CN114507656B - A method for preparing fucotetraose rich in guluronic acid - Google Patents

Abstract

The invention provides a method for preparing fucoidan rich in guluronic acid, which comprises the steps of isomerizing mannuronic acid M in algin into guluronic acid G by using algin isomerase; and then the amino acid sequence is SEQ ID NO:1 to prepare the fucoidan by catalysis. The invention establishes a method for preparing the fucoidan rich in guluronic acid based on the screening to obtain a novel algin lyase, and the prepared fucoidan has definite structure and stable composition; has application prospect in the fields of medicine and the like.

Description

Translated fromChinese

一种制备富含古洛糖醛酸的褐藻四糖的方法A method for preparing fucotetraose rich in guluronic acid

技术领域technical field

本发明涉及从海洋生物中制备能够用于生物医药的褐藻四糖，即一种制备富含古洛糖醛酸的褐藻四糖的方法；属于海洋生物资源海藻多糖制备技术领域。The invention relates to preparing fucotetraose from marine organisms that can be used in biomedicine, that is, a method for preparing fucotetraose rich in guluronic acid; it belongs to the technical field of preparation of seaweed polysaccharides from marine biological resources.

背景技术Background technique

褐藻胶是从海藻中提取的一类由甘露糖醛酸(M)和古洛糖醛酸(G)两种互为差向异构体的糖单元通过β-1,4糖苷键连接而成的多糖。其降解产物褐藻寡糖具有抗氧化、抑菌、抗肿瘤、免疫调节等多种不同的生物学活性在创新海洋药物及特医性食品等领域具有广阔的应用前景。Alginate is a kind of sugar unit extracted from seaweed, which is composed of mannuronic acid (M) and guluronic acid (G), which are epimers of each other, connected by β-1,4 glycosidic bonds. of polysaccharides. Its degradation products, fucoidan oligosaccharides, have various biological activities such as anti-oxidation, antibacterial, anti-tumor, and immune regulation, and have broad application prospects in the fields of innovative marine drugs and special medical foods.

大量研究发现褐藻寡糖的活性与其结构组成直接相关，例如聚合度为5的褐藻寡糖在增强巨噬细胞抗菌活性和促进骨肿瘤病人恢复方面要优于其他聚合度的褐藻寡糖。但如何高效地制备出结构明确、聚合度单一的褐藻寡糖仍是一个难题，导致目前市场上供应的褐藻寡糖种类少且价格高，不利于褐藻寡糖产业链的快速发展。比如按照实验推算褐藻寡糖的推荐日剂量为10mg/天/人，然而D-甘露糖醛酸五糖每10mg的价格是650元，如此高的价格大大限制了均一性褐藻寡糖在医药等领域的广泛应用。A large number of studies have found that the activity of fucoidan oligosaccharides is directly related to its structural composition. For example, fucoidan oligosaccharides with a polymerization degree of 5 are better than fucoidan oligosaccharides with other polymerization degrees in enhancing the antibacterial activity of macrophages and promoting the recovery of bone tumor patients. However, how to efficiently prepare fucoidan oligosaccharides with a clear structure and a single degree of polymerization is still a difficult problem. As a result, there are few types of fucoidan oligosaccharides available in the market and their prices are high, which is not conducive to the rapid development of the fucoidan oligosaccharide industry chain. For example, according to the experimental calculation, the recommended daily dose of fucoidan oligosaccharides is 10 mg/day/person, but the price of D-mannuronic acid pentasaccharide is 650 yuan per 10 mg. Such a high price greatly limits the use of uniform fucoidan oligosaccharides in medicine, etc. wide range of applications in the field.

目前，均一性寡糖制备成本高的主要原因是过程繁琐、重复性差、产物难以纯化。而酶解法制备褐藻寡糖不仅催化效率高、反应条件温和环境友好，并且具有特异性高的优点，是未来均一性褐藻寡糖产业化发展的重要方法。因此迫切需要深入研究可用于均一性寡糖制备的褐藻胶修饰酶。褐藻寡糖的均一性涉及到化学组成和聚合度两个方面。At present, the main reasons for the high cost of preparation of homogeneous oligosaccharides are the cumbersome process, poor repeatability, and difficult purification of the product. The preparation of fucoidan oligosaccharides by enzymatic hydrolysis not only has high catalytic efficiency, mild reaction conditions and environmental friendliness, but also has the advantages of high specificity. It is an important method for the industrial development of uniform fucoidan oligosaccharides in the future. Therefore, it is urgent to study the alginate modification enzymes that can be used in the preparation of uniform oligosaccharides. The uniformity of fucoidan oligosaccharides involves two aspects of chemical composition and degree of polymerization.

发明内容Contents of the invention

本发明的目的是提供一种制备富含古洛糖醛酸的褐藻四糖的方法，能够制备高纯度的褐藻四糖。The purpose of the present invention is to provide a method for preparing fucotetraose rich in guluronic acid, which can prepare high-purity fucotetraose.

本发明提供一种褐藻胶裂解酶，所提供的褐藻胶裂解酶包含：The present invention provides a kind of alginate lyase, the provided alginate lyase comprises:

1)氨基酸序列为SEQ ID NO:1的蛋白酶；1) the amino acid sequence is the protease of SEQ ID NO:1;

MKFKSLFCFILVCSFLLLGLAGCGSSSPTNEATSVDDETQMAQGNEPVTSDLAEDEPYQWINFDFSSPITLSAVHISFYDGDEDAIYFRFESSNDNKNWSTHLNTVNSTSKLEFETFNLDENVTARYFRLASLGTSNNNQTSIAEVTFSATNEQDTFAIPGLVEAEDYSAFYDSTAGNQGGEYRDDDVDIEQTSDITGNYNIDFITDGEWLAYPINVGSGGEYNAKLRVSSANGGGSIIIYVDDLEKGRLAVPATTQWETKNIQLGVLSSGEHTLKVLFTAGEFELNWIELSRATLKASTLDPNLPPSGNFDLSQWYLGAPIDDNADGKSDSISESQLAAGYEHPQWFYTADDGAMVFKVEIDAPKTSTNTSYSRSELREMLRAGDTSISTQGINKNNWVFSTYSSDDKNAAGGIDGELTATLKVDYVTTTGESSQVGRVIIGQIHAKDDEPARLYYRKLKDNSKGSIYLAHEPNGGNDQLYNMIGTSSSTAVDPVDGIELGEIFTYSIKVTGNTLLVTIMRDGKPDVTQSVDMSNSGYHTGYDQYMYFKAGVYNQNNTGDPSDYVQASFYRLFTSHN；MKFKSLFCFILVCSFLLLGLAGCGSSSPTNEATSVDDETQMAQGNEPVTSDLAEDEPYQWINFDFSSPITLSAVHISFYDGDEDAIYFRFESSDNNKNWTHLNTVNSTSKLEFETFNLDENVTARYFRLASLGTSNNNQTSIAEVTFSATNEQDTFAIPGLVEAEDYSAFYDSTAGNQGGEYRDDDDVDIEQTSDITG NYNIDFITDGEWLAYPINVGSGGEYNAKLRVSSANGGGSIIIYVDDLEKGRLAVPATTQWETKNIQLGVLSSGEHTLKVLFTAGEFELNWIELSRATLKASTLDPNLPPSGNFDLSQWYLGAPIDDNADGKSDSISESQLAAGYEHPQWFYTADDGAMVFKVEIDAPKTSTNTSYSRSELREMLRAGDTSIST QGINKNNWVFSTYSSDDKNAAGGIDGELTATLKVDYVTTTGESSQVGRVIIGQIHAKDDEPARLYYRKLKDNSKGSIYLAHEPNGGNDQLYNMIGTSSSTAVDPVDGIELGEIFTYSIKVTGNTLLVTIMRDGKPDVTQSVDMSNSGYHTGYDQYMYFKAGVYNQNNTGDPSDYV QASFYRLFTSHN;

2)在1)中取代、缺失、添加一个或数个氨基酸，由1)衍生的，具有1)中酶解效果的蛋白酶；2) A protease derived from 1) by substituting, deleting, or adding one or several amino acids in 1), and having the enzymolysis effect in 1);

编码上述褐藻胶裂解酶的基因，其一种具体的序列为SEQ ID NO:2；A specific sequence of the gene encoding the above-mentioned alginate lyase is SEQ ID NO: 2;

