WO2021093588A1 - 一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠及其制备方法和应用 - Google Patents

一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠及其制备方法和应用 Download PDF

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WO2021093588A1
WO2021093588A1 PCT/CN2020/124301 CN2020124301W WO2021093588A1 WO 2021093588 A1 WO2021093588 A1 WO 2021093588A1 CN 2020124301 W CN2020124301 W CN 2020124301W WO 2021093588 A1 WO2021093588 A1 WO 2021093588A1
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potato
acyl hydrolase
ester acyl
beads
nanomagnetic
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PCT/CN2020/124301
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English (en)
French (fr)
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吴金鸿
吴乔羽
翁晴
许璟珅
周密
王正武
于颖
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上海交通大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01023—Acylglycerol lipase (3.1.1.23)

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  • This application relates to the technical field of protein purification, in particular to a functional nano magnetic bead for enriching potato ester acyl hydrolase, and a preparation method and application thereof.
  • Patatin molecular weight 40-45kDa
  • protease inhibitors molecular weight 5-25kda
  • other proteins molecular weight above 50kDa.
  • Patain is a group of globular glycoproteins that specifically exist in potato tubers, accounting for about 40% of the total tuber protein. Under natural conditions, they usually exist as dimers. They were first isolated from potato tubers in 1980 by RACUSEN et al. come out. In addition to its antioxidant activity, Patain also has good emulsifying, foaming and gelling properties. These characteristics make it a good food additive to act in food.
  • Patatin has non-specific ester acyl hydrolase activity (LAH), and has certain effects on phospholipids, sugar esters, monoacyl and diacylglycerols, and long-chain fatty acid esters. Specificity. researchers added Patatin to red wine, cheese and other foods to observe its clarification and flavor improvement. The results proved that potato ester acyl hydrolase can play a certain role in the food industry as a food additive.
  • LAH ester acyl hydrolase activity
  • Nano magnetic beads refer to small magnetic particles whose size can be measured in nanometers, generally 1-100nm. This type of magnetic beads has superparamagnetic magnetism and has strong magnetic responsiveness in an external magnetic field. After the magnetic field is removed, the magnetic particles of the magnetic particles disappear immediately and are uniformly dispersed in the solution again. This feature can help nano-magnetic beads absorb a certain component in the liquid, and then magnetically separate the magnetic beads to achieve the purpose of separation and purification.
  • Carboxyl nanomagnetic beads have a large specific surface area and a high content of carboxyl groups on the surface, which can react with primary amine groups or sulfhydryl groups of proteins, peptides, and antibodies, and couple them to the surface of the magnetic beads. When the magnetic microspheres are put into the solution containing the target protein, the target protein will be tightly combined with the magnetic microspheres, which can be separated by using an external magnetic field.
  • Lectin is a glycoprotein or sugar-binding protein that can be purified from plants, invertebrates, and higher animals. Because it can agglutinate red blood cells, it is called lectin. Lectins can recognize glycoproteins and sugar groups on the surface of cell membranes, and a lectin can specifically bind to a specific sugar group. Therefore, lectins play a very important role in scientific research.
  • one aspect of this application provides a method for preparing functional nano magnetic beads for enriching potato ester acyl hydrolase, which includes the following steps:
  • Step 1 Activate carboxyl nanomagnetic beads: transfer the carboxyl nanomagnetic beads to a centrifuge tube, wash the carboxyl nanomagnetic beads with MES buffer and magnetically separate them, then resuspend the carboxyl nanomagnetic beads in MES buffer and add Ethyl-dimethylamine-propylcarbodiimide (EDAC) is mixed, mixed evenly and washed with MES buffer solution, and then precipitated after magnetic separation to obtain activated nano magnetic beads;
  • EDAC Ethyl-dimethylamine-propylcarbodiimide
  • Step 2 Coupling lectin: Dissolve the Reticulosporum aureus aureus lectin in MES buffer, add it to the activated nanomagnetic beads, shake well and spin for 16-24 hours, wash with MES buffer, and take it after magnetic separation Precipitate, add a quenching solution to the precipitate, mix homogeneously and then magnetically separate, and remove the supernatant to obtain functional nano magnetic beads coupled with the Dictyostelium aureum lectin for enriching potato ester acyl hydrolase.
  • step 1 the carboxyl nanomagnetic beads are superparamagnetic beads.
  • the carboxylated magnetic nanobeads contain iron oxide, and the mass ratio of the iron oxide in the carboxylated magnetic nanobeads is greater than 90%.
  • the concentration of the MES buffer is 0.05 mol/L, and the pH is 5.2.
  • step 1 the mass ratio of ethyl-dimethylamine-propylcarbodiimide of the carboxyl nanomagnetic beads is 5:8-5:4.
  • step 2 the mass of the total protein in the Dictyostelium aureus aureus lectin required per milligram of activated nanomagnetic beads is 20-500 ⁇ g.
  • the quenching solution is a 1 mol/L glycine solution with a pH of 8.0.
  • the preservation method of the functional nano magnetic beads for enriching potato ester acyl hydrolase obtained in step 2 is: washing the functional nano magnetic beads with MES buffer for several times and then resuspending in MES buffer.
  • the functional nanomagnetic beads are resuspended in MES buffer at a concentration of 5 mg/mL.
  • the storage temperature of the functional nano magnetic beads is -20°C.
  • Another aspect of the present invention provides a functional nano magnetic bead for enriching potato ester acyl hydrolase prepared according to the above method, and the functional nano magnetic bead is coupled with a Dictyostelium aureus aureus agglutinin.
