Characterization of catechol 1,2-dioxygenase from cell extracts of Sphingomonas xenophaga QYY

Sphingomonas xenophaga QYY, capable of growing significantly on more than ten kinds of aromatic compounds as sole carbon source, was used to study characterization of catechol 1,2-dioxygenase (C12O) in cell extracts. Characterization of the crude C12O showed that the maximum activity was obtained at 40-70℃ and pH 7.8-8.8. Metal ions had different influences on the activity of crude C12O. It was suggested that strain QYY possessed an inducible and ferric-dependent C12O. Kinetic studies showed that the value of Vmax and Km was 0.25 μmol catechol/L/mg protein/min and 52.85 μmol/L, respectively. In addition, the partial purification of C12O was achieved by a HiTrap Q Sepharose column chromatography.

Indoleamine-2,3-dioxygenase 1/cyclooxygenase 2 expression prediction for adverse prognosis in colorectal cancer

AIM To evaluate indoleamine-2,3-dioxygenase 1/cyclooxygenase 2(IDO1/COX2) expression as an independent prognostic biomarker for colorectal cancer(CRC) patients.METHODS We retrospectively studied the medical records of 95 patients who received surgical resection from August 2008 to January 2010. All patients were randomly assigned to adjuvant treatment with or without celecoxib groups after surgery. We performed standard immunohistochemistry to assess the expression levels of IDO1/COX2 and evaluated the correlation of IDO1/COX2 with clinicopathological factors and overall survival(OS) outcomes.RESULTS The expression of nuclear IDO1 was significantly correlated with body mass index(P < 0.001), and IDO1 expression displayed no association with sex, age, tumor differentiation, T stage, N stage, carcinoembryonic antigen, cancer antigen 19-9, CD3+ and CD8+ tumor infiltrating lymphocytes, and COX2. In univariate analysis, we found that nuclear IDO1(P = 0.039), nuclear/cytoplasmic IDO1 [hazard ratio(HR) = 2.044, 95% confidence interval(CI): 0.871-4.798, P = 0.039], nuclear IDO1/COX2(HR = 3.048, 95%CI: 0.868-10.7, P = 0.0049) and cytoplasmic IDO1/COX2(HR = 2.109, 95%CI: 0.976-4.558, P = 0.022) all yielded significantly poor OS outcomes. Nuclear IDO1(P = 0.041), nuclear/cytoplasmic IDO1(HR = 3.023, 95%CI: 0.585-15.61, P = 0.041) and cytoplasmic IDO1/COX2(HR = 2.740, 95%CI: 0.764-9.831, P = 0.038) have significantly poor OS outcomes for the CRC celecoxib subgroup. In our multivariate Cox model, high coexpression of cytoplasmic IDO1/COX2 was found to be an independent predictor of poor outcome in CRC(HR = 2.218, 95%CI: 1.011-4.48, P = 0.047) and celecoxib subgroup patients(HR = 3.210, 95%CI: 1.074-9.590, P = 0.037).CONCLUSION Our results showed that cytoplasmic IDO1/COX2 coexpression could be used as an independent poor predictor for OS in CRC.

1,2-环己二醇脱氢镍基催化剂制备方法

以硝酸镍为镍源,分别以NH3.H2O,NH4HCO3,NH3.H2O+NH4HCO3为沉淀剂采用沉淀法制备镍/硅藻土催化剂,用于1,2-环己二醇脱氢制备邻苯二酚,考察了不同沉淀剂、焙烧温度、沉淀温度制备的催化剂对1,2-环己二醇脱氢反应性能的影响,并通过XRD,BET,CO2-TPD(热设计功耗)等方法对催化剂进行了表征。结果表明:不同的沉淀剂对镍的分散度、孔结构及表面碱量都有影响,以NH3.H2O+NH4HCO3复合沉淀剂制备的镍/硅藻土催化剂上活性组分镍晶粒度小、分散度较高,催化剂平均孔径较大,催化剂表面碱中心数目多、碱量大,表现出良好的催化活性和邻苯二酚选择性。另外,催化剂适宜沉淀温度为90℃、焙烧温度为350℃。上述适宜条件制备的催化剂在320℃下用于1,2-环己二醇脱氢制备邻苯二酚,1,2-环己二醇转化率达到99.1%,邻苯二酚选择性达到86.8%。

