海洋细菌来源的新型糖胺聚糖降解酶的筛
发布时间：2018-07-16 16:20
【摘要】：糖胺聚糖(glycosaminoglycan,GAG)又称为粘多糖,是由重复单位组成的直链多糖。根据其二糖单位的不同,糖胺聚糖又被分为透明质酸(Hyaluronic Acid,HA),肝素/硫酸乙酰肝素(Heparin/HeparanSulfate,Hep/HS),硫酸软骨素/硫酸皮肤素(Chondroitin Sulfate/Dermatan Sulfate,CS/DS),硫酸角质素(Keratan Sulfate,KS)。糖胺聚糖常常由于各种修饰酶的作用使糖链变得异常复杂,主要表现为D-葡萄糖醛酸在C5-差向异构酶的作用下转变为L-艾杜糖醛酸,糖链中不同部位羟基(-OH)和氨基(-NH2)的硫酸化,以及己糖胺二位氨基的乙酰化等。糖胺聚糖结构的复杂性赋予其功能的多样性,不同的结构具有不同的功能。糖胺聚糖广泛存在于动物细胞表面和细胞基质中,常常通过与各种蛋白的相互作用参与了细胞的增殖分化、细胞间的识别、细胞转移、组织形态发生、癌变等各种生理和病理过程。糖胺聚糖所具有的一系列重要的生物学功能使其成为重要的生物活性分子,在医药及功能食品中得到广泛应用,如肝素、硫酸软骨素、透明质酸等。但由于糖胺聚糖结构的不均一性,特别是硫酸化糖胺聚糖结构的高度复杂性,使不同来源和不同批次的同一种糖胺聚糖在活性上存在很大差异。近年来的大量研究表明,糖胺聚糖的生物功能是通过多糖链中具有特殊结构的功能区与特定蛋白的特异相互作用来实现的,同一多糖链中存在不同结构的功能区,与不同的蛋白相互作用可以行使不同的功能。因此通过运用底物特异性的糖胺聚糖内切酶选择性的部分降解糖胺聚糖多糖链,制备结构均一或相对均一的特定功能区寡糖;通过运用底物特异性糖胺聚糖硫酸酯酶可以对特性功能区寡糖进行改造和修饰;通过综合运用底物特异性糖胺聚糖内切酶和外切酶可以对特性功能区寡糖进行测序。这些具有特异性的糖胺聚糖工具酶不仅在糖胺聚糖构效关系研究和寡糖制备中具有重要作用,并且在神经系统损伤治疗中也有重要作用。但是目前可以利用的糖胺聚糖工具酶屈指可数,因此寻找新型糖胺聚糖工具酶对于糖胺聚糖构效关系研究具有重要作用。海洋作为生命的起源,存在大量的生命形式。海洋中存在大量的结构多样的糖胺聚糖,但是之前并没有相关糖胺聚糖降解酶的研究和报道。本论文以硫酸软骨素为唯一碳源,在海泥中筛选出一株高效降解糖胺聚糖的菌株并进行了基因测序。通过生物信息学分析发现了一系列新型糖胺聚糖工具酶基因,并对这些基因进行了深入研究。主要研究成果包括以下几个方面:(a)糖胺聚糖降解菌的筛选:以鲨鱼软骨来源的硫酸软骨素CS-C为唯一碳源,从海泥中筛选得到15株多糖降解菌,其中编号为FC509的菌株表现为对褐藻胶、硫酸软骨素、硫酸皮肤素、透明质酸、肝素等多种多糖的降解和利用活性。因此本论文重点对FC509菌株进行研究,通过对其进行全基因组测序和生物信息学分析,发现了一系列与糖胺聚糖降解利用相关的酶基因,并着重对其中的多个降解酶基因进行了深入研究。(b)内切型糖胺聚糖裂解酶HCLase的相关研究:将HCLase基因构建到大肠杆菌表达载体中异源表达,并利用镍柱进行纯化。底物降解实验表明:该酶具有高效降解HA和CS的活性,因此被命名为HCLase。基本酶学性质研究表明:HCLase与已报道的糖胺聚糖降解酶不同,其具有嗜盐特性,这与它来源于海洋细菌有关,切表现出良好的温度和pH稳定性。底物降解模式分析表明:HCLase是一个内切型糖胺聚糖裂解酶,最小底物是4糖,最小产物是2糖。同时我们对HCLase不能降解的四糖进行了序列测定,其结构为△4,5HexUAα1-3GalNAc(6S)β1-4GlcUA(2S)β1-3GalNAc(6S),这个结果揭示 CS 糖链中葡萄糖醛酸二位羟基的硫酸化会抑制HCLase对糖苷键的有效切割。HCLase的最大的优点是具有超高的酶活和良好的稳定性,这些特性使其成为具有巨大应用潜力的新型糖胺聚糖工具酶。(c)外切型糖胺聚糖裂解酶HCDLase的相关研究:将HCDLase基因构建到大肠杆菌表达载体pET30a中异源表达,并运用亲和层析柱对异源表达的酶进行了纯化。酶学性质研究和底物降解模式分析表明:该酶具有典型的外切型酶活性,可以从糖链的还原端有效降解HA、CS和DS,但不能降解Hep和HS,因此被命名为HCDLase。进一步的研究表明:HCDLase可以降解还原端荧光标记的硫酸化CS寡糖并产生二糖,但是不能降解荧光标记的DS和HA寡糖,这说明糖苷键类型和硫酸化程度影响HCDLase的降解活性。最后我们运用这种酶成功的对系列硫酸软骨素六糖和八糖进行了测序。上述一系列研究结果表明:HCDLase作为一个新型的外切型糖胺聚糖裂解酶,在糖胺聚糖的构效关系研究,特别是复杂CS功能寡糖测序中具有重要的应用价值。(d)新型内切型糖胺聚糖硫酸酯酶4-O-Endosulfatase的相关研究:将该硫酸酯酶基因构建到大肠杆菌表达载体pET30a中,并进行诱导表达和分离纯化。基本酶学性质研究表明:该酶在最适反应条件(30℃,pH8.0)下具表现出强的CS和DS硫酸酯酶活性;底物降解模式分析表明:该酶与已报道的CS/DS硫酸酯酶不同,不仅可以高效去除糖链端基的GalNAc上4位硫酸基团,而且还可以有效去除多糖链内部的4位硫酸根。多底物降解分析显示:该酶可有效去除多种CS和DS多糖链中的4位硫酸根,去除率在17-65%之间,硫酸基团的去除率与糖链的硫酸化模式密切相关。作为第一个被深入研究的CS/DS内切型硫酸酯酶,4-O-Endosulfatase在CS/DS构效关系研究和糖链硫酸化修饰调节中具有重要应用价值。
[Abstract]:Glycosaminoglycan (GAG), also known as mucopolysaccharide, is a straight chain polysaccharide made up of repeated units. According to its disaccharide units, glycosaminoglycan is divided into Hyaluronic Acid (HA), heparin / heparan sulfate (Heparin/HeparanSulfate, Hep/HS), and chondroitin sulfate / derma sulfate (Chondroitin Sulfate/Dermatan). Sulfate, CS/DS), Keratan Sulfate (KS). Glycosaminoglycan often makes sugar chains become very complex because of the action of various modified enzymes. The main manifestation is that D- glucuronic acid changes to L- aluronic acid under the action of C5- differential isomerase, the sulfation of different hydroxyl groups (-OH) and amino (-NH2) in the sugar chain, and hexamines two Acetylation of aminoglycans. The complexity of the structure of glycosaminoglycan gives its functional diversity and different structures have different functions. Glycosaminoglycan exists widely in the surface of animal cells and cell matrix. It often participates in the proliferation and differentiation of cells, identification of cells, cell metastasis and tissue shape by interaction with various proteins. A series of important biological and pathological processes. A series of important biological functions of glycosaminoglycan have made it an important bioactive molecule. It has been widely used in medical and functional