WO2019232929A1 - 一种牛乳中乳过氧化物酶的纯化方法 - Google Patents

一种牛乳中乳过氧化物酶的纯化方法 Download PDF

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WO2019232929A1
WO2019232929A1 PCT/CN2018/100375 CN2018100375W WO2019232929A1 WO 2019232929 A1 WO2019232929 A1 WO 2019232929A1 CN 2018100375 W CN2018100375 W CN 2018100375W WO 2019232929 A1 WO2019232929 A1 WO 2019232929A1
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ammonium sulfate
milk
solution
column
whey
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PCT/CN2018/100375
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French (fr)
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王加启
张养东
郑楠
李松励
赵圣国
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中国农业科学院北京畜牧兽医研究所
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Publication of WO2019232929A1 publication Critical patent/WO2019232929A1/zh

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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase

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  • the present invention relates to a method for rapidly purifying lactoperoxidase in liquid milk (such as cow milk), and more specifically, it uses rapid protein purification chromatography technology to purify lactoperoxidase in liquid milk through one-step hydrophobic chromatography. It belongs to the field of protein purification technology.
  • Lactoperoxidase (LPO, EC 1.11.1.7), as one of the endogenous enzymes in emulsion, is widely present in mammalian glands, salivary glands, lacrimal glands and their respective secretions (milk, saliva, tears).
  • LPO has high thermal stability and can be used as one of the evaluation indexes for ultra pasteurization.
  • LPO is an effective antibacterial agent, which can not only be used to reduce the bacterial flora in milk and dairy products, but also activate the lactoperoxidase system (LPOS) by adding hydrogen peroxide and thiocyanate to extend the frozen raw milk. Shelf life can also be widely used in preserving food, cosmetics and eye drops.
  • LPO has been applied in dentistry and wound treatments, suitable for treating gingivitis and periodontal disease; adding LPO to toothpaste or mouthwash can kill oral bacteria and reduce the acid produced by these bacteria.
  • LPO can be used medically as an antitumor and antiviral agent.
  • the object of the present invention is to provide a method for purifying lactoperoxidase in liquid milk, which is mainly used for extracting and purifying lactoperoxidase from raw milk.
  • Biologically active lactoperoxidase The enzyme protein can be obtained by one-step hydrophobic chromatography, and the protein purity is ⁇ 90%.
  • a method for purifying lactoperoxidase in liquid milk characterized in that an agarose gel hydrophobic chromatography column is used to perform hydrophobic chromatography on ammonium sulfate whey solution, and lactoperoxidase is separated and purified to obtain lactoperoxidase. , Can be further purified to obtain higher purity lactoperoxidase.
  • the liquid milk is human or mammalian milk, for example, human milk, goat milk, cow milk, horse milk, camel milk, or other animal milk, preferably cow milk.
  • the agarose gel hydrophobic chromatography column may be a prepacked column selected from a butyl-sepharose hydrophobic chromatography column (such as a Butyl Sepharose 4 FF prepacked column, a Butyl-S Sepharose 6 FF prepacked column), Phenyl-Sepharose Hydrophobic Chromatography Columns (e.g.
  • Phenyl Sepharose 6FF (LS) Prepacked Columns, Phenyl Sepharose 6 FF (HS) Prepacked Columns) and Octyl-Sepharose Hydrophobic Chromatography Columns (such as Octyl Sepharose FF prepacked column), particularly preferably an octyl-sepharose 4FF (such as Octyl Sepharose 4FF prepacked column) hydrophobic chromatography column.
  • the ammonium sulfate whey solution is a whey solution containing ammonium sulfate, and the solubility saturation of the ammonium sulfate in the solution is 30-70%, preferably 40-60%, and most preferably 45-55%.
  • the solubility saturation of the ammonium sulfate is relative to the saturated solution of ammonium sulfate, and the saturation is calculated based on the saturation of the saturated solution of ammonium sulfate at the same temperature as 100%.
  • the ammonium sulfate saturated solution is correspondingly prepared according to the ambient temperature under the experimental operating conditions. Generally, the ambient temperature is 20-35 ° C.
  • the concentration of the ammonium sulfate is the solubility saturation of the ammonium sulfate.
  • a method for purifying lactoperoxidase in liquid milk comprising: performing ammonium sulfate whey solution chromatography on an agarose gel hydrophobic chromatography column to obtain purified lactoperoxidase, wherein ammonium sulfate whey solution Is a whey solution containing an ammonium sulfate solution, the concentration of ammonium sulfate in the whey solution is 30-70%, preferably 40-60%, and most preferably 45-55%; the purification method includes the following steps:
  • composition and content of the starting buffer are: 50mM NaH 2 PO 4 , 50% (NH 4 ) 2 SO 4 , 2mM CaCl 2 , pH 7.0; the composition and content of the elution buffer are: 50mM NaH 2 PO 4 , 2 mM CaCl 2 , pH 7.0.