1-(1,4-苯并二噁烷-2-甲酰基)哌嗪的合成

1-(1,4-苯并二噁烷-2-甲酰基)哌嗪为甲磺酸多沙唑嗪的重要中间体,以邻苯二酚与环氧氯丙烷为原料,经环合、氧化、氯化、酰化、缩合四步反应合成了治疗高血压和前列腺增生药物甲磺酸多沙唑嗪的关键中间体1-(1,4-苯并二噁烷-2-甲酰基)哌嗪。原料廉价,易得,反应条件温和,操作方便。

苯酚降解菌株GXY-1分离鉴定、降解及其粗酶特性研究

从处理石化废水的活性污泥样品中分离得到一株能以苯酚为唯一碳源生长的菌株GXY-1.通过形态特征、生理生化及16S rDNA序列分析,初步鉴定菌株GXY-1属于布鲁氏杆菌属(Brucella sp.).实验结果表明,菌株GXY-1对土霉素和四环素不敏感,其降解苯酚的最佳条件为温度30℃,pH7.0.在最佳条件下,GXY-1对600mg/L苯酚的降解率在60h时达99%以上.同时GXY-1还可以利用苯胺、萘、氯苯等芳香化合物为唯一碳源生长.测定了降解途径中相关酶的活性,表明菌株GXY-1是通过邻苯二酚1,2双加氧酶催化开环来降解苯酚.该菌株粗酶的最适反应pH为7.8,最适反应温度为40℃.

苯酚降解菌TX1的分离鉴定及其降解特性

从受酚类污染的土壤中分离筛选出1株能够高效降解苯酚的菌株。通过形态学、生理生化以及26S核糖体（rDNA）测序等手段对其进行初步鉴定，确定该菌株为丝孢酵母菌属（Trichosporon sp．），命名为TX1。初步研究结果表明，该菌株对苯酚的代谢途径为将苯酚先转化为邻苯二酚的苯酚羟化酶定位于细胞膜和细胞质；进而通过邻苯二酚1，2-双加氧酶（C120）邻位开环裂解，C120为诱导型胞内酶。通过分批培养得到菌株TX1降解苯酚的生长动力学模型可用霍尔丹（Haldane）方程表述，相关参数μmax=0．667h^-1，Ks=51．14mg／L，Ki=271．7mg／L。

Up-regulation of indoleamine 2,3-dioxygenase 1(IDO1)expression and catalytic activity is associated with immunosuppression and poor prognosis in penile squamous cell carcinoma patients

Background: Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan (Trp)catabolism have been demonstrated to play an important role in tumor immunosuppression. This study examined the expression and catalytic activity of IDO1 in penilesquamous cell carcinoma (PSCC) and explored their clinical significance.Methods: IDO1 expression level, serum concentrations of Trp and kynurenine (Kyn)were examined in 114 PSCC patients by immunohistonchemistry and solid-phaseextraction-liquid chromatography-tandem mass spectrometry. The survival was analyzed using Kaplan-Meier method and the log-rank test. Hazard ratio of death was analyzed via univariate and multivariate Cox regression. Immune cell types were definedby principal component analysis. The correlativity was assessed by Pearson’s correlation analysis.Results: The expression level of IDO1 in PSCC cells was positively correlatedwith serum Kyn concentration and Kyn/Trp radio (KTR;both P < 0.001) but negatively correlated with serum Trp concentration (P = 0.001). Additionally, IDO1 upregulation in cancer cells and the increase of serum KTR were significantly associated with advanced N stage (both P < 0.001) and high pathologic grade (P = 0.008and 0.032, respectively). High expression level of IDO1 in cancer cells and serumKTR were associated with short disease-specific survival (both P < 0.001). However, besides N stage (hazard radio [HR], 6.926;95% confidence interval [CI],2.458-19.068;P < 0.001) and pathologic grade (HR, 2.194;95% CI, 1.021-4.529;P = 0.038), only serum KTR (HR, 2.780;95% CI, 1.066-7.215;P = 0.036) was anindependent predictor for PSCC prognosis. IDO1 expression was positively correlated with the expression of interferon-𝛾 (IFN𝛾, P < 0.001) and immunosuppressivemarkers (programmed cell death protein 1, cytotoxic T-lymphocyte-associated protein 4 and programmed death-ligand 1 and 2;all P < 0.05), and the infiltration ofimmune cells (including cytotoxic T lymphocytes, regulatory T lymphocytes, tumorassociated macrophages, and myeloid-derived suppressor cells;all P < 0.001) inPSCC tissues. Furthermore, the expression of IDO1 was induced by IFN𝛾 in a dosedependent manner in PSCC cells.Conclusions: IFN𝛾-induced IDO1 plays a crucial role in immunoediting andimmunosuppression in PSCC. Additionally, serum KTR, an indicator of IDO1catabolic activity, can be utilized as an independent prognostic factor for PSCC.