ATGAAGTTTAAATCATTGTTTTGTTTTATCTTAGTTTGTAGTTTTTTGCTGTTAGGGCTAGCAGGCTGTGGCTCATCATCACCAACTAATGAGGCTACTTCAGTAGATGATGAAACGCAGATGGCACAAGGTAACGAACCTGTAACAAGTGACTTAGCAGAGGATGAACCTTATCAATGGATTAACTTTGATTTTTCATCGCCTATTACACTAAGTGCTGTGCATATTTCATTTTATGACGGTGACGAGGATGCAATTTATTTTAGATTCGAGTCATCTAATGACAATAAAAATTGGTCTACCCATCTGAATACAGTTAATTCAACGAGTAAGCTCGAATTTGAAACATTCAACTTAGATGAAAATGTAACTGCTCGTTATTTTAGATTAGCCAGCTTGGGTACTTCGAATAACAATCAAACAAGCATAGCGGAAGTTACATTTAGCGCAACCAATGAGCAAGACACTTTTGCTATACCTGGGTTAGTTGAGGCTGAAGATTATAGCGCTTTTTATGACTCAACTGCTGGGAATCAAGGTGGGGAATACCGCGATGATGATGTTGATATAGAGCAGACGTCTGATATCACAGGAAACTATAACATAGATTTTATTACCGATGGAGAGTGGTTAGCGTATCCTATTAACGTGGGCAGTGGTGGTGAGTATAATGCGAAGCTGCGTGTTTCTTCTGCTAATGGCGGTGGTTCAATTATTATTTATGTTGATGACCTAGAAAAAGGTCGCTTAGCAGTACCAGCAACAACACAATGGGAAACTAAAAATATTCAATTAGGTGTATTGTCATCCGGTGAGCATACGCTCAAGGTTTTATTTACAGCTGGTGAATTTGAACTTAATTGGATTGAGTTATCTAGGGCTACTTTAAAAGCATCGACGTTAGATCCGAATTTGCCACCTTCGGGTAATTTTGATTTGTCTCAGTGGTATTTAGGCGCACCCATTGATGACAATGCTGATGGTAAGTCTGATTCTATCTCAGAATCTCAGCTAGCAGCTGGTTATGAGCATCCTCAATGGTTTTATACTGCAGATGATGGGGCAATGGTTTTTAAGGTTGAGATTGATGCTCCAAAGACATCAACTAACACAAGCTATAGCCGTAGTGAACTACGCGAAATGCTTAGAGCGGGTGATACGAGTATCAGCACGCAGGGGATAAACAAAAATAACTGGGTATTTTCTACCTATTCGTCGGATGATAAAAATGCAGCTGGTGGCATTGACGGTGAATTAACAGCGACACTTAAAGTTGATTACGTAACTACAACTGGTGAAAGCTCTCAGGTTGGACGCGTTATTATTGGGCAAATTCATGCAAAAGACGATGAACCAGCGCGTCTGTATTACCGTAAATTAAAAGACAACAGCAAAGGCTCAATTTACCTAGCTCATGAACCTAATGGTGGAAATGATCAGCTTTATAACATGATCGGCACAAGTAGTAGCACAGCAGTTGATCCTGTTGATGGTATTGAACTTGGTGAAATCTTTACTTATTCCATAAAAGTAACTGGAAATACCTTGTTGGTTACTATTATGCGTGATGGTAAGCCTGATGTAACTCAATCAGTTGATATGAGCAATAGCGGGTATCATACCGGCTATGATCAATATATGTATTTTAAAGCAGGTGTATATAATCAAAATAATACTGGTGATCCAAGTGATTATGTACAAGCCTCATTCTATCGCTTATTTACTTCACATAATTAA。ATGAAGTTTAAATCATTGTTTTGTTTTATCTTAGTTTGTAGTTTTTTGCTGTTAGGGCTAGCAGGCTGTGGCTCATCATCACCAACTAATGAGGCTACTTCAGTAGATGATGAAACGCAGATGGCACAAGGTAACGAACCTGTAACAAGTGACTTAGCAGAGGATGAACCTTATCAATGGATTAACTTTGATTTTTCATCGCCTATTACACTAAGTGCTGT GCATATTTCATTTTTATGACGGTGACGAGGATGCAATTTATTTTAGATTCGAGTCATCTAATGACAATAAAAAATTGGTCTACCCATCTGAATACAGTTAATTCAACGAGTAAGCTCGAATTTGAAACATTCAACTTAGATGAAAATGTAACTGCTCGTTATTTTAGATTAGCCAGCTTGGGTACTTCGAATAACAATCAAACAAGCATAGCGGAAGTTACATTTAGCGCAACCA ATGAGCAAGACACTTTTTGCTATACCTGGGTTAGTTGAGGCTGAAGATTATAGCGCTTTTTATGACTCAACTGCTGGGAATCAAGGTGGGGAATACCGCGATGATGATGTTGATATAGAGCAGACGTCTGATATCACAGGAAACTATAACATAGATTTTATTACCGATGGAGAGTGGTTAGCGTATCCTATTAACGTGGGCAGTGGTGGTGAGTATA ATGCGAAGCTGCGTGTTTCTTCTGCTAATGGCGGTGGTTCAATTATTATTTATGTTGATGACCTAGAAAAAGGTCGCTTAGCAGTACCAGCAACAACACAATGGGAAACTAAAAATATTCAATTAGGTGTATTGTCATCCGGTGAGCATACGCTCCAAGGTTTTATTTACAGCTGGTGAATTTGAACTTAATTGGATTGAGTTATTCTAGGGCTACTTTAAAGCA TCGACGTTAGATCCGAATTTGCCACCTTCGGGTAATTTTGATTTGTCTCAGTGGTATTTAGGCGCACCCATTGATGACAATGCTGATGGTAAGTCTGATTCTATCTCAGAATCTCAGCTAGCAGCTGGTTATGAGCATTCCTCAATGGTTTTATACTGCAGATGATGGGGCAATGGTTTTTAAGGTTGAGATTGATGCTCCAAAGACATCAACTAACACAAG CTATAGCCGTAGTGAACTACGCGAAATGCTTAGAGCGGGTGATACGAGTATCAGCACGCAGGGGATAAACAAAAATAACTGGGTATTTTTCTACCTATTCGTCGGATGATAAAAATGCAGCTGGTGGCATTGACGGTGAATTAACAGCGACACTTAAAGTTGATTACGTAACTACAACTGGTGAAAGCTCTCAGGTTGGACGCGTTATTATTGGGC AAATTCATGCAAAAGACGATGAACCAGCGCGTCTGTATTACCGTAAATTAAAAGACAACAGCAAAGGCTCAATTTACCTAGCTCATGAACCTAATGGTGGAAATGATCAGCTTTATAACATGATCGGCACAAGTAGTAGCAGCAGCAGTTGATCCTGTTGATGGTATTGAACTTGGTGAAATCTTACTTATTCCATAAAAGTAACTGGAAATACCTTGTTGGTTACTAT TATGCGTGATGGTAAGCCTGATGTAACTCAATCAGTTGATATGAGCAATAGCGGGTATCATACCGGCTATGATCAATATATGTATTTAAAGCAGGTGTATATAATCAAAATAACTGGTGATCCAAGTGATTATGTACAAGCCTCATTCTATCGCTTATTTACTTCATAATTAA.

本发明还提供一种重组表达载体，所述的重组表达载体中插入有用于编码上述褐藻胶裂解酶基因的核酸片段；The present invention also provides a recombinant expression vector, wherein a nucleic acid fragment for encoding the above-mentioned alginate lyase gene is inserted into the recombinant expression vector;

本发明还提供重组工程菌株，所述的重组工程菌株中包含有上述的重组表达载体。The present invention also provides a recombinant engineering strain, which contains the above-mentioned recombinant expression vector.

本发明还提供上述的褐藻胶裂解酶在制备褐藻四糖中的应用。The present invention also provides the application of the above-mentioned alginate lyase in the preparation of fucotetraose.

本发明还提供一种制备褐藻四糖的方法，是先使用褐藻胶异构酶来使褐藻胶中甘露糖醛酸(M)异构化为古洛糖醛酸(G)；再用上述的褐藻胶裂解酶来催化制备褐藻四糖；The present invention also provides a method for preparing fucotetraose, which is to use alginate isomerase to isomerize mannuronic acid (M) in alginate to guluronic acid (G); Alginate lyase to catalyze the preparation of fucotetraose;

所述的异构酶，作为本发明实施例的具体记载，其为褐藻胶异构酶AlgE4和/或AlgE6；The isomerase, as specifically described in the embodiments of the present invention, is alginate isomerase AlgE4 and/or AlgE6;

其中AlgE4、AlgE6的氨基酸序列分别为SEQ ID NO:3和SEQ ID NO:4。Wherein the amino acid sequences of AlgE4 and AlgE6 are SEQ ID NO:3 and SEQ ID NO:4 respectively.

MDYNVKDFGALGDGVSDDRASIQAAIDAAYAAGGGTVYLPAGEYRVSAAGEPGDGCLMLKDGVYLAGAGMGETVIKLIDGSDQKITGMVRSAYGEETSNFGMRDLTLDGNRDNTSGKVDGWFNGYIPGGDGADRDVTIERVEVREMSGYGFDPHEQTINLTIRDSVAHDNGLDGFVADYLVDSVFENNVAYANDRHGFNVVTSTHDFVMTNNVAYGNGSSGLVVQRGLEDLALPSNILIDGGAYYDNAREGVLLKMTSDITLQNADIHGNGSSGVRVYGAQDVQILDNQIHDNAQAAAVPEVLLQSFDDTAGASGTYYTTLNTRIEGNTISGSANSTYGIQERNDGTDYSSLIDNDIAGVQQPIQLYGPHSTVSGEPGATPQQPSTGSDGEPLVGGDTDDQLQGGSGADRLDGGAGDDILDGGAGRDRLSGGAGADTFVFSAREDSYRTDTAVFNDLILDFEASEDRIDLSALGFSGLGDGYGGTLLLKTNAEGTRTYLKSFEADAEGRRFEVALDGDHTGDLSAANVVFAATGTTTELEVLGDSGTQAGAIV(SEQ ID NO:3)；MDYNVKDFGALGDGVSDDRASIQAAIDAAYAAGGGTVYLPAGEYRVSAAGEPGDGCLMLKDGVYLAGAGMGETVIKLIDGSDQKITGMVRSAYGEETSNFGMRDLTLDGNRDNTSGKVDGWFNGYIPGGDGADRDVTIERVEVREMSGYGFDPHEQTINLTIRDSVAHDNGLDGFVADYLV DSVFENNVAYANDRHGFNVVTSTHDFVMTNNVAYGNGSSGLVVQRGLEDLALPSNILIDGGAYYDNAREGVLLKMTSDITLQNADIHGNGSSGVRVYGAQDVQILDNQIHDNAQAAAVPEVLLQSFDDTAGASGTYYTTLNTRIEGNTISGSANSTYGIQERNDGTDYSSLINDIAGVQQPIQLYGPHST VSGEPGATPQQPSTGSDGEPLVGGDTDDQLQGGSGADRLDGGAGDDILDGGAGRDRLSGGAGADTFVFSAREDSYRTDTAVFNDLILDFEASEDRIDLSALGFSGLGDGYGGTLLLKTNAEGTRTYLKSFEADAEGRRFEVALDGDHTGDLSAANVVFAATGTTTELEVLGDSGTQAGAIV (SEQ ID NO: 3 );

