliphatic hydrocarbons compared to dextrose-grown cells, and (4) mid-log-phase cells were significantly (2-fold) more hydrophobic than stationary phase cells. Based on these results, we hypothesize that growth-associated extracellular biosurfactant production and modulation of cell surface hydrophobicity plays an important role in hydrocarbon assimilation/uptake in Pseudomonas sp. strain PP2.     

An ultraviolet absorbance method for determining acetylsalicylic acid hydrolase activity

An ultraviolet absorbance method for quantitation of acetylsalicylic acid esterase (hydrolase) activity has been developed and validated. The sensitivity of the method was found to be 2.8 nmol/ml-min in the assay cuvette. Linearity of the reaction with enzyme concentration and time has been demonstrated. The product of the enzymatic reaction, salicylic acid, has been identified by thin-layer chromatography using acetyl-[¹⁴C]salicylic acid. The quantities of salicylic acid produced in 5, 10, and 15 min of incubation were equal when assayed by the spectrophotometric method and by the acetyl-[¹⁴C]salicylic acid thin-layer chromatographic method. The time required for assay by ultraviolet absorbance is approximately 3 min/sample.     

Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1

1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoateto 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates. The comparison of the K_mand V_max values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate.The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.     

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.     

Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase

Fatty acyl ethyl esters, previously identified in our laboratory as metabolites of ethanol in human and rabbit myocardium, arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to isolate and purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of rabbit myocardium, as assayed by the rate of synthesis of ethyl [14C]oleate from 0.4 mM [14C]oleic acid and 0.2 M ethanol, was 31 nmol/(g.h), and all of it was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M) separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was then purified over 5000-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-albumin affinity chromatographies with an overall yield of 40%. Up to 45 micrograms of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with Mr 26 000, and gel permeation chromatography under nondenaturing conditions indicated a Mr of 50 000 for the active enzyme. Kinetic analyses using the purified enzyme indicated that greatest rates of ethyl ester synthesis were observed with unsaturated octadecanoic fatty acid substrates [Vmax = 1.9 and 1.5 nmol/(mg.s) for linoleate and oleate, respectively], with lesser rates associated with palmitate, stearate, and arachidonate substrates [0.14, 0.03, and 0.35 nmol/(mg.s), respectively].(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase from Polaromonas naphthalenivorans CJ2

Prior research revealed that Polaromonas naphthalenivorans CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising nagRAaGHAbAcAdBFCQEDJI'-orf1-tnpA and nagR2-orf2I'KL (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the orf2 gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that orf2 does not encode salicylate 5-hydroxylase. Instead, we have found that orf2 encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed orf2 nagX. After expression in Escherichia coli, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of P. naphthalenivorans CJ2 defective in nagX failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because nagX and an adjacent MarR-type regulatory gene are both closely related to homologues in Azoarcus species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.     

Metabolism of 2-, 3- and 4-hydroxybenzoates by soil isolates Alcaligenes sp. strain PPH and Pseudomonas sp. strain PPD

Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3- and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O(2) uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3- and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O(2) uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3- or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.