Indoleamine 2,3-dioxygenase: As a potential prognostic marker and immunotherapeutic target for hepatocellular carcinoma

Tumor cells induce an immunosuppressive microen-vironment which leads towards tumor immune escape. Understanding the intricacy of immunomodulation by tumor cells is essential for immunotherapy. Indoleamine 2,3-dioxygenase(IDO) is an immunosuppressive enzyme which mediates tumor immune escape in various cancers including hepatocellular carcinoma(HCC). IDO up-regulation in HCC may lead to recruitment of regulatory T-cells into tumor microenvironment and therefore inhibit local immune responses and promote metastasis. HCC associated fibroblasts stimulate natural killer cells dysfunction through prostaglandin E2 and subsequently IDO promotes favorable condition for tumor metastasis. IDO up-regulation induces immuno-suppression and may enhance the risk of hepatitis C virus and hepatitis B virus induced HCC. Therefore, IDO inhibitors as adjuvant therapeutic agents may have clinical implications in HCC. This review proposes future prospects of IDO not only as a therapeutic target but also as a prognostic marker for HCC.

Inhibition of allogeneic T-cell response by Kupffer cells expressing indoleamine 2,3-dioxygenase

AIM:To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC)pretreated with interferon-γ(IFN-γ)in vitro. METHODS:The expressions of indoleamine 2,3-dioxygenase(IDO)mRNA and FasL mRNA in KC pretreated with IFN-γwere studied with real-time polymerase chain reaction(PCR).The catabolism of tryptophan by IDO from KC was analyzed by high performance liquid chromatography.Allogeneic T-cell response was used to confirm the inhibition of KC in vitro.The proliferation of lymphocytes was detected using[ 3 H]thymidine incorporation.Cell cycle and lymphocyte apoptosis were evaluated by flow cytometric assay. RESULTS:Real-time PCR revealed IDO mRNA and FasL mRNA expressions in KC pretreated with IFN-γ,and IDO catabolic effect was confirmed by a decrease in tryptophan and increase in kynurenine concentration. KC expressing IDO and FasL in BABL/c mice acquired the ability to suppress the proliferation of T-cells from C57BL/6,which could be blocked by addition of 1-methyl-tryptophan and anti-FasL antibody.KC expressing IDO could induce allogeneic T-cell apoptosisCONCLUSION:In addition to Fas/FasL pathway,IDO may be another mechanism for KC to induce immune tolerance.

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.

Review of 10 years of research on breast cancer patients:Focus on indoleamine 2,3-dioxygenase

Therapeutic manipulation of the immune system in cancer has been an extensive area of research in the field of oncoimmunology.Immunosuppression regulates antitumour immune responses.An immunosuppressive enzyme,indoleamine 2,3-dioxygenase(IDO)mediates tumour immune escape in various malignancies including breast cancer.IDO upregulation in breast cancer cells may lead to the recruitment of regulatory T(T-regs)cells into the tumour microenvironment,thus inhibiting local immune responses and promoting metastasis.Immunosuppression induced by myeloid derived suppressor cells activated in an IDOdependent manner may enhance the possibility of immune evasion in breast cancer.IDO overexpression has independent prognostic significance in a subtype of breast cancer of emerging interest,basal-like breast carcinoma.IDO inhibitors as adjuvant therapeutic agents may have clinical implications in breast cancer.This review proposes future prospects of IDO not only as a therapeutic target but also as a valuable prognostic marker for breast cancer.

Mesenchymal-epithelial Transition Factor Regulates Monocyte Function during Mycobacterial Infection via Indoleamine 2,3-dioxygenase

Objective Mycobacterium tuberculosis(Mtb),the causative agent of tuberculosis(TB),causes an estimated 1.6 million human deaths annually,but the pathogenesis of TB remains unclear.Immunity plays a critical role in the onset and outcome of TB.This study aimed to uncover the roles of innate and adaptive immunity in TB.Methods The gene expression profiles generated by RNA sequencing from human peripheral blood mononuclear cells(PBMCs)stimulated with or without Mtb strain H37Rv antigens were analyzed.A total of 973 differentially expressed mRNAs were identified.Results The differentially expressed genes were enriched in innate immunity signaling functions.The mesenchymal-epithelial transition factor(MET)gene was significantly upregulated in CD14^(+)monocytes.A MET inhibitor improved the uptake of the BCG strain by monocytes and macrophages as well as inhibited the expression of indoleamine 2,3-dioxygenase(IDO).The expression of IDO was increased in PBMCs stimulated with Mtb antigens,and the IDO inhibitor promoted the expression of CD40,CD83,and CD86.Conclusion Our results might provide clues regarding the immunomodulatory mechanisms used by Mtb to evade the host defense system.