MDYNVKDFGALGDGVSDDRVAIQAAIDAAHAAGGGTVYLPPGEYRVSAAGEPSDGCLTLRDNVYLAGAGMGQTVIKLVDGSAQKITGIVRSPFGEETSNFGMRDLTLDGNRANTVDKVDGWFNGYAPGQPGADRNVTIERVEVREMSGYGFDPHEQTINLVLRDSVAHHNGLDGFVADYQIGGTFENNVAYANDRHGFNIVTSTNDFVMRNNVAYGNGGNGLVVQRGSENLAHPENILIDGGSYYDNGLEGVLVKMSNNVTVQNADIHGNGSSGVRVYGAQGVQILGNQIHDNAKTAVAPEVLLQSYDDTLGVSGNYYTTLNTRVEGNTITGSANSTYGVQERNDGTDFSSLVGNTINGVQEAAHLYGPNSTVSGTVSAPPQGTDGNDVLIGSDVGEQISGGAGDDRLDGGAGDDLLDGGAGRDRLTGGLGADTFRFALREDSHRSPLGTFSDLILDFDPSQDKIDVSALGFIGLGNGYAGTLAVSLSADGLRTYLKSYDADAQGRSFELALDGNHAATLSAGNIVFAAATPVDPSAEAQPIVGSDLDDQLHGTLLGEEISGGGGADQLYGYGGGDLLDGGAGRDRLTGGEGADTFRFALREDSHRSAAGTFSDLILDFDPTQDKLDVSALGFTGLGNGYAGTLAVSVSDDGTRTYLKSYETDAEGRSFEVSLQGNHAAALSADNILFATPVPVDPGVEGTPVVGSDLDDELHGTLGSEQILGGGGADQLYGYAGNDLLDGGAGRDKLSGGEGADTFRFALREDSHRSPLGTFGDRILDFDPSQDRIDVSALGFSGLGNGYAGSLAVSVSDDGTRTYLKSYEADAQGLSFEVALEGDHAAALSADNIVFAATDAAAAGELGVIGASGQPDDPAV(SEQ ID NO:4)。MDYNVKDFGALGDGVSDDRVAIQAAIDAAHAAGGGTVYLPPGEYRVSAAGEPSDGCLTLRDNVYLAGAGMGQTVIKLVDGSAQKITGIVRSPFGEETSNFGMRDLTLDGNRANTVDKVDGWFNGYAPGQPGADRNVTIERVEVREMSGYGFDPHEQTINLVLRDSVAHHNGLDGFVADY QIGGTFENNVAYANDRHGFNIVTSTNDFVMRNNVAYGNGGNGLVVQRGSENLAHPENILIDGGSYYDNGLEGVLVKMSNNVTVQNADIHGNGSSGVRVYGAQGVQILGNQIHDNAKTAVAPEVLLQSYDDTLGVSGNYYTTLNTRVEGNTITGSANSTYGVQERNDGTDFSSLVGNTINGVQEAAH LYGPNSTVSGTVSAPPQGTDGNDVLIGSDVGEQISGGAGDDRLDGGAGDDLLDGGAGRDRLTGGLGADTRFALREDSHRSPLGTFSDLILDFDPSQDKIDVSALGFIGLGNGYAGTLAVSLSADGLRTYLKSYDADAQGRSFELALDGNHAATLSAGNIVFAAATPVDPSAEAQPIVGSDLDDQLHGTLLGEEISGGG GADQLYGYGGGDLLDGGAGRDRLTGGEGADTRFALREDSHRSAAGTFSDLILDFDPTQDKLDVSALGFTGLGNGYAGTLAVSVSDDGTRTYLKSYETDAEGRSFEVSLQGNHAAALSADNILFATPVPVDPGVEGTPVVGSDLDDELHGTLGSEQILGGGGADQLYGYAGNDLLDGGAGRDKLSGGEGADTF RFALREDSHRSPLGTFGDRILDFDPSQDRIDVSALGFSGLGNGYAGSLAVSVSDDGTRTYLKSYEADAQGLSFEVALEGDHAAALSADNIVFAATDAAAAGELGVIGASGQPDDPAV (SEQ ID NO: 4).

本发明在筛选获得了一种新型的褐藻胶裂解酶的基础上，建立了一种制备发明富含古洛糖醛酸的褐藻四糖的方法，所制备的褐藻四糖结构明确，组成稳定；具有在医药等领域应用的前景。On the basis of screening and obtaining a novel alginate lyase, the present invention establishes a method for preparing fucotetraose rich in guluronic acid. The prepared fucotetraose has a clear structure and stable composition; It has the prospect of being applied in medicine and other fields.

附图说明Description of drawings

图1：褐藻胶裂解酶AlyPC1结构域示意图；Figure 1: Schematic diagram of the structure domain of alginate lyase AlyPC1;

图2：AlyPC1多序列比对图，其中PA1167,来自于Pseudomonas aeruginosa PAO1(AAG04556.1)；A1-Ⅱ’来自于Sphingomonas sp.A1(BAD16656.1)；AlyA来自Klebsiellapneumoniae(AAA25049.1)；AlyQ来自于Persicobacter sp.CCB-QB2(WP_053404615.1)；AlgAT5、AlyB来自于Vibrio spendidus OU02；Figure 2: Multiple sequence alignment of AlyPC1, in which PA1167 is from Pseudomonas aeruginosa PAO1 (AAG04556.1); A1-Ⅱ' is from Sphingomonas sp.A1 (BAD16656.1); AlyA is from Klebsiellapneumoniae (AAA25049.1); AlyQ From Persicobacter sp.CCB-QB2 (WP_053404615.1); AlgAT5, AlyB from Vibrio spendidus OU02;

图3：AlyPC1基因PCR扩增结果图，其中泳道M：2000bp DNA marker；泳道1：扩增的AlyPC1基因片段。Figure 3: PCR amplification results of AlyPC1 gene, where lane M: 2000bp DNA marker; lane 1: amplified AlyPC1 gene fragment.

图4：AlyPC1蛋白纯化结果图，其中泳道M：蛋白marker；泳道1：经IPTG诱导后菌体；泳道2：AlyPC1超声后上清；泳道3：蛋白酶酶切后的AlyPC1；Figure 4: AlyPC1 protein purification results, in which lane M: protein marker; lane 1: bacteria induced by IPTG; lane 2: AlyPC1 supernatant after sonication; lane 3: AlyPC1 after protease digestion;

图5：AlyPC1对不同底物降解过程图；Figure 5: A diagram of the degradation process of different substrates by AlyPC1;

图6：AlyE4、AlyE6纯化后电泳图，其中泳道1：pGEX-6P-1-AlgE4；泳道2：pGEX-6P-1-AlgE6；Figure 6: Electropherogram of AlyE4 and AlyE6 after purification, where lane 1: pGEX-6P-1-AlgE4; lane 2: pGEX-6P-1-AlgE6;

图7：AlyPC1与Al-E1反应TLC以及HPLC分析图，其中图A中M为分子量已知的AOS(DP2,DP3)；1为TLC分析AlyPC1与Al-E1反应产物；图B为HPLC分析AlyPC1与Al-E1反应产物图；Figure 7: TLC and HPLC analysis diagrams of the reaction between AlyPC1 and Al-E1, where M in Figure A is AOS (DP2, DP3) with known molecular weight; 1 is the TLC analysis of the reaction product of AlyPC1 and Al-E1; Figure B is the HPLC analysis of AlyPC1 Reaction product diagram with Al-E1;

图8：TLC(A)以及HPLC(B)分析凝胶过滤样品组成图。Figure 8: TLC (A) and HPLC (B) analysis of gel filtration sample composition.

具体实施方式Detailed ways

本发明筛选获得了一种新型的褐藻胶裂解酶，能够特异性裂解富含G的褐藻胶，产生褐藻四糖。其具体的氨基酸序列为SEQ ID NO:1。但本领域的普通技术人员能够通过在SEQ ID NO:1的氨基酸上通过取代、缺失、添加一个或数个氨基来获得也具有将甘露糖醛酸(M)异构化为古洛糖醛酸(G)功效的衍生蛋白。The invention screens and obtains a novel alginate lyase, which can specifically crack the G-rich alginate to produce fucotetraose. Its specific amino acid sequence is SEQ ID NO:1. However, those of ordinary skill in the art can obtain the amino acid that also has the ability to isomerize mannuronic acid (M) to guluronic acid by substituting, deleting, or adding one or several amino groups on the amino acid of SEQ ID NO:1. (G) Efficacy of derivative proteins.

编码上述褐藻胶裂解酶的基因，其一种具体的核苷酸序列为SEQ ID NO:2；但还可以是其它编码上述蛋白的具体序列。A specific nucleotide sequence of the gene encoding the above-mentioned alginate lyase is SEQ ID NO: 2; but it can also be other specific sequences encoding the above-mentioned protein.

本发明还提供一种制备富含古洛糖醛酸的褐藻多糖的方法，是将褐藻胶先用藻胶异构酶进行异构化，再用本发明所筛选获得的褐藻胶裂解酶进行处理，酶解产物可通过常规的褐藻寡糖纯化方法，例如胶层析技术来进行纯化，从而制备获得富含古洛糖醛酸G的褐藻四糖。The present invention also provides a method for preparing fucoidan rich in guluronic acid, which is to firstly use alginate isomerase to isomerize alginate, and then use the alginate lyase screened in the present invention to process , the enzymatic hydrolysis product can be purified by conventional fucoidan oligosaccharide purification methods, such as gel chromatography, so as to prepare fucotetraose rich in guluronic acid G.

下面结合实施例和附图对本发明进行详细的描述。The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings.

实施例1：新型褐藻胶裂解酶AlyPC1的筛选及重组表达Example 1: Screening and recombinant expression of a novel alginate lyase AlyPC1

1、褐藻胶裂解酶AlyPC1的序列及相关分析1. Sequence and correlation analysis of alginate lyase AlyPC1

发明人筛选获得了一个新型褐藻胶裂解酶AlyPC1,其具有优先识别polyG(多聚古洛糖醛酸)的功效，其产物分布中四糖含量在特定时间含量很高(>50％)。The inventor screened and obtained a novel alginate lyase AlyPC1, which has the function of preferentially recognizing polyG (polyguluronic acid), and the tetrasaccharide content in the product distribution is very high (>50%) at a specific time.

本发明所提供的新型的褐藻胶裂解酶基因，命名为AlyPC1。AlyPC1的cDNA有1737bp(SEQ ID NO:2)，编码578个氨基酸(SEQ ID NO:1)。The novel alginate lyase gene provided by the present invention is named AlyPC1. The cDNA of AlyPC1 has 1737bp (SEQ ID NO: 2), encoding 578 amino acids (SEQ ID NO: 1).

