途径优化强化枯草芽胞杆菌合成肝素前体

肝素前体是化学酶法合成肝素的起点,肝素前体的微生物高效合成具有重要意义。在已构建的产肝素前体的枯草芽胞杆菌((1.71±0.08)g/L)中,分析了UDP-葡萄糖醛酸(UDP-GlcUA)途径中关键酶基因(pgcA、gtaB、tuaD)以及UDP-乙酰氨基葡糖(UDP-GlcNAc)途径中关键酶基因(glmS、glmM、glmU)的过量表达对肝素前体产量及其分子量的影响。在此基础上,通过共表达tuaD、gtaB、glmU、glmM和glmS基因,摇瓶中肝素前体产量提高至(2.89±0.11)g/L,分子量为(75.90±1.18)kDa。通过在3 L发酵罐中进行补料分批发酵,肝素前体的产量最终积累到(7.25±0.36)g/L,分子量为(46.66±2.71)kDa,为工业化生产肝素奠定了基础。     

低分子肝素与普通肝素在介入手术中安全性与疗效的比较

肝素已经普遍用于冠状动脉介入术中,但是由于在应用过程中抗凝效果个体差异大、出血风险相对较高、并发症等问题的日益显现,已经引起了医生们的注意。相对而言,低分子肝素具有抗凝作用强、出血风险低、并发症发生率相对较低、无需实验室监测等优点,进而越来越广泛的在冠状动脉介入术中担当重要角色。普通肝素与低分子肝素在冠状动脉介入术中的有效性与安全性的比较是值得我们去探讨的,知道普通肝素与低分子肝素的优缺点后,可使我们在冠状动脉介入术中更好的选择抗凝药物,使手术的成功率大大提高,降低术后并发症的发生,减轻患者的痛苦。本文结合普通肝素与低分子肝素的药理作用及相关临床研究的结果,分析比较了普通肝素与低分子肝素在冠状动脉介入术中的有效性与安全性。     

肝素修饰尿激酶某些性质的研究

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。     

辛伐他汀联合低分子肝素对慢性心衰心肺功能及相关指标的影响

目的:探索辛伐他汀联合低分子肝素对慢性心力衰竭(CHF)患者的D-二聚体、N-末端脑钠肽前体(NT-proBNP)、左室射血分数(LVEF)、左室舒末内径、血清反应蛋白(CRP)、血浆纤维蛋白原(FB)、血清总胆固醇(TC)及低密度脂蛋白胆固醇(LDL-C)的影响。方法:选择我院自2010年4月至2014年7月间收治的患有CHF患者80例,按照随机数表法分成治疗组和对照组,各40例。两组均给予常规治疗,治疗组在常规治疗的基础上加用辛伐他汀和低分子肝素,治疗两个月。对比两组患者治疗前后D-二聚体、NT-proBNP、LVEF、左室舒末内径、CRP、FB、TC和LDL-C水平。结果:与治疗前相比,对照组患者治疗后的D-二聚体、NT-proBNP、LVEF、左室舒末内径、CRP、FB、TC和LDL-C水平均无明显改善,而治疗组患者上述各项指标均显著改善,差异均具有统计学意义(均P0.05)。结论:临床上应用辛伐他汀联合低分子肝素可以有效改善CHF患者的相关指标,对于疾病的治疗具有重要作用,值得在临床上应用及推广。     

肝素酶在医药领域应用的研究进展

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。     

HIT抗体检测对下肢动脉硬化性闭塞症患者的临床意义

目的:探讨HIT抗体在HIT中的诊断价值.方法:采用酶联免疫吸附法(ELISA)检测HIT抗体,进一步观测应用肝素的患者HIT抗体的产生情况,临床观察血管外科应用普通肝素治疗49名下肢动脉硬化性闭塞症(ASO)患者.采用自身对照,实验组为应用肝素治疗后的49名患者血清,对照组为应用肝素治疗前该49名患者血清,分别进行HIT抗体检测.结果:实验组中HIT抗体阳性例数为21例,对照组为3例.实验组与对照组差异有统计学意义.实验组中9例患者血小板下降(血小板下降范围23.3％～63.8％),其中8例HIT抗体阳性,1例抗体阴性.该9例患者后经功能性检查确诊为HIT患者.其中2例患者血小板下降早于HIT抗体的出现,其余6例患者血小板下降时间比HIT抗体出现时间晚.HIT发病率为18.37％.结论:HIT抗体检测的实验敏感性较高,且不涉及放射性,操作简单,快捷.与临床表现、血小板计数检测结合可对应用肝素的下肢动脉硬化性闭塞症患者进行初筛.     