  • the third aspect of the present invention provides an application of the above-mentioned functional nano magnetic beads in enriching potato ester acyl hydrolase, including the following steps:
  • Step A Preparation of crude potato ester acyl hydrolase: the crude potato protein precipitate is extracted from potato starch wastewater by acid precipitation, and the obtained crude potato protein precipitate is re-dissolved in PBS buffer, and after impurity removal and elution, use filter Membrane filtration to obtain the first eluate;
  • Step B Enrichment of potato ester acyl hydrolase: Put the above-mentioned functional nano magnetic beads coupled with Dictyostelium aureum lectin in a centrifuge tube, wash with MES buffer for multiple times, and then use PBS buffer for multiple times Washing, adding the above-mentioned first eluent, mixing uniformly and magnetically separating, and removing the separating liquid to obtain nano magnetic beads enriched with potato ester acyl hydrolase;
  • Step C Elute the potato ester acyl hydrolase: Use the PBS buffer containing fucose to elute the nano magnetic beads enriched with the potato ester acyl hydrolase obtained in Step B to obtain the second eluate, and collect the second wash Liquid removal and purification are carried out to obtain potato ester acyl hydrolase.
  • the acid precipitation method is to use HCl to adjust the pH of the potato starch wastewater to 4.0, stir at room temperature and then stand still, and centrifuge to obtain a precipitate.
  • the PBS buffer has a concentration of 0.02 mol/L and a pH of 7
  • the MES buffer has a concentration of 0.050.02 mol/L and a pH of 5.2.
  • step A the impurity removal and elution method is column chromatography.
  • the column chromatography is Q-Sepharose Fast Flow anion column chromatography.
  • the filter membrane is a 0.22 ⁇ m filter membrane.
  • the concentration of fucose is 0.2 mol/L.
  • the purification method of the second eluate is to remove fucose small molecular impurities through a 3000 Da dialysis bag.
  • step C the potato ester acyl hydrolase obtained in step C is vacuum freeze-dried at 5-8 Pa and -60-66°C for 48-72 hours, and then stored at -20°C.
  • the method of the present invention can easily prepare functional nano magnetic beads for enriching potato ester acyl hydrolase.
  • the functional nano magnetic beads can be resuspended in MES buffer for storage, and can be stored at low temperature (- Stable storage at 20°C for 6 months.
  • the prepared functional nano magnetic beads can be used directly, without on-site preparation, which is very convenient.
  • functional nano-magnetic beads coupled with lectins can specifically enrich potato ester acylhydrolase from miscellaneous proteins. While retaining the biological activity of potato ester acylhydrolase, the purity of the obtained potato ester acylhydrolase is Very high, so that the product quality has been further guaranteed.
  • the enrichment or adsorption and immobilization of potato ester acylhydrolase by functional nano magnetic beads coupled with lectin also saves the enrichment time, improves the production efficiency of the extracted enzyme or the catalytic performance of the enzyme in application, and can realize a large-scale process produce.
  • Fig. 1 is a schematic diagram of the process of enriching potato ester acyl hydrolase with functional nano-magnetic beads according to a preferred embodiment of the present application;
  • Figure 2 is a comparison of the response signal intensity of a potato ester acyl hydrolase sample of a preferred embodiment of the present application to different lectins;
  • Fig. 3 is an ion exchange chromatogram of anion column chromatography of potato crude protein according to a preferred embodiment of the present application
  • Figure 4 is a gel electrophoresis diagram of a potato ester acyl hydrolase in a preferred embodiment of the present application.
  • the present invention exemplarily provides a method for preparing functional nano magnetic beads for enriching potato ester acyl hydrolase, which includes the following steps:
  • Step 1 Activate carboxyl nanomagnetic beads
  • the carboxyl nanomagnetic beads are superparamagnetic beads, in which the mass of iron oxide accounts for more than 90%. Take 10mg The carboxyl magnetic beads were transferred to a centrifuge tube, washed several times with 5mL MES buffer and magnetically separated, the particles were resuspended in 5mL MES buffer and added 16mg ethyl-dimethylamine-propylcarbodiimide (EDAC) After vortexing and mixing for 30 minutes, it was repeatedly washed with MES buffer, and the precipitate was collected after magnetic separation to obtain activated nano magnetic beads.
  • EDAC ethyl-dimethylamine-propylcarbodiimide
  • the carboxyl groups on the surface of the magnetic nanobeads are activated by diimine EDAC, take the Reticulosporium aureum agglutinin (AAL) and dissolve it in 5mL MES buffer.
  • AAL Reticulosporium aureum agglutinin
  • the amount of lectin is based on: per mg of activated magnetic nanobeads
  • the total protein content in the lectin used should be 20-500 ⁇ g.
  • Potato ester acylhydrolase samples have different response signals to different lectins. As shown in Figure 2, the response signal of lectin AAL is the strongest, followed by lectin ConA, followed by lectin GNA and lectin RCA- I. Other lectins also have weak corresponding signals to potato ester acylhydrolase.
  • the examples provided by the present invention mainly use the lectin AAL.
  • the obtained functional nano-magnetic beads can be stored separately if they are not used immediately.
  • the storage method is: repeatedly wash the functional nano-magnetic beads with MES buffer, and resuspend the functional nano-magnetic beads in MES buffer after the last wash At this time, the concentration of the functional nanomagnetic beads in the MES buffer is 5mg/mL, and they are stored as a suspension at -20°C, and they still have a certain degree of efficacy after 6 months of storage.
  • Step 2 Use functional nano magnetic beads to enrich potato ester acyl hydrolase
  • the second eluate is purified to remove fucose and other small molecular impurities to obtain potato ester acyl hydrolase.
  • the purified and enriched potato ester acyl hydrolase was vacuum freeze-dried at 5-10 Pa and -55°C for 48-72h, and stored at -20°C.
  • Potato ester acyl hydrolase is a kind of lipase derived from potato with ester acyl hydrolase activity and can specifically hydrolyze fat substrates.
  • Fatty substrates include: monoacylglycerols, diacylglycerols, galactose fats, monoacyl and/or diacyl phosphate fats.