经过SignalP5.0(https://services.healthtech.dtu.dk/service.SignalP-5.0)预测AlyPC1的信号肽，其中AlyPC1前22个氨基酸为信号肽。将去掉信号肽的氨基酸序列在SMART(Letunic,Khedkar,&Bork,2021)以及Pram(Mistry et al.,2021)中进行结构域的预测，如图1所示，AlyPC1由3个结构域构成，包括两个非催化结构域以及一个催化结构域，两个非催化结构域分别是F5_F8_type_C以及CBM6,催化区为PL7家族的褐藻胶裂解酶。The signal peptide of AlyPC1 is predicted by SignalP5.0 (https://services.healthtech.dtu.dk/service.SignalP-5.0), and the first 22 amino acids of AlyPC1 are the signal peptide. The amino acid sequence without the signal peptide was predicted in SMART (Letunic, Khedkar, & Bork, 2021) and Pram (Mistry et al., 2021). As shown in Figure 1, AlyPC1 consists of three domains, including Two non-catalytic domains and one catalytic domain, the two non-catalytic domains are F5_F8_type_C and CBM6 respectively, and the catalytic region is alginate lyase of PL7 family.

将AlyPC1催化结构域与已有晶体结构的褐藻胶裂解酶进行多序列比对，如图2所示，AlyPC1催化区与AlyA的同源性仅为58.5％。蛋白序列NCBI blast结果表明同源性最高的为69％，说明本发明的褐藻胶裂解酶AlyPC1是一种新型的褐藻胶裂解酶。A multiple sequence alignment of the catalytic domain of AlyPC1 and the alginate lyase with the existing crystal structure, as shown in Figure 2, shows that the homology between the catalytic domain of AlyPC1 and AlyA is only 58.5%. The protein sequence NCBI blast results showed that the highest homology was 69%, indicating that the alginate lyase AlyPC1 of the present invention is a novel alginate lyase.

2、AlyPC1酶的制备与活性研究2. Preparation and activity research of AlyPC1 enzyme

1)AlyPC1原核表达系统的构建以及蛋白纯化1) Construction of AlyPC1 prokaryotic expression system and protein purification

以Pseudoalteromonas sp.为模板克隆AlyPC1，扩增引物中分别引入了酶切位点BamH Ⅰ以及Xho Ⅰ，序列如表1所示。首先克隆AlyPC1片段，利用1％的琼脂糖凝胶电泳检测(图3)，克隆正确的片段经过胶回收获得纯度较高的PCR产物。同时提取质粒pET32a，将质粒以及纯化的PCR产物利用BamHⅠ以及XhoⅠ限制性酶进行酶切，酶切体系如表2a、表2b所示，在37℃下反应20h，酶切后按照纯化试剂盒说明书对酶切片段进行纯化，利用T4连接酶将线性pET32a质粒与酶切片段连接，将其转化至E.coli.BL21(DE3)，挑取单克隆菌株，进行测序分析并于-80℃冰箱保存菌株。AlyPC1 was cloned using Pseudoalteromonas sp. as a template, and restriction sites BamH Ⅰ and Xho Ⅰ were introduced into the amplification primers respectively. The sequences are shown in Table 1. Firstly, the AlyPC1 fragment was cloned and detected by 1% agarose gel electrophoresis ( FIG. 3 ). The cloned fragment was recovered by gel to obtain a PCR product with higher purity. Extract the plasmid pET32a at the same time, and digest the plasmid and the purified PCR product with BamHI and XhoI restriction enzymes. The digestion system is shown in Table 2a and Table 2b. React at 37°C for 20 hours. After digestion, follow the instructions of the purification kit. Purify the digested fragment, use T4 ligase to connect the linear pET32a plasmid with the digested fragment, transform it into E.coli.BL21(DE3), pick a monoclonal strain, perform sequencing analysis and store it in a -80°C refrigerator strain.

表1：AlyPC1克隆引物表Table 1: List of primers for AlyPC1 cloning

注：引物F以及R中分别带有BamHⅠ以及XhoⅠ酶切位点Note: Primers F and R have BamHI and XhoI restriction sites respectively

表2A：AlyPC1酶切体系表Table 2A: AlyPC1 digestion system table

表2B:质粒pET32a酶切体系表Table 2B: Plasmid pET32a enzyme digestion system table

将测序正确的菌株经过活化后扩大培养，将活化的菌株按照1％的接种量接种于含有1L液体LB培养基的锥形瓶中，在37℃条件下培养约1.5h，测定培养液在OD₆₀₀的吸收值，待OD₆₀₀值为0.6-0.8时向培养基中加入400μL的IPTG(终浓度为0.1mM)。在16℃，180rpm培养20h。在4℃条件下3500g离心30min收集菌体。使用15mL的缓冲液(20mM Tris-HCl，500mMNaCl，10mM咪唑)重悬菌体，使用超声破碎仪破碎菌体，随后12000g离心30min，收集上清液。将上清液与平衡好的Ni-NTA琼脂糖凝胶柱子混合，在4℃结合约1h，用结合缓冲液冲洗柱子，用考马斯亮蓝检测流出的杂蛋白，至无杂蛋白流出，冲洗结束，使用蛋白酶对AlyPC1上的标签进行酶切，按照目的蛋白与蛋白酶100：1的比例加入蛋白酶，在4℃进行酶切4h，酶切结束后目标蛋白随缓冲液流出，纯化的各步骤均取样进行SDS-PSGE电泳检测。如图4所示，经过Ni-NTA琼脂糖凝胶纯化后可以获得纯度较高的AlyPC1蛋白(图4第3泳道表现为单一条带)，分子量为61kDa，与预测分子量相符。After activation, the strains with correct sequencing were expanded and cultivated. The activated strains were inoculated into Erlenmeyer flasks containing 1L liquid LB medium according to the inoculation amount of 1%, and cultured at 37°C for about 1.5h, and the OD of the culture solution was measured.₆₀₀ absorption value, when the OD₆₀₀ value is 0.6-0.8, add 400 μL of IPTG (final concentration is 0.1 mM) to the medium. Cultivate at 16° C., 180 rpm for 20 h. The cells were collected by centrifugation at 3500 g for 30 min at 4°C. 15 mL of buffer solution (20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole) was used to resuspend the bacterial cells, and the bacterial cells were disrupted using a sonicator, followed by centrifugation at 12000 g for 30 min, and the supernatant was collected. Mix the supernatant with the well-balanced Ni-NTA agarose gel column, bind at 4°C for about 1 hour, wash the column with binding buffer, and detect the outflowing impurity protein with Coomassie brilliant blue, until no impurity protein flows out, the washing is over , Use protease to digest the label on AlyPC1, add protease according to the ratio of 100:1 between the target protein and protease, and carry out enzyme digestion at 4°C for 4 hours. Perform SDS-PSGE electrophoresis detection. As shown in Figure 4, AlyPC1 protein with high purity can be obtained after Ni-NTA agarose gel purification (the third lane in Figure 4 shows a single band), with a molecular weight of 61 kDa, which is consistent with the predicted molecular weight.

实施例2：AlyPC1蛋白酶的底物特异性研究Embodiment 2: The substrate specificity research of AlyPC1 protease

将poly M和poly G两种底物溶解在20mM Tris-HCl(pH8.5)200mM NaCl中(底物浓度是2mg/ml)，在200μL的反应体系中进行反应，其中含有190μL的底物以及10μL(0.02mg/ml)酶AlyPC1，在30℃条件下，在96孔板中使用酶标仪(SYNERGY-H1,Bio-tek)检测反应在235nm的变化。Dissolve poly M and poly G substrates in 20mM Tris-HCl (pH8.5) 200mM NaCl (substrate concentration is 2mg/ml), and react in a 200μL reaction system, which contains 190μL of substrate and 10 μL (0.02 mg/ml) of enzyme AlyPC1 was placed in a 96-well plate at 30° C. using a microplate reader (SYNERGY-H1, Bio-tek) to detect changes in the reaction at 235 nm.

结果如图5所示，结果表明本发明筛选获得的新型褐藻胶裂解酶AlyCP1能特异性的降解ployG底物，对PolyM底物在该实验条件下未检测到降解活性。说明该酶具有很强的底物识别特异性，可以进一步提高降解产物中G的含量。The results are shown in Figure 5, and the results show that the novel alginate lyase AlyCP1 screened by the present invention can specifically degrade the polyG substrate, and no degradation activity was detected for the PolyM substrate under the experimental conditions. It shows that the enzyme has a strong substrate recognition specificity, which can further increase the G content in the degradation product.

实施例3：制备富含古洛糖醛酸的褐藻四糖Example 3: Preparation of Fucotetraose Rich in Guluronic Acid

一、异构酶法制备polyG1. Preparation of polyG by isomerase method

褐藻胶异构酶又称为甘露糖醛酸C-5差向异构酶，是一种具有催化β-D-(1,4)-甘露糖醛酸通过异构化转化为α-L-(1,4)-古洛糖醛酸的酶。Alginate isomerase, also known as mannuronic acid C-5 epimerase, is a kind of epimerase that catalyzes the conversion of β-D-(1,4)-mannuronic acid into α-L- (1,4)-Guluronic acid enzyme.

使用褐藻胶异构酶Alg E4(SEQ ID NO:3)和Alg E6(SEQ ID NO:4),并选择海洋巨藻来源的底物，配合两种异构酶获得了高古洛糖醛酸G含量的褐藻胶样品。Using alginate isomerases Alg E4 (SEQ ID NO: 3) and Alg E6 (SEQ ID NO: 4), and selecting substrates derived from marine macroalgae, the two isomerases were combined to obtain homoguluronic acid Alginate samples with G content.

1、AlgE4、AlgE6重组表达1. Recombinant expression of AlgE4 and AlgE6

AlgE4(1662bp)、AlgE6(2625bp)由上海生工生物工程有限公司合成，与载体pUC57构建质粒，根据片段两端BamH I、Xho I限制性酶切位点进行酶切，并与表达载体pGEX-6P-1，转化E.coli BL21(DE3)，构建原核表达载体。AlgE4 (1662bp) and AlgE6 (2625bp) were synthesized by Shanghai Sangon Bioengineering Co., Ltd., constructed plasmids with vector pUC57, digested according to the BamH I and Xho I restriction enzyme sites at both ends of the fragment, and combined with the expression vector pGEX- 6P-1 was transformed into E.coli BL21(DE3), and the prokaryotic expression vector was constructed.

