首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 140 毫秒
1.
黄杆菌肝素酶Ⅱ(HepⅡ)是一类可特异性切割肝素、硫酸乙酰肝素类分子内连接键的酶。文中对黄杆菌肝素酶Ⅱ重组菌的诱导时机、诱导剂添加量、诱导温度、诱导时间等诱导产酶条件进行优化。经过优化最佳摇瓶发酵产酶条件为:37℃培养重组菌至对数生长前期,添加诱导剂IPTG至终浓度为0.3 g/L,20℃下诱导10 h,酶活达到最高,为570 U/L。在此基础上通过发酵罐高密度培养手段将菌体浓度OD600进一步提高到98,酶活大幅度提高到9 436 U/L,该研究结果为HepⅡ的工业化生产与应用奠定了良好的基础。  相似文献   

2.
本文对比研究了溴化氰活化及高碘酸活化肝素修饰的两种修饰尿激酶的性质。结果表明尿激酶在溴化氰活化肝素(肝素CN),高碘酸钠活化肝素(肝素I_4)的共价修饰后,其残余自由氨基分别是64%和52%;酶活性分别保留94%和90%;抗胃蛋白酶水解以及抗冻融变性的能力均高于天然酶;在离体血浆中的失活速变低于天然酶。本文还对修饰酶进行了萤光及紫外差光谱的分析,讨论了修饰过程对构象的影响。  相似文献   

3.
肝素酶是一类能够特定切割肝素或硫酸乙酰肝素中α-1,4糖苷键并将其裂解成有活性寡糖片段的酶,主要分为真核生物肝素酶(Heparanase)和原核生物肝素酶(Heparinase)。由于原核生物肝素酶是一种高效绿色的生物催化剂,因此近年来在医药领域的应用性研究逐渐被重视。文中结合本课题组相关工作,归纳介绍了原核生物肝素酶通过作用于硫酸肝素蛋白聚糖(HSPGs)生成肝素小分子,抑制肿瘤细胞增殖方面的应用;原核生物肝素酶在制备第三代创新型抗凝血药物低分子量肝素(Low molecular weight heparin,LWMH)和超低分子量肝素(Ultra low molecular weight heparin, ULMWH)方面的应用;原核生物肝素酶作为肝素拮抗药物等医药领域的重要应用;并展望了原核生物肝素酶的未来应用前景及挑战。  相似文献   

4.
将提纯的一种内切型肝素酶固定于聚酯载体上 ,固定化效率达 78 8%。酶活力在pH为 7 5左右时表现最高 ,并且在此条件下固定化酶的稳定性最好。最适反应温度为 4 0℃。热稳定性试验表明 ,固定化酶的稳定性较差。固定化酶的使用半衰期比游离酶延长 4 4倍。固定化酶催化肝素底物反应的Km 值约为 95 4 μmol L而游离酶的Km 值约为 71 2 μmol L。固定化酶可以同时作用于肝素和硫酸乙酰肝素 ,而对硫酸软骨素没有催化能力。肝素经降解后 ,产生一定量的非硫酸化或低硫酸化的二糖和不同聚合度的寡糖混合物。  相似文献   

5.
微生物肝素酶是一类作用于肝素和类肝素的多糖裂解酶,在低分子肝素的制备以及肝素类分子的结构解析等领域具有十分重要的应用前景。本文将结合微生物基因组测序的最新进展,综述微生物肝素酶的来源,并对肝素酶的作用机理加以讨论;结合本实验室研究对肝素酶的重组表达及其在低分子肝素生产的应用最新进展作以综述,并对肝素酶其它潜在的应用作以讨论。  相似文献   

6.
嘧啶核苷磷酸化酶(PyNPase)是嘧啶核苷补救代谢途径中的关键酶,广泛分布于微生物及动物组织细胞中。近几年来,很多学者对PyNPase在抗癌药物合成和癌症治疗方面的作用及其临床应用进行了广泛的研究。本文综述了PyNPase与肿瘤患者临床病理特征、抗癌药物评价等之间的关系。  相似文献   