  • Step 1 Activate carboxyl nanomagnetic beads
  • Step 2 Coupling with Reticulosporium aureum lectin (AAL)
  • Coupling efficiency [(A280 Pre-Coupling Solution x D)–(A280 Post-Coupling Solution x D)]/((A280 Pre-Coupling Solution x D)) ⁇ 100%
  • the coupling efficiency is 95%, which means that 95% of the AAL is adsorbed on the magnetic nano-beads, so the preparation method of the functional magnetic nano-beads is feasible and can be used In the enrichment process of potato ester acyl hydrolase.
  • Step 2 Use functional nano magnetic beads to enrich potato ester acyl hydrolase
  • Example 1 Take the functional nano magnetic beads prepared in Example 1 and place them in a centrifuge tube. After repeated washing three times with MES washing buffer, wash them three times with 5 column volumes of 0.02mol/L PBS buffer (pH7.0). Add 2 mL of the first eluent, and after fully shaking, magnetically separate and remove the separating solution to obtain nano magnetic beads enriched with potato ester acyl hydrolase.
  • the second eluate is purified to remove fucose and other small molecular impurities to obtain potato ester acyl hydrolase.
  • the purified and enriched potato ester acyl hydrolase was vacuum freeze-dried at 5Pa and -66°C for 48h, and stored at -20°C.
  • Step 1 Activate carboxyl nanomagnetic beads
  • Step 2 Coupling with Reticulosporium aureum lectin (AAL)
  • AAL lectin Take 4mg of AAL lectin and dissolve it in 5mL MES buffer, add the lectin solution to activated magnetic beads, shake well, mix and rotate for 24 hours, wash four times with MES buffer after magnetic separation, and take the precipitate after magnetic separation. Add 5mL 1mol/L glycine solution (pH 8.0) to the precipitate, vortex to mix and spin for 30min, remove the supernatant after magnetic separation, and obtain functional nanomagnetic beads.
  • 5mL 1mol/L glycine solution pH 8.0
  • Enriching potato ester acyl hydrolase by using the functional nano magnetic beads prepared in Example 3 includes:
  • Step 2 Use functional nano magnetic beads to enrich potato ester acyl hydrolase
  • Example 3 Take the functional nano magnetic beads prepared in Example 3 and place them in a centrifuge tube. After repeated washing three times with MES washing buffer, wash them three times with 5 column volumes of 0.02mol/L PBS buffer (pH7.0). Add 2 mL of the first eluent, and after fully oscillating, magnetically separate and remove the separating solution to obtain nano magnetic beads enriched with potato ester acyl hydrolase.
  • the second eluate is purified to remove fucose and other small molecular impurities to obtain potato ester acyl hydrolase.
