技术领域Technical field
本发明涉及一种多酶协同降解动物组织制备胶原蛋白寡肽和寡聚糖的方法，属于生物技术领域。The invention relates to a method for preparing collagen oligopeptides and oligosaccharides by synergistically degrading animal tissue with multiple enzymes, and belongs to the field of biotechnology.
背景技术Background technique
胶原蛋白，简称胶原，是一种结构蛋白，大量存在于人体结缔组织如软骨和皮肤等的细胞外基质中，同时也是哺乳动物中含量最丰富的蛋白，约占人体总蛋白的三分之一。胶原蛋白种类繁多，目前已经发现30种不同的胶原蛋白。胶原蛋白是由原胶原也就是单个胶原分子组成的胶原纤维，而胶原分子通常是由三条左螺旋的氨基酸链以右螺旋的方式组成三螺旋结构，最常见的Ⅰ，Ⅱ，Ⅲ型胶原蛋白的三螺旋结构长度约为300nm平均直径为1.5nm，氨基酸的组成却存在共同特点，三条链呈现Gly-X-Y的排列方式，甘氨酸残基占了总氨基酸残基的三分之一，X和Y通常分别为脯氨酸和羟脯氨酸，两者约占总氨基酸组成的四分之一。由于其特殊的三螺旋结构，天然胶原蛋白具有异常的稳定性，很难被一般的蛋白酶所降解。Collagen, referred to as collagen, is a structural protein that is found in large amounts in the extracellular matrix of human connective tissues such as cartilage and skin. It is also the most abundant protein in mammals, accounting for about one-third of the total protein in the human body. . There are many types of collagen, and 30 different types of collagen have been discovered so far. Collagen is a collagen fiber composed of procollagen, that is, a single collagen molecule. Collagen molecules are usually composed of three left-helical amino acid chains in a right-helical manner to form a triple helix structure. The most common collagen types I, II, and III are The length of the triple helix structure is about 300nm and the average diameter is 1.5nm. The composition of the amino acids has common characteristics. The three chains are arranged in a Gly-X-Y arrangement. Glycine residues account for one-third of the total amino acid residues. X and Y are usually They are proline and hydroxyproline respectively, which account for about a quarter of the total amino acid composition. Due to its special triple helix structure, natural collagen has exceptional stability and is difficult to be degraded by general proteases.
低分子量的胶原蛋白寡肽的制备方法主要分为两类：化学降解法和生物酶解法。化学降解法是目前商业化生产胶原蛋白的主要方式，尽管化学法生产低分子量胶原蛋白的工艺已经十分成熟，但也存在比如破坏氨基酸结构，产品组分单一性差、环境污染等问题。相比于化学降解法，生物酶解法具有反应条件温和、无污染、工艺简单、产品分子量易于控制、不会破坏氨基酸活性基团等优势。The preparation methods of low molecular weight collagen oligopeptides are mainly divided into two categories: chemical degradation methods and biological enzymatic hydrolysis methods. Chemical degradation is currently the main way to produce collagen commercially. Although the process of producing low molecular weight collagen through chemical methods is very mature, there are still problems such as damage to the amino acid structure, poor product component unity, and environmental pollution. Compared with chemical degradation methods, biological enzymatic hydrolysis methods have the advantages of mild reaction conditions, no pollution, simple process, easy control of product molecular weight, and will not destroy the active groups of amino acids.
硫酸软骨素，是共价连接在蛋白质上形成蛋白聚糖的一类糖胺聚糖。硫酸软骨素广泛分布于动物组织的细胞外基质和细胞表面，糖链由交替的葡萄糖醛酸和N-乙酰半乳糖胺(又称N-乙酰氨基半乳糖)二糖单位组成，通过一个似糖链接区连接到核心蛋白的丝氨酸残基上。硫酸软骨素表现出高生物活性，如抗癌症、抗炎、免疫调节、抗氧化等不仅在细胞转移、分化、增殖、识别以及组织形成等生物过程中起到了重要的作用，还应用于医药及临床。由于硫酸软骨素的分子量大，生物利用度低，在治疗脊柱损伤、轴突再生、抑制肿瘤发生时，硫酸软骨素的很多功效不能得到有效的发挥。目前降解产生硫酸软骨素的方法主要有：1)酸法降解，酸降解是指将硫酸软骨素溶解于酸溶液(如硫酸、盐酸)中在一定的溫度下进行降解，酸降解由于反应比较剧烈会导致硫酸根含量的降低，而且寡糖的自由基清除的能力也会降低；(2)氧化降解法，氧化降解法中最常用的方法是过氧化氢降解法，采用氧化降解法的优点是产物无毒，不需要特殊的后处理，对双糖的结构破坏比较小；(3)酶解法，酶解法是指利用硫酸软骨素酶或透明质酸酶等软骨素降解酶对硫酸软骨素酶解获得寡糖。其中，酶解法因条件温和、专一性强，是目前最常用的降解硫酸软骨素的方法。硫酸软骨素裂解酶(chondroitinase或chondroitin sulfateyase，简称为“ChSase”)是一类能将硫酸软骨素、软骨素、透明质酸等糖胺聚糖降解为不饱和二糖的裂解酶。其中，硫酸软骨素ABC裂解酶，为特异性最为广泛的一类硫酸软骨素裂解酶。Chondroitin sulfate is a type of glycosaminoglycan covalently linked to proteins to form proteoglycans. Chondroitin sulfate is widely distributed in the extracellular matrix and cell surface of animal tissues. The sugar chain is composed of alternating glucuronic acid and N-acetylgalactosamine (also known as N-acetylgalactosamine) disaccharide units, through a sugar-like The linker region is attached to the serine residues of the core protein. Chondroitin sulfate exhibits high biological activities, such as anti-cancer, anti-inflammatory, immune regulation, antioxidant, etc. It not only plays an important role in biological processes such as cell transfer, differentiation, proliferation, recognition, and tissue formation, but is also used in medicine and clinical. Due to the large molecular weight and low bioavailability of chondroitin sulfate, many of the effects of chondroitin sulfate cannot be effectively exerted when treating spinal injuries, axon regeneration, and inhibiting tumor occurrence. At present, the main methods for producing chondroitin sulfate through degradation are: 1) Acid degradation. Acid degradation refers to dissolving chondroitin sulfate in an