7.
目的:肝素酶在白细胞游走和恶性肿瘤转移的过程中发挥重要作用,肝素酶抗体的制备对于自身免疫病和肿瘤的良恶性鉴别诊断具有重要意义。制备抗人肝素酶单克隆抗体,用于肝素酶的研究及临床恶性肿瘤的鉴别诊断。方法:通过杂交瘤技术将分泌抗人肝素酶单抗的小鼠B细胞与小鼠骨髓瘤细胞Sp2/0融合,获得稳定分泌抗人肝素酶单抗的杂交瘤细胞;用有限稀释法获得单克隆,以重组人肝素酶及含肝素酶的血小板裂解液对抗体进行Western印迹检测。结果:Western印迹结果显示制备的单抗与人肝素酶具有特异性免疫识别特性。结论:获得了能够特异性免疫识别人肝素酶的分泌性抗人肝素酶单克隆抗体。  相似文献   

8.
嘧啶核苷的研究进展   总被引:5,自引:0,他引:5  
王锐 《生物技术通讯》2007,18(3):539-542
嘧啶核苷包括尿嘧啶核苷和胞嘧啶核苷,其在食品工业和医药行业上应用广泛。介绍了嘧啶核苷的用途、测定方法、生产方法等;根据代谢控制发酵原理,以尿苷生产菌的选育为例,详细介绍了嘧啶核苷生产菌的育种策略,并对嘧啶核苷生产菌的育种实例、育种展望进行了综述。  相似文献   

9.
具抗肿瘤活性的乙酰肝素酶抑制剂的研究与开发   总被引:1,自引:0,他引:1  
乙酰肝素酶是一种内源β-D-葡萄糖醛酸酶,通过抑制肝素酶的活性可抑制肿瘤细胞的生成及转移等。肝素酶抑制剂的研究,已成为抗肿瘤药物的研究热点之一。本文对肝素酶抑制剂作用的机理、筛选方法和已分离或合成的抑制剂等进行了综述,并讨论了今后对潜在的乙酰肝素酶抑制剂海洋生物羊栖菜多糖的开发。本文就DLC-1基因的结构及生物学功能、在乳腺癌中失活的机制和在乳腺癌中的表达及其意义作一综述。  相似文献   

10.
目的:克隆肝素酶基因的可变剪接体并测序。方法:根据人肝素酶的cDNA序列设计引物,用RT-PCR方法从正常人外周血白细胞中扩增肝素酶基因的可变剪接体,构建至pGEM-T Easy载体中,转化大肠杆菌DH5α感受态细胞,筛选阳性克隆并进行序列测定。结果:获得了肝素酶基因的3种可变剪接体形式,即5号外显子缺失可变剪接体、6号外显子缺失可变剪接体、5和6号外显子缺失可变剪接体,其中后2种可变剪接体尚未报道。结论:克隆了肝素酶基因的3种可变剪接体,有助于研究各种肝素酶可变剪接体编码蛋白的结构和功能及其在肿瘤发生转移过程中的作用。  相似文献   

11.
Heparinase production by Flavobacterium heparinum.   总被引:11,自引:3,他引:8       下载免费PDF全文
Heparinase production by Flavobacterium heparinum in complex protein digest medium, with heparin employed as the inducer, has been studied and improved. The maximum productivity of heparinase has been increased 156-fold over that achieved by previously published methods to 375 U/liter per h in the complex medium. Rapid deactivation of heparinase activity, both specific and total, was observed at the onset of the stationary phase. Nutritional studies on growth and heparinase production showed an obligate requirement for L-histidine and no vitamin requirement. L-Methionine partially relieved the L-histidine requirement. A defined medium containing glucose, ammonium sulfate, basal salts, L-methionine, and L-histidine was developed for growth and heparinase production. The growth rate in this medium was 0.21 h-1, which is 40%, higher than that in complex medium. The maximum volumetric productivity of heparinase in the defined medium was increased to 1,475 U/liter per h, providing a 640-fold increase over that achieved by previously published methods. No rapid deactivation was observed. An examination of alternate inducers for heparinase showed that heparin degradation products, hyaluronic acid, heparin monosulfate, N-acetyl-D-glucosamine, and maltose, induce heparinase in complex medium. An Azure A assay was modified and fully developed to measure the heparin concentration during fermentation and the heparinase specific activity of crude extracts of F. heparinum obtained from sonication, thus negating the need for further purification to measure activity."  相似文献   