球形红细菌降解2,4-二硝基甲苯的途径及酶学性质

采用气相色谱-质谱联用仪(GC-MS)分析了球形红细菌降解2,4-二硝基甲苯(2,4-DNT)的中间代谢产物,分析了可能的降解途径,讨论了培养基中2,4-DNT的初始质量浓度、溶液pH值及反应温度对3种酶的酶比活力影响。结果表明,当2,4-DNT的初始质量浓度为40 mg/L时,培养72h后,可以检测到5种物质:2,4-二硝基甲苯、4-氨基-2硝基甲苯、2-氨基-4硝基甲苯、4-硝基-1,2-二(三甲基硅烷)苯、1,2,4-三(三甲基硅烷)苯,其可能的降解途径为2,4-DNT首先还原为4-氨基-2-硝基甲苯和2-氨基4-硝基甲苯,再进一步转化为4-硝基-1,2-羟基苯,然后氧化为1,2,4-三羟基苯,之后开环生成β-酮己二酸,最终降解为小分子物质。硝基还原酶、邻苯二酚2,3-双加氧酶和邻苯二酚1,2-双加氧酶的酶比活力最适宜温度分别为35、30、35℃,最适宜pH值分别为7.0、8.0、7.0,最适宜培养基初始2,4-DNT质量浓度均为40mg/L。

Multistep conversion of cresols by phenol hydroxylase and 2,3-dihydroxy-biphenyl 1,2-dioxygenase

A mulfistep conversion system composed of phenol hydroxylase （PHrND） and 2,3-dihydroxy-biphenyl 1,2-dioxygenase （BphCLA_4） was used to synthesize methylcatechols and semialdehydes from o- and m-cresol for the first time. Docking studies displayed by PyMOL predicted that cresols and methylcatechols could be theoretically transformed by this multistep conversion system~ High performance liquid chromatography mass spectrometry （HPLC-MS） analysis also indicated that the products formed from multistep conversion were the corresponding 3-methylcatechol, 4-methylcatechol, 2- hydroxy-3-methyl-6-oxohexa-2,4-dienoic acid （2- hydroxy-3-methyl-ODA） and 2-hydroxy-5-methyl-6-oxo- hexa-2,4-dienoic acid （2-hydroxy-5-methyl-ODA）. The optimal cell concentrations of the recombinant E. coli strain BL21 （DE3） expressing phenol hydroxylase （PHrND） and 2,3-dihydroxy-biphenyl 1,2-dioxygenase （BphCLA_4） and pH for the multistep conversion of o- and m-cresol were 4.0 （g-L-1 cell dry weight） and pH 8.0, respectively. For the first step conversion, the formation rate of 3- methylcatechol （0.29μmol·L-1·min-1·mg-1cell dry weight） from o-cresol was similarly with that ofmethylca- techols （0.28 μmol·L-1·min-1·mg-1 cell dry weight） from m-cresol by strain PHrND. For the second step conversion, strain BphCLA_4 showed higher formation rate （0.83 μmol·L-1·min-1·mg-1 cell dry weight） for 2-hydroxy-3-methyl- ODA and 2-hydroxy-5-methyl-ODA from m-cresol, which was 1.1-fold higher than that for 2-hydroxy-3-methyl- ODA （0.77 μmol·L-1·min-1·mg-1. mglcell dry weight） from ocresol. The present study suggested the potential application of the multistep conversion system for the production of chemical synthons and high-value products.