  • the purified and enriched potato ester acyl hydrolase was vacuum freeze-dried at 8.0 Pa and -60°C for 60 hours, and stored at -20°C.

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Abstract

一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠的制备方法，步骤包括：将羧基纳米磁珠与乙基-二甲基胺-丙基碳化二亚胺混合，活化纳米磁珠后，将活化的纳米磁珠与金橙黄网胞盘菌凝集素耦合，得到功能性纳米磁珠。本发明还提供了一种耦合了金橙黄网胞盘菌凝集素的功能性纳米磁珠在富集马铃薯酯酰基水解酶中的应用，步骤包括：从马铃薯淀粉废水中提取粗蛋白并进行除杂洗脱后得到洗脱液，利用功能性纳米磁珠富集洗脱液中马铃薯酯酰基水解酶，洗脱纯化后得到马铃薯酯酰基水解酶。通过使用本方法制备的功能性纳米磁珠富集或吸附固定马铃薯酯酰基水解酶，可以缩短富集马铃薯酯酰基水解酶的时间，提高马铃薯酯酰基水解酶的纯度和酶催化性能，同时还保留了其生物活性。

Description

一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠及其制备方法和应用 技术领域
本申请涉及蛋白纯化技术领域，尤其涉及一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠及其制备方法和应用。
背景技术
中国作为全球第四大粮食作物，马铃薯广泛分布在世界各大地区。不同的薯产国都有着不同程度的马铃薯加工业，发展十分迅速。随着马铃薯工业的发展，生产过程中越来越多的马铃薯淀粉加工后的废液被排放，然而废液中富含蛋白质，其中干物质含量为20-50g/Kg，粗蛋白的含量占据了1/3，许多优质蛋白资源因此被浪费。
马铃薯蛋白可以分为三大类：Patatin(分子量40-45kDa)，蛋白酶抑制剂(分子量5-25kda)，其他蛋白(分子量50kDa以上)。Patain是一组特异性存在于马铃薯块茎中的球类糖蛋白，占块茎总蛋白的40％左右，在自然条件下通常以二聚体形式存在，1980年首次由RACUSEN等人从马铃薯块茎中分离出来。Patain除具有抗氧化活性外，还具有良好的乳化性、发泡性和凝胶性，这些特性使得它能作为一种良好的食品添加剂作用于食品当中。除此之外，与其他块茎贮藏蛋白不同的是，Patatin具有非特异性酯酰基水解酶活性(LAH)，对于磷脂、糖酯、单酰基和二酰基甘油、长链脂肪酸酯等都具有一定的特异性。研究者们将Patatin添加到红酒、奶酪等食物中观察其澄清性、风味改善性，结果证明，马铃薯酯酰基水解酶作为食品添加剂可以在食品工业中发挥出一定的作用。目前虽然已有较多关于马铃薯酯酰基水解酶提取纯化的方法：膨胀床吸附法、基因表达法、磁性壳聚糖微球法、凝胶色谱法等，但是这些方法需要多个纯化步骤，或者在预处理阶段引入了化学试剂，导致蛋白质容易变性且纯化效率低不利于后续研究及工业化应用。
纳米磁珠是指大小可以用纳米来衡量的小磁性微粒，一般为1-100nm。这类磁珠具有超顺磁的磁性，外加磁场中有较强的磁响应性，撤去磁场后，磁性微粒的磁性马上消失，重新均匀分散于溶液中。这种特性可以帮助纳米磁珠吸附液体中的某种成分，然后通过磁性分离磁珠来达到分离纯化的目的。羧基纳米磁珠比表面积大，表面含有高含量羧基，能够与蛋白、多肽、抗体的伯胺基或巯基反应，将其共价偶联到磁珠表面。将磁性微球放入含有目标蛋白的溶液中时，目标蛋白会与磁性微球紧密结合，利用外部磁场就可以对其进行分离。
凝集素(Lectin)是一种能够从植物、无脊椎动物、高等动物中提纯的糖蛋白或结合糖的蛋白，因为能凝集红血球，所以被称为凝集素。凝集素可以识别糖蛋白和细胞膜表面的糖基， 并且一种凝集素能专一性结合某一种特异性糖基，因此凝集素在科研工作中扮演着十分重要的角色。
本领域的技术人员致力于开发一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠，来快速和有效富集或吸附固定马铃薯淀粉废水中马铃薯酯酰基水解酶。
发明内容
有鉴于现有技术的上述缺陷，本申请所要解决的技术问题是如何方便、快速和有效的回收和富集马铃薯淀粉加工废水中马铃薯酯酰基水解酶。为实现上述目的，本申请一方面提供了一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠的制备方法，包括以下步骤：
步骤1：活化羧基纳米磁珠：将羧基纳米磁珠转移至离心管，使用MES缓冲液清洗该羧基纳米磁珠并磁性分离后，将该羧基纳米磁珠重悬于MES缓冲液中，并加入乙基-二甲基胺-丙基碳化二亚胺(EDAC)混合，混合均匀后使用MES缓冲液清洗，磁性分离后取沉淀，得到活化的纳米磁珠；
步骤2：耦合凝集素：取金橙黄网胞盘菌凝集素溶于MES缓冲液中，加入到活化的纳米磁珠中，摇匀后旋转16～24h，使用MES缓冲液清洗，磁性分离后取沉淀，在沉淀中加入猝灭溶液，混合均匀后磁性分离，去除上清液后得到耦合了金橙黄网胞盘菌凝集素的用于富集马铃薯酯酰基水解酶的功能性纳米磁珠。
进一步地，步骤1中，羧基纳米磁珠为超顺磁珠。
进一步地，羧基纳米磁珠包含氧化铁，氧化铁在羧基纳米磁珠中的质量占比大于90％。
进一步地，MES缓冲液的浓度为0.05mol/L，pH为5.2。
进一步地，步骤1中，羧基纳米磁珠乙基-二甲基胺-丙基碳化二亚胺的质量比为5:8～5:4。
进一步地，步骤2中，每毫克活化的纳米磁珠所需的金橙黄网胞盘菌凝集素中的总蛋白质的质量为20～500μg。
进一步地，步骤2中，猝灭溶液为1mol/L甘氨酸溶液，pH为8.0。
进一步地，步骤2获得的用于富集马铃薯酯酰基水解酶的功能性纳米磁珠的保存方法为：将功能性纳米磁珠用MES缓冲液清洗多次后，重悬于MES缓冲液中。
优选地，该功能性纳米磁珠以5mg/mL的浓度重悬于MES缓冲液中。
优选地，该功能性纳米磁珠的保存温度为-20℃。