Myricetin inhibits interferon-γ-induced programmed death ligand-1 and indoleamine 2,3-dioxygenase 1 expression in lung cancer cells

OBJECTIVE Programmed death ligand-1(PD-L1)and indoleamine 2,3-dioxygenase 1(IDO1)are immune checkpoints which can be induced by interferon-γ(IFN-γ)in the tumor microenvironment,leading to immune escape of tumors.Myricetin(MY)is a flavonoid distributed in many edible and medicinal plants.The aim of this study is to clarify the effect and the mechanism of MY on inhibiting IFN-γ-induced PD-L1 and IDO1 in lung cancer cells.METHODS Expressions of PD-L1 and major histocompatibility complex-I(MHC-I)were evaluated by flow cytometry and Western blotting,and the expression of IDO1 was measured by Western blotting.qRT-PCR was used to detect their mRNA levels.The function of T cells was evaluated using a co-culture system consist of lung cancer cells and the Jurkat-PD-1 T cell line that overexpressing PD-1.Molecular docking analysis,Western blotting and immunofluorescence were used for mechanism study.RESULTS MY potently inhibited IFN-γ-induced PD-L1 and IDO1 expression in human lung cancer cells,while didn't show obvious effect on the expression of MHC-I.In addition,MY restored the survival,proliferation,CD69 expression and interleukin-2(IL-2)secretion of Jurkat-PD-1 T cells suppressed by IFN-γ-treated lung cancer cells in the co-culture system.Mechanistically,IFN-γup-regulated PD-L1 and IDO1 at the transcriptional level through the JAK-STAT-IRF1 axis,which was targeted and inhibited by MY.CONCLUSION Our research revealed a new insight into the anti-tumor effects of MY which inhibited IFN-γ-induced PD-L1 and IDO1 expression,supporting the potential of MY in anti-tumor immunotherapy.

Molecular Cloning and Characterization of Porcine Indoleamine 2,3-Dioxygenase and Its Expression in Various Tissues

In order to confirm the existence of indoleamine 2,3-dioxygenase(IDO) gene in swine,and to clone the novel gene followed by the molecule structure properties and expression pattern analysis,the porcine mRNA sequences homologous to human IDO were obtained from GenBank database by bioinformatics method.By using RT-PCR,the IDO gene was cloned from porcine endothelial cell line and the accuracy of the nucleic acid sequence was confirmed,and the expression pattern of the gene was detected.The three-dimensional structure model of porcine IDO was built referring to the tertiary structure of human IDO using biological sequence analysis software and database.The results showed that the porcine IDO was identified by sequencing.The nucleotide sequences were confirmed as a novel gene after submitted to Genbank.Porcine IDO was expressed in the lung,thymus,epididymis and anterior chamber with a basic level,however in peripheral blood mononuclear cells(PBMCs) the IDO gene was highly expressed.The three-dimensional structure model of porcine IDO was similar to that of human IDO.It was suggested that identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene.Study of IDO on NK cells-mediated xenograft rejection will be a novel therapeutic target for the development of xenotransplantation.

Forkhead box P3 and indoleamine 2,3-dioxygenase co-expression in Pakistani triple negative breast cancer patients

BACKGROUND Forkhead box P3(FOXP3)is a specific marker for immunosuppressive regulatory T(T-reg)cells.T-regs and an immunosuppressive enzyme,indoleamine 2,3-dioxygenase(IDO),are associated with advanced disease in cancer.AIM To evaluate the co-expression of FOXP3 and IDO in triple negative breast cancer(TNBC)with respect to hormone-positive breast cancer patients from Pakistan.METHODS Immunohistochemistry was performed to analyze the expression of FOXP3,IDO,estrogen receptor,progesterone receptor,and human epidermal growth factor receptor on tissues of breast cancer patients(n=100):Hormone-positive breast cancer(n=51)and TNBC(n=49).A total of 100 patients were characterized as FOXP3 negative vs positive and further categorized based on low,medium,and high IDO expression score.Univariate and multivariate logistic regression models were used.RESULTS Out of 100 breast tumors,25%expressed FOXP3 positive T-regs.A significant coexpression of FOXP3 and IDO was observed among patients with TNBC(P=0.01)compared to those with hormone-positive breast cancer.Two variables were identified as significant independent risk factors for FOXP3 positive:IDO expression high(adjusted odds ratio(AOR)5.90;95%confidence interval(CI):1.22-28.64;P=0.03)and TNBC(AOR 2.80;95%CI:0.96-7.95;P=0.05).CONCLUSION Our data showed that FOXP3 positive cells might be associated with high expression of IDO in TNBC patients.FOXP3 and IDO co-expression may also suggest its involvement in disease,and evaluation of FOXP3 and IDO expression in TNBC patients may offer a new therapeutic option.