  • the agarose gel hydrophobic chromatography column is selected from a butyl-agarose gel hydrophobic chromatography column (such as a Butyl, Sepharose, and FF prepacked column, a Butyl-S Sepharose, and a FF prepacked column), and a phenyl-agarose gel column.
  • a butyl-agarose gel hydrophobic chromatography column such as a Butyl, Sepharose, and FF prepacked column, a Butyl-S Sepharose, and a FF prepacked column
  • Gel hydrophobic chromatography columns such as Phenyl Sepharose 6FF (LS) prepacked columns, Phenyl Sepharose 6FF (HS) prepacked columns
  • octyl-sepharose hydrophobic chromatography columns such as Octyl SepharoseFF prepacked columns
  • an octyl-sepharose 4FF such as an Octyl Sepharose 4FF prepacked column
  • the collecting in step e means collecting the protein when the ultraviolet absorption is above 1 mAU.
  • the amounts of ultrapure water, starting buffer and elution buffer are conventional choices in the art, and those skilled in the art can make reasonable selections according to actual needs.
  • the column is washed with 5 column volumes of ultrapure water, and the column is equilibrated with 5 column volumes of starting buffer.
  • the purification method may further include the following steps:
  • the ammonium sulfate whey solution is a conventional ammonium sulfate whey solution with a concentration of 30-70% of ammonium sulfate known to those skilled in the art.
  • the preparation method is not particularly limited, and can be obtained, for example, by the following method.
  • the purification method further includes a step of preparing ammonium sulfate whey solution (hereinafter referred to as "step 2"), including:
  • the step of preparing ammonium sulfate whey further includes:
  • ammonium sulfate whey solution is obtained through treatment and separation through an ultrafiltration membrane or an ultrafiltration tube, and large particles with a particle size greater than 0.2 ⁇ m are removed;
  • step 2-2 further use a 30kDa pore size ultrafiltration membrane or ultrafiltration tube to treat the ammonium sulfate whey solution obtained in step 2-2, separate and remove the impurities less than 30kDa, and concentrate the ammonium sulfate whey solution.
  • the ultrafiltration membrane in the step 2-2 is a hollow fiber column with a pore diameter of 0.2 ⁇ m; and the ultrafiltration membrane in the step 2-3 is a hollow fiber column with a pore diameter of 30kDa.
  • step 2 is further specifically described as:
  • Saturated ammonium sulfate solution was added dropwise to the whey solution at a volume ratio of 1: 1 to prepare a whey solution having a concentration of 50% ammonium sulfate; the whey solution was magnetically stirred at a temperature of 5 ° C for 12 hours.
  • the mixed system was centrifuged at 12,000 rpm and 5 ° C for 30 minutes, and the precipitate was prepared to obtain an ammonium sulfate whey solution; a hollow fiber column with a pore size of 0.2 ⁇ m was used to clarify the ammonium sulfate whey solution after precipitation of ammonium sulfate; a hollow fiber column with a 30 kDa pore size The clear ammonium sulfate whey solution was concentrated.
  • step 2 is further described as:
  • step 2 a saturated ammonium sulfate solution is added to the whey solution.
  • the addition of ammonium sulfate can precipitate and precipitate the heteroproteins in the whey, remove the heteroproteins in advance, and reduce or eliminate its effect on subsequent lactoperoxidase. Disturbance of chromatography separation step.
  • the present invention finds that the concentration of ammonium sulfate in the ammonium sulfate whey solution is 30-70%, which can achieve the maximum removal of foreign proteins and the maximum retention of the target protein, especially 40-60%, especially at 45 The effect is best at -55%.
  • the purification method further includes the step of preparing a whey solution before preparing the ammonium sulfate whey solution, specifically:
  • the rennet solution and skim milk (such as skim milk) are mixed, heated, and centrifuged to prepare a whey solution.
  • the concentration of the rennet solution is 0.05-5 g / ml, preferably 0.1-0.5 g / mL, and particularly preferably 0.2 g / mL.
  • the mass-volume ratio of rennet to skim milk is (0.01-2): 1 (g / L), preferably (0.05-0.2): 1 (g / L), such as 0.08: 1 (g / L).
  • the steps of preparing the whey solution are specifically:
  • the rennet solution and skim milk were mixed, and the casein was solidified and precipitated by heating at 30 ° C for 60 minutes, and centrifuged at 4000 rpm and 25 ° C for 15 minutes to prepare a whey solution.
  • the step of preparing a whey solution is further described as:
  • the skim milk (for example, skim milk) is conventional skim milk known to those skilled in the art, and the preparation method thereof is not particularly limited.
  • whole milk e.g., whole milk
  • the cream is removed to prepare skim milk.
  • the method is further specifically described as follows: centrifuging whole milk fresh milk at 2500 rpm and 25 ° C. for 15 min; discarding the upper separation layer containing cream to prepare skim milk.