foods, such as heparin, chondroitin sulfate, hyaluronic acid, etc. but because of the heterogeneity of the structure of glycosaminoglycan, especially the sulfated glycosaminate The high complexity of the structure of glycosaminoglycan has made a great difference in the activity of the same Glycosaminoglycan from different sources and batches. In recent years, a large number of studies have shown that the biological function of glycosaminoglycan is achieved through the specific interaction of special structures with specific proteins in the polysaccharides chain, and the existence of the same polysaccharide chain. The functional regions of different structures can perform different functions with different proteins. Therefore, by selective partial degradation of glycosaminoglycan polysaccharides chains by using substrate specific glycosaminoglycan endonucleases, a specific functional region of oligosaccharides has been prepared with homogeneous or relative homogeneity, through the use of substrate specific glycosaminoglycan sulfated enzymes. The characteristic functional oligosaccharides can be sequenced by using substrate specific glycosaminoglycan endonucleases and exonucleases. These specific glycosaminoglycan enzymes not only play an important role in the study of glycosaminoglycan structure-activity relationships and oligosaccharide preparation, but also in the nervous system. There are also an important role in the treatment of damage, but the available glycosaminoglycan tool enzymes are very few. Therefore, it is important to find a new glycosaminoglycan tool enzyme for the study of the structure-activity relationship of glycosaminoglycan. As the origin of life, the ocean has a large number of life forms. There are a large number of structural and diverse glycosaminoglycans in the ocean. But there was no research and report on glycosaminoglycan degrading enzymes. In this paper, a strain of highly efficient degradation of glycosaminoglycan was screened in sea mud with chondroitin sulfate as the sole carbon source and the gene was sequenced. A series of new glycosaminoglycan enzyme genes were found through bioinformatics analysis, and these genes were carried out. The main research results included the following aspects: (a) screening of glycosaminoglycan degradation bacteria: 15 strains of polysaccharide degrading bacteria were screened from sea mud with the only carbon source of chondroitin sulfate CS-C from shark cartilage, and the strains with FC509 were displayed as brown alginate, chondroitin sulfate, derma sulfate, hyaluronic acid, heparin Such as the degradation and utilization of various polysaccharides, this paper focuses on the study of FC509 strains. Through the whole genome sequencing and bioinformatics analysis, a series of enzymes related to the degradation and utilization of glycosaminoglycan have been discovered, and the multiple degradation enzyme genes in them have been deeply studied. (b) the endonuclease type glycosaminoglycan polymerization. The related study of glycosylyase HCLase: the HCLase gene was constructed to the heterologous expression vector in the Escherichia coli expression vector and purified by the nickel column. The substrate degradation experiment showed that the enzyme had the activity of highly efficient degradation of HA and CS. Therefore, the study of the basic enzymatic properties of HCLase. showed that HCLase was different from the reported glycosaminoglycan degrading enzyme, It has the characteristics of halophilic, which is related to the marine bacteria, and shows good temperature and pH stability. The analysis of substrate degradation pattern shows that HCLase is an endonuclease type glycosaminoglycan lyase, the minimum substrate is 4 