CN114410611A - Laminaria degrading enzyme OUC-BsLam26 and its application - Google Patents
Laminaria degrading enzyme OUC-BsLam26 and its application Download PDFInfo
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- CN114410611A CN114410611A CN202111568019.2A CN202111568019A CN114410611A CN 114410611 A CN114410611 A CN 114410611A CN 202111568019 A CN202111568019 A CN 202111568019A CN 114410611 A CN114410611 A CN 114410611A
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- CN
- China
- Prior art keywords
- laminarin
- ouc
- bslam26
- degrading enzyme
- enzyme
- Prior art date
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Abstract
本发明公开了一种昆布多糖降解酶OUC‑BsLam26，氨基酸序列如SEQ ID NO.1所示。编码昆布多糖降解酶OUC‑BsLam26的基因，核苷酸序列如SEQ ID NO.2所示。所述昆布多糖降解酶OUC‑BsLam26在降解昆布多糖/制备昆布寡糖中的应用。本发明还公开了一种包含有昆布多糖降解酶OUC‑BsLam26的酶制剂。本发明还公开了携带有编码昆布多糖降解酶OUC‑BsLam26的基因的重组表达载体和重组工程菌。本发明的昆布多糖降解酶OUC‑BsLam26，可作用昆布多糖底物，终产物昆布寡糖聚合度为1～4。本发明的昆布多糖降解酶具有优良的酶学性质和特异性，在酶法制备昆布寡糖中具有重要的工业应用价值以及经济价值。
The invention discloses a laminarin degrading enzyme OUC-BsLam26, the amino acid sequence of which is shown in SEQ ID NO.1. The gene encoding the laminarin degrading enzyme OUC-BsLam26, the nucleotide sequence is shown in SEQ ID NO.2. Application of the laminarin degrading enzyme OUC-BsLam26 in degrading laminarin/preparing lamina oligosaccharide. The invention also discloses an enzyme preparation comprising the laminarin degrading enzyme OUC-BsLam26. The invention also discloses a recombinant expression vector and a recombinant engineering bacteria carrying the gene encoding the laminarin degrading enzyme OUC-BsLam26. The laminarin degrading enzyme OUC-BsLam26 of the present invention can act as a laminarin substrate, and the final product laminarin oligosaccharide has a polymerization degree of 1-4. The laminarin degrading enzyme of the invention has excellent enzymatic properties and specificity, and has important industrial application value and economic value in the enzymatic preparation of laminarin oligosaccharide.
Description
技术领域technical field
本发明涉及一种昆布多糖降解酶OUC-BsLam26的重组表达、制备及其应用，属于功能基 因克隆表达技术领域。The invention relates to the recombinant expression, preparation and application of a laminarin degrading enzyme OUC-BsLam26, and belongs to the technical field of functional gene cloning and expression.
背景技术Background technique
昆布多糖(Laminarin)是一种来源于褐藻的海洋多糖，其结构主要是由D-吡喃葡萄糖 残基通过β-1,3-糖苷键连接与部分β-1,6-糖苷键连接的支链共同构成。昆布寡糖是由昆布 多糖降解得到的聚合度(DP)位于2～10之间的产物，其具有较低的分子量和较高的生物利 用度。近年来经研究证实昆布寡糖具有比昆布多糖更好的生物活性，包括抗氧化、抗凝血、 抗凋亡、调节肠道菌群、骨骼再生修复等众多的生理功能。因此如何高效制备特定聚合度的 昆布寡糖受到了人们的广泛关注。Laminarin is a marine polysaccharide derived from brown algae. Its structure is mainly composed of D-glucopyranose residues connected by β-1,3-glycosidic bonds and partial β-1,6-glycosidic bonds. chain together. Laminaria oligosaccharide is a product with a degree of polymerization (DP) between 2 and 10 obtained from the degradation of laminarin, which has lower molecular weight and higher bioavailability. In recent years, studies have confirmed that lamina oligosaccharides have better biological activities than laminarin, including many physiological functions such as anti-oxidation, anti-coagulation, anti-apoptosis, regulation of intestinal flora, bone regeneration and repair. Therefore, how to efficiently prepare lamina oligosaccharides with a specific degree of polymerization has received extensive attention.
采用昆布多糖制备昆布寡糖的方法主要包括物理法、化学法以及酶解法。物理法主要通 过超声辅助提取以及γ射线等方法降解昆布多糖；化学法主要通过酸溶液和碱溶液对昆布多 糖进行降解。物理法能耗高，产物不均一，而化学法反应条件较为剧烈，且过多使用化学试 剂，不符合绿色环保等理念。而酶解法不仅反应条件温和，特异性较高，而且获得的水解产 物也较为均一。因此，挖掘一种新的昆布多糖酶并用其制备昆布寡糖具有很大的研究价值和 发展潜力。The method for preparing lamina oligosaccharide by using laminarin mainly includes physical method, chemical method and enzymatic hydrolysis method. The physical method mainly degrades laminarin through ultrasonic-assisted extraction and γ-ray methods; the chemical method mainly degrades laminarin through acid solution and alkali solution. The physical method has high energy consumption and non-uniform products, while the chemical method has more severe reaction conditions and excessive use of chemical reagents, which is not in line with the concept of green environmental protection. The enzymatic hydrolysis method not only has mild reaction conditions and high specificity, but also the obtained hydrolysis products are more uniform. Therefore, mining a new laminarin enzyme and using it to prepare lamina oligosaccharides has great research value and development potential.
人类肠道微生物的相关研究成为当前的热点问题。Bacteroides sp.是来自肠道微生物中 与人类炎症、糖尿病等疾病呈负相关的拟杆菌门成员，具有降解多糖和碳水化合物的能力， 是一种理想的挖掘宿主。The related research of human gut microbes has become a hot issue at present. Bacteroides sp. is a member of Bacteroidetes from gut microbes that is negatively related to human inflammation, diabetes and other diseases. It has the ability to degrade polysaccharides and carbohydrates, and is an ideal mining host.
发明内容SUMMARY OF THE INVENTION
针对上述现有技术，本发明提供了一种可以降解昆布多糖产生昆布寡糖的新型降解酶— —昆布多糖降解酶OUC-BsLam26，弥补了现有酶基因库的不足。In view of the above-mentioned prior art, the present invention provides a novel degrading enzyme that can degrade laminarin to produce lamina oligosaccharide - laminarin degrading enzyme OUC-BsLam26, which makes up for the deficiency of the existing enzyme gene bank.
本发明是通过以下技术方案实现的：The present invention is achieved through the following technical solutions:
昆布多糖降解酶OUC-BsLam26，其氨基酸序列如SEQ ID NO.1所示。The amino acid sequence of the laminarin degrading enzyme OUC-BsLam26 is shown in SEQ ID NO.1.