  • the lactoperoxidase purification method of the present invention can dissolve the lactoperoxidase obtained after passing through the hydrophobic chromatography column in an elution buffer with a pH of 7.0, and can be used in subsequent experiments without removing ammonium sulfate.
  • the post-processing of the target protein after purification is simplified.
  • the method for purifying the lactoperoxidase of the present invention is to use a rapid protein purification chromatography technology to purify the lactoperoxidase in liquid milk through one-step hydrophobic chromatography. Utilizing optimized optimal conditions, including the optimal concentration of 50% ammonium sulfate for the precipitation of heteroproteins, the optimal hydrophobic chromatography purification filler Octyl, Sepharose, FF, etc., to purify lactoperoxidase from raw milk, the resulting lactoperoxidase The purity is greater than or equal to 90%.
  • the calculation of the purity is based on the mass ratio of lactoperoxidase to the total protein in the finally obtained solution, which can be calculated by accurate instrument measurement, or can be determined by SDS-PAGE electrophoresis after 10-fold gradient dilution (dilution The brightness of the hybrid protein in the previous protein is not higher than the brightness of the target protein in the diluted protein, which means that the purity of the target protein is greater than or equal to 90%).
  • the Octyl Sepharose FF refers to octyl agarose gel FF; its source is not limited, and it can be purchased through commercial means, for example, it is purchased from GE Company, and its code is GE HiTrap Octyl FF.
  • the lactoperoxidase purification process of the invention omits the disadvantages of a multi-step and complicated extraction and purification method, and adopts one-step hydrophobic chromatography to purify lactoperoxidase in liquid milk, which has the advantages of fast, high efficiency and low cost.
  • the purified lactoperoxidase has a purity of 90% or more, and the protein is dissolved in an elution buffer with a pH of 7.0, and can be used in subsequent experiments or operations without removing ammonium sulfate.
  • Figure 1 Purified lactoperoxidase step eluent with different hydrophobic chromatography purification packings; where: Line 1 is PhenylFF (HS) ultraviolet (UV) peak information; Line 1 'is related to PhenylFF (HS) column Trend of ammonium sulfate content in the eluent (linear change from 100% starting buffer to 100% elution buffer, the same below); line 2 is Butyl FF ultraviolet (UV) peak information; line 2 'is the same as Butyl FF The trend of ammonium sulfate content in the column-related eluent; line 3 is the PhenylFF (LS) ultraviolet (UV) peak information; line 3 'is the trend of ammonium sulfate content in the eluent related to the PhenylFF (LS) column; line 4 is Butyl-S FF ultraviolet (UV) peak information; line 4 'is the change trend of ammonium sulfate content in the eluent associated
  • Figure 2 is a graph showing the results of lactoperoxidase purification in Example 2.
  • Figure 3 SDS-PAGE chart of lactoperoxidase in Example 2, in the figure, 1: protein standard; 2: skim milk; 3: whey; 4: 50% strength ammonium sulfate whey solution; 5: under washing Miscellaneous proteins; 6: Purified lactoperoxidase.
  • rennet Hao Yue
  • color pre-stained protein Standard New England Biolab
  • SurePAGE 4-12% protein pre-made gel Kingsui
  • HiTrap HIC Selection Kit GE Medical
  • the ammonium sulfate concentration in the ammonium sulfate whey solution is the solubility saturation of ammonium sulfate commonly used by those skilled in the art.
  • concentration of the saturated ammonium sulfate solution is 100% saturation.
  • the saturated ammonium sulfate solution is mixed with other solutions (such as pure water) at a volume ratio of 1: 1, the saturation of the ammonium sulfate solution is 50%.
  • the so-called "0% elution buffer to 100% elution buffer linear eluent" starts from 100% starting buffer + 0% elution buffer, and then decreases the starting buffer content and increases the elution buffer. Content until 0% starting buffer + 100% elution buffer.
  • the concentration of ammonium sulfate in the buffer refers to the solubility saturation of the ammonium sulfate.
  • the 50% ammonium sulfate concentration in the starting buffer indicates that the solubility saturation of the ammonium sulfate is 50%.
  • CV means column volume
  • 5CV means the volume of 5 columns.
  • Example 1 Preselected column conditions for hydrophobic chromatography
  • Preparation of skim milk A 500 mL centrifuge bottle was used to centrifuge 1 L of whole milk for 15 min at 2500 rpm and 25 ° C. Discard the upper layer containing the cream and pour the skim milk into a 1 L beaker.
  • Preparation of whey Take 5 mL of ultrapure water to dissolve 1 g of rennet, and prepare a rennet solution at a concentration of 0.2 g / mL. Add 400 ⁇ L of chymosin solution to 1 L of skim milk and mix well. The casein was solidified and precipitated by heating at 30 ° C for 60 minutes, and centrifuged at 4000 rpm and 25 ° C for 15 minutes in a 500 mL centrifuge bottle. Finally, the whey was poured into a clean 2L beaker for use, and the precipitated coagulated casein was discarded.