sugar and the minimum product is 2 sugar. At the same time, we have a sequence determination of the four sugar which is not degraded by HCLase, and its structure is delta 4, 5HexUA a (6S) 1-3GalNAc (6S) beta 1-4GlcUA (2S) beta 1-3GalNAc (6S), the result reveals that the sulfation of two hydroxyl groups of glucuronic acid in the carbohydrate chain of CS can inhibit the effective cutting.HCLase of the glycoside bonds by HCLase, which has high enzyme activity and good stability. These properties make it a new type of glycosamine with great potential for application. The related study on the glycosaminoglycan lyase HCDLase of (c) external tangent glycosaminoglycan lyase: the HCDLase gene was constructed to the heterologous expression in the Escherichia coli expression vector pET30a, and the enzyme was purified by the affinity chromatography column. The enzymatic properties and substrate degradation patterns showed that the enzyme had the typical activity of the exotangent enzyme. In order to degrade HA, CS and DS effectively from the reductive end of the sugar chain, it can not degrade Hep and HS, so a further study named HCDLase. shows that HCDLase can degrade the sulfated CS oligosaccharide and produce two sugars from the reduced terminal fluorescent labeling, but can not degrade the fluorescent labeled DS and HA oligosaccharides, which indicates that the glucoside bond type and the degree of sulfation affect HCDLas. E degradation activity. Finally, we successfully sequenced the series of chondroitin sulfate six sugar and eight sugars using this enzyme. A series of research results showed that as a new type of exosaccharide glycosaminoglycan lyase, the structure activity relationship of glycosaminoglycan, especially the complex CS functional oligosaccharide sequencing, has important application. (d) a study of (d) a novel internal tangential glycosaminoglycan sulfonase 4-O-Endosulfatase: the sulfate esterase gene was constructed into the Escherichia coli expression vector pET30a and was induced and purified. The basic enzymology study showed that the enzyme exhibited a strong CS and DS sulphate enzyme at the optimum reaction condition (30, pH8.0). The degradation mode analysis showed that the enzyme was different from the reported CS/DS sulfate enzyme. It could not only effectively remove the 4 sulphuric acid groups on the GalNAc of the end group of the sugar chain, but also effectively remove the 4 sulfate radicals inside the polysaccharide chain. The multi substrate degradation analysis showed that the enzyme could effectively remove 4 sulfate radicals in the multiple CS and DS polysaccharide chains. The removal rate is between 17-65%, the removal rate of sulphuric acid group is closely related to the sulfation mode of sugar chain. As the first deeply studied CS/DS endonuclease, 4-O-Endosulfatase has important application value in the study of CS/DS structure-activity relationship and in the regulation of sugar chain sulfation modification.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q936
[Abstract]:Glycosaminoglycan (GAG), also known as mucopolysaccharide, is a straight chain polysaccharide made up of repeated units. According to its disaccharide units, glycosaminoglycan is divided into Hyaluronic Acid (HA), heparin / heparan sulfate (Heparin/HeparanSulfate, Hep/HS), and chondroitin sulfate / derma sulfate (Chondroitin Sulfate/Dermatan). Sulfate, CS/DS), Keratan Sulfate (KS). Glycosaminoglycan often makes sugar chains become very complex because of the action of various modified enzymes. The main manifestation is that D- glucuronic acid changes to L- aluronic acid under the action of C5- differential isomerase, the sulfation of different hydroxyl groups (-OH) and amino (-NH2) in the