日本开发透明质酸的发酵生产技术

近年来日本的资生堂日化公司在世界上年首次建立了发酵法大量生产透明质酸的技术。透明质酸是粘多糖的一种,对皮肤有保湿等功效。它广泛分布于自然界,但制制取困难。前几年才开始以鸡、牛等结缔组织为原料,用提取法生产。生物提取法     

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。     

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。     

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。     

浅议透明质酸的生产进展及应用情况

透明质酸是一种广泛分布于人与动物体内的一种大分子酸性粘多糖。从上个世纪30年代Mayer首次从动物眼玻璃体中提取得到HA之后,HA的生产和应用研究到目前为止已经有70多年的悠久历史,本文主要从其独特的制备方法及在各行各业越来越广泛的应用角度进行阐述对其研究的重要性。     

透明质酸制备的研究进展

透明质酸(Hyaluronic acid简称HA)是一种国际上公认的生物大分子保湿剂,用于眼科显微手术、关节炎治疗、高级化妆品等领域.目前,透明质酸的生产方法逐渐由动物组织提取法转向微生物发酵法.细菌发酵法生产透明质酸具有产量不受原料资源限制、成本低、产量高、有较高的相对分子量,分离纯化工艺简便,易于大规模生产等特点成为透明质酸生产的发展方向,应当进一步深入研究.综述了透明质酸的化学结构、理化性质、应用及生产方法等方面的研究现状,并预测了其以后发展趋势.     

生物医用透明质酸多糖的改性和功能化研究

透明质酸(HA)是一种天然聚阴离子粘多糖已广泛用于医药和其它领域。目前已开发出了许多具有新的生物活性和功能性的透明质酸交联产物和衍生物,特别是水凝胶医用材料。本文综述了近年来透明质酸物理和化学改性研究的进展,重点介绍了交联改性、酯化改性、接枝改性和复合改性,以及改性产物在医药、生物组织工程支架等方面的应用前景。     

海萝藻中MAAs吸湿保湿效果分析

类菌胞素氨基酸(MAAs)是一类具有强紫外吸收能力的水溶性氨基酸类物质。本文为研究海萝藻中MAAs的吸湿、保湿性能,采用体外环境保湿测试方法对海萝藻MAAs、丙三醇、透明质酸进行吸湿保湿率测定,对添加不同浓度的海萝藻MAAs浓缩液的爽肤水与乳液进行保湿率测试与吸光度测试。结果表明,海萝藻MAAs的总体吸湿与保湿能力比透明质酸强,但略低于丙三醇;保湿时间长,海萝藻MAAs经过72 h的检测仍表现出比丙三醇和透明质酸高的保湿率;皮肤测试证明添加了60%海萝藻MAAs浓缩液的爽肤水具有最优的保湿效果,经过3 h测试,皮肤含水量较空白区域可提高23.29 c.u.,添加了5%~20%的海萝藻MAAs浓缩液的乳液能显著提高乳液的吸光度值,且添加量越大,吸光值越大。     

裂褶多糖的吸湿和保湿性能初步研究

通过干燥器控制湿度的方法对经冷冻干燥的裂褶多糖（PSG）试样进行吸湿和保湿性能测试,其48h吸湿率和保湿率分别为44．75％、63．06％;168h吸湿率和保湿率分别为112．28％、4．06％。和常用的化妆品保湿剂甘油、透明质酸钠、壳聚糖、聚乙二醇（PEG）进行比较,试验条件下0-168h各试样吸湿能力大小为：PSG〉甘油〉透明质酸钠〉壳聚糖〉PEG10000;48h综合保湿能力大小为：PSG〉甘油〉透明质酸钠〉壳聚糖〉PEG10000;168h综合保湿能力大小为：甘油〉PSG〉壳聚糖〉透明质酸钠〉PEG10000。采用差示扫描量热法（DSC）对裂褶多糖的热力学参数进行了测定,其相变起始温度为53．12℃,高峰温度为97．71℃,终了温度为148．30℃,焓变△H为126．743 J/g。裂褶多糖表现出良好的吸湿和保湿性能,因而是一种很有开发潜力的天然保湿剂。     

Comparative studies of mucopolysaccharides from marine animals. I. Raja eglanteria Bosc.