Crystal structure of thermostable catechol 2,3-dioxygenase determined by multiwavelength anomalous dispersion method

The selenomethionyl derivative of the thermo-stable catechol 2,3-dioxygenase (SeMet-TC23O) is expressed, purified and crystallized. By using multiwave length anoma-lous dispersion (MAD) phasing techniques, the crystal structure of TC23O at 0.3 nm resolutions is determined. TC23O is a homotetramer. Each monomer is composed of N-terminal and C-terminal domains (residues 1-153 and 153-319, respectively). The two domains are proximately symmetric by a non-crystallographic axis. Each domain contains two characteristic motifs which are found in almost all of extradial dioxygenases.

萘降解细菌的分离及其降解和转座基因的分子检测

用富集培养法，从工业废水和活性污泥中分离到9个高效降解萘的细菌菌株（ND7～ND15）．对菌株ND7、ND8、ND9和ND10所进行的16SrDNA序列分析表明，它们都属于假单胞菌属（Pseudomonas）．PCR实验结果表明，上述4个菌株都含有蔡降解基因nahAc、nahG、nahH和catA，ND7和ND8菌株还含有萘降解基因nahU.DNA杂交实验结果表明，上述9个萘降解菌株都含有转座酶基因tnpA1和解体酶基因tnpR．这些结果表明，萘降解细菌的降解基因和转座基因具有高度的保守性．酶学实验证明，ND7、ND8、ND9和ND10菌株都具有儿茶酚1，2-双加氧酶活力和儿萘酚2，3-双加氧酶活力，但在不同菌株中这两种酶的比活力有明显不同．

磷钼钒杂多酸-聚邻苯二胺修饰碳糊电极示差脉冲溶出伏安法测定对苯二酚和邻苯二酚

在由磷钼钒杂多酸和邻苯二胺组成的支持电解质中,以0.100V.s-1扫速在碳糊电极上,于0.800～-0.800V范围内循环扫描20周后,制得磷钼钒杂多酸-聚邻苯二胺修饰碳糊电极。循环伏安法研究发现:对苯二酚(HQ)和邻苯二酚(CC)在该修饰电极上均出现了一对氧化还原峰;示差脉冲溶出伏安法研究发现:HQ和CC分别在0.140V和0.288V处出现二次微分溶出峰,两峰电位差为0.148V。HQ和CC的线性范围均为1.50×10-7～7.50×10-4 mol.L-1,检出限(3S/N)均为6.80×10-8 mol.L-1。据此提出了示差脉冲溶出伏安法测定模拟水样中HQ和CC,测得回收率在94.0%～106.0%之间。

倭蜂猴粪便微生物苯酚羟化酶和邻苯二酚1,2-双加氧酶基因多样性研究

【目的】分析倭蜂猴粪便微生物中苯酚羟化酶(Phenol hydroxylase,PH)和邻苯二酚1,2-双加氧酶(Catechol 1,2-dioxygenase,C12O)的基因多样性。【方法】利用简并引物,以倭蜂猴粪便微生物宏基因组DNA为模板,通过PCR扩增,分别构建PH和C12O基因克隆文库,并对克隆进行测序分析。【结果】倭蜂猴粪便微生物来源的PH和C12O基因序列经BLAST比对分析,与Gen Bank中相应酶的序列一致性分别介于92%-100%和87%-100%。系统进化树分析表明PH基因序列与Neisseria、Burkholderia、Alcaligenes、Acinetobacter 4个属来源的PH序列相关;C12O基因序列全部与Acinetobacter来源的C12O序列相关。序列比对结果表明PH序列具有Lm PH(Largest subunit of multicomponent PH)中高保守的两个DEXRH结构域;C12O序列具有能被Ag+和Hg2+抑制的位点(半胱氨酸)。【结论】倭蜂猴粪便微生物来源的PH为多组分PH,其降解苯酚的中间产物邻苯二酚可以被C12O通过邻位开环途径裂解。