12.
Heparinase I from flavobacterium heparinum has several potential clinical applications; the resulting high demands on protein purity and quantity can be met by recombinant expression in Escherichia coli. Based on laboratory scale experiments with insoluble heparinase I expression followed by renaturation, a process for production of 3 kg/year of heparinase I was designed. We present a comparative analysis of the production costs of soluble and insoluble heparinase I expression, as well as a generalized approach to sensitivity analysis, based on perturbation around a base case design scenario. This may assist focusing further development on process steps for which improvements both are feasible and result in significant cost saving. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 575-582, 1997.  相似文献   

13.
14.
Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.  相似文献   

15.
Sulfur regulation of heparinase and sulfatases in Flavobacterium heparinum   总被引:2,自引:0,他引:2  
Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.  相似文献   

16.
Specific plate assay for bacterial heparinase.   总被引:2,自引:1,他引:1       下载免费PDF全文
A procedure was developed for detecting heparinase activity on heparin agar plates. The method is based on the differential precipitation of heparin and heparinase-generated heparin fragments by protamine sulfate. Heparinase activity is detected by the presence of clear zones against a white background. This method can be used to screen for the expression of recombinant heparinase and to identify Flavobacterium heparinum mutants expressing heparinase constitutively.  相似文献   

17.
Polyclonal IgG rabbit antibodies were prepared against a purified heparinase from Flavobacterium heparinum. Immuno-affinity purification of crude and partially purified heparinase is described. The resulting enzyme was of comparable purity to that prepared using the standard multistep purification scheme. The antibodies prepared were found to increase the activity of bound heparinase.  相似文献   

18.
Upon induction with heparin, Flavobacterium heparinum synthesizes and secretes into its periplasmic space heparinase I (EC 4.2.2.7), heparinase II, and heparinase III (heparitinase; EC 4.2.2.8). Heparinase I degrades heparin, and heparinase II degrades both heparin and heparan sulfate, while heparinase III degrades heparan sulfate predominantly. We isolated the genes encoding heparinases II and III (designated hepB and hepC, respectively). These genes are not contiguous with each other or with the heparinase I gene (designated hepA). hepB and hepC were found to contain open reading frames of 2,316 and 1,980 bp, respectively. Enzymatic removal of pyroglutamate groups permitted sequence analysis of the amino termini of both mature proteins. It was determined that the mature forms of heparinases II and III contain 746 and 635 amino acids, respectively, and have calculated molecular weights of 84,545 and 73,135, respectively. The preproteins have signal sequences consisting of 26 and 25 amino acids. Truncated hepB and hepC genes were used to produce active, mature heparinases II and III in the cytoplasm of Escherichia coli. When these enzymes were expressed at 37 degrees C, most of each recombinant enzyme was insoluble, and most of the heparinase III protein was degraded. When the two enzymes were expressed at 25 degrees C, they were both present predominantly in a soluble, active form.  相似文献   

19.
A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45 degrees C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both heparin and HS, similar to heparinase II from Flavobacterium heparinum.  相似文献   

20.
Purification and characterization of a novel heparinase   总被引:2,自引:0,他引:2  
A unique heparinase was isolated from a recently discovered Gram-negative soil bacterium. The enzyme (heparinase III) was purified by hydroxylapatite chromatography, chromatofocusing, and gel permeation chromatography. The enrichment was 48x, and the specific activity of catalytically pure heparinase was 127 IU/mg of protein. Similar to the heparinase I from Flavobacterium heparinum, heparinase III also degrades heparin to mainly disaccharide fragments. It is specific for heparin and also breaks down heparan sulfate, but not hyaluronic acid and chondroitin sulfate. Heparinase III, however, differs markedly from heparinase I in several other aspects: it has a higher molecular mass (94 versus 43 kDa), pI (9.2 versus 8.5), its Km and kcat are different, and it has a higher energy of activation (15.6 versus 6.3 kcal/mol). Optimal activity was also found at higher pH (7.6 versus 6.5) and temperature (45 versus 37 degrees C). Furthermore, the amino acid composition of heparinase III is quite different from that of heparinase I.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号