正向引物AlgE4 BamH I：CGGGATCCATGGATTACAACGTCAAGGATTTCG、Forward primer AlgE4 BamH I: CGGGATCCATGGATTACAACGTCAAGGATTTCG,

反向引物AlgE4 Xho I：CCCTCGAGCTAGACGATCGCCCCGGCCTG、Reverse primer AlgE4 Xho I: CCCTCGAGCTAGACGATCGCCCCGGCCTG,

正向引物AlgE6 BamH I：CGGGATCCATGGATTACAACGTCAAGGATTTCG、Forward primer AlgE6 BamH I: CGGGATCCATGGATTACAACGTCAAGGATTTCG,

反向引物AlgE6 Xho I：CCCTCGAGTCAGACGGCCGGATCGTCCG。Reverse primer AlgE6 Xho I: CCCTCGAGTCAGACGGCCGGATCGTCCG.

2、AlgE4、AlgE6的纯化2. Purification of AlgE4 and AlgE6

0.5L LB液体培养基，1％接菌量，37℃培养至OD₆₀₀达到0.4-0.6时，在培养基中加入IPTG至终浓度为0.2mM，16℃诱导表达过夜。4℃，3000g离心30min收集菌体，使用1x PBS缓冲液重悬菌体，于冰水浴环境下进行超声破碎15min(超声2s，间隔4s，50％功率)，破碎后的混合液在4℃，12 000g条件下离心30min，收集上清液。0.5L LB liquid medium, 1% inoculum, culture at 37°C until OD₆₀₀ reaches 0.4-0.6, add IPTG to the medium to a final concentration of 0.2mM, induce expression overnight at 16°C. Collect the cells by centrifugation at 3000g for 30 minutes at 4°C, resuspend the cells in 1x PBS buffer, and perform ultrasonic crushing for 15 minutes in an ice-water bath environment (sonication for 2 s, interval of 4 s, 50% power). Centrifuge at 12 000 g for 30 min, and collect the supernatant.

运用亲和层析、柱上酶切的方法对目的蛋白进行纯化。取适量GST-Sepharose 4B凝胶填料加入层析柱中，1x PBS缓冲液平衡柱子，将超声后上清与凝胶填料于4℃环境中充分结合。结合完成后，将未与凝胶填料结合的蛋白流出，加入40倍填料体积的1×PBS缓冲液洗涤凝胶填料，去除剩余的未与凝胶填料结合的蛋白和杂蛋白。在凝胶填料中加入适量1×PBS缓冲液与PreScission酶(酶与目的蛋白质量比约为1:80)，在4℃的条件下进行柱上酶切3h。待酶切反应完成后，收集流出液，即得到纯化后的目的蛋白，结果如图6所示。其中pGEX-6P-1-AlgE4蛋白大小约为84kDa,pGEX-6P-1-AlgE6蛋白大小约为116kDa。从图6中可以看出，经Prescission蛋白酶酶切后，在预期目的蛋白大小处获得了特异性条带。The target protein was purified by affinity chromatography and on-column enzyme digestion. Take an appropriate amount of GST-Sepharose 4B gel filler and add it to the chromatography column, equilibrate the column with 1x PBS buffer, and fully combine the supernatant after ultrasonication with the gel filler at 4°C. After the binding is completed, the protein that is not bound to the gel packing is flowed out, and 40 times the packing volume of 1×PBS buffer is added to wash the gel packing to remove the remaining unbound protein and miscellaneous proteins. Add an appropriate amount of 1×PBS buffer and PreScission enzyme (the ratio of enzyme to target protein is about 1:80) to the gel filler, and perform on-column digestion at 4°C for 3 hours. After the enzyme cleavage reaction was completed, the effluent was collected to obtain the purified target protein, and the result is shown in Figure 6. The pGEX-6P-1-AlgE4 protein size is about 84kDa, and the pGEX-6P-1-AlgE6 protein size is about 116kDa. It can be seen from Figure 6 that after digestion with Prescission protease, a specific band was obtained at the expected size of the target protein.

3、异构化反应3. Isomerization reaction

反应缓冲液为20mM Tris-HCl，20mM NaCl，pH 7.5，底物为1％(m/v)褐藻胶10mL，加入预先添加Ca2+冰上孵育30min的1.5mL AlgE4(0.23mg/mL)和0.5mL AlgE6(0.94mg/mL)，反应体系Ca2+终浓度为1mM，在37℃，120rpm条件下进行反应，并于21h补加0.9mLAlgE4和0.2mL AlgE6，于45h补加0.6mL AlgE4和0.2mL AlgE6(AlgE4浓度为0.23mg/mL，AlgE6浓度为0.94mg/mL)。取不同反应时间的产物于100℃加热5min终止反应，以检测反应过程中的异构效果。将反应后的褐藻胶样品于12000g离心10min后，室温下透析2.5～3h，透析外液为去离子水，透析结束后将样品冻干。The reaction buffer is 20mM Tris-HCl, 20mM NaCl, pH 7.5, the substrate is 1% (m/v) alginate 10mL, add 1.5mL AlgE4 (0.23mg/mL) and 0.5mL AlgE6 (0.94mg/mL), the final concentration of Ca2+ in the reaction system was 1mM, the reaction was carried out at 37°C and 120rpm, and 0.9mL AlgE4 and 0.2mL AlgE6 were added at 21h, and 0.6mL AlgE4 and 0.2mL AlgE6 were added at 45h ( AlgE4 concentration is 0.23mg/mL, AlgE6 concentration is 0.94mg/mL). The products with different reaction times were heated at 100°C for 5 minutes to terminate the reaction, so as to detect the isomerization effect during the reaction. After the reacted alginate sample was centrifuged at 12000 g for 10 minutes, it was dialyzed at room temperature for 2.5-3 hours, and the dialyzed external fluid was deionized water, and the sample was freeze-dried after the dialyzing.

4、异构化效率的检测4. Detection of isomerization efficiency

将异构化反应前后的褐藻胶样品，分别制备为20mL的1mg/mL褐藻胶水溶液。将两份褐藻胶溶液分别用HCl(1mol/L,0.1mol/L)调pH为5.6，将其置于100℃水浴中反应1h；之后用HCl(1mol/L,0.1mol/L)调pH为3.8，将其置于100℃水浴中反应30min；接着再用NaOH(1mol/L,0.1mol/L)调pH为7～8后，将所得样品溶液冻干(48h)；冻干样品再用99.9％的D₂O(1mL)溶解后，溶液再次冷冻干燥(约20h)。取所得冻干样品粉末10mg，用D₂O(1mL)溶清，再加入事先配制的TTHA(三乙基四胺六乙酸，在D₂O中按0.3mol/L的浓度配制，并用DCl或NaOD调节pH值至5～5.5，溶清。)溶液。在BRUKER AVANCE III 400核磁共振波谱仪上进行检测。测试温度353K。The alginate samples before and after the isomerization reaction were prepared as 20 mL of 1 mg/mL alginate aqueous solution. Use HCl (1mol/L, 0.1mol/L) to adjust the pH of the two alginate solutions to 5.6, and place them in a water bath at 100°C for 1 hour; then use HCl (1mol/L, 0.1mol/L) to adjust the pH to 3.8, put it in a water bath at 100°C for 30 minutes; then use NaOH (1mol/L, 0.1mol/L) to adjust the pH to 7-8, then freeze-dry the obtained sample solution (48h); After dissolving with 99.9%_D2O (1 mL), the solution was freeze-dried again (about 20 h). Take 10 mg of the obtained freeze-dried sample powder, dissolve it with D₂ O (1 mL), then add TTHA (triethylenetetraminehexaacetic acid prepared in advance, prepared at a concentration of 0.3 mol/L in D₂ O, and dissolve it with DCl or NaOD to adjust the pH value to 5 ~ 5.5, dissolve clear.) solution. Detection was performed on a BRUKER AVANCE III 400 NMR spectrometer. The test temperature is 353K.

¹H-NMR数据计算(具体数据计算方法参照中华人民共和国医药行业标准《YY/T1654-2019组织工程医疗器械产品海藻酸钠》)结果，异构化反应前褐藻胶样品中G的含量是M含量的2.3倍；而经过异构化反应后，褐藻胶样品中G的含量是M含量的4.8倍，G的含量有明显升高，说明异构化反应使褐藻胶中部分M转化为G，异构化反应有显著效果。¹ H-NMR data calculation (the specific data calculation method refers to the pharmaceutical industry standard of the People's Republic of China "YY/T1654-2019 Sodium Alginate for Tissue Engineering Medical Devices") results, the G content in the alginate sample before the isomerization reaction is M content of 2.3 times; and after the isomerization reaction, the G content in the alginate sample is 4.8 times the M content, and the G content has increased significantly, indicating that the isomerization reaction converts part of the M in the alginate into G. The isomerization reaction has a significant effect.

二、褐藻四糖的制备与表征2. Preparation and Characterization of Fucotetraose

1、酶解制备褐藻四糖1. Preparation of fucoidose by enzymatic hydrolysis

100mg的异构化后的褐藻胶溶液溶解于4mL的去离子水中，加入浓度为1.9mg/mL的AlyPC1蛋白酶进行30min反应。反应结束分别利用TLC以及HPLC法对于降解产物进行分析。100 mg of the isomerized alginate solution was dissolved in 4 mL of deionized water, and AlyPC1 protease with a concentration of 1.9 mg/mL was added to react for 30 min. After the reaction, the degradation products were analyzed by TLC and HPLC respectively.

1)TLC检测：取合适大小的硅胶板，将样品均匀点样至距离硅胶板下端1.5cm处，随后硅胶板放置于盛有展开剂(展开剂为正丁醇，甲酸，水按照4:6:1(v:v:v)加入)的层析杠中，待层析液展开至距离硅胶板最上端1cm处，取出硅胶板，待样品吹干后，将硅胶板放置于显色剂中显色10s，显色剂中含有二苯胺(2g)，苯胺(2mL)，丙酮(100mL),磷酸(10mL),浓盐酸(1mL)，在高温下(>100℃)烘烤10min，观察产物分布。1) TLC detection: take a suitable size silica gel plate, spot the sample evenly to a distance of 1.5cm from the lower end of the silica gel plate, then place the silica gel plate in a developing agent (developing agent is n-butanol, formic acid, water according to the ratio of 4:6 :1 (v:v:v) into the chromatographic bar, wait for the chromatographic solution to expand to 1cm from the top of the silica gel plate, take out the silica gel plate, and after the sample is dried, place the silica gel plate in the developer Color development for 10s, the color developer contains diphenylamine (2g), aniline (2mL), acetone (100mL), phosphoric acid (10mL), concentrated hydrochloric acid (1mL), bake at high temperature (>100°C) for 10min, and observe the product distributed.

