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1.
裂褶菌产纤维二糖脱氢酶条件优化及部分酶学性质研究   总被引:3,自引:0,他引:3  
方靖  刘稳 《菌物系统》2000,19(1):107-110
研究了裂褶菌Schizophyllum communeAS 5.391产纤维二糖胶氢酶的条件。该酶是诱导酶棉花是最好的诱导底物。培养基中加入琥珀酸纳缓冲液和土温80利于酶的合成。而木素相关物质藜芦醇或愈创木酚对酶的生成没有影响。该酶在pH3.5~10.5和45℃以下保持稳定,其最适作用温度为30℃,最适pH为4.5。Zn^2+和Ag^+对该酶活性有很大的抑制作用,而叠氮化钠和氰化钾对酶活力没有影响  相似文献   

2.
青霉胞外菊粉酶的纯化和性质研究   总被引:1,自引:0,他引:1  
青霉菌(PenicilliumSP.91-4)产生的胞外菊粉酶(extracellularinulinase)粗酶液,经硫酸铵沉淀,超滤浓缩,Sephadex-G-100凝胶过滤,DEAE-Sephacel离子交换柱层析等步骤,得到提纯30倍的酶E。酶E反应时最适pH4.5,最适温度为50℃,在pH4.7~7.6范围内,温度50℃以下酶E活性稳定;其活性受Ag^+,Cu^2+PCMB强烈抑制。采用  相似文献   

3.
地衣芽孢杆菌β-甘露聚糖酶的纯化及酶学性质   总被引:6,自引:0,他引:6  
地衣芽孢杆菌(Bacilluslicheniformis)NK-27菌株发酵产生的β-甘露聚糖酶(β-mannanase)经硫酸铵盐析沉淀,两次DEAE纤维素和SephadexG-100离子交换柱层析以及制备PAGE筹步骤,获得了凝胶电泳均一的样品。用SDS-凝胶电泳测得纯化后的β-甘露聚糖酶分子量为26kD,用凝胶聚焦电泳测得等电点PI为5.0。酶反应的最适pH为9.0,最后温度为60℃,稳定pH为6.0—9.0,稳定温度为40℃。金属离子中Mg ̄(2+)、Ca ̄(2+)、Fe ̄(2+)、Ni ̄(2+)对该酶有一定的激活作用;而Sn ̄(2+)、Zn ̄(2+)、Al ̄(3+)、Ag ̄+和Hg ̄(2+)对该酶有强烈的抑制作用。NK-27菌株的β-甘露聚糖酶对魔芋葡萄甘露聚糖和角豆胶半乳甘露聚糖的Km值分别为7.14和5.56mg·ml ̄(-1);V_(max)分别为200.53和157.45μmol·mg ̄(-1)·min ̄(-1)。  相似文献   

4.
菜豆幼苗EPSP合成酶的分离纯化和它的部分性质   总被引:3,自引:0,他引:3  
利用硫酸铵分级沉淀,Sephedex G-50凝胶柱层析,FPLC Mono-Q和磷酸纤维素离子层析法从菜豆幼苗中分离提纯了EPSP合成酶。该酶被纯化2961.6倍,比活性达到6219.4nmolmg^-1蛋白min^-1。该酶分子量经SDS-PAGE检测为51kD,等电点为pH5.7,酶促反应最适pH7.5,最适温度45℃。6.2μmol/L的除草剂草甘膦能抑制EPSP合成酶活性的50%。  相似文献   

5.
报道了弹性蛋白亲和层析分离纯化芳香黄杆菌产胞外弹性蛋白酶。经一步柱层析可获聚丙烯酰胺凝胶电泳均一的酶制品,收率可达50%,并制备了酶的结晶。该酶在SDS-PAGE上测得分子量为21380,IEF-PAGE测得等电点8.9,最适作用温度为50℃,最适作用pH为7.4,在40℃以下热稳定性良好,pH4.5-9.5范围内稳定,重金属离子Fe3+、Zn2+、Cr3+、Co2+、Hg2+、Ni2+、Ag+、Cu2+等严重抑制酶活性,黄杆菌弹性蛋白酶除了特异水解弹性蛋白外,对干酪素、血纤维蛋白、白蛋白、明胶、血红蛋白等多种蛋白质均能水解。  相似文献   