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.

AncPhore: A versatile tool for anchor pharmacophore steered drug discovery with applications in discovery of new inhibitors targeting metallo-β-lactamases and indoleamine/tryptophan 2,3-dioxygenases

We herein describe AncPhore,a versatile tool for drug discovery,which is characterized by pharmacophore feature analysis and anchor pharmacophore(i.e.,most important pharmacophore features)steered molecular fitting and virtual screening.Comparative analyses of numerous protein-ligand complexes using AncPhore revealed that anchor pharmacophore features are biologically important,commonly associated with protein conservative characteristics,and have significant contributions to the binding affinity.Performance evaluation of AncPhore showed that it had substantially improved prediction ability on different types of target proteins including metalloenzymes by considering the specific contributions and diversity of anchor pharmacophore features.To demonstrate the practicability of AncPhore,we screened commercially available chemical compounds and discovered a set of structurally diverse inhibitors for clinically relevant metallo-β-lactamases(MBLs);of them,4 and 6 manifested potent inhibitory activity to VIM-2,NDM-1 and IMP-1 MBLs.Crystallographic analyses of VIM-2:4 complex revealed the precise inhibition mode of 4 with VIM-2,highly consistent with the defined anchor pharmacophore features.Besides,we also identified new hit compounds by using AncPhore for indoleamine/tryptophan 2,3-dioxygenases(IDO/TDO),another class of clinically relevant metalloenzymes.This work reveals anchor pharmacophore as a valuable concept for target-centered drug discovery and illustrates the potential of AncPhore to efficiently identify new inhibitors for different types of protein targets.

Expression and Prognostic Value of Indoleamine 2,3-dioxygenase in Pancreatic Cancer

Background： Indoleamine 2,3-dioxygenase （IDO）, an enzyme for tryptophan metabolism through the kynurenine pathway, exhibits an immunosuppressive effect and induces immune tolerance in tumor cells. The effects of IDO on pancreatic cancer are poorly understood. This study aimed to investigate the expression and prognostic significance of IDO in pancreatic cancer. Methods： We evaluated the protein expression of IDO in PANC-1, CFPAC-I, and BxPC-3 cell lines with or without 48 11 treatment by 500 U/ml interferon-,/（IFN-y）. We performed immunollistochemical staining and Western blot analysis lbr IDO expression in both pancreatic cancer and normal pancreas tissues obtained from Chinese PLA General Hospital from July 2012 to December 2013. Survival analysis was performed to correlate IDO expression and histopathologic parameters with overall survival. The Kaplan-Meier method and Cox proportional hazards regression model were conducted. Results＂ PANC-I, CFPAC-I, and BxPC-3 cell lines expressed IDO at the protein level, and the relative expression amount increased after stimulation with 500 U/ml IFN-y. Immunohistochemical analysis results revealed that high IDO expression was observed in 59% of pancreatic adenocarcinoma tissues. Compared with normal pancreatic tissues, pancreatic adenocarcinoma showed significantly higher IDO expression levels, especially among patients with high tumor node metastasis （TNM） stages （X2= 4.550, P = 0.030）, poor histological differentiation （X2=5.690, P = 0.017）, and lymph node metastasis （X2=4.340 P = 0.037）. Kaplan-Meier survival curves showed that high 1DO expression was correlated with low survival rates （hazard ratio [HR] = 0.49 P = 0.009）. Multivariate analysis using Cox proportional hazards model indicated that lymph node metastasis （HR = 0.35 P= 0.010） and IDO expression （HR = 0.42 P = 0.020） were two independent prognostic predictors of pancreatic adenocarcinoma. Conclusions： The study confirmed that high IDO expression in pancreatic adenocarcinoma was related to poor prognosis of patients. These findings provided evidence that IDO was involved in pancreatic adenocarcinoma progression and might serve as a relevant therapeutic target.

Indoleamine 2,3-dioxygenase in tumor induced tolerance

Objective To review the recent studies about the role of indoleamine 2,3-dioxygenase （IDO） in tumor induced tolerance. Data sources Published articles （1978-2009） on IDO and tumor induced tolerance were selected from Medline. Study selection Articles selected were relevant to development of IDO in tumor induced tolerance. Of all originally identified articles, 50 specially addressed the stated purpose. Results Recent work has revealed IDO at high levels in tumors and in tumor-draining lymph nodes and a close relationship between IDO activity and the regulatory T cells. Conclusion Up-regulation of IDO is proven to be a mechanism of acquired tolerance in tumors, in which the closely coupled positive feedback system between IDO and reclulatorv T cells may be considered to play an important role.