A mucopolysaccharide (MPS) was extracted from the pectoral fins of the skate, Raja eglanteria Bosc. The purified MPS had an anticoagulant activity of 28 IU/mg and an optical rotation value, [α]_D²⁵, of ?9°. The MPS was analyzed for uronic acid, hexosamine, N-sulfate, O-sulfate, O-sulfate, and neutral sugar. This analysis suggests that this MPS is an over-sulfated chondroitin sulfate, the electrophoretic pattern and infra-red spectrum of which are similar to chondroitin sulfate.     

Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.     

Isolation and partial characterization of proteoglycans from sheep lung parenchyma.

Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE-cellulose in urea and eluted with increasing concentrations of NaCl. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaCl and further purified by Sepharose Cl-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminoglycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single-chain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.     

Partial characterization of dermatan sulfate by proton nuclear magnetic resonance spectroscopy

The degree of galactosamine N-acetylation, iduronic acid composition, and total uronic acid/hexosamine ratios of the three dermatan sulfates of human skin, DS18, DS28, and DS35 (M. O. Longas et al. (1987) Carbohydr. Res. 159, 127-136), were determined by Fourier transform, proton nuclear magnetic resonance (FT 1H NMR) spectroscopy. Analysis of DS of varying ages was conducted at 400 MHz and 60 degrees C. Chemical shifts for H-1, H-2, H-4, and H-5 of L-IdUA were independent of those for the respective protons of D-GalNAc and D-GlcUA. The resonance intensities of H-1 and acetamido methyl protons of D-GalNac did not display the expected 1:3 ratios. Therefore, their integration values were employed to estimate the percentage N-acetylation (N-CH3/3 H-1) which was corroborated chemically. The L-IdUA content, relative to total uronic acid, was calculated from signal intensities of H-1 of L-IdUA and D-GlcUA and ascertained by quantitative chemical methods. Total uronic acid/hexosamine ratios were determined from both 1H NMR spectroscopy and chemical analyses. The data show the following N-acetylation (N-CH3/3 H-1) of galactosamine in DS:DS18, 61-72% between 17 and 60 years, unaffected by senescence; DS28, 78-86% with no age-related trend; DS35, 101% at 19 years. Furthermore, in all ages investigated, the percentage (wt/wt) L-IdUA relative to total uronic acid was 42-44% for DS18 and 37-40% for DS28. At age 19 years, DS35 had a 29% (wt/wt) L-IdUA. The total uronic acid/hexosamine ratios for DS18 and DS28 varied from 1.40:1.0 to 1.70:1.0 irrespective of age.     

ACID MUCOPOLYSACCHARIDE (GLYCOSAMINOGLYCAN) AT THE EPITHELIAL-MESENCHYMAL INTERFACE OF MOUSE EMBRYO SALIVARY GLANDS

Acid mucopolysaccharide (glycosaminoglycan) has been demostrated at the epithelial-mesenchymal interface of mouse embryo submandibular glands by (a) specific staining for polymeric sulfate with Alcian blue 8 GX at various magnesium concentrations, (b) specific staining for polymeric uronic acid by selective oxidation of these residues to Schiff-reactive compounds, (c) electron microscope localization of ruthenium red staining, (d) radioautographic localization of glucosamine-³H and ³⁵SO₄, and (e) by susceptibility of the glucosamine radioactivity at the interface to digestion with protease-free hyaluronidase. Moreover, material labeled with glucosamine-³H and ³⁵SO₄ and with chemical characteristics identical with those of acid mucopolysaccharide were isolated from the glands. Acid mucopolysaccharide is distributed over the entire epithelial surface. The amount of acid mucopolysaccharide, as revealed by the staining procedures, is nearly equivalent at all sites. In contrast, the rate of accumulation of glucosamine-labeled mucopolysaccharide is greater at the surface of the distal ends of the growing and branching lobules. This distribution of newly synthesized acid mucopolysaccharide at the sites of incipient cleft formation suggests that surface-associated acid mucopolysaccharide is involved in the morphogenetic process. A mechanism of branching morphogenesis is proposed which accounts for the distribution of collagen fibers and total and newly synthesized acid mucopolysaccharide at the epithelial surface.     