acid solution (such as sulfuric acid, hydrochloric acid) and degrading it at a certain temperature. Acid degradation has a violent reaction due to the It will lead to a reduction in sulfate content, and the free radical scavenging ability of oligosaccharides will also be reduced; (2) Oxidative degradation method. The most commonly used method in oxidative degradation method is hydrogen peroxide degradation method. The advantages of using oxidative degradation method are The product is non-toxic, does not require special post-processing, and has relatively little structural damage to the disaccharide; (3) Enzymatic hydrolysis. The enzymatic hydrolysis method refers to the use of chondroitin degrading enzymes such as chondroitin sulfate or hyaluronidase to treat chondroitin sulfate. Solve to obtain oligosaccharides. Among them, enzymatic hydrolysis is currently the most commonly used method to degrade chondroitin sulfate due to its mild conditions and strong specificity. Chondroitin sulfate lyase (chondroitinase or chondroitin sulfateyase, referred to as "ChSase") is a type of lytic enzyme that can degrade glycosaminoglycans such as chondroitin sulfate, chondroitin, and hyaluronic acid into unsaturated disaccharides. Among them, chondroitin sulfate ABC lyase is the most specific type of chondroitin sulfate lyase.
我国是农产品生产大国，每年会产生大量不能利用的农副产品，其中动物来源的蛋白质，如软骨、角、皮肤、骨头和鳞片生产和加工会产生许多副产品。这些副产品中不能食用或不能在市场上出售，进而导致环境问题，与涉及极端条件(高温、高压、腐蚀性化学品)的化学过程相比，温和条件下的酶水解提供了一种更可接受的方法来获得低毒、低环境影响的高质量生物活性提取物，可以从这些副产物中提取硫酸软骨素和胶原蛋白，从而减轻环境污染提高产品附加值。但目前采用酶解方法处理动物组织时，通常需要先对动物组织进行预处理(如酸处理或碱处理)，而后再加入酶体系进行酶解，这种方法不仅工艺繁琐，且水解效果有限，大大增加了成本，限制了对农产品的生产及加工。Our country is a major producer of agricultural products and produces a large amount of unusable agricultural and sideline products every year. Among them, the production and processing of animal-derived proteins, such as cartilage, horns, skin, bones and scales, will produce many by-products. None of these by-products are edible or commercially available, leading to environmental concerns. Compared to chemical processes involving extreme conditions (high temperature, pressure, corrosive chemicals), enzymatic hydrolysis under mild conditions provides a more acceptable This method is used to obtain high-quality bioactive extracts with low toxicity and low environmental impact. Chondroitin sulfate and collagen can be extracted from these by-products, thereby reducing environmental pollution and increasing the added value of products. However, when enzymatic hydrolysis is currently used to treat animal tissue, it is usually necessary to pre-treat the animal tissue first (such as acid treatment or alkali treatment), and then add an enzyme system for enzymatic hydrolysis. This method is not only cumbersome, but also has limited hydrolysis effect. This greatly increases the cost and limits the production and processing of agricultural products.
发明内容Contents of the invention
为解决上述问题，本发明以来自Bacillus cereus VD021的胶原蛋白水解酶(Protein ID：R8HPH3)为出发序列进行突变改造，随后优化其在大肠杆菌重组表达，得到的胶原蛋白水解酶可高效水解胶原蛋白，将其全部降解成胶原蛋白寡肽。与此同时，本发明还基于来自P.vulgaris(GenBank:E08025.1)的硫酸软骨素裂解酶，提供了一种硫酸软骨素ABC裂解酶突变体，通过大肠杆菌重组表达得到，该突变体酶可高效水解硫酸软骨素，具有重要的经济价值和社会意义。In order to solve the above problems, the present invention uses the collagen hydrolase (Protein ID: R8HPH3) from Bacillus cereus VD021 as the starting sequence for mutation transformation, and then optimizes its recombinant expression in E. coli. The obtained collagen hydrolase can efficiently hydrolyze collagen. , which will all be degraded into collagen oligopeptides. At the same time, the present invention also provides a chondroitin sulfate ABC lyase mutant based on the chondroitin sulfate lyase from P. vulgaris (GenBank: E08025.1), which is obtained by recombinant expression in Escherichia coli. The mutant enzyme It can efficiently hydrolyze chondroitin sulfate and has important economic value and social significance.
本发明的第一个目的是提供一种降解动物组织的方法，包括以下步骤：The first object of the present invention is to provide a method for degrading animal tissue, comprising the following steps:
以未经处理的动物组织为底物，加入胶原蛋白水解酶突变体和/或硫酸软骨素ABC裂解酶突变体进行酶解反应，酶解一段时间后，得到降解后的产物；Using untreated animal tissue as the substrate, add a collagen hydrolase mutant and/or a chondroitin sulfate ABC lyase mutant to perform an enzymatic hydrolysis reaction. After a period of enzymatic hydrolysis, a degraded product is obtained;
所述胶原蛋白水解酶突变体的氨基酸序列如SEQ ID NO.2所示，所述硫酸软骨素ABC裂解酶突变体的氨基酸序列如SEQ ID NO.4所示。The amino acid sequence of the collagen hydrolase mutant is shown in SEQ ID NO.2, and the amino acid sequence of the chondroitin sulfate ABC lyase mutant is shown in SEQ ID NO.4.