昆布多糖降解酶OUC-BsLam26的氨基酸序列(SEQ ID NO.1)：Amino acid sequence of laminarin degrading enzyme OUC-BsLam26 (SEQ ID NO.1):
MKKIFYSLILSTLMFGACSASDDGVPQTPEDPDGTQEPQEEPVTYTGIQKRSVKRGVSYSFQLPKEDTQMLGSAIS WAYNWGTEISSDLSTEFSKQQIDYCPMSWNANPDIAPKIRRYVNAHPECKYLLAYNEPNLTDQARMTPQEAAAQWPALRA LASELNLKLISPAMNYGTLPDYSDPIKWLDEFFSNVPLSDVDGIAIHCYMESPSSLINYVSMFKKYGKPIWLTEFCAWPS SDKISISSQMNYMSETLHYLESDPDVFRYAWFIPRGIGPTDPSTGTTSNSLLPGKPNALTDLGTVFVNMSTLDKTAYYNK NQVIPAEHYSSINTVENLTSVSAHLRPTTDISGILDVYDLKQEQWLKYQLDAPKAGTYQLDIRYTSFRDNKVEITIDKGT AATLDLPNVDSKWTTVTTNIQLKAGKQTLRIKVTTGNIALNWLRFKD。MKKIFYSLILSTLMFGACSASDDGVPQTPEDPDGTQEPQEEPVTYTGIQKRSVKRGVSYSFQLPKEDTQMLGSAIS WAYNWGTEISSDLSTEFSKQQIDYCPMSWNANPDIAPKIRRYVNAHPECKYLLAYNEPNLTDQARMTPQEAAAQWPALRA LASELNLKLISPAMNYGTLPDYSDPIKWLDEFFSNVPLSDVDGIAIHCYMESPSSLINYVSMFKKYGKPIWLTEFCAWPS SDKISISSQMNYMSETLHYLESDPDVFRYAWFIPRGIGPTDPSTGTTSNSLLPGKPNALTDLGTVFVNMSTLDKTAYYNK NQVIPAEHYSSINTVENLTSVSAHLRPTTDISGILDVYDLKQEQWLKYQLDAPKAGTYQLDIRYTSFRDNKVEITIDKGT AATLDLPNVDSKWTTVTTNIQLKAGKQTLRIKVTTGNIALNWLRFKD。
一种编码上述昆布多糖降解酶OUC-BsLam26的基因，其核苷酸序列如SEQ ID NO.2所示。A gene encoding the laminarin degrading enzyme OUC-BsLam26, the nucleotide sequence of which is shown in SEQ ID NO.2.
编码昆布多糖降解酶OUC-BsLam26的基因的核苷酸序列(SEQ ID NO.2)：Nucleotide sequence of gene encoding laminarin degrading enzyme OUC-BsLam26 (SEQ ID NO. 2):
5’-ATGAAAAAGATATTCTATTCACTCATACTATCCACACTGATGTTCGGAGCCTGTTCCGCTTCGGATGACGGA GTTCCTCAAACACCTGAAGACCCCGACGGGACACAGGAACCGCAGGAAGAGCCGGTCACCTACACCGGTATACAGAAACG TAGTGTAAAACGTGGAGTCAGCTATAGTTTCCAGCTCCCCAAAGAAGACACGCAAATGTTGGGAAGTGCCATATCCTGGG CTTACAACTGGGGGACAGAGATTTCTTCCGATTTAAGTACTGAGTTCAGCAAGCAGCAAATCGACTATTGCCCAATGTCA TGGAATGCCAATCCAGACATCGCTCCCAAGATTCGCAGGTACGTCAACGCACACCCGGAATGTAAATATCTACTTGCATA CAATGAGCCTAATCTGACAGACCAGGCAAGAATGACACCCCAGGAAGCTGCCGCACAATGGCCTGCACTGCGTGCATTAG CTTCTGAACTAAATCTGAAGCTCATATCTCCGGCTATGAACTACGGAACATTGCCGGATTATAGTGACCCGATCAAATGG TTGGACGAGTTTTTCAGCAACGTTCCCCTCAGTGATGTAGACGGTATAGCTATCCATTGTTACATGGAAAGCCCTTCTTC TCTTATCAACTATGTAAGCATGTTCAAAAAATATGGAAAACCTATTTGGTTAACAGAATTCTGTGCATGGCCCAGTAGTG ATAAGATTAGCATATCAAGCCAGATGAACTATATGAGCGAGACTCTTCATTATCTTGAGTCCGATCCTGACGTATTCCGC TATGCCTGGTTCATCCCAAGAGGTATTGGACCGACCGATCCAAGTACCGGTACAACCAGCAACAGCTTATTACCCGGTAA GCCCAATGCATTGACTGACCTCGGAACAGTGTTTGTGAATATGTCCACTCTGGATAAAACTGCTTATTACAATAAGAATC AAGTTATTCCCGCCGAACACTACAGTTCAATAAACACAGTTGAGAATTTAACATCAGTCAGCGCACACCTGCGCCCTACA ACCGATATTTCGGGCATATTGGATGTTTATGATTTGAAACAGGAACAATGGCTGAAATATCAACTGGATGCTCCTAAAGC CGGAACTTACCAGTTGGATATCCGCTATACCAGTTTCAGAGATAACAAGGTTGAGATAACTATAGACAAAGGTACGGCAGCAACACTAGACCTGCCTAATGTAGACAGCAAATGGACTACTGTCACTACCAACATCCAACTAAAAGCAGGTAAGCAAACG CTACGTATAAAAGTCACTACAGGCAATATTGCATTAAATTGGCTGCGTTTCAAAGACTAA-3’。5’-ATGAAAAAGATATTCTATTCACTCATACTATCCACACTGATGTTCGGAGCCTGTTCCGCTTCGGATGACGGA GTTCCTCAAACACCTGAAGACCCCGACGGGACACAGGAACCGCAGGAAGAGCCGGTCACCTACACCGGTATACAGAAACG TAGTGTAAAACGTGGAGTCAGCTATAGTTTCCAGCTCCCCAAAGAAGACACGCAAATGTTGGGAAGTGCCATATCCTGGG CTTACAACTGGGGGACAGAGATTTCTTCCGATTTAAGTACTGAGTTCAGCAAGCAGCAAATCGACTATTGCCCAATGTCA TGGAATGCCAATCCAGACATCGCTCCCAAGATTCGCAGGTACGTCAACGCACACCCGGAATGTAAATATCTACTTGCATA CAATGAGCCTAATCTGACAGACCAGGCAAGAATGACACCCCAGGAAGCTGCCGCACAATGGCCTGCACTGCGTGCATTAG CTTCTGAACTAAATCTGAAGCTCATATCTCCGGCTATGAACTACGGAACATTGCCGGATTATAGTGACCCGATCAAATGG TTGGACGAGTTTTTCAGCAACGTTCCCCTCAGTGATGTAGACGGTATAGCTATCCATTGTTACATGGAAAGCCCTTCTTC TCTTATCAACTATGTAAGCATGTTCAAAAAATATGGAAAACCTATTTGGTTAACAGAATTCTGTGCATGGCCCAGTAGTG ATAAGATTAGCATATCAAGCCAGATGAACTATATGAGCGAGACTCTTCATTATCTTGAGTCCGATCCTGACGTATTCCGC TATGCCTGGTTCATCCCAAGAGGTATTGGACCGACCGATCCAAGTACCGGTACAACCAGCAACAGCTTATTACCCGGTAA GCCCAATGCATTGACTGACCTCGGAACAGTGTTTGTGAATATGTCCACTCTGGATAAAACTGCTTATTACAATAAGAATC AAGTTATTCCCGCCGAACACTACAGTTCAATAA ACACAGTTGAGAATTTAACATCAGTCAGCGCACACCTGCGCCCTACA ACCGATATTTCGGGCATATTGGATGTTTATGATTTGAAACAGGAACAATGGCTGAAATATCAACTGGATGCTCCTAAAGC CGGAACTTACCAGTTGGATATCCGCTATACCAGTTTCAGAGATAACAAGGTTGAGATAACTATAGACAAAGGTACGGCAGCAACACTAGACCTGCCTAATGTAGACAGCAAATGGACTACTGTCACTACCAACATCCAACTAAAAGCAGGTAAGCAAACG CTACGTATAAAAGTCACTACAGGCAATATTGCATTAAATTGGCTGCGTTTCAAAGACTAA-3’。
所述昆布多糖降解酶OUC-BsLam26在降解昆布多糖/制备昆布寡糖中的应用。Application of the laminarin degrading enzyme OUC-BsLam26 in degrading laminarin/preparing laminarin oligosaccharides.