6.
经过硫酸铵30%~50%分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达53%,并制备了酶的结晶,该酶以SephadexG100柱层析及SDS-PAGE测得分子量约为81000,该酶的最适pH为7.0,最适温度为45℃,Zn2+、Mn2+、Fe2+、Cu2+、Co2+等重金属离子不同程度地抑制酶活性。  相似文献   

7.
适于非水相催化用细菌脂肪酶基本性质的研究   总被引:3,自引:0,他引:3  
对假单胞菌产脂肪酶的基本酶学性质进行了研究.该酶水解油脂时的最适作用pH为9.0,最适作用温度为45℃;在pH7.0~10.0范围内稳定,在60℃以下热稳定性良好.K+、Ca2+、Mg2+等金属离子对酶有明显的激活作用,而Hg2+、Cu2+、Sn2+等重金属离子却对酶有较强的抑制作用;几种表面活性剂和胆汁盐均使酶发生不同程度的失活.该酶在水解油脂时表现出1,3-位置专一性,且对不同种类和来源的油脂水解作用速率不同  相似文献   

8.
麻蝇幼虫肠道蛋白酶BGP的分离纯化及性质   总被引:1,自引:0,他引:1  
棕尾别麻蝇幼虫肠液经SDS-PAGE后,X光片显影,呈现两条蛋白酶活性带.IEF后,两条蛋白酶活性带的等电点分别为pH7.7和6.8.麻蝇幼虫肠液经55%~75%硫酸铵沉淀,以及连续两次制备等电聚焦,分离纯化出等电点约为pH7.7,分子量约为35kD的蛋白酶BGP.该酶能分解酪蛋白和类胰蛋白酶专一底物Bz-Phe-Val-ArgNA,不能分解弹性蛋白酶专一底物elastin-CongoRed和类胰凝乳蛋白酶专一底物Suc(Ala)2Pro-PheNA.SBBI,Leupeptin和PMSF能强烈抑制其活性.专一底物和抑制剂的结果表明,BGP是一种类胰蛋白酶.其最适反应温度为50℃,最适作用pH为8.5.不耐高温,50℃保温30min活性急剧下降.Hg2+,Zn2+和Cu2+能抑制酶活性.Ca2+,Mg2+对酶无激活作用,EDTA无抑制作用.  相似文献   

9.
豆壳过氧化物酶的分离纯化及其性质研究   总被引:28,自引:2,他引:28  
从豆壳抽提液经硫酸铵分级沉淀,DEAE-SephadexA-50离子交换层析,ConA-Sepharose4B亲合层析和Bio-GelP-60凝胶过滤,纯化了豆壳过氧化物酶(soybeanhulper-oxidase,ShP).纯化酶的比活力为7077U/mg,在SDS-PAGE上显示出一条蛋白质带.ShP分子量为38000,等电点为3.9;ShP为一含血红素的糖蛋白,含糖量为18.7%,光谱学分析揭示,在406nm处有一典型的Soret带,在510nm和640nm处有特征吸收峰.酶反应的最适pH在4.0附近,最适温度为45℃;在pH2.5~12.0之间较稳定,75℃,保温60min,酶活力残余68%,ShP是一种良好的耐酸碱、耐热过氧化物酶.动力学分析求得ShP的表观Km(愈创木酚)为1.62mmol/L,表现Km(H2O2)为0.34mmol/L.在所测定的化学试剂中,N-3、CN-、Fe3+、Fe2+和Sn2+对酶有较强烈的抑制作用,而重金属离子Ag+、Hg2+、Pb2+、Cu2+、Cr3+以及SDS和EDTA对酶活力无显著影响  相似文献   