三光气在羰基化环合反应中的研究

以三光气为环合试剂、三乙胺为碱,由邻氨基苯酚、邻苯二胺、邻苯二酚分别合成苯并噁唑酮、苯并咪唑酮、1,3苯并二氧戊环2酮。化合物结构经IR、MS和NMR确证。合成方法具有操作简便、所得产品纯度高等特点。

超声浸渍法制备负载型镍基催化剂及其催化性能

分别以硅藻土、γ-AL_2O_3和MgO为载体,采用均相沉淀-超声浸渍法,制备用于1,2-环己二醇脱氢制备邻苯二酚的镍基催化剂,利用SEM和BET对催化剂进行表征,并对催化活性和选择性进行考察。研究表明,超声浸渍法制备的催化剂,表观形貌规整,颗粒大小均匀,具有较大比例利于生成邻苯二酚的中孔结构,表现出良好的活性和选择性。在超声频率28 kHz、超声声强300 W·m^(-2)和超声时间60 min制备的Ni/硅藻土催化剂,于320℃、101.3 kPa、空速2.4 h^(-1)、N_2流量30 mL·min^(-1)和n(1,2-环己二醇):n(水)=1:4条件下反应,1,2-环己二醇转化率达96.7%,邻苯二酚选择性为92.3%。

石油烃降解菌对邻苯二酚、苯甲酸钠降解特性的研究

邻苯二酚和苯甲酸是苯类化合物代谢过程中两种常见的中间产物,也是工业废水中常见的环境污染物。为了高效去除苯类污染物,考察了高效降解石油中芳香烃组分的菌株 Acinetobacter sp. Tust-DM21 对邻苯二酚和苯甲酸钠的降解,并通过邻苯二酚 1, 2-双加氧酶(C12O)的粗酶特性和苯甲酸 1, 2-双加氧酶的酶活初步研究其代谢过程。在不含碳源无机盐培养基中,菌株 Tust-DM21 能够分别以邻苯二酚和苯甲酸钠为唯一碳源进行生长。通过改变接菌量、底物浓度、pH、温度等方面对该菌降解这两类苯类物质的特性进行研究,对 C12O 的最适 pH、温度和动力学参数以及苯甲酸 1, 2-双加氧酶的酶活进行分析。菌株对邻苯二酚的最大耐受浓度为 700 mg/L,菌株降解 600 mg/L 邻苯二酚的最佳条件为:接菌量 5%、pH6.0、温度 35℃,降解率达 92%以上。菌株可耐受 4 500 mg/L 苯甲酸钠,降解浓度为 1 500 mg/L 苯甲酸钠的最佳条件为:接菌量 3%、pH8.0、温度 35℃,降解率达 95%以上。C12O 的最适 pH 为 8.0,温度 35℃,其动力学参数为 Km=25.68 μmol/L,Vmax=0.131 mol/(mL min);苯甲酸 1, 2-双加氧酶的酶活为 3.53 U。该菌对邻苯二酚、苯甲酸钠有良好的降解效果,具有潜在的应用前景。

Phenol Biodegradation by <i>Corynebacterium glutamicum</i>Encapsulated in Electrospun Fibers

In Northern Israel, olive mills discharge liquid waste causing contamination of subterranean aquifers with phenol, rendering them albeit temporarily, unfit for both drinking and irrigation. The impact of groundwater pollution due to phenol spillage can be extensive. We developed a model system for the biodegradation of phenol-contaminated wastewater by the bacterium Corynebacterium glutamicum. Experiments consisting of suspended cultures demonstrated the native ability of this organism to utilize phenol for its metabolic pathways enabling degradation, at levels of nearly 100 ppm within 24 hours. With the use of bioinformatic data, a complete degradation pathway was constructed. Quantitative Real Time PCR analysis of the first two enzymes in this pathway revealed very distinct expression patterns and two different regulation mechanisms were postulated. Additionally, an electrospinning core-shell system was used to assemble electrospun microtubes containing bacteria on porous metallic carriers. We used these carriers as a new immobilization technique and demonstrated their significant phenol degrading capacity in a batch bioreactor configuration. This system demonstrates the feasibility of constructing a water treatment system for the management of phenol-contaminated water.