一种降解昆布多糖/制备昆布寡糖的方法：采用上述昆布多糖降解酶OUC-BsLam26降解昆 布多糖，得到昆布寡糖产物，产物中包含葡萄糖、昆布二糖、昆布三糖以及昆布四糖。A method for degrading laminarin/preparing laminarin oligosaccharide: adopting the above-mentioned laminarin degrading enzyme OUC-BsLam26 to degrade laminarin to obtain a laminarin oligosaccharide product, the product comprising glucose, kelp disaccharide, kelp trisaccharide and kelp tetrasaccharide.
进一步地，所述降解的条件为：昆布多糖溶液的浓度为0.2％～0.3％(m/v，单位g/ml)， 温度30～50℃，pH值3.0～8.0，时间10分钟以上，优选0.5小时以上。优选的，降解条件为：温度45℃，pH值6.0，时间0.5小时以上。优选的降解条件还可以为：在35～40℃、pH 3.0～10.0的条件下酶解48小时以上。Further, the degradation conditions are: the concentration of the laminarin solution is 0.2%-0.3% (m/v, unit g/ml), the temperature is 30-50°C, the pH value is 3.0-8.0, and the time is more than 10 minutes, preferably 0.5 hours or more. Preferably, the degradation conditions are: the temperature is 45°C, the pH value is 6.0, and the time is more than 0.5 hours. The preferred degradation conditions may also be: enzymatic hydrolysis for more than 48 hours under the conditions of 35-40° C. and pH 3.0-10.0.
进一步地，所述昆布寡糖为单一的昆布四糖。酶解后，采用制备型硅胶柱层析进行分离， 流动相为：正丁醇：乙酸：水＝2：1：1(v/v/v)，得到洗脱液，经检测，洗脱液中，得到了单一的昆布四糖，实现了昆布四糖的分离制备。Further, the laminaria oligosaccharide is a single laminaria tetrasaccharide. After enzymatic hydrolysis, preparative silica gel column chromatography was used for separation. The mobile phase was: n-butanol: acetic acid: water = 2:1:1 (v/v/v) to obtain an eluent. After detection, the eluent was , obtained a single kelp tetrasaccharide, and realized the separation and preparation of kelp tetrasaccharide.
所述编码昆布多糖降解酶OUC-BsLam26的基因，在制备降解昆布多糖/制备昆布寡糖的酶 制剂中的应用。The application of the gene encoding the laminarin degrading enzyme OUC-BsLam26 in the preparation of an enzyme preparation for degrading laminarin/preparing laminarin oligosaccharide.
一种酶制剂，包括上述昆布多糖降解酶OUC-BsLam26。An enzyme preparation, comprising the above-mentioned laminarin degrading enzyme OUC-BsLam26.
所述酶制剂在降解昆布多糖/制备昆布寡糖中的应用。Application of the enzyme preparation in degrading laminarin/preparing lamina oligosaccharide.
一种重组表达载体，其携带有上述编码昆布多糖降解酶OUC-BsLam26的基因。A recombinant expression vector carrying the above-mentioned gene encoding the laminarin degrading enzyme OUC-BsLam26.
一种重组工程菌，其基因组中插入有上述编码昆布多糖降解酶OUC-BsLam26的基因，能 表达昆布多糖降解酶OUC-BsLam26。A kind of recombinant engineering bacteria, the gene of above-mentioned encoding laminarin degrading enzyme OUC-BsLam26 is inserted in its genome, can express laminarin degrading enzyme OUC-BsLam26.
所述重组工程菌在制备昆布多糖酶OUC-Bs-26中的应用。The application of the recombinant engineering bacteria in the preparation of laminarinase OUC-Bs-26.
本发明的昆布多糖降解酶OUC-BsLam26来源于人体肠道微生物Bacteroides sp.，经鉴 定属于糖苷水解酶GH128家族。本发明构建了含昆布多糖酶基因的重组载体，实现了在大肠 杆菌中的异源表达，同时为该酶的工业化生产和应用提供了良好的基础。该酶在45℃和pH 6.0 条件下具有较高的催化活性，经过镍柱纯化后的比酶活可达3.42U/mg。该酶能够高效水解 昆布多糖，水解产物主要为昆布四糖，昆布三糖，昆布二糖以及葡萄糖。本发明的昆布多糖 酶制剂，具有较高的活性和稳定性(在40℃下放置48h，酶活仍可保留90％以上；在pH 3.0～ 10.0的缓冲液中保留48h，酶活也能保留50％以上)，且效率高、纯度高、产量高，制备的 寡糖可用于调节肠道菌群、肥胖治疗等方面，具有实现工业化利用的潜力。The laminarin degrading enzyme OUC-BsLam26 of the present invention is derived from human intestinal microorganism Bacteroides sp., and is identified as belonging to the GH128 family of glycoside hydrolase. The invention constructs a recombinant vector containing laminarinase gene, realizes heterologous expression in E. coli, and provides a good foundation for the industrial production and application of the enzyme. The enzyme has high catalytic activity at 45℃ and pH 6.0, and the specific enzyme activity after purification by nickel column can reach 3.42U/mg. The enzyme can efficiently hydrolyze laminarin, and the hydrolyzed products are mainly laminarin tetrasaccharide, kelp trisaccharide, lamina disaccharide and glucose. The laminarin enzyme preparation of the present invention has high activity and stability (the enzyme activity can still retain more than 90% when placed at 40° C. for 48 hours; the enzyme activity can also be retained in a buffer of pH 3.0-10.0 for 48 hours. More than 50%), and high efficiency, high purity, high yield, the prepared oligosaccharide can be used for regulating intestinal flora, obesity treatment, etc., and has the potential to achieve industrialized utilization.