10.
柑叶片蔗糖酶的分离纯化及其部分性质的研究   总被引:8,自引:0,他引:8  
柑(Citrusreticulata Blanco)幼叶中存在高活性的酸性蔗糖酶。经硫酸铵盐析、DEAE-琼脂糖离子交换层析、SephacrylS-200 凝胶层析纯化,活性回收率6.4% ,纯化倍数179.2 倍。纯化的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,SDS-PAGE显示1 条蛋白带,其亚基分子量40 kD。用SephacrylS-200 凝胶层析法测得分子量为80 kD。推测该酶由两个相同亚基构成。以蔗糖为底物测定该酶的表观Km 为1.6×10- 2 m ol·L- 1,Vm ax为100 m g 还原糖·m g- 1蛋白质·h- 1。最适pH 5.0,酸碱稳定区在pH 4.5—5.5 之间。最适温度55℃  相似文献   

11.
The pathophysiology of congenital diaphragmatic hernia (CDH) is constituted by pulmonary hypoplasia and pulmonary hypertension (PH). We previously reported successful treatment with imatinib of a patient with CDH. This study examines the effect of antenatal imatinib administration on the pulmonary vasculature in a rat model of CDH. Pregnant rats were given nitrofen to induce CDH. Controls were given olive oil. Half of the CDH fetuses and half of the controls were treated with imatinib antenatally E17-E21, rendering four groups: Control, Control+Imatinib, CDH, and CDH+Imatinib. Lung sections were obtained for morphometry and immunohistochemistry, and protein was purified for Western blot. Effects of nitrofen and imatinib on Ki-67, caspase-3, PDGF-B, and PDGF receptors were analyzed. Imatinib significantly reduced medial wall thickness in pulmonary arteries of rats with CDH. It also normalized lumen area and reduced the proportion of fully muscularized arteries. Imatinib also caused medial thinning in the control group. Cell proliferation was increased in CDH, and this proliferation was significantly reduced by imatinib. PDGF-B and PDGFR-β were upregulated in CDH, and imatinib treatment resulted in a downregulation. PDGFR-α remained unchanged in CDH but was significantly downregulated by imatinib. Antenatal imatinib treatment reduces development of medial wall thickness and restores lumen area in pulmonary arteries in nitrofen-induced CDH. The mechanism is reduced cell proliferation. Imatinib is an interesting candidate for antenatal therapy for PH in CDH, but potential side effects need to be investigated and more specific targeting of PDGF signaling is needed.  相似文献   

12.
The molecular basis of the pathogenesis of pulmonary hypertension (PH) associated with congenital diaphragmatic hernia (CDH) is poorly understood. Variation in responses to therapeutic strategies such as nitric oxide (NO) inhalation and extracorporeal membrane oxygenation (ECMO) in patients with CDH remains a major problem in pediatric critical care. We investigated the expression pattern of NO-generating enzyme nitricoxide synthase (NOS) (both endothelial [eNOS] and inducible [iNOS] isoforms) in the lungs of CDH patients with PH and evaluated the influence of ECMO on the expression levels of these genes in an attempt to understand the underlying molecular mechanisms. Lung autopsy specimens from 23 cases of CDH not treated by ECMO and 10 ECMO-treated CDH cases were studied and compared with 11 age-matched controls. Expression of iNOS and eNOS was assessed by immunohistochemistry and video-image analysis. Expression of iNOS in the endothelium of small pulmonary arteries (external diameter≤200 μm) was significantly lower in CDH cases that had not received ECMO treatment (p=0.04). ECMO-treated CDH cases did not differ from controls in iNOS expression. Alveclar macrophages (CD68+ cells), of which the number also was increased, showed significantly enhanced staining for iNOS in CDH cases (p=0.03) compared with controls. The observed decrease in pulmonary expression of iNOS in patients with CDH suggests a potential role in the pathogenesis of pulmonary hypertension in newborns with CDH. ECMO treatment was correlated with induction of this enzyme, which may result in NO-mediated vasodilatation and thereby transiently reduce the pulmonary hypertension in CDH.  相似文献   

13.