sugar chain, and hexamines two Acetylation of aminoglycans. The complexity of the structure of glycosaminoglycan gives its functional diversity and different structures have different functions. Glycosaminoglycan exists widely in the surface of animal cells and cell matrix. It often participates in the proliferation and differentiation of cells, identification of cells, cell metastasis and tissue shape by interaction with various proteins. A series of important biological and pathological processes. A series of important biological functions of glycosaminoglycan have made it an important bioactive molecule. It has been widely used in medical and functional foods, such as heparin, chondroitin sulfate, hyaluronic acid, etc. but because of the heterogeneity of the structure of glycosaminoglycan, especially the sulfated glycosaminate The high complexity of the structure of glycosaminoglycan has made a great difference in the activity of the same Glycosaminoglycan from different sources and batches. In recent years, a large number of studies have shown that the biological function of glycosaminoglycan is achieved through the specific interaction of special structures with specific proteins in the polysaccharides chain, and the existence of the same polysaccharide chain. The functional regions of different structures can perform different functions with different proteins. Therefore, by selective partial degradation of glycosaminoglycan polysaccharides chains by using substrate specific glycosaminoglycan endonucleases, a specific functional region of oligosaccharides has been prepared with homogeneous or relative homogeneity, through the use of substrate specific glycosaminoglycan sulfated enzymes. The characteristic functional oligosaccharides can be sequenced by using substrate specific glycosaminoglycan endonucleases and exonucleases. These specific glycosaminoglycan enzymes not only play an important role in the study of glycosaminoglycan structure-activity relationships and oligosaccharide preparation, but also in the nervous system. There are also an important role in the treatment of damage, but the available glycosaminoglycan tool enzymes are very few. Therefore, it is important to find a new glycosaminoglycan tool enzyme for the study of the structure-activity relationship of glycosaminoglycan. As the origin of life, the ocean has a large number of life forms. There are a large number of structural and diverse glycosaminoglycans in the ocean. But there was no research and report on glycosaminoglycan degrading enzymes. In this paper, a strain of highly efficient degradation of glycosaminoglycan was screened in sea mud with chondroitin sulfate as the sole carbon source and the gene was sequenced. A series of new glycosaminoglycan enzyme genes were found through bioinformatics analysis, and these genes were carried out. The main research results included the following aspects: (a) screening of glycosaminoglycan degradation bacteria: 15 strains of polysaccharide degrading bacteria were screened from sea mud with the only carbon source of chondroitin sulfate CS-C from shark cartilage, and the strains with FC509 were displayed as brown alginate, chondroitin sulfate, derma sulfate, hyaluronic acid, heparin Such as the degradation and utilization of various polysaccharides, this paper focuses on the study of FC509 strains. Through the whole genome sequencing and bioinformatics analysis, a series of enzymes related to the degradation and utilization of glycosaminoglycan have been discovered, and the multiple degradation enzyme genes in them have been deeply studied. (b) the endonuclease type glycosaminoglycan polymerization. The related study of glycosylyase HCLase: the HCLase gene was constructed to