此外，目前的文献仅能够检测到昆布多糖的水解产物，而没有将特定聚合度的寡糖制备 出来，与此相比，本发明的昆布多糖酶不仅能够检测到水解产物，而且能够将特定聚合度的 寡糖分离制备出来，对于后续的工业化应用以及研究特定聚合度寡糖的活性具有重要的意义。In addition, the current literature can only detect the hydrolyzed product of laminarin without preparing oligosaccharides with a specific degree of polymerization. Compared with this, the laminarinase of the present invention can not only detect the hydrolyzed product, but also can convert the specific polymerized oligosaccharide. The separation and preparation of oligosaccharides with a specific degree of polymerization is of great significance for subsequent industrial applications and research on the activity of oligosaccharides with a specific degree of polymerization.
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。Various terms and phrases used herein have their ordinary meanings as known to those skilled in the art.
附图说明Description of drawings
图1：本发明的昆布多糖降解酶纯化后的纯酶SDS-PAGE电泳图，其中，M为标准蛋白Marker；1为粗酶蛋白；2为纯化后的昆布多糖酶蛋白。Figure 1: SDS-PAGE electrophoresis image of pure enzyme after purification of the laminarin degrading enzyme of the present invention, wherein, M is the standard protein Marker; 1 is the crude enzyme protein; 2 is the purified laminarin enzyme protein.
图2：温度和pH变化对相对酶活的影响示意图，其中，A：温度对相对酶活的影响；B：在不同温度下放置48h对相对酶活的影响；C：pH对相对酶活的影响；D：在不同pH下放置 48h对相对酶活的影响。Figure 2: Schematic diagram of the effect of temperature and pH changes on the relative enzyme activity, where A: the effect of temperature on the relative enzyme activity; B: the effect of placing at different temperatures for 48 hours on the relative enzyme activity; C: the effect of pH on the relative enzyme activity Effect; D: The effect of placing at different pH for 48h on the relative enzyme activity.
图3：金属离子和化学试剂以及底物特异性对相对酶活的影响示意图，其中，A；金属离 子和化学试剂对相对酶活的影响；B：底物特异性研究的结果示意图。Figure 3: Schematic diagram of the influence of metal ions and chemical reagents and substrate specificity on the relative enzyme activity, wherein, A; the influence of metal ions and chemical reagents on the relative enzyme activity; B: Schematic diagram of the results of the substrate specificity study.
图4：本发明的昆布多糖降解酶解产物的TLC图。Figure 4: TLC chart of the enzymatic hydrolysis product of laminarin degradation of the present invention.
图5：本发明的昆布多糖降解酶酶解产物的液相图。Figure 5: The liquid phase diagram of the enzymatic hydrolysis product of the laminarin degrading enzyme of the present invention.
图6：本发明的昆布多糖降解酶解产物的质谱图。Figure 6: Mass spectrum of the enzymatic hydrolysis product of laminarin degradation of the present invention.
图7：本发明的昆布多糖降解酶分离纯化产物的TLC图。Figure 7: TLC chart of the isolated and purified product of the laminarin degrading enzyme of the present invention.
图8：本发明的昆布多糖降解酶分离纯化产物的TLC图（实施例13）。Figure 8: TLC chart of the isolated and purified product of the laminarin degrading enzyme of the present invention (Example 13).
具体实施方式Detailed ways
下面结合实施例对本发明作进一步的说明。然而，本发明的范围并不限于下述实施例。 本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各 种变化和修饰。The present invention will be further described below in conjunction with the examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the inventions.
下述实施例中所涉及的仪器、试剂、材料等，若无特别说明，均为现有技术中已有的常 规仪器、试剂、材料等，可通过正规商业途径获得。下述实施例中所涉及的实验方法，检测 方法等，若无特别说明，均为现有技术中已有的常规实验方法，检测方法等。The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are conventional instruments, reagents, materials, etc. existing in the prior art, and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. existing in the prior art.
实施例1昆布多糖降解酶基因OUC-BsLam26的克隆Example 1 Cloning of laminarin degrading enzyme gene OUC-BsLam26
本发明的昆布多糖降解酶基因OUC-BsLam26为全基因合成所得，从NCBI文库中进行挖掘 得到的(昆布多糖酶的来源主要是微生物，还有的来源于动物、高等植物以及病毒等。但植 物来源的昆布多糖酶在正常的生长环境下含量较少，且相较于微生物来源的昆布多糖酶活性 低。相关实验证明来源于细菌的芽孢杆菌属、杆菌属、黄杆菌属、纤维菌属、类芽孢杆菌等 均具有昆布多糖酶活性，此外，实验证明肠道微生物来源的Akkermanisamuciniphila菌具 有编码昆布多糖酶的基因，能够实现在大肠杆菌中的异源表达，因此本发明从肠道微生物菌 群中挖掘到一株菌，验证其是否具有可能表达昆布多糖活性的蛋白)，其来源于肠道微生物 Bacteroides sp.CBA7301，序列号为QIU93826.1。该片段包含了1332个碱基序列，如SEQ ID NO.2所示，编码443个氨基酸序列，如SEQ ID NO.1所示。根据序列比对和进化树分析，其 与来自Pseudomonas viridiflava CFBP 1590的endo-β-1,3-glucanase同源性最高，预测 蛋白序列相似值为39.70％，该昆布多糖酶OUC-Bs-26属于糖苷水解酶第128家族(GH128)。 本发明首次将该酶表达、纯化并进行制备相关研究。The laminarin degrading enzyme gene OUC-BsLam26 of the present invention is obtained by whole gene synthesis, and is obtained by mining from the NCBI library (the source of laminarinase is mainly microorganisms, and some are derived from animals, higher plants and viruses, etc. But plants The laminarinase from the source has less content under normal growth environment, and the activity of laminarinase is lower than that of the microbial source. Relevant experiments have proved that the bacteria-derived Bacillus, Bacillus, Flavobacterium, Cellulobacterium, Paenibacillus and the like all have laminarinase activity. In addition, experiments have shown that Akkermanisamuciniphila, which is derived from intestinal microorganisms, has a gene encoding laminarinase and can achieve heterologous expression in Escherichia coli. Therefore, the present invention is derived from intestinal microbial flora. A strain of bacteria was excavated in , to verify whether it has a protein that may express the activity of laminarin), which is derived from the intestinal microorganism Bacteroides sp. CBA7301, and the serial number is QIU93826.1. The fragment contains 1332 base sequences, as shown in SEQ ID NO. 2, and encodes 443 amino acid sequences, as shown in SEQ ID NO. 1. According to sequence alignment and phylogenetic tree analysis, it has the highest homology with endo-β-1,3-glucanase from Pseudomonas viridiflava CFBP 1590, and the predicted protein sequence similarity value is 39.70%. The laminarinase OUC-Bs-26 belongs to Glycoside hydrolase family 128 (GH128). The present invention expresses, purifies and conducts relevant research on preparation of the enzyme for the first time.
