海洋細(xì)菌來源的新型糖胺聚糖降解酶的篩
發(fā)布時(shí)間：2018-07-16 16:20
【摘要】：糖胺聚糖(glycosaminoglycan,GAG)又稱為粘多糖,是由重復(fù)單位組成的直鏈多糖。根據(jù)其二糖單位的不同,糖胺聚糖又被分為透明質(zhì)酸(Hyaluronic Acid,HA),肝素/硫酸乙酰肝素(Heparin/HeparanSulfate,Hep/HS),硫酸軟骨素/硫酸皮膚素(Chondroitin Sulfate/Dermatan Sulfate,CS/DS),硫酸角質(zhì)素(Keratan Sulfate,KS)。糖胺聚糖常常由于各種修飾酶的作用使糖鏈變得異常復(fù)雜,主要表現(xiàn)為D-葡萄糖醛酸在C5-差向異構(gòu)酶的作用下轉(zhuǎn)變?yōu)長(zhǎng)-艾杜糖醛酸,糖鏈中不同部位羥基(-OH)和氨基(-NH2)的硫酸化,以及己糖胺二位氨基的乙�；�。糖胺聚糖結(jié)構(gòu)的復(fù)雜性賦予其功能的多樣性,不同的結(jié)構(gòu)具有不同的功能。糖胺聚糖廣泛存在于動(dòng)物細(xì)胞表面和細(xì)胞基質(zhì)中,常常通過與各種蛋白的相互作用參與了細(xì)胞的增殖分化、細(xì)胞間的識(shí)別、細(xì)胞轉(zhuǎn)移、組織形態(tài)發(fā)生、癌變等各種生理和病理過程。糖胺聚糖所具有的一系列重要的生物學(xué)功能使其成為重要的生物活性分子,在醫(yī)藥及功能食品中得到廣泛應(yīng)用,如肝素、硫酸軟骨素、透明質(zhì)酸等。但由于糖胺聚糖結(jié)構(gòu)的不均一性,特別是硫酸化糖胺聚糖結(jié)構(gòu)的高度復(fù)雜性,使不同來源和不同批次的同一種糖胺聚糖在活性上存在很大差異。近年來的大量研究表明,糖胺聚糖的生物功能是通過多糖鏈中具有特殊結(jié)構(gòu)的功能區(qū)與特定蛋白的特異相互作用來實(shí)現(xiàn)的,同一多糖鏈中存在不同結(jié)構(gòu)的功能區(qū),與不同的蛋白相互作用可以行使不同的功能。因此通過運(yùn)用底物特異性的糖胺聚糖內(nèi)切酶選擇性的部分降解糖胺聚糖多糖鏈,制備結(jié)構(gòu)均一或相對(duì)均一的特定功能區(qū)寡糖;通過運(yùn)用底物特異性糖胺聚糖硫酸酯酶可以對(duì)特性功能區(qū)寡糖進(jìn)行改造和修飾;通過綜合運(yùn)用底物特異性糖胺聚糖內(nèi)切酶和外切酶可以對(duì)特性功能區(qū)寡糖進(jìn)行測(cè)序。這些具有特異性的糖胺聚糖工具酶不僅在糖胺聚糖構(gòu)效關(guān)系研究和寡糖制備中具有重要作用,并且在神經(jīng)系統(tǒng)損傷治療中也有重要作用。但是目前可以利用的糖胺聚糖工具酶屈指可數(shù),因此尋找新型糖胺聚糖工具酶對(duì)于糖胺聚糖構(gòu)效關(guān)系研究具有重要作用。海洋作為生命的起源,存在大量的生命形式。海洋中存在大量的結(jié)構(gòu)多樣的糖胺聚糖,但是之前并沒有相關(guān)糖胺聚糖降解酶的研究和報(bào)道。本論文以硫酸軟骨素為唯一碳源,在海泥中篩選出一株高效降解糖胺聚糖的菌株并進(jìn)行了基因測(cè)序。通過生物信息學(xué)分析發(fā)現(xiàn)了一系列新型糖胺聚糖工具酶基因,并對(duì)這些基因進(jìn)行了深入研究。主要研究成果包括以下幾個(gè)方面:(a)糖胺聚糖降解菌的篩選:以鯊魚軟骨來源的硫酸軟骨素CS-C為唯一碳源,從海泥中篩選得到15株多糖降解菌,其中編號(hào)為FC509的菌株表現(xiàn)為對(duì)褐藻膠、硫酸軟骨素、硫酸皮膚素、透明質(zhì)酸、肝素等多種多糖的降解和利用活性。因此本論文重點(diǎn)對(duì)FC509菌株進(jìn)行研究,通過對(duì)其進(jìn)行全基因組測(cè)序和生物信息學(xué)分析,發(fā)現(xiàn)了一系列與糖胺聚糖降解利用相關(guān)的酶基因,并著重對(duì)其中的多個(gè)降解酶基因進(jìn)行了深入研究。(b)內(nèi)切型糖胺聚糖裂解酶HCLase的相關(guān)研究:將HCLase基因構(gòu)建到大腸桿菌表達(dá)載體中異源表達(dá),并利用鎳柱進(jìn)行純化。