透明质酸分离纯化研究进展

微生物发酵法是生产透明质酸的主要方法;分离纯化是发酵法制备高分子量、高纯度透明质酸的关键环节。本文探讨了透明质酸发酵液的特性和分离纯化过程中工艺条件对透明质酸分子量和结构的影响;对预处理、分离、纯化各阶段的工艺方法进行了系统比较和分析;指出了透明质酸分离纯化工艺中存在的问题;并提出了今后分离纯化研究的重点。     

透明质酸的产需情况及市场分析

介绍了透明质酸的性能、生产方法、用途和市场情况。透明质酸(HA)是近年来较为热点的生化产品之一,由于具有优良的保湿、润滑和生物相容性能,被应用于日化、医药、保健食品等领域。目前,全球透明质酸的年生产能力达到20t/a,2006年国内发酵法透明质酸的生产厂家达到8家,年生产能力约8t/a,大约40%的产品出口美国、日本等地。     

透明质酸的制备及其应用进展

综述了近年来透明质酸在制备和应用方面的研究现状,并预测了其发展趋势。     

Hyaluronan Synthase and Hyaluronidase Expression in Serous Ovarian Carcinoma is Related to Anatomic Site and Chemotherapy Exposure

The present study investigated the expression and clinical role of hyaluronan synthases (HAS1-3) and hyaluronidases (Hyal1-3) in serous ovarian carcinoma. HAS and HYAL mRNA expression was analyzed in 97 tumors (61 effusions, 27 primary carcinomas, 9 solid metastases) using PCR and further studied for association with clinicopathologic parameters, including survival. HAS1 mRNA was overexpressed in effusions compared to primary carcinomas and solid metastases (p < 0.001), and an alternatively spliced HAS1 was expressed only in effusions. HAS2 mRNA was overexpressed in solid metastases and primary carcinomas compared to effusions (p = 0.043), and HAS3 mRNA was overexpressed in primary carcinomas and effusions compared to solid metastases (p = 0.008). HYAL1 mRNA was absent in all specimens, whereas HYAL2 was expressed as two splice variants, of which HYAL2-var2 was overexpressed in solid metastases compared to effusions and primary carcinomas (p < 0.001). HYAL3 mRNA was expressed as wild-type and variant 1–3 form, the latter more highly in primary carcinomas and effusions compared to solid metastases (p = 0.006). HAS1 mRNA was overexpressed in pre- compared to post-chemotherapy effusions (p < 0.001), with opposite finding for HYAL2-var1 and HYAL3-WT (p = 0.016 and p = 0.024, respectively). Higher HYAL2-var1 and HAS1 splice variant mRNA expression in effusions was associated with longer (p = 0.033) and shorter (p = 0.047) overall survival, respectively. These data are the first to document a role for HAS and Hyal members in tumor progression in ovarian carcinoma, as evidenced by their differential expression as function of anatomic site and chemotherapy exposure, with a possible prognostic role for patients with malignant effusions.     

膜蒸馏方法分离浓缩透明质酸水溶液的实验研究

本文介绍了应用减压膜蒸馏分离技术对透明质酸热敏性水溶液的浓缩分离情况,结果表明,使用膜孔直径为０．１５μｍ的聚丙烯微孔疏水膜可使原料液的浓度提高１．８倍以上,透明质酸截留率为８５％,并探讨了循环时间,料液温度,真空度等条件对分离效果的影响。     

微生物发酵装置的工业化设计

用微生物发酵法来生产透明质酸 (HA)、聚羟基脂肪酸酯 (PHAs)等产品具有极大的优越性。从设计角度对实验室生物技术的工业化放大的经验进行总结。     

添加表面活性剂促进兽疫链球菌高产透明质酸

研究了不同类型表面活性剂对兽疫链球菌Streptococcus zooepidemicus H24菌体生长和透明质酸发酵的影响,在此基础上,进一步研究了2.5 L发酵罐中阳离子型表面活性剂十六烷基三甲基溴化铵（CTAB）的添加对透明质酸发酵的影响。研究结果表明：摇瓶发酵时,发酵中后期（12 h）添加离子型表面活性剂有利于透明质酸产量的提高,在摇瓶中添加20 mg/L阳离子型表面活性剂CTAB使HA比对照组提高20%;发酵罐上8～12 h流加CTAB可使兽疫链球菌细胞荚膜脱落,促使透明质酸的产量由5.1 g/L提高到5.7 g/L,增加11.8%。本研究结果为进一步提高发酵法生产其他黏多糖的产量提供了一定的借鉴。     