实施例2含昆布多糖酶降解酶基因的表达载体构建Example 2 Construction of expression vector containing laminarin degrading enzyme gene
将基因片段与pET-28a克隆载体采用无缝克隆技术进行连接，将连接产物转入E.coli DH5 α感受态细胞，涂布于LB培养基固体平板(含有50μg/mL卡那霉素)上，37℃温箱中培 养16小时后，挑取单克隆至含有50μg/mL卡那霉素LB液体培养基中，在转速为220rpm的37℃摇床培养12小时。将单克隆进行阳性验证后测序。将验证成功的质粒提取出来并命名为pET28a-OUC-BsLam26，将其保存在-20℃备用。The gene fragment was connected with the pET-28a cloning vector using seamless cloning technology, and the ligated product was transferred into E.coli DH5 alpha competent cells and spread on a solid plate of LB medium (containing 50 μg/mL kanamycin). , After culturing in a 37°C incubator for 16 hours, pick a single clone into LB liquid medium containing 50 μg/mL kanamycin, and cultivate for 12 hours at a 37°C shaker with a rotational speed of 220 rpm. Monoclones were sequenced after positive validation. The successfully verified plasmid was extracted and named pET28a-OUC-BsLam26, which was stored at -20°C for future use.
实施例3含昆布多糖降解酶基因的重组质粒及工程菌的构建Example 3 Construction of recombinant plasmids and engineered bacteria containing laminarin degrading enzyme genes
将实施例2中抽提的pET28a-OUC-BsLam26质粒转化至宿主E.coli BL21感受态细胞中, 构建好的工程菌在硫酸卡那霉素抗性平板上长出，得到重组表达菌株。The pET28a-OUC-BsLam26 plasmid extracted in Example 2 was transformed into the host E.coli BL21 competent cells, and the constructed engineered bacteria were grown on kanamycin sulfate-resistant plates to obtain recombinant expression strains.
实施例4利用大肠杆菌工程菌制备重组昆布多糖降解酶Example 4 Preparation of recombinant laminarin degrading enzyme using Escherichia coli engineering bacteria
大肠杆菌重组菌株经过在5ml LB液体培养基(含有50μg/mL卡那霉素)中活化12h后，按1％的接种量接入含有硫酸卡那霉素(50μg/mL)的ZYP-5052培养基中，20℃，200rpm低温培养48h，自诱导表达昆布多糖降解酶。The recombinant Escherichia coli strains were activated in 5ml LB liquid medium (containing 50μg/mL kanamycin) for 12h, and then cultured in ZYP-5052 containing kanamycin sulfate (50μg/mL) at a 1% inoculum amount In the base, 20 ℃, 200 rpm low temperature culture for 48 h, auto-induced expression of laminarin degrading enzyme.
发酵结束后，菌液于8000g，4℃，离心10分钟后收集菌体，细胞重悬于50mM的pH7.0 的Tirs-HCl缓冲液中，然后置于冰水浴中超声破碎30min(40％功率,3s开，3s关)，然后8000g，4℃，再次离心10min，收集到的上清液即为粗酶液。昆布多糖酶上含6个His纯 化标签，粗酶液使用Ni-NTA柱进行亲和层析纯化，首先使用平衡缓冲溶液(500mM NaCl,50mMTris-HCl)平衡柱子，然后用20mM咪唑溶液(20mM咪唑，500mM NaCl,50mM Tris-HCl)洗 脱结合力较差的杂蛋白，100mM咪唑溶液(100mM咪唑，500mM NaCl,50mM Tris-HCl)洗脱 目的蛋白，收集此部分的缓冲液洗脱成分，得到纯化的重组昆布多糖降解酶溶液(蛋白含量 为0.3mg/mL)。经SDS-PAGE检测蛋白纯度和分子量，验证条带是否单一，大小是否准确(图 1)，结果显示重组蛋白分子量大小约为63KDa，和预测结果保持一致，即获得了目的蛋白。After the fermentation, the bacterial liquid was collected by centrifugation at 8000g, 4°C for 10 minutes, and the cells were resuspended in 50mM Tirs-HCl buffer at pH 7.0, and then placed in an ice-water bath for sonication for 30min (40% power). , 3s on, 3s off), then centrifuge again at 8000g, 4°C for 10min, and the collected supernatant is the crude enzyme solution. The laminarinase contains 6 His purification tags, and the crude enzyme solution is purified by affinity chromatography using a Ni-NTA column. First, the column is equilibrated with an equilibration buffer solution (500mM NaCl, 50mM Tris-HCl), and then a 20mM imidazole solution (20mM imidazole) is used to equilibrate the column. , 500mM NaCl, 50mM Tris-HCl) to elute the impurity protein with poor binding ability, 100mM imidazole solution (100mM imidazole, 500mM NaCl, 50mM Tris-HCl) to elute the target protein, collect this part of the buffer elution components, get Purified recombinant laminarin degrading enzyme solution (protein content of 0.3 mg/mL). The purity and molecular weight of the protein were detected by SDS-PAGE to verify whether the band was single and the size was accurate (Figure 1).
实施例5重组昆布多糖酶比酶活测定
昆布多糖降解酶OUC-BsLam26活性的标准测定方法为：250微升的反应体系中，包含50μL 酶液、200μL pH 6.0的柠檬酸缓冲液配制的0.2％(m/v)的昆布多糖底物，在35℃下反应 10min，沸水浴10min对酶灭活后在小型离心机12000rpm下离心1min。之后取出180μL上 清溶液与270μL的DNS试剂混合，并在沸水浴中煮沸5min进行显色。之后在小型离心机12000rpm下离心1min后在540nm处测定吸光值。酶活力定义为在标准条件下每min产生1μM还原糖所需要的酶量。经测定，纯化后的昆布多糖降解酶纯酶活力可达3.42U/mg。相比而言，来自产酶溶杆菌Lysobacter enzymogenes的昆布多糖酶GluB的酶活为0.7U/mg，来自栉孔扇贝的昆布多糖酶Lcf在pH 6.0，44℃的条件下酶活可以达到1.67U/mg，本发明的昆布多糖降解酶OUC-BsLam26的酶活明显更高。在同等的酶解效率下，本发明的酶解速度更快， 所需时间更短(时间不超过10分钟)。The standard assay method for the activity of laminarin degrading enzyme OUC-BsLam26 is as follows: 250 μL reaction system contains 50 μL enzyme solution, 200 μL pH 6.0 citrate buffer prepared 0.2% (m/v) laminarin substrate, The reaction was carried out at 35°C for 10 min, the enzyme was inactivated in a boiling water bath for 10 min, and then centrifuged at 12000 rpm in a small centrifuge for 1 min. Afterwards, 180 μL of the supernatant solution was taken out and mixed with 270 μL of DNS reagent, and boiled in a boiling water bath for 5 min for color development. Then, the absorbance was measured at 540 nm after centrifugation at 12,000 rpm in a small centrifuge for 1 min. Enzyme activity was defined as the amount of enzyme required to produce 1 μM reducing sugar per min under standard conditions. It was determined that the pure enzyme activity of the purified laminarin degrading enzyme could reach 3.42U/mg. In contrast, the enzyme activity of laminarinase GluB from Lysobacter enzymogenes was 0.7U/mg, and the enzyme activity of laminarinase Lcf from scallop scallops at pH 6.0 and 44°C could reach 1.67U /mg, the enzyme activity of the laminarin degrading enzyme OUC-BsLam26 of the present invention is significantly higher. Under the same enzymatic hydrolysis efficiency, the enzymatic hydrolysis speed of the present invention is faster and the required time is shorter (the time does not exceed 10 minutes).