2)HPLC检测2) HPLC detection

将YMC-Pack ODS-AM 150x 4.6mmI.D.柱连接到UltiMate 3000系统中，洗脱缓冲液为5nm的四丁基溴化铵(TBAB)(A)和乙腈(B)，流速为1mL/min，样品经过醇沉后过滤有机滤膜(0.22μM),取5μL上样，洗脱从20％B的等梯度洗脱5min开始，然后从20％到45％B的13min梯度洗脱样品。然后在7min内使用55％B的梯度清洗柱上的剩余化合物，然后用90％B洗脱5min。Connect the YMC-Pack ODS-AM 150x 4.6mmI.D. column to the UltiMate 3000 system, the elution buffer is 5nm tetrabutylammonium bromide (TBAB) (A) and acetonitrile (B), the flow rate is 1mL/ min, the sample was filtered through an organic filter (0.22 μM) after alcohol precipitation, and 5 μL of the sample was taken, and the elution started from the isocratic elution of 20% B for 5 minutes, and then the sample was eluted gradually from 20% to 45% B for 13 minutes. The remaining compound on the column was then washed with a gradient of 55% B over 7 min, followed by elution with 90% B for 5 min.

结果：综合TLC和HPLC检测的结果，发现酶解产物是以DP4为主的褐藻寡糖的混合物，其中DP4的比例约为50％(图7)。Results: Based on the results of TLC and HPLC detection, it was found that the enzymatic hydrolysis product was a mixture of fucoidan oligosaccharides dominated by DP4, and the proportion of DP4 was about 50% (Fig. 7).

2、褐藻四糖样品纯化2. Fucotetraose sample purification

将Bio-Gel P-2Gel柱(柱体积是500ml)与ATKA pure蛋白纯化仪连接，使用0.2MNH₄HCO₃作为流动性去分离纯化酶解产物，流速为0.7mL/min，通过检测235nm处的吸收值收集洗脱峰。纯化后样品的TLC和HPLC分析，可以获得纯度＞95％的AOS。Connect the Bio-Gel P-2Gel column (column volume: 500ml) to the ATKA pure protein purifier, use 0.2MNH₄ HCO₃ as the fluidity to separate and purify the enzymatic hydrolyzate, the flow rate is 0.7mL/min, by detecting the Absorbance values were collected for the elution peaks. According to TLC and HPLC analysis of the purified sample, AOS with a purity >95% can be obtained.

3、NMR测试与数据分析3. NMR test and data analysis

将所得四糖样品溶液冻干，之后用D₂O溶解样品，在BRUKER AVANCE III 400核磁共振波谱仪上进行检测。测试温度为353K。The obtained tetrasaccharide sample solution was lyophilized, and then the sample was dissolved with D₂ O, and detected on a BRUKER AVANCE III 400 nuclear magnetic resonance spectrometer. The test temperature is 353K.

¹H-NMR数据计算结果显示，所得褐藻四糖中，与Δ(Δ表示α-L-古洛糖醛酸或β-D-甘露糖醛酸的4,5位因裂解酶酶解而发生β-消除，生成4,5位为共轭双键的不饱和单糖)相连单糖的古洛糖醛酸G的含量是甘露糖醛酸M含量的8倍；还原端单糖中古洛糖醛酸G的含量是甘露糖醛酸M含量的13.5倍；这说明酶解后寡糖中古洛糖醛酸G的含量较所使用的异构化褐藻胶中古洛糖醛酸G的含量又有明显上升，结果表明本发明的新型褐藻胶裂解酶AlyPC1在高古洛糖醛酸G含量褐藻四糖制备中具有重要应用开发前景。¹ H-NMR data calculation results show that in the obtained fucotetraose, the difference with Δ(Δ indicates that the 4 and 5 positions of α-L-guluronic acid or β-D-mannuronic acid are hydrolyzed by lyase β-elimination, generating unsaturated monosaccharides with conjugated double bonds atpositions 4 and 5) The content of guluronic acid G in linked monosaccharides is 8 times that of mannuronic acid M; in the reducing end monosaccharides, guluose The content of uronic acid G is 13.5 times that of mannuronic acid M; this shows that the content of guluronic acid G in the oligosaccharide after enzymolysis is more than that in the used isomerized alginate. The results show that the novel alginate lyase AlyPC1 of the present invention has important application and development prospects in the preparation of fucoidose with high guluronic acid G content.

序列表sequence listing

<110> 中国海洋大学<110> Ocean University of China

<120> 一种制备富含古洛糖醛酸的褐藻四糖的方法<120> A method for preparing fucotetraose rich in guluronic acid

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Thr Ser Asp Leu Ala Glu Asp Glu Pro Tyr Gln Trp Ile Asn Phe AspThr Ser Asp Leu Ala Glu Asp Glu Pro Tyr Gln Trp Ile Asn Phe Asp

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Gly Leu Val Glu Ala Glu Asp Tyr Ser Ala Phe Tyr Asp Ser Thr AlaGly Leu Val Glu Ala Glu Asp Tyr Ser Ala Phe Tyr Asp Ser Thr Ala

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Gly Asn Gln Gly Gly Glu Tyr Arg Asp Asp Asp Val Asp Ile Glu GlnGly Asn Gln Gly Gly Glu Tyr Arg Asp Asp Asp Val Asp Ile Glu Gln

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Thr Ser Asp Ile Thr Gly Asn Tyr Asn Ile Asp Phe Ile Thr Asp GlyThr Ser Asp Ile Thr Gly Asn Tyr Asn Ile Asp Phe Ile Thr Asp Gly

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Pro Ile Asp Asp Asn Ala Asp Gly Lys Ser Asp Ser Ile Ser Glu SerPro Ile Asp Asp Asn Ala Asp Gly Lys Ser Asp Ser Ile Ser Glu Ser

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Gln Leu Ala Ala Gly Tyr Glu His Pro Gln Trp Phe Tyr Thr Ala AspGln Leu Ala Ala Gly Tyr Glu His Pro Gln Trp Phe Tyr Thr Ala Asp

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Asp Gly Ala Met Val Phe Lys Val Glu Ile Asp Ala Pro Lys Thr SerAsp Gly Ala Met Val Phe Lys Val Glu Ile Asp Ala Pro Lys Thr Ser

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Thr Asn Thr Ser Tyr Ser Arg Ser Glu Leu Arg Glu Met Leu Arg AlaThr Asn Thr Ser Tyr Ser Arg Ser Glu Leu Arg Glu Met Leu Arg Ala

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Gly Asp Thr Ser Ile Ser Thr Gln Gly Ile Asn Lys Asn Asn Trp ValGly Asp Thr Ser Ile Ser Thr Gln Gly Ile Asn Lys Asn Asn Trp Val

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Phe Ser Thr Tyr Ser Ser Asp Asp Lys Asn Ala Ala Gly Gly Ile AspPhe Ser Thr Tyr Ser Ser Asp Asp Lys Asn Ala Ala Gly Gly Ile Asp

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Met Asp Tyr Asn Val Lys Asp Phe Gly Ala Leu Gly Asp Gly Val SerMet Asp Tyr Asn Val Lys Asp Phe Gly Ala Leu Gly Asp Gly Val Ser

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Asp Asp Arg Ala Ser Ile Gln Ala Ala Ile Asp Ala Ala Tyr Ala AlaAsp Asp Arg Ala Ser Ile Gln Ala Ala Ile Asp Ala Ala Tyr Ala Ala

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Gly Gly Gly Thr Val Tyr Leu Pro Ala Gly Glu Tyr Arg Val Ser AlaGly Gly Gly Thr Val Tyr Leu Pro Ala Gly Glu Tyr Arg Val Ser Ala

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Ala Gly Glu Pro Gly Asp Gly Cys Leu Met Leu Lys Asp Gly Val TyrAla Gly Glu Pro Gly Asp Gly Cys Leu Met Leu Lys Asp Gly Val Tyr

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Leu Ala Gly Ala Gly Met Gly Glu Thr Val Ile Lys Leu Ile Asp GlyLeu Ala Gly Ala Gly Met Gly Glu Thr Val Ile Lys Leu Ile Asp Gly

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Thr Ser Asn Phe Gly Met Arg Asp Leu Thr Leu Asp Gly Asn Arg AspThr Ser Asn Phe Gly Met Arg Asp Leu Thr Leu Asp Gly Asn Arg Asp

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Asn Thr Ser Gly Lys Val Asp Gly Trp Phe Asn Gly Tyr Ile Pro GlyAsn Thr Ser Gly Lys Val Asp Gly Trp Phe Asn Gly Tyr Ile Pro Gly

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Gly Asp Gly Ala Asp Arg Asp Val Thr Ile Glu Arg Val Glu Val ArgGly Asp Gly Ala Asp Arg Asp Val Thr Ile Glu Arg Val Glu Val Arg

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Glu Met Ser Gly Tyr Gly Phe Asp Pro His Glu Gln Thr Ile Asn LeuGlu Met Ser Gly Tyr Gly Phe Asp Pro His Glu Gln Thr Ile Asn Leu

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Thr Ile Arg Asp Ser Val Ala His Asp Asn Gly Leu Asp Gly Phe ValThr Ile Arg Asp Ser Val Ala His Asp Asn Gly Leu Asp Gly Phe Val

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Ala Asp Tyr Leu Val Asp Ser Val Phe Glu Asn Asn Val Ala Tyr AlaAla Asp Tyr Leu Val Asp Ser Val Phe Glu Asn Asn Val Ala Tyr Ala

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Asn Asp Arg His Gly Phe Asn Val Val Thr Ser Thr His Asp Phe ValAsn Asp Arg His Gly Phe Asn Val Val Thr Ser Thr His Asp Phe Val

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Met Thr Asn Asn Val Ala Tyr Gly Asn Gly Ser Ser Gly Leu Val ValMet Thr Asn Asn Val Ala Tyr Gly Asn Gly Ser Ser Gly Leu Val Val

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Gln Arg Gly Leu Glu Asp Leu Ala Leu Pro Ser Asn Ile Leu Ile AspGln Arg Gly Leu Glu Asp Leu Ala Leu Pro Ser Asn Ile Leu Ile Asp