进一步地，当加入胶原蛋白水解酶突变体进行降解时，酶解至少4小时，优选为12-24h。Further, when a collagen hydrolase mutant is added for degradation, the enzymatic hydrolysis is performed for at least 4 hours, preferably 12-24 hours.
进一步地，当加入硫酸软骨素ABC裂解酶突变体进行降解时，酶解至少4小时。Further, when chondroitin sulfate ABC lyase mutant is added for degradation, the enzyme is hydrolyzed for at least 4 hours.
进一步地，当加入胶原蛋白水解酶突变体时，降解后的产物中含有胶原蛋白寡肽(分子量<1kDa，尤其是分子量为30-25Da的短肽)。Furthermore, when a collagen hydrolase mutant is added, the degraded product contains collagen oligopeptides (molecular weight <1kDa, especially short peptides with a molecular weight of 30-25Da).
进一步地，当加入硫酸软骨素ABC裂解酶突变体时，降解后的产物中含有透明质酸寡聚糖和硫酸软骨素寡聚糖。Further, when a chondroitin sulfate ABC lyase mutant is added, the degraded product contains hyaluronic acid oligosaccharides and chondroitin sulfate oligosaccharides.
进一步地，当向体系中加入两种酶时，胶原蛋白水解酶突变体和硫酸软骨素ABC裂解酶突变体酶量比例为1-5:1-2，2.5:1时降解效果最佳。Furthermore, when two enzymes are added to the system, the enzyme amount ratio of collagen hydrolase mutant and chondroitin sulfate ABC lyase mutant is 1-5:1-2, and the best degradation effect is 2.5:1.
进一步地，酶解温度为30-37℃，优选为37℃。Further, the enzymatic hydrolysis temperature is 30-37°C, preferably 37°C.
进一步地，酶解pH为7.0-8.0，本发明实施例优选为7.5。Further, the pH of enzymatic hydrolysis is 7.0-8.0, and in the embodiment of the present invention, the pH is preferably 7.5.
进一步地，所述动物组织来源包括但不限于鱼、牛、猪、鸡、鸭、鹅等的软骨组织或者富含硫酸软骨素、胶原蛋白或透明质酸的组织或底物。Further, the animal tissue sources include, but are not limited to, cartilage tissues of fish, cattle, pigs, chickens, ducks, geese, etc., or tissues or substrates rich in chondroitin sulfate, collagen or hyaluronic acid.
本发明的第二个目的是提供一种酶组合物，所述组合物中含有胶原蛋白水解酶突变体和/或硫酸软骨素ABC裂解酶突变体；所述胶原蛋白水解酶突变体的氨基酸序列如SEQID NO.2所示，所述硫酸软骨素ABC裂解酶突变体的氨基酸序列如SEQ ID NO.4所示。The second object of the present invention is to provide an enzyme composition, which contains a collagen hydrolase mutant and/or a chondroitin sulfate ABC lyase mutant; the amino acid sequence of the collagen hydrolase mutant As shown in SEQ ID NO.2, the amino acid sequence of the chondroitin sulfate ABC lyase mutant is as shown in SEQ ID NO.4.
本发明的第三个目的是提供上述酶组合物在制备化妆品、功能食品或保健品中的应用，如将酶组合物水解得到的寡肽、寡聚糖添加进上述产品中。The third object of the present invention is to provide the application of the above-mentioned enzyme composition in the preparation of cosmetics, functional foods or health products, such as adding oligopeptides and oligosaccharides obtained by hydrolysis of the enzyme composition into the above-mentioned products.
本发明的第四个目的是提供一种胶原蛋白水解酶突变体，以SEQ ID NO.1所示的氨基酸序列为出发序列进行突变，所述突变为：将第74位苯丙氨酸突变为丝氨酸，并将第169位精氨酸突变为苏氨酸(F74S/R169T)，突变后的氨基酸序列如SEQ ID NO.2所示。The fourth object of the present invention is to provide a collagen hydrolase mutant, which uses the amino acid sequence shown in SEQ ID NO. 1 as the starting sequence for mutation, and the mutation is: mutating the phenylalanine at position 74 to Serine, and the arginine at position 169 was mutated into threonine (F74S/R169T). The amino acid sequence after mutation is shown in SEQ ID NO. 2.
本发明还提供编码上述胶原蛋白水解酶突变体的核酸。The present invention also provides nucleic acids encoding the above-mentioned collagen hydrolase mutants.
本发明还提供携带上述核酸的重组质粒。The present invention also provides recombinant plasmids carrying the above nucleic acids.
本发明还提供表达上述胶原蛋白水解酶突变体的宿主细胞。The present invention also provides host cells expressing the above-mentioned collagen hydrolase mutants.
进一步地，所述宿主细胞为细菌、真菌、植物细胞或动物细胞。Further, the host cell is a bacterium, fungus, plant cell or animal cell.
本发明还提供上述胶原蛋白水解酶突变体、基因、重组质粒或宿主细胞在制备含胶原蛋白寡肽的产品中的应用。The present invention also provides the use of the above collagen hydrolase mutants, genes, recombinant plasmids or host cells in the preparation of products containing collagen oligopeptides.
本发明还提供一种生产胶原蛋白寡肽的方法，通过上述胶原蛋白水解酶突变体水解含胶原蛋白的物质得到。The present invention also provides a method for producing collagen oligopeptide, which is obtained by hydrolyzing collagen-containing substances with the above-mentioned collagen hydrolase mutant.