本发明另一方面提供了根据上述方法制备的用于富集马铃薯酯酰基水解酶的功能性纳米磁珠，该功能性纳米磁珠耦合了金橙黄网胞盘菌凝集素。
本发明第三方面提供了一种如上所述的功能性纳米磁珠在富集马铃薯酯酰基水解酶中的应用，包括以下步骤：
步骤A：制备马铃薯酯酰基水解酶粗品：采用酸沉淀法从马铃薯淀粉废水中提取马铃薯粗蛋白沉淀，将得到的马铃薯粗蛋白沉淀复溶于PBS缓冲液中，进行除杂洗脱后，使用滤膜过滤获得第一洗脱液；
步骤B：富集马铃薯酯酰基水解酶：将上述耦合了金橙黄网胞盘菌凝集素的功能性纳米磁珠置于离心管中，使用MES缓冲液多次清洗，再使用PBS缓冲液多次清洗，加入上述第一洗脱液，混合均匀并磁性分离后去除分离液，获得富集了马铃薯酯酰基水解酶的纳米磁珠；
步骤C：洗脱马铃薯酯酰基水解酶：使用含有岩藻糖的PBS缓冲液洗脱步骤B得到的富集了马铃薯酯酰基水解酶的纳米磁珠，得到第二洗脱液，收集第二洗脱液并进行纯化，获得马铃薯酯酰基水解酶。
进一步地，步骤A中，酸沉淀法为使用HCl将马铃薯淀粉废水的pH调节至4.0，室温下搅拌后静置，离心后获得沉淀。
进一步地，PBS缓冲液浓度为0.02mol/L，pH为7，MES缓冲液的浓度为0.050.02mol/L，pH为5.2。
进一步地，步骤A中，除杂洗脱方式为柱层析。
优选地，该柱层析为Q-Sepharose Fast Flow阴离子柱层析。
优选地，步骤A中，滤膜是0.22μm滤膜。
优选地，步骤C中，岩藻糖的浓度为0.2mol/L。
优选地，步骤C中，第二洗脱液的纯化方法为通过3000Da透析袋脱除岩藻糖小分子杂质。
进一步地，步骤C中得到的马铃薯酯酰基水解酶在5～8Pa、-60～-66℃条件下真空冷冻干燥48～72h后，在-20℃下保存。
通过本发明的方法可以很容易制备得到用于富集马铃薯酯酰基水解酶的功能性纳米磁珠，该功能性纳米磁珠可以重悬于MES缓冲液中进行保存，最长可在低温(-20℃)下稳定保存6个月。在需要富集马铃薯酯酰基水解酶的时候可以直接将制备好的功能性纳米磁珠拿来使用即可，不需要现场配制，非常方便。
另外，耦合了凝集素的功能性纳米磁珠可以从杂蛋白中特异性富集马铃薯酯酰基水解酶，在保留了马铃薯酯酰基水解酶的生物活性的同时，获得的马铃薯酯酰基水解酶的纯度很高，使产品质量得到了进一步的保障。此外，通过耦合了凝集素的功能性纳米磁珠富集或吸附固定马铃薯酯酰基水解酶还节约了富集时间，提高了提取酶的生产效率或酶在应用时催化性能，可以实现大规模工艺生产。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本申请的一个较佳实施例的功能性纳米磁珠富集马铃薯酯酰基水解酶的流程示意图；
图2是本申请一个较佳实施例的马铃薯酯酰基水解酶样品对不同凝集素响应信号强度的比较；
图3是本申请一个较佳实施例的阴离子柱层析马铃薯粗蛋白的离子交换色谱图；
图4是本申请一个较佳实施例的马铃薯酯酰基水解酶的凝胶电泳图。
具体实施方式
以下参考说明书附图介绍本申请的多个优选实施例，使其技术内容更加清楚和便于理解。本申请可以通过许多不同形式的实施例来得以体现，本申请的保护范围并非仅限于文中提到的实施例。
本发明示例性提供了一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠的制备方法，包括以下步骤：
步骤1、活化羧基纳米磁珠
Figure PCTCN2020124301-appb-000001
羧基纳米磁珠为超顺磁珠，其中氧化铁的质量占比大于90％。取10mg
Figure PCTCN2020124301-appb-000002
羧基磁珠转移至离心管，用5mL MES缓冲液清洗多次并磁性分离，将颗粒重悬于5mL MES缓冲液中并加入16mg乙基-二甲基胺-丙基碳化二亚胺(EDAC)涡旋混合30min后，用MES缓冲液反复清洗，磁性分离后取沉淀，得到活化的纳米磁珠。
步骤2、耦合凝集素
纳米磁珠表面的羧基经过二亚胺EDAC活化后，取金橙黄网胞盘菌凝集素(AAL)溶解于5mL MES缓冲液中，凝集素用量的依据为：每毫克活化的纳米磁珠颗粒，使用的凝集素中的总蛋白质含量应该为20-500μg。将凝集素溶液添加到活化的纳米磁珠中，摇匀后旋转16-24h，磁性分离后用MES缓冲液反复清洗，磁性分离后取沉淀，在沉淀中加入猝灭溶液，涡旋混合后旋转30min，磁性分离后去除上清液获得用于富集马铃薯酯酰基水解酶的功能性纳米磁珠。
马铃薯酯酰基水解酶样品对不同的凝集素有不同的响应信号，如图2所示，凝集素AAL的响应信号最强，凝集素ConA次之，接下来分别是凝集素GNA和凝集素RCA-I，其他凝集素对马铃薯酯酰基水解酶也有微弱的相应信号，本发明提供的实施例主要使用凝集素AAL。
获得的功能性纳米磁珠不立即使用可以进行另外保存，保存方式为：用MES缓冲液反复清洗功能性纳米磁珠，并在最后一次洗涤后将功能性纳米磁珠重悬于MES缓冲液中，此时的功能性纳米磁珠在MES缓冲液中的浓度为5mg/mL，以悬浮液的方式保存于-20℃温度下，保存6个月后依旧具备一定的功效性。
如图1所示，使用功能性纳米磁珠富集马铃薯酯酰基水解酶的过程如下：
步骤1、制备马铃薯酯酰基水解酶粗品
使用1mol/L HCl将马铃薯淀粉废水的pH调节至4.0，室温下搅拌10min后，静置1h，离心后得到马铃薯粗蛋白沉淀，将马铃薯粗蛋白沉淀复溶于0.02mol/L PBS溶液(pH7.0)中。然后通过Q-Sepharose Fast Flow阴离子柱层析进行除杂洗脱，洗脱液经0.22μm滤膜过滤后获得第一洗脱液。
步骤2、使用功能性纳米磁珠富集马铃薯酯酰基水解酶
取制备好的功能性纳米磁珠置于离心管中，用MES清洗缓冲液反复清洗以后，用0.02mol/L PBS缓冲液(pH7.0)清洗三次，加入第一洗脱液，充分震荡后，磁性分离，去除分离液，得到富集了马铃薯酯酰基水解酶的纳米磁珠。
步骤3、洗脱马铃薯酯酰基水解酶
用含有0.2mol/L岩藻糖的0.02mol/L PBS缓冲液(pH7.0)磁性洗脱富集了马铃薯酯酰基水解酶的纳米磁珠，获得第二洗脱液，利用3000Da透析袋对第二洗脱液进行纯化，脱除岩藻糖等小分子杂质后得到马铃薯酯酰基水解酶。