  • a 0.2 ⁇ m pore size hollow fiber column was used to clarify the ammonium sulfate whey solution after ammonium sulfate precipitation.
  • a 30 kDa pore size hollow fiber column was used to concentrate the clear ammonium sulfate whey solution to 50 mL.
  • Elution buffer 50mM NaH 2 PO 4 , 2mM CaCl 2 , pH 7.0
  • C.AKTA purification program is set to:
  • Table 1 LPO parameter setting for GE 1mL hydrophobic chromatography prepacked column purification
  • Example 2 Purification method of lactoperoxidase in cow milk
  • sample pretreatment and (2) ammonium sulfate precipitation of heteroprotein in Example 1 sample pretreatment and ammonium sulfate precipitation of heteroprotein were performed.
  • Elution buffer 50mM NaH 2 PO 4 , 2mM CaCl 2 , pH 7.0;
  • a hydrophobic chromatography prepacked column, Octyl, Sepharose, and FF purchased from GE, codenamed GE HiTrap, Octyl, FF was used as purification media.
  • the purification process was:
  • Figures 2 and 3 show the lactoperoxidase purification results and the purified lactoperoxidase SDS-PAGE.
  • the LPO of the protein of interest has a single peak type, indicating that it can elute well from the heteroproteins.
  • the isolated LPO is a single band with a relative molecular weight of about 80 kDa.
  • lactoperoxidase can be purified by pretreatment of raw cow's milk, precipitation of hybrid protein with 50% ammonium sulfate, and then one-step hydrophobic chromatography using Octyl Sepharose FF prepacked column.
  • the protein purity is greater than 90%. It can be used in subsequent experiments without removing ammonium sulfate.
  • the lactoperoxidase purification method established by the present invention is fast, efficient, and low-cost, and provides a basis for lactoperoxidase purification technology and applications in various fields.

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Abstract