本发明还提供一种生产上述胶原蛋白水解酶突变体的方法，包括以下步骤：以含有野生型胶原蛋白水解酶编码基因的质粒为模版，通过定点突变获得含有编码突变体基因的质粒，将该质粒转入大肠杆菌中，培养重组菌得到胶原蛋白水解酶突变体。The present invention also provides a method for producing the above-mentioned collagen hydrolase mutant, which includes the following steps: using a plasmid containing a wild-type collagen hydrolase coding gene as a template, obtaining a plasmid containing a coding mutant gene through site-directed mutagenesis, and converting the The plasmid was transferred into Escherichia coli, and the recombinant bacteria were cultured to obtain a collagen hydrolase mutant.
进一步地，以E.coli BL21(DE3)为出发菌株。Further, E. coli BL21 (DE3) was used as the starting strain.
进一步地，以pET系列质粒为表达载体。Further, pET series plasmids were used as expression vectors.
本发明的第五个目的是提供一种硫酸软骨素ABC裂解酶突变体，以SEQ ID NO.3所示的氨基酸序列为出发序列进行突变，所述突变为：将第107位谷氨酸突变为精氨酸，并将第264位丝氨酸突变为苯丙氨酸(E107R/S264F)，突变后的氨基酸序列如SEQ ID NO.4所示。The fifth object of the present invention is to provide a chondroitin sulfate ABC lyase mutant, using the amino acid sequence shown in SEQ ID NO. 3 as the starting sequence for mutation, and the mutation is: mutating glutamic acid at position 107 is arginine, and the serine at position 264 is mutated into phenylalanine (E107R/S264F). The amino acid sequence after mutation is shown in SEQ ID NO.4.
本发明还提供编码上述硫酸软骨素ABC裂解酶的核酸。The present invention also provides nucleic acids encoding the above-mentioned chondroitin sulfate ABC lyase.
本发明还提供携带上述核酸的重组质粒。The present invention also provides recombinant plasmids carrying the above nucleic acids.
本发明还提供表达上述硫酸软骨素ABC裂解酶突变体的宿主细胞。The present invention also provides host cells expressing the above-mentioned chondroitin sulfate ABC lyase mutants.
进一步地，所述宿主细胞为细菌、真菌、植物细胞或动物细胞。Further, the host cell is a bacterium, fungus, plant cell or animal cell.
本发明还提供上述硫酸软骨素ABC裂解酶突变体、基因、重组质粒或宿主细胞在制备含硫酸软骨素寡聚糖和/或含透明质酸寡聚糖的产品中的应用。The present invention also provides the use of the above chondroitin sulfate ABC lyase mutants, genes, recombinant plasmids or host cells in the preparation of products containing chondroitin sulfate oligosaccharides and/or hyaluronic acid oligosaccharides.
本发明还提供一种生产硫酸软骨素寡聚糖和/或含透明质酸寡聚糖的方法，通过上述硫酸软骨素ABC裂解酶突变体水解含硫酸软骨素和/或含透明质酸的物质(如动物组织)得到。The present invention also provides a method for producing chondroitin sulfate oligosaccharides and/or hyaluronic acid-containing oligosaccharides, by hydrolyzing chondroitin sulfate-containing and/or hyaluronic acid-containing substances through the above-mentioned chondroitin sulfate ABC lyase mutant. (such as animal tissue) obtained.
本发明还提供一种生产上述硫酸软骨素ABC裂解酶突变体的方法，包括以下步骤：以含有野生型硫酸软骨素ABC裂解酶编码基因的质粒为模版，通过定点突变获得含有编码突变体基因的质粒，将该质粒转入大肠杆菌中，培养重组菌得到硫酸软骨素ABC裂解酶突变体。The invention also provides a method for producing the above-mentioned chondroitin sulfate ABC lyase mutant, which includes the following steps: using a plasmid containing a wild-type chondroitin sulfate ABC lyase encoding gene as a template, obtaining a plasmid containing the encoding mutant gene through site-directed mutagenesis. The plasmid is transferred into Escherichia coli, and the recombinant bacteria are cultured to obtain the chondroitin sulfate ABC lyase mutant.
进一步地，以E.coli BL21(DE3)为出发菌株。Further, E. coli BL21 (DE3) was used as the starting strain.
进一步地，以pRSF质粒为表达载体。Further, pRSF plasmid was used as the expression vector.
本发明的有益效果：Beneficial effects of the present invention:
本发明提供了一种可高效降解底物的水解酶突变体和硫酸软骨素裂解酶ABC突变体，用获得的胶原蛋白水解酶突变体F74S/R169T和硫酸软骨素ABC裂解酶突变体E107R/S264F降解新鲜的未经处理纯化的动物组织制备低分子量胶原蛋白、低分子量硫酸软骨素、低分子量透明质酸，证明其具有良好的降解效果。The invention provides a hydrolase mutant and a chondroitin sulfate lyase ABC mutant that can efficiently degrade substrates, using the obtained collagen hydrolase mutant F74S/R169T and chondroitin sulfate ABC lyase mutant E107R/S264F. Degrading fresh unprocessed and purified animal tissues to prepare low molecular weight collagen, low molecular weight chondroitin sulfate, and low molecular weight hyaluronic acid proved to have good degradation effects.
附图说明Description of the drawings
图1为胶原蛋白水解酶突变体F74S/R169T降解鸡胸软骨中胶原蛋白的时间过程。Figure 1 shows the time course of collagen hydrolase mutant F74S/R169T degrading collagen in chicken breast cartilage.
图2为硫酸软骨素ABC裂解酶突变体E107R/S264F降解鸡胸软骨中硫酸软骨素的时间过程。Figure 2 shows the time course of chondroitin sulfate sulfate degradation in chicken breast cartilage by the chondroitin sulfate ABC lyase mutant E107R/S264F.