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Gly Gly Ala Tyr Tyr Asp Asn Ala Arg Glu Gly Val Leu Leu Lys MetGly Gly Ala Tyr Tyr Asp Asn Ala Arg Glu Gly Val Leu Leu Lys Met

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Gln Ile His Asp Asn Ala Gln Ala Ala Ala Val Pro Glu Val Leu LeuGln Ile His Asp Asn Ala Gln Ala Ala Ala Val Pro Glu Val Leu Leu

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Gln Ser Phe Asp Asp Thr Ala Gly Ala Ser Gly Thr Tyr Tyr Thr ThrGln Ser Phe Asp Asp Thr Ala Gly Ala Ser Gly Thr Tyr Tyr Thr Thr

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Leu Asn Thr Arg Ile Glu Gly Asn Thr Ile Ser Gly Ser Ala Asn SerLeu Asn Thr Arg Ile Glu Gly Asn Thr Ile Ser Gly Ser Ala Asn Ser

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Thr Tyr Gly Ile Gln Glu Arg Asn Asp Gly Thr Asp Tyr Ser Ser LeuThr Tyr Gly Ile Gln Glu Arg Asn Asp Gly Thr Asp Tyr Ser Ser Ser Leu

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Ile Asp Asn Asp Ile Ala Gly Val Gln Gln Pro Ile Gln Leu Tyr GlyIle Asp Asn Asp Ile Ala Gly Val Gln Gln Pro Ile Gln Leu Tyr Gly

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Pro His Ser Thr Val Ser Gly Glu Pro Gly Ala Thr Pro Gln Gln ProPro His Ser Thr Val Ser Gly Glu Pro Gly Ala Thr Pro Gln Gln Pro

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Ser Thr Gly Ser Asp Gly Glu Pro Leu Val Gly Gly Asp Thr Asp AspSer Thr Gly Ser Asp Gly Glu Pro Leu Val Gly Gly Asp Thr Asp Asp

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Gln Leu Gln Gly Gly Ser Gly Ala Asp Arg Leu Asp Gly Gly Ala GlyGln Leu Gln Gly Gly Ser Gly Ala Asp Arg Leu Asp Gly Gly Ala Gly

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Asp Asp Ile Leu Asp Gly Gly Ala Gly Arg Asp Arg Leu Ser Gly GlyAsp Asp Ile Leu Asp Gly Gly Ala Gly Arg Asp Arg Leu Ser Gly Gly

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Ala Gly Ala Asp Thr Phe Val Phe Ser Ala Arg Glu Asp Ser Tyr ArgAla Gly Ala Asp Thr Phe Val Phe Ser Ala Arg Glu Asp Ser Tyr Arg

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Thr Asp Thr Ala Val Phe Asn Asp Leu Ile Leu Asp Phe Glu Ala SerThr Asp Thr Ala Val Phe Asn Asp Leu Ile Leu Asp Phe Glu Ala Ser

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Glu Asp Arg Ile Asp Leu Ser Ala Leu Gly Phe Ser Gly Leu Gly AspGlu Asp Arg Ile Asp Leu Ser Ala Leu Gly Phe Ser Gly Leu Gly Asp

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Gly Tyr Gly Gly Thr Leu Leu Leu Lys Thr Asn Ala Glu Gly Thr ArgGly Tyr Gly Gly Thr Leu Leu Leu Lys Thr Asn Ala Glu Gly Thr Arg

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Thr Tyr Leu Lys Ser Phe Glu Ala Asp Ala Glu Gly Arg Arg Phe GluThr Tyr Leu Lys Ser Phe Glu Ala Asp Ala Glu Gly Arg Arg Phe Glu

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Val Phe Ala Ala Thr Gly Thr Thr Thr Glu Leu Glu Val Leu Gly AspVal Phe Ala Ala Thr Gly Thr Thr Thr Glu Leu Glu Val Leu Gly Asp

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Ser Gly Thr Gln Ala Gly Ala Ile ValSer Gly Thr Gln Ala Gly Ala Ile Val

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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

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Met Asp Tyr Asn Val Lys Asp Phe Gly Ala Leu Gly Asp Gly Val SerMet Asp Tyr Asn Val Lys Asp Phe Gly Ala Leu Gly Asp Gly Val Ser

1 5 10 151 5 10 15

Asp Asp Arg Val Ala Ile Gln Ala Ala Ile Asp Ala Ala His Ala AlaAsp Asp Arg Val Ala Ile Gln Ala Ala Ile Asp Ala Ala His Ala Ala

20 25 30 20 25 30

Gly Gly Gly Thr Val Tyr Leu Pro Pro Gly Glu Tyr Arg Val Ser AlaGly Gly Gly Thr Val Tyr Leu Pro Pro Gly Glu Tyr Arg Val Ser Ala

35 40 45 35 40 45

Ala Gly Glu Pro Ser Asp Gly Cys Leu Thr Leu Arg Asp Asn Val TyrAla Gly Glu Pro Ser Asp Gly Cys Leu Thr Leu Arg Asp Asn Val Tyr

50 55 60 50 55 60

Leu Ala Gly Ala Gly Met Gly Gln Thr Val Ile Lys Leu Val Asp GlyLeu Ala Gly Ala Gly Met Gly Gln Thr Val Ile Lys Leu Val Asp Gly

65 70 75 8065 70 75 80

Ser Ala Gln Lys Ile Thr Gly Ile Val Arg Ser Pro Phe Gly Glu GluSer Ala Gln Lys Ile Thr Gly Ile Val Arg Ser Pro Phe Gly Glu Glu

85 90 95 85 90 95

Thr Ser Asn Phe Gly Met Arg Asp Leu Thr Leu Asp Gly Asn Arg AlaThr Ser Asn Phe Gly Met Arg Asp Leu Thr Leu Asp Gly Asn Arg Ala

100 105 110 100 105 110

Asn Thr Val Asp Lys Val Asp Gly Trp Phe Asn Gly Tyr Ala Pro GlyAsn Thr Val Asp Lys Val Asp Gly Trp Phe Asn Gly Tyr Ala Pro Gly

115 120 125 115 120 125

Gln Pro Gly Ala Asp Arg Asn Val Thr Ile Glu Arg Val Glu Val ArgGln Pro Gly Ala Asp Arg Asn Val Thr Ile Glu Arg Val Glu Val Arg

130 135 140 130 135 140

Glu Met Ser Gly Tyr Gly Phe Asp Pro His Glu Gln Thr Ile Asn LeuGlu Met Ser Gly Tyr Gly Phe Asp Pro His Glu Gln Thr Ile Asn Leu

145 150 155 160145 150 155 160

Val Leu Arg Asp Ser Val Ala His His Asn Gly Leu Asp Gly Phe ValVal Leu Arg Asp Ser Val Ala His His Asn Gly Leu Asp Gly Phe Val

165 170 175 165 170 175

Ala Asp Tyr Gln Ile Gly Gly Thr Phe Glu Asn Asn Val Ala Tyr AlaAla Asp Tyr Gln Ile Gly Gly Thr Phe Glu Asn Asn Val Ala Tyr Ala

180 185 190 180 185 190

Asn Asp Arg His Gly Phe Asn Ile Val Thr Ser Thr Asn Asp Phe ValAsn Asp Arg His Gly Phe Asn Ile Val Thr Ser Thr Asn Asp Phe Val

195 200 205 195 200 205

Met Arg Asn Asn Val Ala Tyr Gly Asn Gly Gly Asn Gly Leu Val ValMet Arg Asn Asn Val Ala Tyr Gly Asn Gly Gly Asn Gly Leu Val Val

210 215 220 210 215 220

Gln Arg Gly Ser Glu Asn Leu Ala His Pro Glu Asn Ile Leu Ile AspGln Arg Gly Ser Glu Asn Leu Ala His Pro Glu Asn Ile Leu Ile Asp

225 230 235 240225 230 235 240

Gly Gly Ser Tyr Tyr Asp Asn Gly Leu Glu Gly Val Leu Val Lys MetGly Gly Ser Tyr Tyr Asp Asn Gly Leu Glu Gly Val Leu Val Lys Met

245 250 255 245 250 255

Ser Asn Asn Val Thr Val Gln Asn Ala Asp Ile His Gly Asn Gly SerSer Asn Asn Val Thr Val Gln Asn Ala Asp Ile His Gly Asn Gly Ser

260 265 270 260 265 270

Ser Gly Val Arg Val Tyr Gly Ala Gln Gly Val Gln Ile Leu Gly AsnSer Gly Val Arg Val Tyr Gly Ala Gln Gly Val Gln Ile Leu Gly Asn

275 280 285 275 280 285

Gln Ile His Asp Asn Ala Lys Thr Ala Val Ala Pro Glu Val Leu LeuGln Ile His Asp Asn Ala Lys Thr Ala Val Ala Pro Glu Val Leu Leu

290 295 300 290 295 300

Gln Ser Tyr Asp Asp Thr Leu Gly Val Ser Gly Asn Tyr Tyr Thr ThrGln Ser Tyr Asp Asp Thr Leu Gly Val Ser Gly Asn Tyr Tyr Thr Thr Thr

305 310 315 320305 310 315 320

Leu Asn Thr Arg Val Glu Gly Asn Thr Ile Thr Gly Ser Ala Asn SerLeu Asn Thr Arg Val Glu Gly Asn Thr Ile Thr Gly Ser Ala Asn Ser

325 330 335 325 330 335

Thr Tyr Gly Val Gln Glu Arg Asn Asp Gly Thr Asp Phe Ser Ser LeuThr Tyr Gly Val Gln Glu Arg Asn Asp Gly Thr Asp Phe Ser Ser Leu

340 345 350 340 345 350

Val Gly Asn Thr Ile Asn Gly Val Gln Glu Ala Ala His Leu Tyr GlyVal Gly Asn Thr Ile Asn Gly Val Gln Glu Ala Ala His Leu Tyr Gly

355 360 365 355 360 365

Pro Asn Ser Thr Val Ser Gly Thr Val Ser Ala Pro Pro Gln Gly ThrPro Asn Ser Thr Val Ser Gly Thr Val Ser Ala Pro Pro Gln Gly Thr

370 375 380 370 375 380

Asp Gly Asn Asp Val Leu Ile Gly Ser Asp Val Gly Glu Gln Ile SerAsp Gly Asn Asp Val Leu Ile Gly Ser Asp Val Gly Glu Gln Ile Ser