步骤4、样品干燥和保存
将经过纯化富集得到的马铃薯酯酰基水解酶在5-10Pa、-55℃条件下真空冷冻干燥48-72h，在-20℃条件下保存。
马铃薯酯酰基水解酶是一种来源于马铃薯的脂肪酶，具有酯酰基水解酶活性，能特异水解脂肪底物。脂肪底物包括：单酰基甘油、二酰基甘油、半乳糖脂肪、单酰基和/或二酰基磷酸脂肪。
实施例1：
制备用于富集马铃薯酯酰基水解酶的功能性纳米磁珠，包括：
步骤1、活化羧基纳米磁珠
取10mg
Figure PCTCN2020124301-appb-000003
羧基纳米磁珠转移至离心管，用5mL MES缓冲液清洗至少一次以上并磁性分离四次，将羧基纳米磁珠颗粒重悬于5mL MES缓冲液中并加入16mg乙基-二甲基胺-丙基碳化二亚胺(EDAC)涡旋混合30min，随后用MES缓冲液反复清洗四次，磁性分离后取沉淀，得到活化的纳米磁珠。
步骤2、耦合金橙黄网胞盘菌凝集素(AAL)
取2mg AAL凝集素溶解于5mL MES缓冲液中，将凝集素溶液添加到活化的纳米磁珠中，摇匀后混合旋转24h，磁性分离后用MES缓冲液反复清洗四次，磁性分离后取沉淀，在沉淀中加入5mL 1mol/L甘氨酸溶液(pH为8.0)涡旋混合后旋转30min，磁性分离后去除上清液获得功能性纳米磁珠。
纳米磁珠耦合效率的测定：
制备凝集素AAL溶液时，预留50μL添加到950μL MES缓冲液中，标记为预耦合溶液。将剩余的凝集素溶液添加到耦合颗粒中，摇匀后在室温下混合旋转24h。磁性分离，将上清液标记为后耦合溶液。用紫外分光光度计测量与耦合溶液和后耦合溶液在280nm波长下的吸光度值，结果如图3所示。并运用以下公式计算耦合效率：
耦合效率＝[(A280 Pre-Coupling Solution x D)–(A280 Post-Coupling Solution x D)]/((A280 Pre-Coupling Solution x D))×100％
经过测量计算，得知耦合效率为95％，表示有95％的金橙黄网胞盘菌凝集素(AAL)吸附于纳米磁珠颗粒上，所以该功能性纳米磁珠的制备方法可行，可以运用于马铃薯酯酰基水解酶的富集过程当中。
实施例2
通过利用实施例1中制备的功能性纳米磁珠富集马铃薯酯酰基水解酶，包括：
步骤1、制备马铃薯酯酰基水解酶粗品
使用1mol/L HCl将马铃薯淀粉废水的pH调节至4.0，室温下搅拌10min后，静置1h，离心后得到马铃薯粗蛋白沉淀，将马铃薯粗蛋白沉淀复溶于0.02mol/L PBS溶液(pH7.0)中。然后通过Q-Sepharose Fast Flow阴离子柱层析进行除杂洗脱，洗脱液经0.22μm滤膜过滤后获得第一洗脱液。
步骤2、使用功能性纳米磁珠富集马铃薯酯酰基水解酶
取实施例1中制备好的功能性纳米磁珠置于离心管中，用MES清洗缓冲液反复清洗三次以后，用5倍柱体积的0.02mol/L PBS缓冲液(pH7.0)清洗三次，加入2mL第一洗脱液，充分震荡后，磁性分离，去除分离液，得到富集了马铃薯酯酰基水解酶的纳米磁珠。
步骤3、洗脱马铃薯酯酰基水解酶
用含有0.2mol/L岩藻糖的0.02mol/L PBS缓冲液(pH7.0)磁性洗脱富集了马铃薯酯酰基水解酶的纳米磁珠，获得第二洗脱液，利用3000Da透析袋对第二洗脱液进行纯化，脱除岩藻糖等小分子杂质后得到马铃薯酯酰基水解酶。
步骤4、样品干燥和保存
将经过纯化富集得到的马铃薯酯酰基水解酶在5Pa、-66℃条件下真空冷冻干燥48h，在-20℃条件下保存。
实施例3
制备和保存用于富集马铃薯酯酰基水解酶的功能性纳米磁珠，包括：
步骤1、活化羧基纳米磁珠
取20mg
Figure PCTCN2020124301-appb-000004
羧基纳米磁珠转移至离心管，用5mL MES缓冲液清洗至少一次以上并磁性分离四次，将羧基纳米磁珠颗粒重悬于5mL MES缓冲液中并加入 16mg乙基-二甲基胺-丙基碳化二亚胺(EDAC)涡旋混合30min后，用MES缓冲液反复清洗四次，磁性分离后取沉淀，得到活化的纳米磁珠。
步骤2、耦合金橙黄网胞盘菌凝集素(AAL)
取4mg AAL凝集素溶解于5mL MES缓冲液中，将凝集素溶液添加到活化磁珠中，摇匀后混合旋转24h，磁性分离后用MES缓冲液反复清洗四次，磁性分离后取沉淀，在沉淀中加入5mL 1mol/L甘氨酸溶液(pH为8.0)，涡旋混合后旋转30min，磁性分离后去除上清液，获得功能性纳米磁珠。
步骤3、保存功能性纳米磁珠
用MES缓冲液反复清洗功能性纳米磁珠，并在最后一次洗涤后将功能性纳米磁珠重悬于MES缓冲液中，功能性纳米磁珠在MES缓冲液中的浓度为5mg/mL，以悬浮液的方式在-20℃下保存。
实施例4
通过利用实施例3中制备的功能性纳米磁珠富集马铃薯酯酰基水解酶，包括：
步骤1、制备马铃薯酯酰基水解酶粗品
使用1mol/L HCl将马铃薯淀粉废水的pH调节至4.0，室温下搅拌20min后，静置1h，离心后得到马铃薯粗蛋白沉淀，将马铃薯粗蛋白沉淀复溶于0.02mol/L PBS溶液(pH7.0)中，然后通过Q-Sepharose Fast Flow阴离子柱层析进行除杂洗脱，洗脱液经0.22μm滤膜过滤后获得第一洗脱液。
步骤2、使用功能性纳米磁珠富集马铃薯酯酰基水解酶
取实施例3中制备好的功能性纳米磁珠置于离心管中，用MES清洗缓冲液反复清洗三次以后，用5倍柱体积的0.02mol/L PBS缓冲液(pH7.0)清洗三次，加入2mL第一洗脱液，充分震荡后，磁性分离，去除分离液，得到得到富集了马铃薯酯酰基水解酶的纳米磁珠。
步骤3、洗脱马铃薯酯酰基水解酶
用含有0.2mol/L岩藻糖的0.02mol/L PBS缓冲液(pH7.0)磁性洗脱富集了马铃薯酯酰基水解酶的纳米磁珠，获得第二洗脱液，利用3000Da透析袋对第二洗脱液进行纯化，脱除岩藻糖等小分子杂质后得到马铃薯酯酰基水解酶。
步骤4、样品干燥和保存
将经过纯化富集得到的马铃薯酯酰基水解酶在8.0Pa、-60℃条件下真空冷冻干燥60h，在-20℃条件下保存。
SDS-PAGE凝胶电泳分析