图3为硫酸软骨素ABC裂解酶突变体E107R/S264F降解鸡胸骨软骨中透明质酸的时间过程。Figure 3 shows the time course of degradation of hyaluronic acid in chicken sternal cartilage by the chondroitin sulfate ABC lyase mutant E107R/S264F.
图4为胶原蛋白水解酶突变体F74S/R169T和硫酸软骨素ABC裂解酶突变体E107R/S264F协同降解新鲜未经加工的鸡胸软骨的时间过程。Figure 4 shows the time course of the synergistic degradation of fresh unprocessed chicken breast cartilage by collagen hydrolase mutant F74S/R169T and chondroitin sulfate ABC lyase mutant E107R/S264F.
图5为野生型胶原蛋白水解酶和硫酸软骨素ABC裂解酶协同降解新鲜未经加工的鸡胸软骨的时间过程。Figure 5 shows the time course of the synergistic degradation of fresh unprocessed chicken breast cartilage by wild-type collagen hydrolase and chondroitin sulfate ABC lyase.
具体实施方式Detailed ways
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.
下述实施例涉及的材料及方法如下：The materials and methods involved in the following examples are as follows:
1、质粒构建试剂及测序验证均在上海生物生工公司购买和完成；各种分析纯试剂购自国药集团；鸡胸软骨购自农贸市场。1. Plasmid construction reagents and sequencing verification were purchased and completed at Shanghai Biotech Company; various analytical pure reagents were purchased from Sinopharm Group; chicken breast cartilage was purchased from the farmer's market.
2、培养基2. Culture medium
LB培养基：10g·L-1NaCl，10g·L-1胰蛋白胨，5g·L-1酵母粉。LB medium: 10g·L-1 NaCl, 10g·L-1 tryptone, 5g·L-1 yeast powder.
TB培养基：2.31g·L-1KH2PO4，12.54g·L-1K2HPO4，12g·L-1胰蛋白胨，24g·L-1酵母粉，4mL·L-1甘油。TB medium: 2.31g·L-1 KH2 PO4 , 12.54g·L-1 K2 HPO4 , 12g·L-1 tryptone, 24g·L-1 yeast powder, 4mL·L-1 glycerol.
3、胶原蛋白水解酶突变体F74S/R169T摇瓶培养3. Collagen hydrolase mutant F74S/R169T culture in shake flask
将pET-28a-ColVD021质粒与突变体F74S/R169T编码基因连接，构建重组质粒pET-28a-ColVD021-F74S/R169T，导入Escherichia coli BL21(DE3)中得到工程菌F74S/R169T，用于摇瓶培养；The pET-28a-ColVD021 plasmid was connected to the mutant F74S/R169T coding gene to construct the recombinant plasmid pET-28a-ColVD021-F74S/R169T, which was introduced into Escherichia coli BL21 (DE3) to obtain the engineering strain F74S/R169T for shake flask culture. ;
摇瓶培养具体步骤：在含有终浓度50μg·mL-1硫酸卡那霉素LB平板上划线，放37℃培养箱过夜培养，挑取单菌落到添加终浓度50μg·mL-1硫酸卡那霉素的5mL液体LB培养基里，放置弹簧摇床过夜培养10h左右，离心收集菌体。Specific steps for shake flask culture: draw a line on an LB plate containing kanamycin sulfate at a final concentration of 50 μg·mL-1 , place it in a 37°C incubator for overnight cultivation, pick a single colony and add kanamycin sulfate at a final concentration of 50 μg·mL-1 In 5 mL liquid LB medium containing mycomycin, place the culture medium on a spring shaker overnight for about 10 hours, and centrifuge to collect the cells.
4、硫酸软骨素ABC裂解酶突变体E107R/S264F摇瓶培养4. Chondroitin sulfate ABC lyase mutant E107R/S264F shake flask culture
将pRSF-csABC I质粒与突变体E107R/S264F编码基因连接，构建重组质粒pRSF-csABC I-E107R/S264F，导入E.coli BL21(DE3)菌中得到工程菌E107R/S264F，用于摇瓶培养；The pRSF-csABC I plasmid was connected to the mutant E107R/S264F coding gene to construct the recombinant plasmid pRSF-csABC I-E107R/S264F, which was introduced into E.coli BL21 (DE3) bacteria to obtain the engineering strain E107R/S264F, which was used for shake flask culture. ;
摇瓶培养具体步骤：接种于3mL含有50μg·mL-1卡那霉素的新鲜LB液体培养基，37℃、220rpm培养至对数中期(8-10h)，按1％(v/v)的接种量将种子液重新转接到30mL含有50μg·mL-1卡那霉素的新鲜LB液体培养基，30℃、220rpm培养3h，加入终浓度为0.2mM的IPTG诱导，25℃、220rpm继续培养8h，离心收集菌体。Specific steps for shake flask culture: inoculate into 3 mL of fresh LB liquid medium containing 50 μg·mL-1 kanamycin, culture at 37°C, 220 rpm to mid-logarithmic phase (8-10 h), and use 1% (v/v) The inoculum amount was transferred back to 30 mL of fresh LB liquid medium containing 50 μg·mL-1 kanamycin, and cultured at 30°C and 220rpm for 3 hours. IPTG with a final concentration of 0.2mM was added for induction, and the culture was continued at 25°C and 220rpm. 8h, centrifuge to collect the bacteria.
5、胶原蛋白水解酶酶活测定方法5. Method for measuring collagen hydrolase activity
本发明采用茚三酮显色法测定胶原酶的活性。The present invention adopts ninhydrin colorimetric method to measure the activity of collagenase.