Background

Pulmonary hypertension (PH) is a lethal disease that is associated with characteristic histological abnormalities of the lung vasculature and defects of angiopoetin-1 (ANG-1), TIE-2 and bone morphogenetic protein receptor (BMPR)-related signalling. We hypothesized that if these signalling defects cause PH generically, they will be readily identifiable perinatally in congenital diaphragmatic hernia (CDH), where the typical pulmonary vascular changes are present before birth and are accompanied by PH after birth.

Methods

CDH (predominantly left-sided, LCDH) was created in Sprague-Dawley rat pups by e9.5 maternal nitrofen administration. Left lungs from normal and LCDH pups were compared at fetal and postnatal time points for ANG-1, TIE-2, phosphorylated-TIE-2, phosphorylated-SMAD1/5/8 and phosphorylated-ERK1/2 by immunoprecipitation and Western blotting of lung protein extracts and by immunohistochemistry on lung sections.

Results

In normal lung, pulmonary ANG-1 protein levels fall between fetal and postnatal life, while TIE-2 levels increase. Over the corresponding time period, LCDH lung retained normal expression of ANG-1, TIE-2, phosphorylated-TIE-2 and, downstream of BMPR, phosphorylated-SMAD1/5/8 and phosphorylated-p44/42.

Conclusion

In PH and CDH defects of ANG-1/TIE-2/BMPR-related signalling are not essential for the lethal vasculopathy.  相似文献   

14.
BACKGROUND : Pulmonary hypoplasia (PH) is the main cause of mortality in newborns with congenital diaphragmatic hernia (CDH). Prenatal administration of retinoic acid (RA) stimulates alveologenesis in the nitrofen‐induced pulmonary hypoplasia. Insulin‐like growth factor receptors (IGFRs) play a crucial role in alveologenesis during lung development. We recently demonstrated that IGFRs were downregulated in later stages of lung development in the nitrofen CDH model. Several studies suggest the ability of RA to regulate insulin‐like growth factor signaling. We hypothesized that IGFRs pulmonary gene expression is upregulated after the administration of RA in the nitrofen‐induced CDH model. METHODS : Pregnant rats were exposed to either olive oil or nitrofen on day 9 (D9) of gestation. RA was given intraperitoneally on days D18, D19, and D20. Fetal lungs were dissected on D21 and divided into control, control + RA, CDH, and CDH + RA group. IGFRs gene and protein expression were determined using RT‐PCR and immunohistochemistry. RESULTS : mRNA expression levels of IGFRs were significantly increased in control + RA and CDH + RA compared with CDH group. Immunoreactivity of IGFRs was markedly increased in control + RA and CDH + RA compared with CDH lungs. CONCLUSIONS : Upregulation of pulmonary gene and protein expression of IGFRs after prenatal RA treatment in the nitrofen model suggests that RA may promote lung growth by stimulating IGFRs mediated alveologenesis. Birth Defects Res (Part B) 92:148–151, 2011. © 2011 Wiley‐Liss, Inc.  相似文献   