一种液态乳中乳过氧化物酶的纯化方法，使用琼脂糖凝胶疏水层析柱对硫酸铵乳清溶液进行疏水层析，分离纯化。得到的乳过氧化物酶纯度大于等于90％，蛋白溶解于pH＝7.0的洗脱缓冲液中，无需去除硫酸铵即可用于后续实验或操作。

Description

一种牛乳中乳过氧化物酶的纯化方法
本申请要求2018年6月6日向中国国家知识产权局提交的专利申请号为201810575168.3，发明名称为“一种牛乳中乳过氧化物酶的纯化方法”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及一种液态乳(例如牛乳)中乳过氧化物酶的快速纯化方法，更具体地说是采用快速蛋白纯化层析技术，经一步疏水层析纯化液态乳中乳过氧化物酶，属于蛋白纯化技术领域。
背景技术
乳过氧化物酶(lactoperoxidase,LPO,EC 1.11.1.7)作为乳液中的内源酶之一，广泛存在于哺乳动物的乳腺、唾液腺、泪腺及其各自分泌物(乳、唾液、眼泪)中。LPO有较高的热稳定性，可作为超巴氏杀菌的评定指标之一。LPO是一种有效的抗菌剂，不仅可以用来减少奶及奶制品中的细菌菌群，通过添加过氧化氢和硫氰酸盐来活化乳过氧化物酶体系(LPOS)延长冷冻原料乳的保存期限，还可以在保存食品，化妆品和滴眼液中有着广泛的应用。同时，LPO已经在牙科和伤口治疗中得到应用，适用于治疗牙龈炎和牙周病等；牙膏或漱口水中加入LPO可以杀死口腔细菌，并减少由这些细菌产生的酸。此外，LPO在医学上可以用作抗肿瘤和抗病毒剂。
开发LPO的最大问题是其分离过程烦琐、纯化困难、制造成本较高， 难以实现商业化，所以使用不同的分离纯化技术来提取LPO一直是许多研究的重点，探索操作简单方便、纯化快速、成本低、效率高、开发潜力大的新型分离提纯方法是今后要努力发展的方向。目前已有研究用不同的纯化方法来提取LPO，如CM-Sephadex离子交换色谱法、Sephadex g-100凝胶过滤色谱法、苯基-琼脂糖疏水亲和层析、Toyopearl-SP阳离子交换层析法等。然而这些研究的纯化方法由于一般至少使用两种不同的交换柱，操作比较复杂、耗时长、成本高。目前还有一些方法可以分离LPO，但操作方法更加繁琐，如分离纯化LPO可在粗制凝乳酶乳清中添加1.9～2.5mol·L -1(NH 4) 2SO 4盐析出来，接着通过磷酸钙凝胶色谱，在二氧化硅方解石凝胶中可进一步纯化，最后从4mol·L -1K 2HPO 4中结晶析出。
显然，还需要更多的、更快捷的分离和纯化LPO的方法。
发明内容
为了改善现有技术的不足，本发明的目的是提供一种液态乳中乳过氧化物酶的纯化方法，主要用于从生乳中提取纯化乳过氧化物酶，具有生物学活性的乳过氧化物酶蛋白经一步疏水层析即可获得，且蛋白纯度≥90％。
为了实现上述发明目的，本发明提供了以下技术方案：
一种液态乳中乳过氧化物酶的纯化方法，其特征在于，使用琼脂糖凝胶疏水层析柱对硫酸铵乳清溶液进行疏水层析，分离纯化得到乳过氧化物酶，非必要地，可进一步纯化得到纯度更高的乳过氧化物酶。
所述液态乳为人或哺乳动物的乳汁，例如，人乳、羊乳、牛乳、马乳、骆驼乳或其它动物乳，优选牛乳。
所述琼脂糖凝胶疏水层析柱可以为预装柱，选自丁基-琼脂糖凝胶疏水层析柱(如Butyl Sepharose 4 FF预装柱、Butyl-S Sepharose 6 FF预装 柱)、苯基-琼脂糖凝胶疏水层析柱(如Phenyl Sepharose 6 FF(LS)预装柱、Phenyl Sepharose 6 FF(HS)预装柱)和辛基-琼脂糖凝胶疏水层析柱(如Octyl Sepharose FF预装柱)，特别优选为辛基-琼脂糖凝胶4 FF(如Octyl Sepharose 4 FF预装柱)疏水层析柱。
所述硫酸铵乳清溶液为含有硫酸铵的乳清溶液，所述溶液中硫酸铵的溶解饱和度为30-70％，优选40-60％，最优选45-55％。所述硫酸铵的溶解饱和度是相对于硫酸铵饱和溶液而言的，以相同温度条件下硫酸铵饱和溶液的饱和度为100％为基准换算的。在本发明中，硫酸铵饱和溶液根据实验操作条件下的环境温度进行相应的配制，一般情况下环境温度为20-35℃。在本发明中，除特别限定之外，所述硫酸铵的浓度均为硫酸铵的溶解饱和度。
一种液态乳中乳过氧化物酶的纯化方法，包括：将硫酸铵乳清溶液通过琼脂糖凝胶疏水层析柱进行层析，得到纯化的乳过氧化物酶，其中硫酸铵乳清溶液为含有硫酸铵溶液的乳清液，所述乳清液中硫酸铵的浓度为30-70％，优选40-60％，最优选45-55％；所述纯化方法包括如下步骤：
a.使用超纯水清洗层析柱；
b.使用100％起始缓冲液平衡层析柱；
c.将澄清的硫酸铵乳清溶液加到层析柱；