标准曲线的绘制：Drawing of standard curve:
取11个5mL离心管，依次加入浓度为0mmol·L-1、0.06mmol·L-1、0.12mmol·L-1、0.18mmol·L-1、0.24mmol·L-1、0.30mmol·L-1、0.36mmol·L-1、0.42mmol·L-1、0.48mmol·L-1、0.54mmol·L-1、0.60mmol·L-1的甘氨酸标准溶液0.3mL。先后加入等体积的乙酸缓冲液和茚三酮显色液，混匀后放入沸水中，反应15min，随后用冷水冷却5min，加入等体积的60％乙醇，充分混合后吸取200μL到96孔板中，使用酶标仪测定样品在570nm下的吸光值，以甘氨酸浓度为横坐标，以减去空白对照后的吸光值为纵坐标，绘制标准曲线。Take 11 5mL centrifuge tubes and add them in sequence with concentrations of 0mmol·L-1 , 0.06mmol·L-1 , 0.12mmol·L-1 , 0.18mmol·L-1 , 0.24mmol·L-1 , and 0.30mmol·L- 1. 0.3 mL of glycine standard solution of 0.36mmol·L-1 , 0.42mmol·L-1, 0.48mmol·L-1, 0.54mmol·L -1 and 0.60mmol·L-1 . Add an equal volume of acetic acid buffer and ninhydrin chromogenic solution, mix well, put it into boiling water, react for 15 minutes, then cool with cold water for 5 minutes, add an equal volume of 60% ethanol, mix thoroughly, and pipet 200 μL into a 96-well plate. , use a microplate reader to measure the absorbance value of the sample at 570 nm, use the glycine concentration as the abscissa, and use the absorbance value after subtracting the blank control as the ordinate to draw a standard curve.
以来源于鱼鳞皮肤的胶原蛋白为底物，反应体系为：1.3mL胶原蛋白溶液，0.2mL粗酶液，在37℃水浴锅反应0.5h，加入1.5mL 10％三氯乙酸溶液终止反应。随后混匀并取样0.3mL至新的5mL离心管中，加入0.3mL乙酸缓冲液，混匀再加入0.3mL茚三酮显色液，混匀。具体操作同标准曲线的绘制，不同之处在于最终的反应液需要用60％乙醇适当稀释。Using collagen derived from fish scale skin as the substrate, the reaction system is: 1.3 mL collagen solution, 0.2 mL crude enzyme solution, react in a 37°C water bath for 0.5 h, and add 1.5 mL 10% trichloroacetic acid solution to terminate the reaction. Then mix and take a 0.3 mL sample into a new 5 mL centrifuge tube, add 0.3 mL acetic acid buffer, mix well, and then add 0.3 mL ninhydrin chromogenic solution and mix well. The specific operation is the same as that of drawing the standard curve, except that the final reaction solution needs to be appropriately diluted with 60% ethanol.
酶活力单位定义：Enzyme activity unit definition:
在37℃，pH 7.5(钙离子存在)条件下反应0.5h，每分钟水解胶原蛋白产生1μmol甘氨酸所需的酶量为1个酶活力单位U。When reacting for 0.5 hours at 37°C and pH 7.5 (in the presence of calcium ions), the amount of enzyme required to hydrolyze collagen to produce 1 μmol of glycine per minute is 1 unit of enzyme activity U.
6、胶原蛋白分子量测定6. Determination of collagen molecular weight
使用凝胶排阻色谱检测胶原蛋白的降解以及测定胶原蛋白肽的分子量。测定分子量时，使用50mM Tris-Hcl，5mM CaCl2，pH 7.5缓冲液溶解胶原蛋白，底物浓度为20mg·mL-1，加入不同终酶活的胶原蛋白水解酶，50℃反应，反应过程中定时取样，煮沸样品使蛋白灭活，12000rpm离心10min，上清液使用0.22μm水系滤膜去除杂质后测定样品分子量。高效液相色谱条件：色谱柱为TSK-G2000SWxl(7.8×300mm，5μm)，柱温为25℃；以浓度0.1M的NaNO3溶液为流动相，设定流速0.8mL·min-1；进样量为20μL；使用示差检测器检测。以不同分子量的胶原蛋白肽作为标准样品，制作不同标样洗脱时间与分子量之间的标准曲线。Gel exclusion chromatography was used to detect collagen degradation and determine the molecular weight of collagen peptides. When measuring the molecular weight, use 50mM Tris-Hcl, 5mM CaCl2 , pH 7.5 buffer to dissolve collagen, with a substrate concentration of 20mg·mL-1 , add collagen hydrolase with different final enzyme activities, and react at 50°C. During the reaction Take samples regularly, boil the sample to inactivate the protein, centrifuge at 12,000 rpm for 10 min, use a 0.22 μm water-based filter membrane to remove impurities from the supernatant, and then measure the molecular weight of the sample. High performance liquid chromatography conditions: the chromatographic column is TSK- G2000SWxl (7.8×300mm, 5μm), the column temperature is 25°C; NaNO3 solution with a concentration of 0.1M is used as the mobile phase, and the flow rate is set to 0.8mL·min-1 ; the injection volume is 20μL; a differential detector is used for detection. Use collagen peptides of different molecular weights as standard samples to create a standard curve between the elution time and molecular weight of different standard samples.
7、硫酸软骨素ABC裂解酶酶活测定方法7. Method for determination of chondroitin sulfate ABC lyase activity
采用动力学的方法表征硫酸软骨素裂解酶的酶活。硫酸软骨素裂解酶将硫酸软骨素和肝素降解成4,5-不饱和糖醛酸，该化合物在232nm处有最高吸收峰，摩尔消光系数为3800L·mol-1cm-1。Kinetic methods were used to characterize the enzymatic activity of chondroitin sulfate lyase. Chondroitin sulfate lyase degrades chondroitin sulfate and heparin into 4,5-unsaturated uronic acid. This compound has the highest absorption peak at 232nm and a molar extinction coefficient of 3800L·mol-1cm-1 .