取适量马铃薯粗蛋白样品和通过实施例4富集得到的马铃薯酯酰基水解酶样品分别配制成蛋白液，从中取40uL蛋白液，加入10uL上样缓冲液于EP管中，于沸水中 孵育5min，然后用12％聚丙烯酰胺凝胶分离蛋白样品，电泳缓冲液为1×Tris-甘氨酸电极缓冲液，浓缩胶电压为80V，分离胶电压为120V。电泳结束后，取出分离胶，用考马斯亮蓝染液染色30min,随后用脱色液脱色至透明。在全自动化学发光图像分析系统上拍照分析。结果如图4所示，其中编号1、12的条带为marker，编号2、11的条带为马铃薯粗蛋白，编号3-10的条带为马铃薯酯酰基水解酶。通过图4可知，通过本方法富集得到的马铃薯酯酰基水解酶的纯度很高，可高达95％以上。
以上详细描述了本申请的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (20)

  1. 一种用于富集马铃薯酯酰基水解酶的功能性纳米磁珠的制备方法，其中，所述方法包括以下步骤：
    步骤1：活化羧基纳米磁珠：将羧基纳米磁珠转移至离心管，使用MES缓冲液清洗所述羧基纳米磁珠并磁性分离后，将所述羧基纳米磁珠重悬于所述MES缓冲液中，并加入乙基-二甲基胺-丙基碳化二亚胺(EDAC)混合，混合均匀后使用所述MES缓冲液清洗，磁性分离后取沉淀，得到活化的纳米磁珠；
    步骤2：耦合凝集素：取金橙黄网胞盘菌凝集素溶于所述MES缓冲液中，加入到所述活化的纳米磁珠中，摇匀后旋转16～24h，使用所述MES缓冲液清洗，磁性分离后取沉淀，在沉淀中加入猝灭溶液，混合均匀后磁性分离，去除上清液后得到耦合了金橙黄网胞盘菌凝集素的用于富集马铃薯酯酰基水解酶的功能性纳米磁珠。
  2. 如权利要求1所述的制备方法，其中，步骤1中，所述羧基纳米磁珠为超顺磁珠。
  3. 如权利要求2所述的制备方法，其中，所述羧基纳米磁珠包含氧化铁，所述氧化铁在所述羧基纳米磁珠中的质量占比大于90％。
  4. 如权利要求1所述的制备方法，其中，所述MES缓冲液的浓度为0.05mol/L，pH为5.2。
  5. 如权利要求1所述的制备方法，其中，步骤1中，所述羧基纳米磁珠与所述乙基-二甲基胺-丙基碳化二亚胺的质量比为5:8～5:4。
  6. 如权利要求1所述的制备方法，其中，步骤2中，每毫克所述活化的纳米磁珠所需的所述金橙黄网胞盘菌凝集素中的总蛋白质的质量为20～500μg。
  7. 如权利要求1所述的制备方法，其中，步骤2中，所述猝灭溶液为1mol/L甘氨酸溶液，pH为8.0。
  8. 如权利要求1所述的制备方法，其中，步骤2获得的所述用于富集马铃薯酯酰基水解酶的功能性纳米磁珠的保存方法为：将所述功能性纳米磁珠用MES缓冲液清洗多次后，重悬于MES缓冲液中。
  9. 如权利要求8所述的制备方法，其中，所述功能性纳米磁珠以5mg/mL的浓度重悬于MES缓冲液中。
  10. 如权利要求8所述的制备方法，其中，所述功能性纳米磁珠的保存温度为-20℃。
  11. 一种如权利要求1-10中任一项所述的方法制备的用于富集马铃薯酯酰基水解酶的功能性纳米磁珠，其中，所述功能性纳米磁珠与金橙黄网胞盘菌凝集素耦合。
  12. 一种如权利要求11所述的功能性纳米磁珠在富集马铃薯酯酰基水解酶中的应用，其中，包括以下步骤：
    步骤A：制备马铃薯酯酰基水解酶粗品：采用酸沉淀法从马铃薯淀粉废水中提取马铃薯粗蛋白沉淀，将得到的所述马铃薯粗蛋白沉淀复溶于PBS缓冲液中，进行除杂洗脱后，使用滤膜过滤获得第一洗脱液；
    步骤B：富集马铃薯酯酰基水解酶：将如权利要求11所述的功能性纳米磁珠置于离心管中，使用MES缓冲液多次清洗，再使用PBS缓冲液多次清洗，加入所述第一洗脱液，混合均匀并磁性分离后去除分离液，获得富集了马铃薯酯酰基水解酶的纳米磁珠；
    步骤C：洗脱马铃薯酯酰基水解酶：使用含有岩藻糖的PBS缓冲液洗脱步骤B得到的富集了马铃薯酯酰基水解酶的纳米磁珠，得到第二洗脱液，收集所述第二洗脱液并进行纯化，获得所述马铃薯酯酰基水解酶。
  13. 如权利要求12所述的应用，其中，步骤A中，所述酸沉淀法为使用HCl将所述马铃薯淀粉废水的pH调节至4.0，室温下搅拌后静置，离心后获得所述沉淀。
  14. 如权利要求12所述的应用，其中，所述PBS缓冲液浓度为0.02mol/L，pH为7，所述MES缓冲液的浓度为0.05mol/L，pH为5.2。
  15. 如权利要求12所述的应用，其中，步骤A中，所述除杂洗脱方式为柱层析。
  16. 如权利要求15所述的应用，其中，所述柱层析为Q-Sepharose Fast Flow阴离子柱层析。
  17. 如权利要求12所述的应用，其中，步骤A中，所述滤膜是0.22μm滤膜。
  18. 如权利要求12所述的应用，其中，步骤C中，所述岩藻糖的浓度为0.2mol/L。
  19. 如权利要求12所述的应用，其中，步骤C中，所述第二洗脱液的纯化方法为通过3000Da透析袋脱除岩藻糖小分子杂质。
  20. 如权利要求12所述的应用，其中，步骤C中得到的所述马铃薯酯酰基水解酶在5～8Pa、-60～-66℃条件下真空冷冻干燥48～72h后，在-20℃下保存。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120258484A1 (en) * 2009-12-29 2012-10-11 Korea Basic Science Institute Cancer diagnosis marker using the aberrant glycosylation of a protein
CN104772124A (zh) * 2015-04-27 2015-07-15 新疆大学 一种用于富集糖蛋白的凝集素磁性纳米材料及其制备方法
CN106093021A (zh) * 2016-06-03 2016-11-09 浙江省农业科学院 酸度调控和凝集素识别的大肠杆菌可视化生物传感方法
CN107430126A (zh) * 2014-11-17 2017-12-01 昆士兰大学 食管腺癌和巴雷特食管的糖蛋白生物标志物及其用途