the heterologous expression vector in the Escherichia coli expression vector and purified by the nickel column. The substrate degradation experiment showed that the enzyme had the activity of highly efficient degradation of HA and CS. Therefore, the study of the basic enzymatic properties of HCLase. showed that HCLase was different from the reported glycosaminoglycan degrading enzyme, It has the characteristics of halophilic, which is related to the marine bacteria, and shows good temperature and pH stability. The analysis of substrate degradation pattern shows that HCLase is an endonuclease type glycosaminoglycan lyase, the minimum substrate is 4 sugar and the minimum product is 2 sugar. At the same time, we have a sequence determination of the four sugar which is not degraded by HCLase, and its structure is delta 4, 5HexUA a (6S) 1-3GalNAc (6S) beta 1-4GlcUA (2S) beta 1-3GalNAc (6S), the result reveals that the sulfation of two hydroxyl groups of glucuronic acid in the carbohydrate chain of CS can inhibit the effective cutting.HCLase of the glycoside bonds by HCLase, which has high enzyme activity and good stability. These properties make it a new type of glycosamine with great potential for application. The related study on the glycosaminoglycan lyase HCDLase of (c) external tangent glycosaminoglycan lyase: the HCDLase gene was constructed to the heterologous expression in the Escherichia coli expression vector pET30a, and the enzyme was purified by the affinity chromatography column. The enzymatic properties and substrate degradation patterns showed that the enzyme had the typical activity of the exotangent enzyme. In order to degrade HA, CS and DS effectively from the reductive end of the sugar chain, it can not degrade Hep and HS, so a further study named HCDLase. shows that HCDLase can degrade the sulfated CS oligosaccharide and produce two sugars from the reduced terminal fluorescent labeling, but can not degrade the fluorescent labeled DS and HA oligosaccharides, which indicates that the glucoside bond type and the degree of sulfation affect HCDLas. E degradation activity. Finally, we successfully sequenced the series of chondroitin sulfate six sugar and eight sugars using this enzyme. A series of research results showed that as a new type of exosaccharide glycosaminoglycan lyase, the structure activity relationship of glycosaminoglycan, especially the complex CS functional oligosaccharide sequencing, has important application. (d) a study of (d) a novel internal tangential glycosaminoglycan sulfonase 4-O-Endosulfatase: the sulfate esterase gene was constructed into the Escherichia coli expression vector pET30a and was induced and purified. The basic enzymology study showed that the enzyme exhibited a strong CS and DS sulphate enzyme at the optimum reaction condition (30, pH8.0). The degradation mode analysis showed that the enzyme was different from the reported CS/DS sulfate enzyme. It could not only effectively remove the 4 sulphuric acid groups on the GalNAc of the end group of the sugar chain, but also effectively remove the 4 sulfate radicals inside the polysaccharide chain. The multi substrate degradation analysis showed that the enzyme could effectively remove 4 sulfate radicals in the multiple CS and DS polysaccharide chains. The removal rate is between 17-65%, the removal rate of sulphuric acid group is closely related to the sulfation mode of sugar chain. As the first deeply studied CS/DS endonuclease, 4-O-Endosulfatase has important application value in the study of CS/DS structure-activity relationship and in the regulation of sugar chain sulfation modification.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q936
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