硫酸软骨素裂解酶酶活单位定义:在30℃条件下，每分钟产生1μmol4,5-不饱和糖醛酸所需要的酶量。具体步骤:将40μL适量的酶液加入到960μL预热的硫酸软骨素底物溶液(20mM Tris-HCl，10mg·mL-1CSA，pH 7.4)中，吹吸混匀后，采用分光光度计进行检测，计算ΔA232/Δt。以热处理失活的酶按照上述操作作为空白对照。The definition of chondroitin sulfate lyase enzyme activity unit is: the amount of enzyme required to produce 1 μmol of 4,5-unsaturated uronic acid per minute at 30°C. Specific steps: Add 40 μL of appropriate amount of enzyme solution to 960 μL of preheated chondroitin sulfate substrate solution (20mM Tris-HCl, 10mg·mL-1CSA, pH 7.4), pipe and mix, and use a spectrophotometer for detection. , calculate ΔA232 /Δt. The enzyme inactivated by heat treatment was used as a blank control according to the above operation.
8、寡糖分子量测定8. Determination of oligosaccharide molecular weight
凝胶渗透色谱-液相色谱(GPC-HPLC)技术用于寡糖分子量的测定。分析条件:色谱柱:Linear column 7.8×300mm；流动相:0.1mol·L-1NaNO3溶液；流速:0.9mL·min-1；柱温:40℃；进样量:40μL。根据葡聚糖标准样品的洗脱体积，使用凝胶渗透色谱分析法制作出分子量和洗脱体积之间的标准曲线。在相同的条件下，测出每个样品的洗脱体积，GPC软件可计算出每个样品的重均分子量、数均分子量及分子量分布。Gel permeation chromatography-liquid chromatography (GPC-HPLC) technology is used to determine the molecular weight of oligosaccharides. Analysis conditions: chromatographic column: Linear column 7.8×300mm; mobile phase: 0.1mol·L-1NaNO3 solution; flow rate: 0.9mL·min-1 ; column temperature: 40°C; injection volume: 40μL. According to the elution volume of the dextran standard sample, a standard curve between molecular weight and elution volume was produced using gel permeation chromatography. Under the same conditions, the elution volume of each sample is measured, and the GPC software can calculate the weight average molecular weight, number average molecular weight and molecular weight distribution of each sample.
实施例1：胶原蛋白水解酶F74S/R169T突变后酶活Example 1: Enzyme activity of collagen hydrolase F74S/R169T mutation
为了考察突变后胶原蛋白水解酶酶活，降解10g/L鱼鳞胶原蛋白酶活为8.96±0.21U/mL，酶活相比野生型提高125％。In order to examine the enzyme activity of collagen hydrolase after mutation, the enzyme activity of degrading 10g/L fish scale collagen was 8.96±0.21U/mL, and the enzyme activity was increased by 125% compared to the wild type.
实施例2：硫酸软骨素ABC裂解酶E107R/S264F突变后酶活Example 2: Enzyme activity of chondroitin sulfate ABC lyase E107R/S264F mutation
为了考察突变后硫酸软骨素ABC裂解酶E107R/S264F，降解10g/L CSA突变体酶活为1.4×103U·L-1，酶活相比野生型提高46％。In order to examine the mutated chondroitin sulfate ABC lyase E107R/S264F, the enzyme activity of the mutant degrading 10g/L CSA was 1.4×103 U·L-1 , and the enzyme activity increased by 46% compared with the wild type.
实施例3：胶原蛋白水解酶F74S/R169T降解未经加工鸡胸软骨水解过程Example 3: Hydrolysis process of unprocessed chicken breast cartilage degraded by collagen hydrolase F74S/R169T
为了考察上述制备的胶原蛋白水解酶突变体水解制备胶原蛋白的效率，向2g未经加工的农产品鸡软骨中加入60U胶原蛋白水解酶突变体F74S/R169T并在降解过程中定时取样分析反应体系中胶原蛋白分子量分布的变化情况。In order to examine the efficiency of hydrolyzing collagen by the above-prepared collagen hydrolase mutant, 60 U of collagen hydrolase mutant F74S/R169T was added to 2 g of unprocessed agricultural product chicken cartilage, and samples were taken regularly during the degradation process to analyze the reaction system. Changes in collagen molecular weight distribution.
结果见图1。在反应的初期，胶原蛋白水解的底物的分子量迅速降低，之后逐渐趋于不变。大分子(分子量>5kDa)分布比例分别从3.02％降至0.5％，小分子(分子量<1kDa)比例分别从81.64％升高至98.01，其平均分子量为30-25Da，效果优于未经突变的胶原蛋白水解酶。The results are shown in Figure 1. In the early stage of the reaction, the molecular weight of the collagen hydrolyzed substrate decreased rapidly, and then gradually became constant. The distribution proportion of large molecules (molecular weight >5kDa) dropped from 3.02% to 0.5%, and the proportion of small molecules (molecular weight <1kDa) increased from 81.64% to 98.01 respectively. Its average molecular weight is 30-25Da, and the effect is better than that without mutation. Collagen hydrolase.
实施例4：硫酸软骨素ABC裂解酶E107R/S264F降解未经加工鸡胸软骨水解过程Example 4: Hydrolysis process of unprocessed chicken breast cartilage degraded by chondroitin sulfate ABC lyase E107R/S264F
为了考察上述制备的硫酸软骨素ABC裂解酶突变体水解制备寡聚糖的效率，向2g未经加工的农产品鸡软骨中加入60U硫酸软骨素ABC裂解酶突变体E107R/S264F，并在降解过程中定时取样分析反应体系中胶原蛋白分子量分布的变化情况。In order to examine the efficiency of hydrolysis of the chondroitin sulfate ABC lyase mutant prepared above to prepare oligosaccharides, 60 U of chondroitin sulfate ABC lyase mutant E107R/S264F was added to 2 g of unprocessed agricultural product chicken cartilage, and during the degradation process Regular sampling was performed to analyze changes in the molecular weight distribution of collagen in the reaction system.
