微生物发酵法生产透明质酸

透明质酸(Hyaluronic acid,以下简称HA)是一种高分子粘多糖,具有良好的亲水性、生物相容性和保湿功能,广泛应用于食品、化妆品和医药领域.透明质酸的传统生产方法是主要从动物组织中提取,用微生物发酵法生产透明质酸正逐步取代传统生产方法,其优点是原料易得、成本低、所产透明质酸有更高的产量和分子量等原因.     

透明质酸生物合成途径及基因工程研究进展

透明质酸是由N-乙酰氨基葡萄糖和葡萄糖醛酸组成的双糖单位聚合而成的直链酸性黏多糖,已被广泛应用于药物、化妆品和食品添加剂。微生物发酵法是目前生产透明质酸最有效的方法。生物体内透明质酸的合成途径基本一致,均为Leloir途径。透明质酸合成操纵子由透明质酸合酶基因、尿苷二磷酸葡萄糖脱氢酶基因和尿苷二磷酸葡萄糖焦磷酸化酶基因组成,其表达受Cov S/CovR和Lux S等多种调控系统调控。随着分子生物学技术的迅速发展以及对透明质酸合成相关基因了解的不断深入,人们从提高透明质酸安全性、提高透明质酸产量和调控透明质酸分子质量三个方面出发,通过基因工程手段构建出了高产、安全、一定分子质量范围的透明质酸生产菌株。就有关透明质酸生物合成途径、合成相关基因表达调控及生产菌株分子生物学改造的策略与研究进展进行综述和展望。     

微生物发酵法生产高分子量透明质酸的研究进展

透明质酸是一种线性大分子黏多糖,分子量定义其流变性能,影响生理反应,并决定合适的用途。传统研究通过优化发酵提高透明质酸的产量已取得显著成效,近年来,研究重点逐渐转向如何提高透明质酸产品的分子量。目前,高分子量透明质酸具有良好的黏弹性、保湿性、黏附性,在医药中的应用是中、低分子量透明质酸不可替代的。概述了透明质酸分子量的调控机制,以及利用微生物发酵法生产高分子量透明质酸的研究进展,并对其发展方向进行了展望。     

浅议透明质酸发酵条件的优化

现阶段透明质酸已广泛应用于化妆品、美容行业、保健食品、医药等很多领域。国内市场及国际市场得扩展速度都非常快,随之而来的透明质酸的市场竞争也日趋激烈。因此,为了满足国内日益增长的市场需求,我国应大力发展微生物发酵法制备透明质酸的技术,选育高产菌种,完善发酵工艺,以提高工业化的产量与质量。为进一步提高兽疫链球菌透明质酸的产量,优化其发酵条件,采用15L-30L×3的发酵系统进行试验。     

发酵透明质酸寡糖兽疫链球菌工程菌株的构建

透明质酸(HA)广泛应用于医学、化妆品、食品等领域。HA的生物活性取决于其分子量(M_w)。透明质酸寡糖由于具有重要的生理活性与特殊生理功能,在医药领域具有重要的应用前景。兽疫链球菌因其发酵周期短、生产强度较强的特点,在商业生产HA上具有广泛的应用。为了高效发酵合成透明质酸寡糖和解决发酵过程的溶氧问题,文中通过在兽疫链球菌WSH-24中过表达透明质酸合酶HasA以及优化表达水蛭来源的透明质酸酶LHAase。重组菌株摇瓶发酵24h,透明质酸寡糖积累至0.97g/L,比野生菌提高了182.0%。在3L发酵罐中发酵24 h,透明质酸寡糖生产强度为294.2 mg/(L·h),HA积累至7.06 g/L,比野生菌的罐上水平提高了112.4%。文中所构建的发酵合成透明质酸寡糖的兽疫链球菌重组菌株具有重要的应用前景。     

透明质酸的制备与鉴定

透明质酸是一种酸性粘多糖,其特有的粘弹性和保湿性已被用于医药与化妆品的生产中。本文以黄牛眼睛为材料,通过提取,除杂质、有机溶剂沉淀等步骤进行透明质酸的制备,并对其性质进行鉴定,表明用此方法制备的透明质酸基本达到了市场的要求,而且此工艺简单,成本低廉。     

透明质酸酶的研究进展

透明质酸酶是能降解透明质酸及部分糖胺聚糖的一类糖苷酶,可应用于医疗和美容等领域。透明质酸酶也可用于制备小分子糖胺寡糖,许多研究发现小分子糖胺寡糖具有比大分子糖胺聚糖更高的生物免疫活性。为便于研究人员对透明质酸酶进行进一步的基础研究及应用研究,本文介绍了透明质酸和透明质酸酶,梳理了透明质酸酶的分类、结构和催化机理,归纳总结了透明质酸酶的酶活力测定方法、重组表达、酶学性质和应用,展望了透明质酸酶的研究方向。     