实施例6测定昆布多糖降解酶的最适反应条件和稳定性Example 6 Determination of optimal reaction conditions and stability of laminarin degrading enzymes
将实施例4中得到的纯化昆布多糖降解酶在不同温度和pH下反应，测定温度和pH对酶 活力的影响。选取25℃、30℃、35℃、40℃、45℃、50℃的温度，按照实施例5昆布多糖降解酶比酶活测定方法反应10min测定最适温度，用DNS法测定酶活。在45℃下，选用pH为 3.0～10.0的缓冲液作为酶反应的不同测定pH缓冲液，根据昆布多糖降解酶的酶活力，确定 昆布多糖降解酶的最适pH。在35℃、40℃、45℃、50℃的温度下对酶进行孵育，在最适条件 45℃，pH 6.0下测定其残留酶活，得到其温度稳定性。分别与不同pH的缓冲液混合，并于4℃中孵育，在最适温度下测定其残留酶活，得到其pH稳定性。以最高酶活力为100％，计算在不同条件下的相对酶活力，结果如图2所示，重组昆布多糖降解酶的最适反应温度为45℃，最适pH为6.0，同时其最适温度测定实验结果显示该重组昆布多糖降解酶在40～50℃酶活均 比较高。重组昆布多糖降解酶在35℃和40℃下放置48h，酶活仍可保留90％以上；在pH3.0～ 10.0的缓冲液中保留48h，酶活也能保留50％以上，说明酶活稳定性较好。The purified laminarin degrading enzyme obtained in Example 4 was reacted at different temperatures and pHs, and the effects of temperature and pH on the enzyme activity were determined. Select the temperature of 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C, and react for 10 min according to the method for determination of specific enzyme activity of laminarin degrading enzyme in Example 5 to determine the optimum temperature, and measure the enzyme activity by DNS method. At 45 °C, a buffer with a pH of 3.0 to 10.0 was selected as the pH buffer for different determinations of the enzyme reaction, and the optimum pH of the laminarin degrading enzyme was determined according to the enzymatic activity of the laminarin degrading enzyme. The enzyme was incubated at temperatures of 35°C, 40°C, 45°C, and 50°C, and its residual enzyme activity was measured at the optimum condition of 45°C and pH 6.0 to obtain its temperature stability. They were mixed with buffers of different pH and incubated at 4°C, and their residual enzyme activities were measured at the optimum temperature to obtain their pH stability. Taking the highest enzyme activity as 100%, the relative enzyme activity under different conditions was calculated. The results are shown in Figure 2. The optimal reaction temperature of the recombinant laminarin degrading enzyme is 45 °C, the optimal pH is 6.0, and the optimal temperature is The assay results showed that the recombinant laminarin degrading enzyme had relatively high enzymatic activity at 40-50℃. Recombinant laminarin degrading enzyme was placed at 35°C and 40°C for 48h, and the enzyme activity could still retain more than 90%; if it was retained in a buffer of pH 3.0 to 10.0 for 48h, the enzyme activity could also retain more than 50%, indicating that the enzyme activity was stable Sex is better.
实施例7测定金属离子和化学试剂对重组昆布多糖降解酶活性的影响Example 7 Determination of the effects of metal ions and chemical reagents on the activity of recombinant laminarin degrading enzymes
将不同的金属离子及化学试剂SDS和Na2EDTA分别加入昆布多糖降解酶OUC-BsLam26纯 酶中，使其终体系的浓度分别为1mM和10mM，在25℃条件下，放置1h，然后分别加入 使用pH 6.0柠檬酸盐配制的0.2％(w/v)的昆布多糖底物，置于45℃条件下，反应30min，用DNS法进行酶活的测定。结果如图3A所示，大部分的试剂对酶活有一定的促进作用，但是高浓度的Fe3+和Cu2+均抑制酶的活性。Different metal ions and chemical reagents SDS and Na2EDTA were added to the laminarin degrading enzyme OUC-BsLam26 pure enzyme, respectively, so that the final concentration of the system was 1mM and 10mM, respectively, at 25 ℃, placed for 1h, and then added respectively using pH 6.0 0.2% (w/v) laminarin substrate prepared with citrate was placed under the condition of 45°C, reacted for 30min, and the enzyme activity was measured by DNS method. The results are shown in Figure 3A, most of the reagents have a certain promoting effect on the enzyme activity, but high concentrations of Fe 3+ and Cu 2+ both inhibited the enzyme activity.
实施例8测定昆布多糖降解酶OUC-BsLam26的底物选择性研究Example 8 Determination of substrate selectivity of laminarin degrading enzyme OUC-BsLam26
分别用pH 6.0的柠檬酸盐缓冲液配制0.2％(w/v)的昆布多糖、茯苓多糖、酵母β-葡 聚糖、和凝胶多糖。分别取不同反应底物200μL，然后加入50μL的昆布多糖降解酶 OUC-BsLam26纯酶混合均匀，在最适条件下反应，用DNS法测定酶活。结果如图3B所示，昆 布多糖降解酶OUC-BsLam26对昆布多糖的催化活性最强，其次是凝胶多糖，酵母β-葡聚糖， 对茯苓多糖、羧甲基纤维素钠(CMC-Na)和琼脂糖没有活性。0.2% (w/v) laminarin, tuckahoe polysaccharide, yeast β-glucan, and curdlan were formulated in citrate buffer pH 6.0, respectively. Take 200 μL of different reaction substrates respectively, then add 50 μL of laminarin degrading enzyme OUC-BsLam26 pure enzyme and mix well, react under optimal conditions, and measure the enzyme activity by DNS method. The results are shown in Fig. 3B, the laminarin-degrading enzyme OUC-BsLam26 has the strongest catalytic activity on laminarin, followed by curdlan, yeast β-glucan, tuckahoe polysaccharide, sodium carboxymethyl cellulose (CMC-Na ) and agarose are inactive.