在反应的初期，鸡胸软骨底物中的大分子硫酸软骨素分子量(图2)和透明质酸(图3)迅速降低，之后逐渐趋于不变。大分子使用GPC-HPLC的方法分析分子量随时间变化规律。从图2-3均可以看出，解聚的初始反应速率较快，分子量迅速降低。酶活越高，分子量降低速率越快。随后反应速率逐渐降低，直至分子量几乎不发生变化。通过控制硫酸软骨素ABC裂解酶E107R/S264F加酶量可以从未经加工的鸡胸软骨中提取出小分子量硫酸软骨素和硫透明质酸，效果优于未经突变的硫酸软骨素ABC裂解酶。In the early stage of the reaction, the molecular weight of macromolecules chondroitin sulfate (Figure 2) and hyaluronic acid (Figure 3) in the chicken breast cartilage substrate decreased rapidly, and then gradually became unchanged. Macromolecules use the GPC-HPLC method to analyze the changes in molecular weight over time. It can be seen from Figures 2-3 that the initial reaction rate of depolymerization is fast and the molecular weight decreases rapidly. The higher the enzyme activity, the faster the molecular weight decreases. The reaction rate then gradually decreases until there is almost no change in molecular weight. By controlling the amount of chondroitin sulfate ABC lyase E107R/S264F, small molecular weight chondroitin sulfate and sulfur hyaluronic acid can be extracted from unprocessed chicken breast cartilage, and the effect is better than that of unmutated chondroitin sulfate ABC lyase.
实施例5：胶原蛋白水解酶F74S/R169T和硫酸软骨素ABC裂解酶E107R/S264F组合降解未经加工鸡胸软骨水解过程Example 5: Hydrolysis process of unprocessed chicken breast cartilage by combining collagen hydrolase F74S/R169T and chondroitin sulfate ABC lyase E107R/S264F
采用胶原蛋白降解酶与硫素软骨素降解酶协同酶解动物软骨组织制备蛋白寡肽和寡聚糖的体系为：加入胶原蛋白水解酶突变体F74S/R169T和硫酸软骨素ABC裂解酶突变体E107R/S264F酶活为2.5:1时，降解效果最佳。因此以该比例添加突变体或野生酶进行鸡软骨的水解。The system of using collagen degrading enzyme and thiochondroitin degrading enzyme to enzymatically hydrolyze animal cartilage tissue to prepare protein oligopeptides and oligosaccharides is as follows: adding collagen hydrolase mutant F74S/R169T and chondroitin sulfate ABC lyase mutant E107R /S264F enzyme activity is 2.5:1, the degradation effect is the best. Therefore, the mutant or wild enzyme was added at this ratio to hydrolyze chicken cartilage.
突变体组合的酶解结果见图4，野生型组合的酶解结果见图5(其中，低分子量胶原蛋白指分子量<1kDa)。在反应的初期，鸡胸软骨底物中的硫酸软骨素大分子量、透明质酸大分子量迅速降低且胶原蛋白小分子量迅速上升，随后反应速率逐渐降低，直至分子量几乎不发生变化。大分子使用GPC-HPLC的方法分析分子量随时间变化规律。而从未经突变的胶原蛋白水解酶和硫酸软骨素ABC裂解酶降解鸡胸软骨效果来看，其反应速率、低分子量胶原蛋白含量以及硫酸软骨素分子量等都明显差于突变体协同酶解。The enzymatic hydrolysis results of the mutant combination are shown in Figure 4, and the enzymatic hydrolysis results of the wild-type combination are shown in Figure 5 (wherein, low molecular weight collagen refers to molecular weight <1kDa). In the early stage of the reaction, the macromolecular weight of chondroitin sulfate and hyaluronic acid in the chicken breast cartilage substrate decreased rapidly and the small molecular weight of collagen increased rapidly. Then the reaction rate gradually decreased until the molecular weight almost did not change. Macromolecules use the GPC-HPLC method to analyze the changes in molecular weight over time. Judging from the degradation of chicken breast cartilage by unmutated collagen hydrolase and chondroitin sulfate ABC lyase, the reaction rate, low molecular weight collagen content and chondroitin sulfate molecular weight were significantly worse than the mutant synergistic enzymatic hydrolysis.
综上，通过控制胶原蛋白水解酶F74S/R169T和硫酸软骨素ABC裂解酶E107R/S264F加酶量并按照一定比例添加胶原蛋白水解酶F74S/R169T和硫酸软骨素ABC裂解酶E107R/S264F可以从未经加工的鸡胸软骨中提取出小分子量硫酸软骨素、胶原蛋白寡肽和透明质酸。In summary, by controlling the amount of collagen hydrolase F74S/R169T and chondroitin sulfate ABC lyase E107R/S264F and adding collagen hydrolase F74S/R169T and chondroitin sulfate ABC lyase E107R/S264F according to a certain ratio, it can be achieved from never. Small molecular weight chondroitin sulfate, collagen oligopeptides and hyaluronic acid were extracted from processed chicken breast cartilage.
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or modifications may be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.
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|---|---|---|---|---|
| CN119570766A (en)* | 2025-02-08 | 2025-03-07 | 成都恒福禧生物技术有限公司 | Collagenase mutant, gene fragment, recombinant plasmid, recombinant expression system, collagen peptide, and preparation method and use thereof |
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