一株苯酚降解菌的筛选鉴定及响应面法优化其降解

从某化工厂污水处理车间活性污泥中分离、筛选到一株能以苯酚为唯一碳源和能源生长的菌株YH8.基于形态特征、生理生化特性、BIOLOG细菌自动鉴定系统、16S rDNA和gyrB基因序列同源性分析鉴定菌株YH8,鉴定菌株YH8为Acinetobacter guillouiae.在苯酚浓度低于1200 mg·L-1,温度为26~34℃,pH为7.0~10.0时,菌株YH8培养60 h对苯酚的降解率达70%以上.运用单因素实验初步确定苯酚降解的最适外加碳源和氮源分别为山梨醇和NaNO3,最适温度为30℃,最适初始pH为9.0,最适接种量为5%.为了提高菌株YH8的降解率,首先利用Plackett-Burman实验设计评估并筛选出影响苯酚降解的3个关键因素为初始pH、苯酚浓度、山梨醇浓度.用最陡爬坡实验逼近以上3个因子的最大响应区域,采用Box-Behnken实验设计及响应面法分析,确定其最优降解条件为初始pH 9.26、苯酚浓度1163.63 mg·L-1、山梨醇浓度7.81%、接种量5%、NaNO3浓度2%、温度30℃、培养时间96 h,在此条件下苯酚降解率可达98.95%.苯酚降解酶活性及酶定域实验表明,菌株YH8相关降解酶为胞内酶,且苯酚可诱导苯酚羟化酶（LmPH）和邻苯二酚1,2-双加氧酶（C（12）O）的合成.通过降解酶特异性引物从菌株YH8扩增得到LmPH和C12O基因片段,经质粒检测和消除实验发现菌株YH8相关降解基因位于质粒上.此外,菌株YH8能耐受高浓度NaCl和多种重金属离子,对多种抗生素具有抗性.

石墨烯/聚邻苯二胺膜修饰碳糊电极电催化性能的研究

研制了一种新型的石墨烯／聚邻苯二胺膜饰碳糊电极（GR／PPD／CPE）。在Nail2P04-NazHP04缓冲溶液（pH7．3）中，利用循环伏安法研究了对苯二酚（HQ）和邻苯二酚（cc）在该修饰电极上的电化学行为，利用线性扫描溶出伏安法研究了扫速与峰电流或峰电位的关系，利用微分脉冲溶出伏安法测定了HQ和CC的含量，线性范围分别为6．5×10-7～5．0×10-4mol／L和1．0×10-7～5．0×10-4mol／L，检出限（S／N=3）分别为8．0×10-8mol／L和2．0×10-7mol／L。方法用于自来水样中的HQ和CC的测定，回收率在96．5％～102．5％之间。

Pseudomonas putida ND6中两个高度同源catA基因的分离、克隆及比较

【目的】分别对恶臭假单胞菌(Pseudomonas putida)ND6基因组中基因高度同源的儿茶酚1,2-双加氧酶基因catA_1、catA_2进行克隆和原核表达,并进行生物信息学分析和酶学性质比较。【方法】通过生物信息学软件对catA_1和catA_2进行序列分析和进化树构建,利用经典的酶切连接方法和核酸外切酶III介导的无缝连接两种方法分别对catA_1和catA_2进行了表达载体的构建,对表达产物的酶学性质(最适反应温度,最适反应p H,耐热性,底物特异性等)进行了研究。【结果】catA_1和catA_2在基因序列上相似性为73.57%,两个酶的最适反应p H均为7.4,最适反应温度分别为45℃、50℃,Km值分别为1.28、1.66μmol/L,酶活力单位分别为9.00、8.35 U/mg,两个酶对儿茶酚和4-甲基儿茶酚具有较好的反应活性,在C12O分类中属于同一类型。【结论】本研究通过两种方式成功构建了高度同源性两个基因的表达载体,对于分离高同源性基因有一定的参考意义,同时通过对ND6菌株中同工酶的酶学性质进行研究和比较,加深了对ND6萘降解机制的理解,进一步探讨了冗余降解基因存在的意义。