透明质酸产生菌的选育及发酵培养基的优化

将实验室保存UVD-34透明质酸产生菌再进行超声波处理,挑选出了一株透明质酸产量较高的菌,并对其发酵培养基进行初步优化。结果表明当可溶性淀粉质量分数为2%,复合氮源为3%,碳酸氢钠为0.1%时,其透明质酸的产量可达4.59 g.L-1,比优化前提高了1.29倍。该突变株值得进一步研究。     

海豚链球菌诱变发酵法制备透明质酸及其在动物皮肤修复中的应用

透明质酸(HA)是一种非常重要的生物材料,是体内广泛存在的细胞外基质成分之一。为了获得产量、分子量及纯度较高的透明质酸,并研究透明质酸水凝胶在动物皮肤修复中的潜在作用。通过紫外诱变的方法对海豚链球菌进行诱变,并对此突变菌发酵后产物的蛋白含量及HA分子量进行了测定,通过CTAB法对发酵产物进行提纯,运用物理冻融法将透明质酸制成水凝胶后,用于兔背部全层皮肤修复的初探。结果表明通过诱变海豚链球菌产透明质酸的能力从(82.3±3.3)mg/L增加到(120±10.6)mg/L,增加了46.4%;产物经纯化后蛋白含量从(0.178±0.011)mg/L减少到(0.032±0.017)mg/L,减少了82.02%;所制得透明质酸的分子量约为3.0×105 Da;透明质酸水凝胶对兔全层皮肤缺损的修复有较明显的促进作用,能减轻炎症和伤口瘢痕的形成。     

超声波对发酵液中提取透明质酸的影响

目的：提高发酵液中透明质酸的提取率。方法：应用超声波预处理发酵液的方法对醇沉法提取透明质酸的影响进行研究。结果：在超声波功率100W、处理温度15℃、处理时间10min时,透明质酸提取率相对于未经超声波处理的发酵液提高了38.6%。因此,超声波预处理透明质酸发酵液可以达到提高透明质酸提取率的目的。     

透明质酸的制备及应用研究进展

透明质酸(hyaluronic acid,HA)是一种高分子量的直链酸性粘多糖。由于其具有特殊的生理作用、独特的流变学性质和极强的持水保湿能力,在化妆品工业、医学研究、临床治疗等领域有着广泛的应用。概述了透明质酸的制备及其在化妆品、保健食品和医药方面的应用研究进展。     

由透明质酸发酵液制备高纯度透明质酸的方法

为简化由透明质酸发酵液制备高纯度透明质酸的过程,本实验利用氯苯除去菌体,由乙醇分离得到透明质酸提取物,向提取物中加入由4.2 g/L碳酸氢钠和5.3 g/L碳酸钠以体积比为45:5组成的水溶液及胰蛋白酶进行水解.水解后所得分离物在磷酸二氢钠含量为0.45 g/L,磷酸氢二钠含量为0.05 g/L及氯化钠含量为11.6 g/L的水溶液中溶解,向其中加入3倍体积的95%乙醇,得到沉淀,绝干的沉淀物中透明质酸质量比为98.9%.随后,经活性炭以及透析处理.最终透明质酸质量比为99.996%,相对分子量5.8×105.总回收率96.17%.     

Structure for Hyaluronic Acid

THE mucopolysaccharide hyaluronic acid exists naturally as a hydrated gel. It is the constituent of synovial fluid which acts as a lubricant; it also occurs in the vitreous humour where its function is probably to maintain the precise shape of the eye. Chemically it is a regular, unbranched polymer with a repeating unit of the type (-N-G-)_n where N is N-acetylglucosamine and G is glucuronic acid. The glycosidic linkages N to G and G to N are 1e, 4e and le, 3e respectively (Fig. 1). Our arguments are based on the postulate that both glucopyranose rings are in the C1 chain conformation.     