底物降解實(shí)驗(yàn)表明:該酶具有高效降解HA和CS的活性,因此被命名為HCLase�；久笇W(xué)性質(zhì)研究表明:HCLase與已報(bào)道的糖胺聚糖降解酶不同,其具有嗜鹽特性,這與它來源于海洋細(xì)菌有關(guān),切表現(xiàn)出良好的溫度和pH穩(wěn)定性。底物降解模式分析表明:HCLase是一個(gè)內(nèi)切型糖胺聚糖裂解酶,最小底物是4糖,最小產(chǎn)物是2糖。同時(shí)我們對(duì)HCLase不能降解的四糖進(jìn)行了序列測(cè)定,其結(jié)構(gòu)為△4,5HexUAα1-3GalNAc(6S)β1-4GlcUA(2S)β1-3GalNAc(6S),這個(gè)結(jié)果揭示 CS 糖鏈中葡萄糖醛酸二位羥基的硫酸化會(huì)抑制HCLase對(duì)糖苷鍵的有效切割。HCLase的最大的優(yōu)點(diǎn)是具有超高的酶活和良好的穩(wěn)定性,這些特性使其成為具有巨大應(yīng)用潛力的新型糖胺聚糖工具酶。(c)外切型糖胺聚糖裂解酶HCDLase的相關(guān)研究:將HCDLase基因構(gòu)建到大腸桿菌表達(dá)載體pET30a中異源表達(dá),并運(yùn)用親和層析柱對(duì)異源表達(dá)的酶進(jìn)行了純化。酶學(xué)性質(zhì)研究和底物降解模式分析表明:該酶具有典型的外切型酶活性,可以從糖鏈的還原端有效降解HA、CS和DS,但不能降解Hep和HS,因此被命名為HCDLase。進(jìn)一步的研究表明:HCDLase可以降解還原端熒光標(biāo)記的硫酸化CS寡糖并產(chǎn)生二糖,但是不能降解熒光標(biāo)記的DS和HA寡糖,這說明糖苷鍵類型和硫酸化程度影響HCDLase的降解活性。最后我們運(yùn)用這種酶成功的對(duì)系列硫酸軟骨素六糖和八糖進(jìn)行了測(cè)序。上述一系列研究結(jié)果表明:HCDLase作為一個(gè)新型的外切型糖胺聚糖裂解酶,在糖胺聚糖的構(gòu)效關(guān)系研究,特別是復(fù)雜CS功能寡糖測(cè)序中具有重要的應(yīng)用價(jià)值。(d)新型內(nèi)切型糖胺聚糖硫酸酯酶4-O-Endosulfatase的相關(guān)研究:將該硫酸酯酶基因構(gòu)建到大腸桿菌表達(dá)載體pET30a中,并進(jìn)行誘導(dǎo)表達(dá)和分離純化�；久笇W(xué)性質(zhì)研究表明:該酶在最適反應(yīng)條件(30℃,pH8.0)下具表現(xiàn)出強(qiáng)的CS和DS硫酸酯酶活性;底物降解模式分析表明:該酶與已報(bào)道的CS/DS硫酸酯酶不同,不僅可以高效去除糖鏈端基的GalNAc上4位硫酸基團(tuán),而且還可以有效去除多糖鏈內(nèi)部的4位硫酸根。多底物降解分析顯示:該酶可有效去除多種CS和DS多糖鏈中的4位硫酸根,去除率在17-65%之間,硫酸基團(tuán)的去除率與糖鏈的硫酸化模式密切相關(guān)。作為第一個(gè)被深入研究的CS/DS內(nèi)切型硫酸酯酶,4-O-Endosulfatase在CS/DS構(gòu)效關(guān)系研究和糖鏈硫酸化修飾調(diào)節(jié)中具有重要應(yīng)用價(jià)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q936
【相似文獻(xiàn)】
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1 張?zhí)烀?凌沛學(xué);;糖胺聚糖研究新進(jìn)展[J];食品與藥品;2008年09期
2 樊繪曾,T3世馥,李毅敏;刺參糖胺聚糖的亞分級(jí)和各級(jí)分的理化特征與抗凝性質(zhì)[J];生物化學(xué)雜志;1993年02期
3 馮伯森,孫穎,仝允栩;硫酸糖胺聚糖(Sulfated Glycosaminoglycans)在花背蟾蜍眼早期形態(tài)發(fā)生中的合成[J];動(dòng)物學(xué)報(bào);1989年01期
4 肖英,賀艷麗,王e，
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