d.使用20％起始缓冲液和80％洗脱缓冲液清洗层析柱至紫外吸收为0mAU，其中所述百分比为体积百分比；
e.使用100％洗脱缓冲液洗脱并收集乳过氧化物酶。
所述起始缓冲液组成及含量为：50mM NaH 2PO 4,50％(NH 4) 2SO 4,2mM CaCl 2,pH 7.0；所述洗脱缓冲液组成及含量为：50mM NaH 2PO 4,2mM CaCl 2,pH 7.0。
所述琼脂糖凝胶疏水层析柱选自丁基-琼脂糖凝胶疏水层析柱(如Butyl Sepharose 4 FF预装柱、Butyl-S Sepharose 6 FF预装柱)、苯基-琼脂糖凝胶疏水层析柱(如Phenyl Sepharose 6 FF(LS)预装柱、Phenyl Sepharose 6 FF(HS)预装柱)和辛基-琼脂糖凝胶疏水层析柱(如Octyl Sepharose FF预装柱)，特别优选为辛基-琼脂糖凝胶4 FF(如Octyl Sepharose 4 FF预装柱)疏水层析柱。
根据本发明，步骤e的收集是指收集紫外吸收在1mAU以上时的蛋白。
根据本发明，所述纯化方法中，超纯水、起始缓冲液和洗脱缓冲液的用量为本领域的常规选择，本领域技术人员可以根据实际需要进行合理的选择。如使用1-50个柱体积，优选3-10个柱体积的纯水清洗层析柱；使用1-50个柱体积，优选3-10个柱体积的缓冲液平衡层析柱。示例性地，使用5倍柱体积的超纯水对层析柱进行清洗，使用5倍柱体积的起始缓冲液对层析柱进行平衡处理等等。
非必要的，所述纯化方法还可以包括如下步骤：
f.使用100％洗脱缓冲液平衡层析柱；
g.使用20％乙醇水溶液保存层析柱。
根据本发明，所述硫酸铵乳清溶液为本领域技术人员知晓的常规的硫酸铵的浓度在30-70％范围内的硫酸铵乳清溶液。制备方法没有特别的限定，例如可以通过如下的方法制备得到。
根据本发明，所述纯化方法还包括，硫酸铵乳清溶液制备步骤(以下代称为“步骤2”)，包括：
2-1.将饱和硫酸铵溶液加入乳清溶液后混合，制备30-70％，优选40-60％，最优选45-55％，例如50％硫酸铵浓度的乳清溶液，搅拌，离心或过滤，分离得到清液，即为硫酸铵乳清溶液。
非必要地，所述硫酸铵乳清制备步骤还包括：
2-2.通过超滤膜或超滤管，处理分离得到硫酸铵乳清溶液，去除粒径大于0.2μm的大颗粒杂质；
2-3.如果需要，进一步使用30kDa孔径的超滤膜或超滤管处理2-2步骤得到的硫酸铵乳清溶液，分离去除小于30kDa的杂蛋白，同时浓缩硫酸铵乳清溶液。
优选，所述2-2步骤的超滤膜为孔径为0.2μm的中空纤维柱；2-3步骤的超滤膜为孔径为30kDa的中空纤维柱。
根据本发明，所述步骤2进一步具体描述为：
按照1:1的体积比将饱和硫酸铵溶液滴加到乳清溶液中，制备50％硫酸铵浓度的乳清溶液；在5℃温度下磁力搅拌乳清溶液12小时，将搅拌后的硫酸铵混合体系在12000rpm、5℃条件下离心30min，去沉淀，制备得到硫酸铵乳清溶液；使用0.2μm孔径的中空纤维柱澄清硫酸铵沉淀后的硫酸铵乳清溶液；使用30kDa孔径的中空纤维柱浓缩澄清的硫酸铵乳清溶液。
根据本发明，所述步骤2更进一步描述为：
取1L去离子水溶解750g硫酸铵制备饱和硫酸铵溶液；按照1:1的体积比缓慢滴加饱和硫酸铵溶液到乳清溶液中，制备50％硫酸铵浓度的乳清溶液；在5℃温度下磁力搅拌乳清溶液12小时；硫酸铵乳清溶液倒入250mL高速离心瓶，在12000rpm、5℃条件下离心30min；上清液倒入2L容积的烧杯中，丢弃沉淀的蛋白；使用0.2μm孔径的中空纤维柱澄清硫酸铵沉淀后的硫酸铵乳清溶液；使用30kDa孔径的中空纤维柱浓缩澄清的硫酸铵乳清溶液至50mL。
本发明中，步骤2中，向乳清溶液中加入饱和硫酸铵溶液，硫酸铵的加入可以使乳清中的杂蛋白沉淀析出，提前去除杂蛋白，降低或消除其 对后续乳过氧化物酶层析析出步骤的干扰。通过调整加入后的硫酸铵浓度实现去除杂蛋白和保留目的蛋白之间的最佳平衡。本发明通过实验研究发现，硫酸铵乳清溶液中的硫酸铵浓度为30-70％能达到最大限度的去除杂蛋白同时最大限度的保留目的蛋白的效果，特别优选40-60％，尤其在45-55％时效果最佳。
根据本发明，所述纯化方法还包括在制备硫酸铵乳清溶液前制备乳清溶液的步骤，具体是：
将凝乳酶溶液和脱脂乳(例如脱脂牛奶)混合，加热，离心，制备得到乳清溶液。
其中，凝乳酶溶液的浓度为0.05-5g/ml，优选0.1-0.5g/mL，特别优选0.2g/mL。
凝乳酶与脱脂乳的质量体积比为(0.01-2):1(g/L)，优选(0.05-0.2):1(g/L)，如0.08:1(g/L)。