以玻璃酸钠为载体改进利福平滴眼液的配制方法

目的:改进利福平滴眼液的配制方法。方法:以计算量的盐酸溶解利福平,然后用等摩尔量的氢氧化钠中和,生成等处方量的氯化钠;并以玻璃酸钠为载体,配制利福平滴眼液。结果:该滴眼液质量稳定、可控,对眼睛无刺激性。结论:本办法设计巧妙,切实可行,既克服了的利福平的溶解困难,又解决了用乙醇作溶剂所产生的眼睛刺激性问题。     

Hyaluronic Acid in Synovial Fluid

Twelve horses with traumatic arthritis were treated with intraarticular injection of hyaluronic acid mixed with cortisone and the results compared with 6 horses treated only with cortisone. There was a significantly better improvement in the group injected with a mixture of hyaluronic acid and cortisone. Further studies have given the same results in traumatic arthritis in horses if hyaluronic acid alone is injected. After injection of hyaluronic acid a large number of granulated monocytes appeared in the synovial fluid, but no inflammatory signs were observed. It is possible that this macrophage invasion is instrumental in producing improvement in the condition of the joint. The injected hyaluronic acid may also adhere to the surface of articular cartilage producing an “clastic cushion” protecting the cartilage surface. Experimental mechanical damage was also inflicted on the surface of articular cartilage in dogs and monkeys, and smoother healing was achieved if hyaluronic acid was injected into the joints after the damage. Injections of hyaluronic acid seem to be of value in treating traumatic arthritis or similar conditions.     

透明质酸及其衍生物研究进展

透明质酸是一种以葡萄糖醛酸和N-乙酰氨基葡萄糖胺为双糖单位交替连接而成的黏多糖。由于其具有独特的分子结构和理化性质,已被成功应用于化妆品、医学美容、眼科手术、关节炎治疗等。对最近几年国内外透明质酸及其衍生物,尤其是海洋生物来源的透明质酸的制备以及将不同分子质量的透明质酸作为新型药用载体及其他方面的研究进展进行了综述。     

Hyaluronic Acid Production in Bacillus subtilis

The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.     

Hyaluronic Acid Degradation by Ascorbic Acid and Influence of Iron

The effects of ascorbic acid, iron and ADP on hyaluronic acid, a compound present in inflamed joints, were investigated in an in vitro system. Ascorbic acid induces degradation of hyaluronic acid which increased in the presence of FeCl, and which is additionally stimulated by ADP chelated ferric ions. The hyaluronic acid degrading reactions induced by the Fe-III/ADP/ascorbic acid system were inhibited by catalase and formate to various extents whereas the presence of superoxide dismutase did not exert any inhibitory effect. Desferrioxamine, a specific iron chelator, completely inhibited hyaluronic acid depolymerisation by ascorbic acid as well as in combination with FeCl₃ or FeCl₃/ADP, respectively. We suggest that the ultimate hyaluronic acid degrading species is OH', generated via the Fe-III/ADP catalysed Haber Weiss reaction. There is also an indication for the involvement of perferryl or/and ferryl species in the degradation process.     

Interaction of Hyaluronectin with Hyaluronic Acid Oligosaccharides

Hyaluronic acid was digested by bovine testicular hyaluronidase, and oligomers were fractionated by gel permeation using AcA 202 Ultrogel, an acrylamide-agarose matrix. Oligosaccharides composed of from two to six disaccharide repeating units were isolated. Two nonasaccharides were prepared by enzymatic or chemical modification of the decasaccharide. Oligosaccharides were compared by a competitive inhibition in the enzyme-linked immunosorbent assay for their ability to inhibit the interaction of hyaluronectin (a hyaluronic acid-binding brain glycoprotein) with hyaluronic acid. Among these oligosaccharides, decasaccharides were the smallest fragments that strongly inhibited the interaction. Octasaccharides inhibited with 700-fold lower affinity than decasaccharides. Dodecasaccharides had the same effect as decasaccharides. Nonasaccharides obtained by beta-glucuronidase splitting of decasaccharides inhibited the interaction more than nonasaccharides prepared by an alkaline treatment.     

Hyaluronic Acid Degradation by Ascorbic Acid and Influence of Iron

The effects of ascorbic acid, iron and ADP on hyaluronic acid, a compound present in inflamed joints, were investigated in an in vitro system. Ascorbic acid induces degradation of hyaluronic acid which increased in the presence of FeCl, and which is additionally stimulated by ADP chelated ferric ions. The hyaluronic acid degrading reactions induced by the Fe-III/ADP/ascorbic acid system were inhibited by catalase and formate to various extents whereas the presence of superoxide dismutase did not exert any inhibitory effect. Desferrioxamine, a specific iron chelator, completely inhibited hyaluronic acid depolymerisation by ascorbic acid as well as in combination with FeCl₃ or FeCl₃/ADP, respectively. We suggest that the ultimate hyaluronic acid degrading species is OH', generated via the Fe-III/ADP catalysed Haber Weiss reaction. There is also an indication for the involvement of perferryl or/and ferryl species in the degradation process.