实施例9TLC法鉴定昆布多糖降解酶OUC-BsLam26的降解产物Example 9 Identification of the degradation products of laminarin degrading enzyme OUC-BsLam26 by TLC
将实施例4中所得昆布多糖降解酶OUC-BsLam26与0.2％的昆布多糖在45℃下反应不同 时间，然后通过薄层色谱层析法(TLC)检测产物。具体方法如下：展开剂(正丁醇：乙酸： 水＝2：1：1)，显色剂为0.2％(w/v)3,5-二羟基甲苯溶于10％(v/v)H2SO4中于75℃显色。以葡萄糖为参照对降解产物进行TLC分析，如图4所示，昆布多糖酶OUC-BsLam26的酶解产物主要由葡萄糖、昆布二糖、昆布三糖和昆布四糖四部分组成。The laminarin degrading enzyme OUC-BsLam26 obtained in Example 4 was reacted with 0.2% laminarin at 45° C. for different times, and then the product was detected by thin layer chromatography (TLC). The specific method is as follows: developing agent (n-butanol:acetic acid:water=2:1:1), color developer is 0.2% (w/v) 3,5-dihydroxytoluene dissolved in 10% (v/v) H Developed in 2 SO 4 at 75°C. Using glucose as a reference, the degradation products were analyzed by TLC. As shown in Figure 4, the enzymatic hydrolysis products of laminarinase OUC-BsLam26 were mainly composed of four parts: glucose, laminabiose, laminarin trisaccharide and laminarin tetrasaccharide.
实施例10液相检测鉴定昆布多糖降解酶OUC-BsLam26的降解产物Example 10 Liquid-phase detection and identification of degradation products of laminarin degrading enzyme OUC-BsLam26
将实施例4中所得昆布多糖降解酶OUC-BsLam-26与0.2％的昆布多糖在45℃下分别孵育 不同的时间，然后通过高效液相色谱法对酶解产物进行检测。如图5所示，昆布多糖酶OUC-BsLam26的酶解产物主要是昆布二糖、昆布三糖、昆布四糖以及昆布五糖。The laminarin degrading enzyme OUC-BsLam-26 obtained in Example 4 was incubated with 0.2% laminarin at 45°C for different times, and then the enzymatic hydrolysis products were detected by high performance liquid chromatography. As shown in Figure 5, the enzymatic hydrolysis products of laminarinase OUC-BsLam26 are mainly lamina disaccharide, lamina trisaccharide, lamina tetrasaccharide and lamina pentasaccharide.
实施例11定义重组昆布多糖降解酶产物聚合度组成Example 11 Defining the composition of the degree of polymerization of recombinant laminarin degrading enzyme products
将实施例4中所得昆布多糖降解酶OUC-BsLam26与0.2％的昆布多糖在45℃下反应，然后 通过ESI-MS检测产物。如图6所示，结果表明产物中含有的昆布寡糖有昆布三糖、昆布四糖、 昆布五糖。The laminarin degrading enzyme OUC-BsLam26 obtained in Example 4 was reacted with 0.2% laminarin at 45°C, and then the product was detected by ESI-MS. As shown in Figure 6, the results show that the kelp oligosaccharides contained in the product include kelp trisaccharide, kelp tetrasaccharide, and kelp pentasaccharide.
实施例12鉴定重组昆布多糖降解酶完全转化后的产物Example 12 Identification of the product after complete conversion of recombinant laminarin degrading enzyme
将实施例4中所得昆布多糖降解酶OUC-BsLam26与1％的昆布多糖在45℃下反应6h，然 后通过TLC检测产物。如图7所示，结果表明酶解产物为葡萄糖、昆布二糖、昆布三糖以及 昆布四糖。The laminarin-degrading enzyme OUC-BsLam26 obtained in Example 4 was reacted with 1% laminarin at 45°C for 6 h, and then the product was detected by TLC. As shown in Figure 7, the results showed that the enzymatic hydrolysis products were glucose, laminabiose, laminatriose and laminatetraose.
实施例13定义昆布多糖降解酶制备昆布寡糖Example 13 Definition of laminarin degrading enzymes to prepare lamina oligosaccharides
将实施例4中所得昆布多糖降解酶OUC-BsLam26与1％浓度的昆布多糖在45℃下反应6h， 采用制备型硅胶柱层析进行分离和制备，流动相为：正丁醇：乙酸：水＝2：1：1(v/v/v)， 然后通过TLC检测产物。如图8所示，结果表明酶解产物有葡萄糖、昆布二糖、昆布三糖以 及昆布四糖，4中包含4种寡糖，5和6中有昆布三糖和昆布四糖，而7和8中制备出了单 一的昆布四糖，实现了昆布四糖的分离制备。The laminarin degrading enzyme OUC-BsLam26 obtained in Example 4 was reacted with 1% laminarin at 45° C. for 6 h, and the separation and preparation were carried out by preparative silica gel column chromatography. The mobile phase was: n-butanol: acetic acid: water =2:1:1 (v/v/v), then the product was detected by TLC. As shown in Figure 8, the results showed that the enzymatic hydrolysis products were glucose, laminabiose, laminatrisaccharide and laminarin tetrasaccharide, 4 contained 4 kinds of oligosaccharides, 5 and 6 contained lamina trisaccharides and lamina tetrasaccharides, and 7 and 6 contained laminarin tetrasaccharides. In 8, a single kelp tetrasaccharide was prepared, and the separation and preparation of kelp tetrasaccharide was realized.
实施例14利用重组昆布多糖降解酶制备酶制剂Example 14 Preparation of enzyme preparation using recombinant laminarin degrading enzyme
利用实施例4制备的重组昆布多糖降解酶制备酶制剂：将发酵破碎后的溶液纯化后，用 缓冲液置换咪唑，冻干后保存酶粉。Utilize the recombinant laminarin degrading enzyme prepared in Example 4 to prepare an enzyme preparation: after purifying the fermented and broken solution, replace imidazole with buffer, and store the enzyme powder after freeze-drying.
实施例15利用重组昆布多糖降解酶制备昆布寡糖Example 15 Preparation of lamina oligosaccharides using recombinant laminarin degrading enzymes
利用实施例4制备的重组昆布多糖降解酶制备昆布寡糖：将酶解后的寡糖分离纯化后， 制备昆布寡糖，冻干后保存昆布寡糖粉末。Preparation of lamina oligosaccharides by using the recombinant laminarin degrading enzyme prepared in Example 4: after separating and purifying the oligosaccharides after enzymatic hydrolysis, lamina oligosaccharides were prepared, and lamina oligosaccharide powder was stored after freeze-drying.
给本领域技术人员提供上述实施例，以完全公开和描述如何实施和使用所主张的实施方 案，而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权 利要求的范围内。The foregoing examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications obvious to those skilled in the art are intended to be within the scope of the appended claims.