根据本发明，制备乳清溶液的步骤具体是：
将凝乳酶溶液和脱脂乳混合，在30℃条件下加热60min使酪蛋白凝固沉淀，在4000rpm、25℃下离心15min，制备得到乳清溶液。
根据本发明，制备乳清溶液的步骤进一步描述为：
取5mL超纯水溶解1g的凝乳酶，制备0.2g/mL浓度的凝乳酶溶液；加入400μL凝乳酶溶液到1L的脱脂乳中并混匀；在30℃条件下加热60min使酪蛋白凝固沉淀，使用500mL离心瓶在4000rpm、25℃下离心15min；最后将乳清倒入2L容积的烧杯中备用，丢弃沉淀的凝固酪蛋白，制备得到乳清溶液。
根据本发明，所述脱脂乳(例如脱脂牛奶)为本领域技术人员知晓的常规脱脂乳，其制备方法没有特别的限定。
例如可以通过如下方法制备得到：将全脂乳(如全脂牛奶)离心分 离，去除奶油，制备得到脱脂乳。
所述方法进一步具体描述如下：在2500rpm、25℃的条件下离心全脂鲜牛奶15min；丢弃含有奶油的上分离层，制备得到脱脂牛奶。
本发明的乳过氧化物酶的纯化方法可以将经过所述疏水层析柱后获得的乳过氧化物酶溶解于pH＝7.0的洗脱缓冲液中，无需再去除硫酸铵即可用于后续实验或操作中，简化了纯化后目的蛋白的后处理。
本发明的乳过氧化物酶的纯化方法是使用快速蛋白纯化层析技术，经一步疏水层析纯化液态乳中的乳过氧化物酶。利用已优化的最优条件，包括硫酸铵沉淀杂蛋白的最佳浓度50％，最优的疏水层析纯化填料Octyl Sepharose FF等，从生乳中纯化乳过氧化物酶，所得乳过氧化物酶的纯度大于等于90％。
所述纯度的计算是基于最终获得的溶液中乳过氧化物酶相对于全部蛋白质的质量比例计算得到，可以通过精确仪器测定计算，也可以通过十倍梯度稀释后SDS-PAGE电泳法测定(稀释前的蛋白中杂蛋白亮度不高于稀释后蛋白中目的蛋白的亮度，即可认为目的蛋白的纯度大于等于90％)。
本发明中，所述的Octyl Sepharose FF是指辛基琼脂糖凝胶FF；其来源没有限定，可以是商业途径购买，例如购买自GE公司，代号为GE HiTrap Octyl FF。
本文所谓“非必要地”是指根据原料不同或操作条件不同，非必要地选择，使用或不使用。
本发明的有益效果在于：
本发明的乳过氧化物酶纯化工艺省略了多步骤、复杂的提取纯化方法的弊端，采用一步疏水层析法纯化液态乳中的乳过氧化物酶，具有快速、高效、低成本的优点，且纯化得到的乳过氧化物酶纯度大于等于90％， 蛋白溶解于pH＝7.0的洗脱缓冲液中，无需去除硫酸铵即可用于后续实验或操作。
附图说明
图1不同疏水层析纯化填料纯化乳过氧化物酶阶梯洗脱液效果图；其中：线1为Phenyl FF(HS)紫外(UV)峰信息；线1’为与Phenyl FF(HS)柱相关洗脱液中硫酸铵含量变化趋势(从100％起始缓冲液至100％洗脱缓冲液线性变化，下同)；线2为Butyl FF紫外(UV)峰信息；线2’为与Butyl FF柱相关洗脱液中硫酸铵含量变化趋势；线3为Phenyl FF(LS)紫外(UV)峰信息；线3’为与Phenyl FF(LS)柱相关洗脱液中硫酸铵含量变化趋势；线4为Butyl-S FF紫外(UV)峰信息；线4’为与Butyl-S FF柱相关洗脱液中硫酸铵含量变化趋势；线5为Octyl FF(HS)紫外(UV)峰信息；线5’为与Octyl FF(HS)柱相关洗脱液中硫酸铵含量变化趋势。
图2实施例2的乳过氧化物酶纯化结果图。
图3实施例2的乳过氧化物酶SDS-PAGE图谱，图中，1：蛋白标准；2：脱脂牛奶；3：乳清；4：50％浓度硫酸铵乳清溶液；5：清洗下的杂蛋白；6：纯化的乳过氧化物酶。
具体实施方式
下文将结合具体实施例对本发明的制备方法做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径得到。
实施例中使用的仪器与试剂：
仪器：AKTA Pure蛋白纯化仪(GE医疗)、磁力搅拌器(SCILOGEX)、蠕动泵(兰格)、Discover12m-PES 0.2μm中空纤维柱(WaterSep)、Discover12m-PES 30K Dalton中空纤维柱(WaterSep)、250mL高速离心瓶(Nalgene)、500mL高速离心瓶(Nalgene)、500mL烧杯(北玻)、2L烧杯(北玻)
试剂：凝乳酶(昊岳)、彩色预染蛋白Standard(New England Biolab)、SurePAGE 4-12％蛋白预制胶(金斯瑞)、HiTrap HIC Selection Kit(GE医疗)