肝素C5异构酶的表达优化及分子改造

肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase,C5,EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶,催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5号位羧基翻转生成L-艾杜糖醛酸 (L-iduronic acid,L-IdoA)。文中以大肠杆菌Escherichia coli为宿主对斑马鱼来源的肝素葡萄糖醛酸C5异构酶基因Glce进行重组表达优化与分子改造。比较了3种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5表达的差异情况,其中以嗜冷启动型载体pCold Ⅲ表达酶活最高,达到(1 873.61±5.42) U/L。为了进一步提高C5的可溶表达量,在N端融合促溶标签SET2后,可溶蛋白表达量比对照提高了50%,酶活达到 (2 409.25±6.43) U/L。在此基础上,通过理性设计对底物结合口袋进行定点突变,获得最优突变体 (V153R) 的酶活和比酶活分别为 (5 804.32±5.63) U/L和(145.14±2.33) U/mg,是原始酶的2.41倍和2.28倍。肝素C5异构酶改造与表达优化为酶法催化合成肝素奠定了基础。     

抗人肝素酶单克隆抗体的制备与鉴定

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。     

细菌素的作用机制及在生产中的应用

细菌素是一类由微生物产生的具有抑菌活性的多肽或前体多肽类物质。本文主要介绍了细菌素的概念、分类、作用机制,细菌素与抗生素的区别及在生产中的应用。同时阐述了细菌素潜在应用价值。     

重组黄杆菌肝素酶III的纯化、表征及培养条件对酶生产的影响

肝素酶III是一种特异性地裂解乙酰肝素的酶,在大肠杆菌中表达时容易形成包涵体。为实现肝素酶III的可溶性表达,利用谷胱甘肽-S-转移酶(GST)与肝素酶III融合性能,通过构建相应的表达质粒pGEX-heparinaseIII,在大肠杆菌中实现了肝素酶Ⅲ的可溶性表达。粗酶通过一步亲和纯化其纯度可达95%以上。通过对LB培养基摇瓶培养Escherichia coli BL21的诱导时机、诱导剂用量、诱导时间等培养条件的优化,确定了该可溶性肝素酶III融合蛋白的最适生产条件。通过对纯酶的最适反应温度、pH、Ca2+浓度等一系列性质研究,确定了该酶的最适反应条件。     

Controlled Photochemical Depolymerization of K5 Heparosan, a Bioengineered Heparin Precursor

Heparosan is a polysaccharide, which serves as the critical precursor in heparin biosynthesis and chemoenzymatic synthesis of bioengineered heparin. Because the molecular weight of microbial heparosan is considerably larger than heparin, the controlled depolymerization of microbial heparosan is necessary prior to its conversion to bioengineered heparin. We have previously reported that other acidic polysaccharides could be partially depolymerized with maintenance of their internal structure using a titanium dioxide-catalyzed photochemical reaction. This photolytic process is characterized by the generation of reactive oxygen species that oxidize individual saccharide residues within the polysaccharide chain. Using a similar approach, a microbial heparosan from Escherichia coli K5 of molecular weight >15,000 was depolymerized to a heparosan of molecular weight 8,000. The (1)H-NMR spectra obtained showed that the photolyzed heparosan maintained the same structure as the starting heparosan. The polysaccharide chains of the photochemically depolymerized heparosan were also characterized by electrospray ionization-Fourier-transform mass spectrometry. While the chain of K5 heparosan starting material contained primarily an even number of saccharide residues, as a result of coliphage K5 lyase processing, both odd and even chain numbers were detected in the photochemically-depolymerized heparosan. These results suggest that the photochemical depolymerization of heparosan was a random process that can take place at either the glucuronic acid or the N-acetylglucosamine residue within the heparosan polysaccharide.     

Escherichia coli K5 heparosan fermentation and improvement by genetic engineering

N-acetyl heparosan is the precursor for the biosynthesis of the important anticoagulant drug heparin. The E. coli K5 capsular heparosan polysaccharide provides a promising precursor for in vitro chemoenzymatic production of bioengineered heparin. This article explores the improvements of heparosan production for bioengineered heparin by fermentation process engineering and genetic engineering.     

Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida,PmHS1

Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

     

Quantitation of heparosan with heparin lyase III and spectrophotometry

Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected.     

Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor

As a precursor of bioengineered heparin, heparosan is currently produced from Escherichia coli K5, which is pathogenic bacteria potentially causing urinary tract infection. Thus, it would be advantageous to develop an alternative source of heparosan from a non-pathogeneic strain. In this work we reported the biosynthesis of heparosan via the metabolic engineering of non-pathogenic E. coli BL21 as a production host. Four genes, KfiA, KfiB, KfiC and KfiD, encoding enzymes for the biosynthesis of heparosan in E. coli K5, were cloned into inducible plasmids pETDuet-1 and pRSFDuet-1 and further transformed into E. coli BL21, yielding six recombinant strains as follows: sA, sC, sAC, sABC, sACD and sABCD. The single expression of KfiA (sA) or KfiC (sC) in E. coli BL21 did not produce heparosan, while the co-expression of KfiA and KfiC (sAC) could produce 63mg/L heparosan in shake flask. The strain sABC and sACD could produce 100 and 120mg/L heparosan, respectively, indicating that the expression of KfiB or KfiD was beneficial for heparosan production. The strain sABCD could produce 334mg/L heparosan in shake flask and 652mg/L heparosan in 3-L batch bioreactor. The heparosan yield was further increased to 1.88g/L in a dissolved oxygen-stat fed-batch culture in 3-L bioreactor. As revealed by the nuclear magnetic resonance analysis, the chemical structure of heparosan from recombinant E. coli BL21 and E. coli K5 was identical. The weight average molecular weight of heparosan from E. coli K5, sAC, sABC, sACD, and sABCD was 51.67, 39.63, 91.47, 64.51, and 118.30kDa, respectively. This work provides a viable process for the production of heparosan as a precursor of bioengineered heparin from a safer bacteria strain.     

Effects of carbon sources and feeding strategies on heparosan production by Escherichia coli K5

This work aimed to develop an optimal carbon source feeding strategy to achieve maximal production of heparosan as a precursor of bioengineered heparin by Escherichia coli K5. Glycerol gave higher heparosan titer and productivity compared to glucose. The maximum heparosan production (187 mg/L) and heparosan productivity (5.19 mg/L/h) in glycerol-defined medium were 26.4% higher than the heparosan production (148 mg/L) and heparosan productivity (4.11 mg/L/h) in glucose-defined medium. DO-stat feeding approach as compared to pH-stat feeding, exponential feeding, exponential combined with pH-stat feeding, and constant rate feeding gave the highest heparosan titer at 8.63 g/L, which was nine times that of batch culture. The obtained optimal glycerol feeding strategy may be useful for the scaling-up of microbial heparosan production.     

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.     

Heparosan Chain Characterization: Sequential Depolymerization of E. Coli K5 Heparosan by a Bacterial Eliminase Heparin Lyase III and a Bacterial Hydrolase Heparanase Bp to Prepare Defined Oligomers

Heparosan is a non-sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered-cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using heparin lyase III (Hep-3, an eliminase from Flavobacterium heparinum) and heparanase Bp (Hep-Bp, a hydrolase from Burkholderia pseudomallei). The partial digestion of E. coli K5 heparosan with purified His-tagged Hep-3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2-DP8) are completely digested with purified GST-tagged Hep-Bp and analyzed using gel permeation chromatography. Hep-Bp specifically cleaves the linkage between d -glucuronic acid (GlcA) and N-acetyl-d -glucosamine (GlcNAc) but not the linkage between 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc-GlcA-GlcNAc). This method successfully demonstrated the substrate selectivity of Hep-BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.     

E. coli K5 fermentation and the preparation of heparosan,a bioengineered heparin precursor

Heparosan is an acidic polysaccharide natural product, which serves as the critical precursor in heparin biosynthesis and in the chemoenzymatic synthesis of bioengineered heparin. Heparosan is also the capsular polysaccharide of Escherichia coli K5 strain. The current study was focused on the examination of the fermentation of E. coli K5 with the goal of producing heparosan in high yield and volumetric productivity. The structure and molecular weight properties of this bacterial heparosan were determined using polyacrylamide gel electrophoresis (PAGE) and Fourier transform mass spectrometry. Fermentation of E. coli K5 in a defined medium using exponential fed‐batch glucose addition with oxygen enrichment afforded heparosan at 15 g/L having a number average molecular weight of 58,000 Da and a weight average molecular weight of 84,000 Da. Biotechnol. Bioeng. 2010;107: 964–973. © 2010 Wiley Periodicals, Inc.