Composition of proteoglycans from rabbit aorta in the experimentally induced atherosclerosis.

Proteoglycans (PG) from normal and atherosclerotic rabbits aortas were extracted with 4 M guanidine hydrochloride and digested with collagenase in the presence of protease inhibitors. The contents of uronic acid and hexosamine from PG fractions purified by isopycnic CsCl gradient ultracentrifugation under associative and dissociative conditions were significantly higher in the atherosclerotic aortas (up to 40%) than in the control tissue. The uronic acid/protein ratio increased from 0.7 to 1.3 in the monomers PG fraction of atherosclerotic aortas. Chromatographic separation and electrophoretic analysis of PG monomers indicated the presence of three different subfractions PGI, PGII and PGIII in both groups of animals. The uronic acid/protein ratio in PGI from experimental aorta was increased whereas this ratio in PGIII was decreased compared to contrast tissue. The observed increase of sugar component in the core proteins suggests their over glycosylation.     

Structure of heparan sulphate oligosaccharides and their degradation by exo-enzymes.

Oligosaccharides obtained from heparan sulphate by nitrous acid degradation were shown to be degraded sequentially by beta-D-glucuronidase or alpha-L-iduronidase followed by alpha D-N-acetylglucosaminidase. Structural analysis of the tetrasaccharide fraction showed the following. (1) N-Acetylglucosamine is preceded by a non-sulphated uronic acid residue that can be either D-glucuronic of L-iduronic acid, but followed by a glucuronic acid residue. (2) The N-acetylglucosamine in the major fraction is sulphated. (3) Very few if any of the uronic acid residues are sulphated (4). The results indicate that the area of the heparan sulphate chain where disaccharides containing N-acetylglucosamine and N-sulphated glucosamine residues alternate is higher in sulphate content than expected and that the sulphate groups are mainly located on the hexosamine units.     

Studies on Cell Wall of Alkalophilic Bacillus

Cell walls of alkalophilic Bacillus No. C-125 and No. A-59 which grew in different pH conditions were prepared and analyzed. In the walls from cells grown at pH 10.3 (pH 10.3-cell wall) and the walls from cells grown at pH 7.5 (pH 7.5-cell wall) of the alkalophilic bacilli, the contents of neutral sugar and phosphorus were low as compared with those of Bacillus subtilis 6160, while uronic acid and amino acids were abundant. The uronic acid content of the pH 10.3-cell walls was higher than that of the pH 7.5-cell walls in both strains. The insoluble fraction (peptidoglycan) of cell walls of Bacillus No. C-125 consisted of muramic acid, glutamic acid, alanine, diaminopimelic acid and glucosamine as in neutrophilic bacilli. In the TCA soluble fraction of pH 10.3-cell walls of Bacillus No. C-125, uronic acid was a polymer of glucuronic acid containing a small amount of hexosamine, and 2/3 of the ninhydrin positive material was glutamic acid which was derived mainly from poly γ-L-glutamic acid.     

Acceptor specificity of the Pasteurella hyaluronan and chondroitin synthases and production of chimeric glycosaminoglycans

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (approximately 10(2-4) sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.     

A defect in exodegradative pathways provides insight into endodegradation of heparan and dermatan sulfates

Within cells, dermatan sulfate (DS) and heparan sulfate (HS) are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis (MPS) type II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2-sulfatase (I2S). Consequently, partially degraded fragments of DS and HS have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a MPS II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). These oligosaccharides were shown to have non-reducing terminal iduronate-2-sulfate residues by digestion with recombinant I2S. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N-acetylhexosamine residues was identified and suggested to originate from DS. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues was also identified and on the basis of the presence of unacetylated hexosamine; these oligosaccharides are proposed to derive from HS. The presence of both odd and even-length oligosaccharides suggests both endo-beta-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative HS oligosaccharide structures identified indicate that heparanase activities are directed toward regions of both low and high sulfation, while the N-acetylhexosaminidase activity acted only in regions of low sulfation in this polysaccharide.