Tryptophan 2,3-dioxygenase 2 controls M2 macrophages polarization to promote esophageal squamous cell carcinoma progression via AKT/GSK3β/IL-8 signaling pathway

Tryptophan 2,3-dioxygnease 2(TDO2) is specific for metabolizing tryptophan to kynurenine(KYN),which plays a critical role in mediating immune escape of cancer.Although accumulating evidence demonstrates that TDO2 overexpression is implicated in the development and progression of multiple cancers,its tumor-promoting role in esophageal squamous cell carcinoma(ESCC) remains unclear.Here,we observed that TDO2 was overexpressed in ESCC tis sues and correlated significantly with lymph node metastasis,advanced clinical stage,and unfavorable prognosis.Functional experiments showed that TDO2 promoted tumor cell proliferation,migration,and colony formation,which could be prevented by inhibition of TDO2 and aryl hydrocarb on receptor(AHR).Further experimentation demonstrated that TDO2 could promote the tumor growth of KYSE150 tumor-bearing model,tumor burden of C57 BL/6 mice with ESCC induced by 4-NQO,enhance the expression of phosphorylated AKT,with subsequent pho sphorylation of GSK3β,and polarization of M2 macrophages by upregulating interleukin-8(IL-8) to accelerate tumor progression in the tumor microenvironment(TME).Collectively,our results discovered that TDO2 could upregulate IL-8 through AKT/GSK3β to direct the polarization of M2 macrophages in ESCC,and suggested that TDO2 could represent as an attractive therapeutic target and prognostic marker to ESCC.

Feiji Recipe inhibits the growth of lung cancer by modulating T-cell immunity through indoleamine-2,3-dioxygenase pathway in an orthotopic implantation model

Objective： Escape from the body＇s immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase （IDO） plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe. Methods： An orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein （2LL-EGFP） and 2LL-EGFP-IDO were investi- gated, and the apoptosis of T-cells was examined by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetra zolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high- performance liquid chromatography. Results： Compared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged （P〈0.0074）, and the IDO protein level decreased （P=0.0072）; moreover, the percentages of CD4＋CD25＋ T-cells and Foxp3＋ T-cells were significantly decreased （P〈0.05）. The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and reg- ulatory T-cells. Conclusion： The molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.

Indoleamine 2,3-dioxygenase and regulatory dendritic cells contribute to the allograft protection induced by infusion of donor-specific splenic stromal cells

It has been reported that splenic stromal cells(SSCs)are capable of directly supporting the development of CD11c ^(lo)CD45RB^(+) IL-10-producing dendritic cells(DCs)from lineage-negative c-kit^(+) progenitor cells in the absence of exogenous cytokines.In vitro,DCs that differentiate on stromal cells suppress mixed leukocyte reaction responses and induce primary alloreactive CD4^(+) T cells to differentiate into IL-10-producing Tr1 cells.However,the precise mechanisms by which these SSCs exert their regulatory functions in vivo remain undefined.Furthermore,their possible contribution to the development of allograft transplantation tolerance has yet to be examined.Here,we have used both murine skin and cardiac allograft transplantation models to explore whether in vivo alloresponses can be regulated by infusion with donor-derived SSCs and to investigate the possible mechanisms by which SSCs exert regulatory effects to prevent allograft rejection.We show that intravenous SSC infusion prolonged murine skin allograft survival.The prolonged graft survival is associated with augmentation of the generation of regulatory DC subsets and CD4^(+) CD25^(+) Foxp3^(+) regulatory T cells(Tregs),as well as upregulation of the production of suppressive cytokines IL-10 and transforming growth factor(TGF)-b.Moreover,we found that indoleamine 2,3-dioxygenase and SSC-derived regulatory DCs contribute to allograft protection by infusion of donor-specific SSCs.Our data suggest that donor-derived SSCs could be used as a therapeutic target to promote transplantation tolerance.

Indoleamine 2,3-Dioxygenase in Endometriosis

Endometriosis(EMS)is a chronic inflammatory and estrogen-dependent gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity.Although it is a benign disease,EMS is tumor-like in several aspects,which include unrestrained growth,decreased apoptosis,and aggressive invasion.EMS involves endocrine disorders and immunological factors.Indoleamine 2,3-dioxygenase(IDO)is an intracellular enzyme that catalyzes the initial and rate-limiting step of the metabolism of tryptophan.IDO is a potential candidate facilitating EMS development.Increased IDO expression in both eutopic and ectopic endometria of women with EMS is biologically important in aspects,which include regulation of endometrial stromal cell function and modulation of adjacent local immunocytes to generate a supportive microenvironment.In turn,the expression of IDO can be regulated by the complex endocrine-immune microenvironment networks in endometrial lesions.Here,we systematically review the roles of IDO in EMS to explore its pathological implications and treatment potential.