15.
Pulmonary vascular diseases of the newborn comprise a wide range of pathological conditions with developmental abnormalities in the pulmonary vasculature. Clinically, pulmonary arterial hypertension (PH) is characterized by persistent increased resistance of the vasculature and abnormal vascular response. The classification of PH is primarily based on clinical parameters instead of morphology and distinguishes five groups of PH. Congenital lung anomalies, such as alveolar capillary dysplasia (ACD) and PH associated with congenital diaphragmatic hernia (CDH), but also bronchopulmonary dysplasia (BPD), are classified in group three. Clearly, tight and correct regulation of pulmonary vascular development is crucial for normal lung development. Human and animal model systems have increased our knowledge and make it possible to identify and characterize affected pathways and study pivotal genes. Understanding of the normal development of the pulmonary vasculature will give new insights in the origin of the spectrum of rare diseases, such as CDH, ACD, and BPD, which render a significant clinical problem in neonatal intensive care units around the world. In this review, we describe normal pulmonary vascular development, and focus on four diseases of the newborn in which abnormal pulmonary vascular development play a critical role in morbidity and mortality. In the future perspective, we indicate the lines of research that seem to be very promising for elucidating the molecular pathways involved in the origin of congenital pulmonary vascular disease. Birth Defects Research (Part C) 102:343–358, 2014. © 2014 Wiley Periodicals, Inc.  相似文献   

16.
纤维二糖脱氢酶的纤维素降解中的作用研究   总被引:5,自引:0,他引:5  
裂褶菌纤维二糖脱氢酶(cellobiose dehydrogenase,CDH)可以提高纤维素酶对纤维素的降解。以纤维二糖为电子供体,CDH作用于羧甲基纤维可降低其溶液的粘度,作用纤维素CF11和磷酸膨胀纤维素,分别使其悬浊液的浊度提高7%和14.4%。CDH与纤维二糖水解酶或切纤维素酶在降解棉花纤维素时没有表现出协同作用。但若棉花事先在纤维二糖存在下用CDH预处理,则变得易于被水解。  相似文献   

17.
Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyze the four-electron oxidation of proline to glutamate via the intermediates P5C and l-glutamate-γ-semialdehyde (GSA). In Gram-negative bacteria, PRODH and P5CDH are fused together in the bifunctional enzyme proline utilization A (PutA) whereas in other organisms PRODH and P5CDH are expressed as separate monofunctional enzymes. Substrate channeling has previously been shown for bifunctional PutAs, but whether the monofunctional enzymes utilize an analogous channeling mechanism has not been examined. Here, we report the first evidence of substrate channeling in a PRODH-P5CDH two-enzyme pair. Kinetic data for the coupled reaction of PRODH and P5CDH from Thermus thermophilus are consistent with a substrate channeling mechanism, as the approach to steady-state formation of NADH does not fit a non-channeling two-enzyme model. Furthermore, inactive P5CDH and PRODH mutants inhibit NADH production and increase trapping of the P5C intermediate in coupled assays of wild-type PRODH-P5CDH enzyme pairs, indicating that the mutants disrupt PRODH-P5CDH channeling interactions. A dissociation constant of 3 μm was estimated for a putative PRODH-P5CDH complex by surface plasmon resonance (SPR). Interestingly, P5CDH binding to PRODH was only observed when PRODH was immobilized with the top face of its (βα)8 barrel exposed. Using the known x-ray crystal structures of PRODH and P5CDH from T. thermophilus, a model was built for a proposed PRODH-P5CDH enzyme channeling complex. The structural model predicts that the core channeling pathway of bifunctional PutA enzymes is conserved in monofunctional PRODH-P5CDH enzyme pairs.  相似文献   

18.
无花果蛋白酶通过8%戊二醛活化载体,共价结合到聚苯乙烯阴离子交换树脂GM201上,固定化作用在pH7.7,酶浓度0.8mg/g树脂,4℃下进行6h。得到的固定化酶表观K_m值(酪蛋白,1.11×10~(-4)mol/L)小于溶液酶K_m值(1.96×10~(-4)mol/L);固定化酶活性在pH6~8保持稳定,溶液酶最适pH为7.2;固定化酶最适温度由溶液酶的50~60℃移至37℃;固定化酶25℃保持7d,重复水解酪蛋白7次后,保留83.3%活性。固定化酶对酪蛋白水解度达47.5%,对大豆球蛋白达11.6%。  相似文献   

19.
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