In Vitro Investigation of Self-Assembled Nanoparticles Based on Hyaluronic Acid-Deoxycholic Acid Conjugates for Controlled Release Doxorubicin: Effect of Degree of Substitution of Deoxycholic Acid

Self-assembled nanoparticles based on a hyaluronic acid-deoxycholic acid (HD) chemical conjugate with different degree of substitution (DS) of deoxycholic acid (DOCA) were prepared. The degree of substitution (DS) was determined by titration method. The nanoparticles were loaded with doxorubicin (DOX) as the model drug. The human cervical cancer (HeLa) cell line was utilized for in vitro studies and cell cytotoxicity of DOX incorporated in the HD nanoparticles was accessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, cellular uptake of fluorescently labeled nanoparticles was also investigated. An increase in the degree of deoxycholic acid substitution reduced the size of the nanoparticles and also enhanced their drug encapsulation efficiency (EE), which increased with the increase of DS. A higher degree of deoxycholic acid substitution also lead to a lower release rate and an initial burst release of doxorubicin from the nanoparticles. In summary, the degree of substitution allows the modulation of the particle size, drug encapsulation efficiency, drug release rate, and cell uptake efficiency of the nanoparticles. The herein developed hyaluronic acid-deoxycholic acid conjugates are a good candidate for drug delivery and could potentiate therapeutic formulations for doxorubicin–mediated cancer therapy.     

基于发酵液特性的透明质酸提取预处理工艺

为进一步提高透明质酸成品质量和提取收率,针对透明质酸发酵液中菌体和蛋白质的物化特性,采用25 g/L三氯乙酸灭活菌体并使蛋白质变性沉淀,以发酵液质量1.2%的混合硅藻土(粗细硅藻土比为1.5:1)为过滤助剂,过滤温度60℃,pH 7.0的发酵液预处理工艺提取透明质酸成品. 再配以酒精沉淀、气浮、酒精造粒的后处理工艺,所得成品透明质酸中葡萄糖醛酸含量达46.39%,蛋白质含量仅为0.047%,分子量为190万,提取收率为91.3%.     

透明质酸的制备及应用研究进展

介绍了透明质酸的结构、理化性质,并对透明质酸的制备及其在医药、化妆品和食品领域中的应用进行了综述。     

透明质酸发酵过程控制的研究

进行了兽疫链球菌NW-162高密度发酵HA的工艺研究,通过发酵过程中不同饱和度的溶解氧和不同pH调控方法对该菌株发酵生产透明质酸的影响进行了研究。实验表明,采用30%饱和度的溶解氧和在发酵过程的0~30 h,调pH 7.2,发酵后期调pH 6.8的调控方法,OD660可达0.84,透明质酸产量为0.532 g/L。在此条件下,有利于细胞生长和产物表达,降低乙酸等有害代谢副产物积累。     

N-马来酰化壳聚糖的合成和性能

通过酰化反应,制备一系列取代度不同的N-马来酰化壳聚糖。测定了产物的取代度、特性黏度、相对分子质量、吸湿保湿性,并用IR、NMR进行结构表征。研究发现高取代度高相对分子质量高黏度产物(M-11)的吸湿保湿性、抑菌性均优于透明质酸,有望替代昂贵的透明质酸,开辟壳聚糖新的应用范围。     

透明质酸在电-膜分离过程中的迁移规律

对电-膜分离技术用于透明质酸分离过程的可行性进行了初步研究. 着重研究了透明质酸在电场作用下的行为,并考察了料液中透明质酸浓度、溶液pH值、离子强度、电流强度等因素对迁移过程的影响. 结果表明,透明质酸透过膜的速度在一定范围内与料液的浓度和电流强度成正比,在pH 7~9范围内透明质酸的电迁移率基本保持恒定. 膜孔径对迁移速度没有明显影响,单位膜面积的产量也与电流密度成正比. 该方法能有效地将透明质酸与蛋白质分离.     

透明质酸发酵过程控制的研究

进行了兽疫链球菌NW-162高密度发酵HA的工艺研究,通过优化了发酵过程中不同饱和度的溶解氧和不同PH调控方法对该菌株发酵生产透明质酸的影响。试验表明:采用30%饱和度的溶解氧和在发酵过程的0～30小时,调pH 7.2,发酵后期调pH 6.8的调控方法,OD660可达0.84,透明质酸产量为0.532 g/L。在此条件下,有利于细胞生长和产物表达,降低乙酸等有害代谢副产物积累。