385 390 395 400385 390 395 400

Gly Gly Ala Gly Asp Asp Arg Leu Asp Gly Gly Ala Gly Asp Asp LeuGly Gly Ala Gly Asp Asp Arg Leu Asp Gly Gly Ala Gly Asp Asp Leu

405 410 415 405 410 415

Leu Asp Gly Gly Ala Gly Arg Asp Arg Leu Thr Gly Gly Leu Gly AlaLeu Asp Gly Gly Ala Gly Arg Asp Arg Leu Thr Gly Gly Leu Gly Ala

420 425 430 420 425 430

Asp Thr Phe Arg Phe Ala Leu Arg Glu Asp Ser His Arg Ser Pro LeuAsp Thr Phe Arg Phe Ala Leu Arg Glu Asp Ser His Arg Ser Pro Leu

435 440 445 435 440 445

Gly Thr Phe Ser Asp Leu Ile Leu Asp Phe Asp Pro Ser Gln Asp LysGly Thr Phe Ser Asp Leu Ile Leu Asp Phe Asp Pro Ser Gln Asp Lys

450 455 460 450 455 460

Ile Asp Val Ser Ala Leu Gly Phe Ile Gly Leu Gly Asn Gly Tyr AlaIle Asp Val Ser Ala Leu Gly Phe Ile Gly Leu Gly Asn Gly Tyr Ala

465 470 475 480465 470 475 480

Gly Thr Leu Ala Val Ser Leu Ser Ala Asp Gly Leu Arg Thr Tyr LeuGly Thr Leu Ala Val Ser Leu Ser Ala Asp Gly Leu Arg Thr Tyr Leu

485 490 495 485 490 495

Lys Ser Tyr Asp Ala Asp Ala Gln Gly Arg Ser Phe Glu Leu Ala LeuLys Ser Tyr Asp Ala Asp Ala Gln Gly Arg Ser Phe Glu Leu Ala Leu

500 505 510 500 505 510

Asp Gly Asn His Ala Ala Thr Leu Ser Ala Gly Asn Ile Val Phe AlaAsp Gly Asn His Ala Ala Thr Leu Ser Ala Gly Asn Ile Val Phe Ala

515 520 525 515 520 525

Ala Ala Thr Pro Val Asp Pro Ser Ala Glu Ala Gln Pro Ile Val GlyAla Ala Thr Pro Val Asp Pro Ser Ala Glu Ala Gln Pro Ile Val Gly

530 535 540 530 535 540

Ser Asp Leu Asp Asp Gln Leu His Gly Thr Leu Leu Gly Glu Glu IleSer Asp Leu Asp Asp Gln Leu His Gly Thr Leu Leu Gly Glu Glu Ile

545 550 555 560545 550 555 560

Ser Gly Gly Gly Gly Ala Asp Gln Leu Tyr Gly Tyr Gly Gly Gly AspSer Gly Gly Gly Gly Ala Asp Gln Leu Tyr Gly Tyr Gly Gly Gly Asp

565 570 575 565 570 575

Leu Leu Asp Gly Gly Ala Gly Arg Asp Arg Leu Thr Gly Gly Glu GlyLeu Leu Asp Gly Gly Ala Gly Arg Asp Arg Leu Thr Gly Gly Glu Gly

580 585 590 580 585 590

Ala Asp Thr Phe Arg Phe Ala Leu Arg Glu Asp Ser His Arg Ser AlaAla Asp Thr Phe Arg Phe Ala Leu Arg Glu Asp Ser His Arg Ser Ala

595 600 605 595 600 605

Ala Gly Thr Phe Ser Asp Leu Ile Leu Asp Phe Asp Pro Thr Gln AspAla Gly Thr Phe Ser Asp Leu Ile Leu Asp Phe Asp Pro Thr Gln Asp

610 615 620 610 615 620

Lys Leu Asp Val Ser Ala Leu Gly Phe Thr Gly Leu Gly Asn Gly TyrLys Leu Asp Val Ser Ala Leu Gly Phe Thr Gly Leu Gly Asn Gly Tyr

625 630 635 640625 630 635 640

Ala Gly Thr Leu Ala Val Ser Val Ser Asp Asp Gly Thr Arg Thr TyrAla Gly Thr Leu Ala Val Ser Val Ser Asp Asp Gly Thr Arg Thr Tyr

645 650 655 645 650 655

Leu Lys Ser Tyr Glu Thr Asp Ala Glu Gly Arg Ser Phe Glu Val SerLeu Lys Ser Tyr Glu Thr Asp Ala Glu Gly Arg Ser Phe Glu Val Ser

660 665 670 660 665 670

Leu Gln Gly Asn His Ala Ala Ala Leu Ser Ala Asp Asn Ile Leu PheLeu Gln Gly Asn His Ala Ala Ala Leu Ser Ala Asp Asn Ile Leu Phe

675 680 685 675 680 685

Ala Thr Pro Val Pro Val Asp Pro Gly Val Glu Gly Thr Pro Val ValAla Thr Pro Val Pro Val Asp Pro Gly Val Glu Gly Thr Pro Val Val

690 695 700 690 695 700

Gly Ser Asp Leu Asp Asp Glu Leu His Gly Thr Leu Gly Ser Glu GlnGly Ser Asp Leu Asp Asp Glu Leu His Gly Thr Leu Gly Ser Glu Gln

705 710 715 720705 710 715 720

Ile Leu Gly Gly Gly Gly Ala Asp Gln Leu Tyr Gly Tyr Ala Gly AsnIle Leu Gly Gly Gly Gly Ala Asp Gln Leu Tyr Gly Tyr Ala Gly Asn

725 730 735 725 730 735

Asp Leu Leu Asp Gly Gly Ala Gly Arg Asp Lys Leu Ser Gly Gly GluAsp Leu Leu Asp Gly Gly Ala Gly Arg Asp Lys Leu Ser Gly Gly Glu

740 745 750 740 745 750

Gly Ala Asp Thr Phe Arg Phe Ala Leu Arg Glu Asp Ser His Arg SerGly Ala Asp Thr Phe Arg Phe Ala Leu Arg Glu Asp Ser His Arg Ser

755 760 765 755 760 765

Pro Leu Gly Thr Phe Gly Asp Arg Ile Leu Asp Phe Asp Pro Ser GlnPro Leu Gly Thr Phe Gly Asp Arg Ile Leu Asp Phe Asp Pro Ser Gln

770 775 780 770 775 780

Asp Arg Ile Asp Val Ser Ala Leu Gly Phe Ser Gly Leu Gly Asn GlyAsp Arg Ile Asp Val Ser Ala Leu Gly Phe Ser Gly Leu Gly Asn Gly

785 790 795 800785 790 795 800

Tyr Ala Gly Ser Leu Ala Val Ser Val Ser Asp Asp Gly Thr Arg ThrTyr Ala Gly Ser Leu Ala Val Ser Val Ser Asp Asp Gly Thr Arg Thr

805 810 815 805 810 815

Tyr Leu Lys Ser Tyr Glu Ala Asp Ala Gln Gly Leu Ser Phe Glu ValTyr Leu Lys Ser Tyr Glu Ala Asp Ala Gln Gly Leu Ser Phe Glu Val

820 825 830 820 825 830

Ala Leu Glu Gly Asp His Ala Ala Ala Leu Ser Ala Asp Asn Ile ValAla Leu Glu Gly Asp His Ala Ala Ala Leu Ser Ala Asp Asn Ile Val

835 840 845 835 840 845

Phe Ala Ala Thr Asp Ala Ala Ala Ala Gly Glu Leu Gly Val Ile GlyPhe Ala Ala Thr Asp Ala Ala Ala Ala Gly Glu Leu Gly Val Ile Gly

850 855 860 850 855 860

Ala Ser Gly Gln Pro Asp Asp Pro Ala ValAla Ser Gly Gln Pro Asp Asp Pro Ala Val

865 870865 870

Claims (10)

Translated fromChinese

1.一种褐藻胶裂解酶，其特征在于，所述的褐藻胶裂解酶的氨基酸序列为SEQ ID NO:1。1. A alginate lyase, characterized in that the amino acid sequence of the alginate lyase is SEQ ID NO:1.2.一种基因，其特征在于，所述基因编码权利要求1所述的褐藻胶裂解酶。2. A gene, characterized in that the gene encodes the alginate lyase according to claim 1.3.如权利要求2所述的基因，其特征在于，所述的基因的核苷酸序列为SEQ ID NO:2。3. The gene according to claim 2, wherein the nucleotide sequence of the gene is SEQ ID NO:2.4.一种重组表达载体，其特征在于，所述的重组表达载体中插入有权利要求2所述的基因的核酸片段。4. A recombinant expression vector, characterized in that the nucleic acid fragment of the gene according to claim 2 is inserted into the recombinant expression vector.5.一种重组工程菌株，其特征在于，所述的重组工程菌株中包含有权利要求4所述的重组表达载体。5. A recombinant engineering strain, characterized in that the recombinant expression vector according to claim 4 is included in the recombinant engineering strain.6.权利要求1所述的褐藻胶裂解酶在制备褐藻寡糖中的应用。6. The application of the alginate lyase described in claim 1 in the preparation of alginate oligosaccharides.7.如权利要求6所述的应用，其特征在于，所述的褐藻寡糖为褐藻四糖。7. The application according to claim 6, wherein the fucoidan oligosaccharide is fucotetraose.8.一种制备褐藻四糖的方法，其特征在于，所述的方法是先使用褐藻胶异构酶来使褐藻胶中甘露糖醛酸异构化为古洛糖醛酸；再用权利要求1所述的褐藻胶裂解酶来催化制备褐藻四糖。8. A method for preparing fucoidose, characterized in that, the method is to first use algin isomerase to isomerize mannuronic acid in algin to guluronic acid; The alginate lyase described in 1 is used to catalyze the preparation of fucotetraose.9.如权利要求8所述的方法，其特征在于，所述的褐藻胶异构酶的氨基酸序列为SEQ IDNO:3。9. The method according to claim 8, wherein the amino acid sequence of the alginate isomerase is SEQ ID NO:3.10.如权利要求8所述的方法，其特征在于，所述的褐藻胶异构酶的氨基酸序列为SEQID NO:4。10. The method according to claim 8, wherein the amino acid sequence of the alginate isomerase is SEQ ID NO:4.

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