序列表 sequence listing
<110> 中国海洋大学<110> Ocean University of China
<120> 昆布多糖降解酶OUC-BsLam26及其应用<120> Laminaria degrading enzyme OUC-BsLam26 and its application
<141> 2021-12-16<141> 2021-12-16
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Pro Gly Lys Pro Asn Ala Leu Thr Asp Leu Gly Thr Val Phe Val AsnPro Gly Lys Pro Asn Ala Leu Thr Asp Leu Gly Thr Val Phe Val Asn
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Met Ser Thr Leu Asp Lys Thr Ala Tyr Tyr Asn Lys Asn Gln Val IleMet Ser Thr Leu Asp Lys Thr Ala Tyr Tyr Asn Lys Asn Gln Val Ile
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Pro Ala Glu His Tyr Ser Ser Ile Asn Thr Val Glu Asn Leu Thr SerPro Ala Glu His Tyr Ser Ser Ile Asn Thr Val Glu Asn Leu Thr Ser
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Val Ser Ala His Leu Arg Pro Thr Thr Asp Ile Ser Gly Ile Leu AspVal Ser Ala His Leu Arg Pro Thr Thr Asp Ile Ser Gly Ile Leu Asp
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gcacacccgg aatgtaaata tctacttgca tacaatgagc ctaatctgac agaccaggca 420gcacacccgg aatgtaaata tctacttgca tacaatgagc ctaatctgac agaccaggca 420
agaatgacac cccaggaagc tgccgcacaa tggcctgcac tgcgtgcatt agcttctgaa 480agaatgacac cccaggaagc tgccgcacaa tggcctgcac tgcgtgcatt agcttctgaa 480
ctaaatctga agctcatatc tccggctatg aactacggaa cattgccgga ttatagtgac 540ctaaatctga agctcatatc tccggctatg aactacggaa cattgccgga ttatagtgac 540
ccgatcaaat ggttggacga gtttttcagc aacgttcccc tcagtgatgt agacggtata 600ccgatcaaat ggttggacga gtttttcagc aacgttcccc tcagtgatgt agacggtata 600
gctatccatt gttacatgga aagcccttct tctcttatca actatgtaag catgttcaaa 660gctatccatt gttacatgga aagcccttct tctcttatca actatgtaag catgttcaaa 660
aaatatggaa aacctatttg gttaacagaa ttctgtgcat ggcccagtag tgataagatt 720aaatatggaa aacctatttg gttaacagaa ttctgtgcat ggcccagtag tgataagatt 720
agcatatcaa gccagatgaa ctatatgagc gagactcttc attatcttga gtccgatcct 780agcatatcaa gccagatgaa ctatatgagc gagactcttc attatcttga gtccgatcct 780
gacgtattcc gctatgcctg gttcatccca agaggtattg gaccgaccga tccaagtacc 840gacgtattcc gctatgcctg gttcatccca agaggtattg gaccgaccga tccaagtacc 840
ggtacaacca gcaacagctt attacccggt aagcccaatg cattgactga cctcggaaca 900ggtacaacca gcaacagctt attacccggt aagcccaatg cattgactga cctcggaaca 900
gtgtttgtga atatgtccac tctggataaa actgcttatt acaataagaa tcaagttatt 960gtgtttgtga atatgtccac tctggataaa actgcttatt acaataagaa tcaagttatt 960
cccgccgaac actacagttc aataaacaca gttgagaatt taacatcagt cagcgcacac 1020cccgccgaac actacagttc aataaacaca gttgagaatt taacatcagt cagcgcacac 1020
ctgcgcccta caaccgatat ttcgggcata ttggatgttt atgatttgaa acaggaacaa 1080ctgcgcccta caaccgatat ttcgggcata ttggatgttt atgatttgaa acaggaacaa 1080
tggctgaaat atcaactgga tgctcctaaa gccggaactt accagttgga tatccgctat 1140tggctgaaat atcaactgga tgctcctaaa gccggaactt accagttgga tatccgctat 1140
accagtttca gagataacaa ggttgagata actatagaca aaggtacggc agcaacacta 1200accagtttca gagataacaa ggttgagata actatagaca aaggtacggc agcaacacta 1200
gacctgccta atgtagacag caaatggact actgtcacta ccaacatcca actaaaagca 1260gacctgccta atgtagacag caaatggact actgtcacta ccaacatcca actaaaagca 1260
ggtaagcaaa cgctacgtat aaaagtcact acaggcaata ttgcattaaa ttggctgcgt 1320ggtaagcaaa cgctacgtat aaaagtcact acaggcaata ttgcattaaa ttggctgcgt 1320
ttcaaagact aa 1332ttcaaagact aa 1332
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| CN115927508A (en) * | 2022-08-29 | 2023-04-07 | 中国海洋大学 | Application of lamina polysaccharide degrading enzyme OUC-ScLam39 in the preparation of lamina oligosaccharides |
| CN116083402A (en) * | 2023-02-02 | 2023-05-09 | 江苏大学 | A kind of β-1,3-glucanase PeBgl1 and its application |
| CN117778354A (en) * | 2024-02-23 | 2024-03-29 | 中国海洋大学 | Laminarin degrading enzyme OUC-ScLam39 mutant and encoding gene and application thereof |
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| CN112708609A (en) * | 2021-02-22 | 2021-04-27 | 中国海洋大学 | Chitosanase OUC-CsnPa and application thereof |
| CN113667661A (en) * | 2021-07-26 | 2021-11-19 | 青岛大学 | Beta-glucosidase and application thereof in preparation of glucose and laminarin oligosaccharide |
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| CN112708609A (en) * | 2021-02-22 | 2021-04-27 | 中国海洋大学 | Chitosanase OUC-CsnPa and application thereof |
| CN113667661A (en) * | 2021-07-26 | 2021-11-19 | 青岛大学 | Beta-glucosidase and application thereof in preparation of glucose and laminarin oligosaccharide |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927508A (en) * | 2022-08-29 | 2023-04-07 | 中国海洋大学 | Application of lamina polysaccharide degrading enzyme OUC-ScLam39 in the preparation of lamina oligosaccharides |
| CN116083402A (en) * | 2023-02-02 | 2023-05-09 | 江苏大学 | A kind of β-1,3-glucanase PeBgl1 and its application |
| CN116083402B (en) * | 2023-02-02 | 2024-05-10 | 江苏大学 | Beta-1, 3-glucanase PeBgl-1 and application thereof |
| CN117778354A (en) * | 2024-02-23 | 2024-03-29 | 中国海洋大学 | Laminarin degrading enzyme OUC-ScLam39 mutant and encoding gene and application thereof |
| CN117778354B (en) * | 2024-02-23 | 2024-04-26 | 中国海洋大学 | Laminarin degrading enzyme OUC-ScLam39 mutant and encoding gene and application thereof |
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