邻苯二酚2,3-双加氧酶基因克隆、定位和高效表达

采用特异性引物 ,以菲、芘降解菌株ZL5的代谢性质粒为模板 ,扩增出邻苯二酚 2 ,3-双加氧酶 (C2 3O)基因 .将该基因和表达载体pET - 30a(+)连接 ,转化E .coliJM10 9(DE3) ,获得了高效表达的转化子 .SDS -PAGE结果表明 ,转化子的C2 3O蛋白不仅在细胞内存在 ,而且能被分泌到胞外 ,薄层扫描显示 ,转化子细胞内和细胞外表达蛋白总量占细胞总蛋白的 4 2 % .酶活分析表明 ,分布在转化子细胞内、外的表达蛋白都具有较高的C2 3O比活力 .Southern杂交将菌株ZL5的C2 3O基因定位在内生质粒的不同酶切片段上 .图 5表 1参

石油污染土壤中芳烃降解菌及邻苯二酚2,3双加氧酶的克隆

石油污染土壤中的芳烃降解菌是进行土壤修复的主要生物资源,本研究对某炼油厂附近土壤中的芳烃降解菌及邻苯二酚2,3双加氧酶基因进行了研究。结果表明,部分石油烃污染土壤中存在着大量的芳烃降解菌;对其中一个土壤样本中的邻苯二酚2,3双加氧酶基因进行克隆,获得了7个不同的邻苯二酚双加氧酶基因序列,序列分析表明这些基因可能来源于土壤中的假单胞菌,且该基因在土壤中的丰度与污染水平及芳烃降解菌的数量相关。可见,土壤中芳烃降解菌数量及降解基因的丰度和多样性,可以对石油污染土壤的生物修复进行监控并为生物修复提供丰富的微生物资源。

L68菌株邻苯二酚2,3-双加氧酶纯化及性质

L68菌株中的邻苯二酚 2 ,3 双加氧酶经硫酸铵分级沉淀、DEAE SepharoseFastFlow柱层析、Hydroxyapatite柱层析、Sephadex G15 0凝胶过滤分离 ,并经SDS 聚丙烯酰胺凝胶电泳检测 ,达到了电泳纯 .提纯倍数为 2 34 .1倍 ,收率为 2 0 .2 % .酶相对分子质量为 (132± 10 )kDa ,由 4个相对分子质量相同的亚基组成 .最适反应温度 35℃ ,且酶活力在 15～ 65℃比较稳定 ;最适 pH为 8.0 ,在 pH 7.0～ 10 .5酶活力较高且比较稳定 .某些金属离子尤其是Fe3+ 对酶活力有强烈的抑制作用 .以邻苯二酚为底物 ,Km 为 2 0 .63μmol/L ,Vmax为 2 .82 μmol/ (min·mg)

邻苯二酚2,3-双加氧酶在恶臭假单胞杆菌整细胞催化中的酶活检测方法

Catechol 2,3-dioxygenase(C23O)is the key enzyme of aromatic substance degradation by Pseudomonas sp..In order to establish a simple assay of C23O activity during the whole-cell catalysis of Pseudomonas putida mt-2,C23O was induced by utilizing sodium benzoate acid as the sole carbon source,and its activity was determined in whole cells by the amended protocol of pure enzyme assay.After suspending the cells with potassium phosphate buffer,the substrate was added and the accumulation of 2-hydroxymuconic semialdehyde was measured by a UV757CRT spectrophotometer at 375 nm.The activity of C23O was evaluated by the climbing slope of time course curve of the UV absorption.By this means,the Km for catechol and C23O in whole cells was 34.67 μmol·L-1,while Vmax was 0.29 μmol·min-1·(mg dry cell)-1,both of which differed from those for pure enzyme by 2—3 orders of magnitude.To eliminate the cell wall barrier for substrate permeation,a cationic surfactant,n-dodecyltrimethylammonium bromide,was used to pre-treat the cells.With 0.1 g·L-1 dodecyl trimethyl ammonium bromide(DTAB) treated for 30 min,the maximum C23O activity could be achieved,which was consistent with the result of treated cells by beads milling.In the present study,a feasible and simple method was put forward for the apparent enzyme activity assay intracells which could be conveniently applied to the whole-cell biocatalysis or to environmental bioremediation.