CN110846295A (zh) * 2019-11-13 2020-02-28 上海交通大学 一种基于功能性纳米磁珠富集马铃薯酯酰基水解酶的方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1217890A (zh) * 1997-11-26 1999-06-02 中国科学院新疆化学研究所 从马铃薯淀粉制取后废水中回收蛋白质的方法
US6649419B1 (en) * 2000-11-28 2003-11-18 Large Scale Proteomics Corp. Method and apparatus for protein manipulation
CN103674918A (zh) * 2013-12-12 2014-03-26 复旦大学 一种基于凝集素液相悬浮芯片检测糖蛋白糖链结构的方法
CN103864888B (zh) * 2014-04-08 2016-03-23 江南大学 一种从马铃薯淀粉加工废水中回收糖蛋白的方法
CN107903301A (zh) * 2017-12-22 2018-04-13 广州誉嘉生物科技有限公司 一种糖基化蛋白的分离方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120258484A1 (en) * 2009-12-29 2012-10-11 Korea Basic Science Institute Cancer diagnosis marker using the aberrant glycosylation of a protein
CN107430126A (zh) * 2014-11-17 2017-12-01 昆士兰大学 食管腺癌和巴雷特食管的糖蛋白生物标志物及其用途
CN104772124A (zh) * 2015-04-27 2015-07-15 新疆大学 一种用于富集糖蛋白的凝集素磁性纳米材料及其制备方法
CN106093021A (zh) * 2016-06-03 2016-11-09 浙江省农业科学院 酸度调控和凝集素识别的大肠杆菌可视化生物传感方法
CN110846295A (zh) * 2019-11-13 2020-02-28 上海交通大学 一种基于功能性纳米磁珠富集马铃薯酯酰基水解酶的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AHN YEONG HEE, SHIN PARK MIN, KIM YONG-SAM, OH NA REE, JI EUN SUN, KIM KWANG HOE, LEE YEON JUNG, KIM SUNG HO, YOO JONG SHIN: "Quantitative analysis of aberrant protein glycosylation in liver cancer plasma by AAL-enrichment and MRM mass spectrometry", ANALYST, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 138, no. 21, 1 January 2013 (2013-01-01), UK, pages 6454, XP055812170, ISSN: 0003-2654, DOI: 10.1039/c3an01126g *
LI, FENG ET AL.: "Enrichment of Glycoproteins in Human Serum Using Concanavalin A-Functionalized Magnetic Nanoparticles and Identification by Mass Spectrometry)", CHINESE JOURNAL OF CHROMATOGRAPHY, vol. 32, no. 4, 30 April 2014 (2014-04-30), pages 369 - 375, XP055811686 *
NORTON PAMELA, COMUNALE MARY ANN, HERRERA HARMIN, WANG MENGJUN, HOUSER JOSEF, WIMMEROVA MICHAELA, ROMANO PATRICK R., MEHTA ANAND: "Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma", PROTEOMICS, WILEY-VCH VERLAG , WEINHEIM, DE, vol. 16, no. 24, 1 December 2016 (2016-12-01), DE, pages 3126 - 3136, XP055811692, ISSN: 1615-9853, DOI: 10.1002/pmic.201600064 *
SCHMIDT JESPER MALLING; DAMGAARD HENRIETTE; GREVE-POULSEN MATHIAS; LARSEN LOTTE BACH; HAMMERSHØJ MARIANNE: "Foam and emulsion properties of potato protein isolate and purified fractions", FOOD HYDROCOLLOIDS, ELSEVIER BV, NL, vol. 74, 7 August 2017 (2017-08-07), NL, pages 367 - 378, XP085202090, ISSN: 0268-005X, DOI: 10.1016/j.foodhyd.2017.07.032 *
WU, QIAOYU ET AL.: "Investigation on the Characteristics of the Activity of Potato Lipid Acyl Hydrolase", FOOD AND MACHINERY, vol. 35, no. 11, 17 October 2019 (2019-10-17), pages 41 - 46, XP055811683 *

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