实施例中，硫酸铵乳清溶液中硫酸铵浓度为本领域技术人员常用的硫酸铵的溶解饱和度。例如饱和硫酸铵溶液浓度为100％饱和度，当饱和硫酸铵溶液与其他溶液(如纯水)按照1:1体积比混合时，该硫酸铵溶液的饱和度为50％。
所谓“0％洗脱缓冲液至100％洗脱缓冲液线性洗脱液”，由100％起始缓冲液+0％洗脱缓冲液开始，依次降低起始缓冲液含量并增加洗脱缓冲液含量，直至0％起始缓冲液+100％洗脱缓冲液为止。缓冲液中硫酸铵的浓度指硫酸铵的溶解饱和度，如起始缓冲液中50％的硫酸铵浓度表明硫酸铵的溶解饱和度为50％。
CV含义为柱容积，5CV表示5个柱容积大小。
实施例1：疏水层析预装柱条件选择
(1)样品前处理
制备脱脂牛奶：使用500mL离心瓶，在2500rpm、25℃的条件下离心1L的全脂鲜牛奶15min。丢弃含有奶油的上分离层，脱脂牛奶倒入1L容积的烧杯中。
制备乳清：取5mL超纯水溶解1g的凝乳酶，制备0.2g/mL浓度的凝 乳酶溶液。加入400μL凝乳酶溶液到1L的脱脂牛奶中并混匀。在30℃条件下加热60min使酪蛋白凝固沉淀，使用500mL离心瓶在4000rpm、25℃下离心15min。最后将乳清倒入干净的2L容积的烧杯中备用，丢弃沉淀的凝固酪蛋白。
(2)硫酸铵沉淀杂蛋白
取1L去离子水溶解750g硫酸铵制备饱和硫酸铵溶液。按照1:1的体积比缓慢滴加饱和硫酸铵溶液到乳清溶液中，制备50％硫酸铵浓度的乳清溶液。在5℃温度下磁力搅拌乳清溶液12小时。硫酸铵乳清溶液倒入250mL高速离心瓶，在12000rpm、5℃条件下离心30min。上清液倒入干净的2L容积的烧杯中，丢弃沉淀的蛋白。使用0.2μm孔径的中空纤维柱澄清硫酸铵沉淀后的硫酸铵乳清溶液。使用30kDa孔径的中空纤维柱浓缩澄清的硫酸铵乳清溶液至50mL。
(3)疏水层析纯化
A.制备起始缓冲液和洗脱缓冲液
起始缓冲液：50mM NaH 2PO 4,50％(NH 4) 2SO 4,2mM CaCl 2,pH 7.0
洗脱缓冲液：50mM NaH 2PO 4,2mM CaCl 2,pH 7.0
B.分别使用1ml Phenyl Sepharose 6 FF(HS)、Phenyl Sepharose 6 FF(LS)、Butyl Sepharose 4 FF、Butyl-S Sepharose 6 FF和Octyl Sepharose 4 FF预装柱，纯化1mL的澄清硫酸铵乳清溶液。
C.AKTA纯化程序设置为：
a.5个柱体积超纯水清洗层析柱；
b.5个柱体积100％起始缓冲液平衡层析柱；
c.上样1mL澄清硫酸铵乳清溶液到层析柱；
d.100％起始缓冲液清洗层析柱至紫外吸收为0mAU；
e.0％洗脱缓冲液至100％洗脱缓冲液10CV线性洗脱挂柱蛋白，5 CV 100％洗脱缓冲液继续洗脱挂柱蛋白。
1ml疏水层析预装柱纯化LPO参数设置见表1。
表1：GE 1mL疏水层析预装柱纯化LPO参数设置
Figure PCTCN2018100375-appb-000001
结果如图1所示，5种琼脂糖凝胶柱均显示一定的分离作用，可利用不同浓度梯度的洗脱缓冲液将LPO与杂蛋白分开。从分离效率看，辛基-琼脂糖凝胶柱Octyl Sepharose 4 FF分离效果最佳，能在硫酸铵浓度为20％左右时将LPO洗脱与杂蛋白分开。
实施例2：牛乳中乳过氧化物酶的纯化方法
按照实施例1中(1)样品前处理和(2)硫酸铵沉淀杂蛋白的相同条件，进行样品前处理和硫酸铵沉淀杂蛋白。
(3)制备起始缓冲液和洗脱缓冲液
起始缓冲液：50mM NaH 2PO 4,50％(NH 4) 2SO 4,2mM CaCl 2,pH7.0；
洗脱缓冲液：50mM NaH 2PO 4,2mM CaCl 2,pH 7.0；
(4)纯化乳过氧化物酶
使用疏水层析预装柱Octyl Sepharose 4 FF(购买自GE公司，代号为GE HiTrap Octyl FF)为纯化填料，纯化流程为：
A.5个柱体积超纯水清洗层析柱；
B.5个柱体积100％起始缓冲液平衡层析柱；
C.上样澄清的硫酸铵乳清溶液到层析柱；
D.20％起始缓冲液和80％洗脱缓冲液(体积比)清洗层析柱至紫外吸收为0mAU；
E.10个柱体积100％洗脱缓冲液洗脱并收集目的蛋白；
F.5个柱体积100％洗脱缓冲液平衡层析柱；
G.5个柱体积20％乙醇水溶液保存层析柱；
乳过氧化物酶纯化结果图和纯化后的乳过氧化物酶SDS-PAGE图谱见图2和图3。目的蛋白LPO为单一峰型，说明可以与杂蛋白较好的分开洗脱出来。分离出的LPO为单一条带，其相对分子量在80kDa左右。通过进行十倍梯度稀释后SDS-PAGE电泳，原浓度蛋白中杂蛋白亮度不高于稀释后蛋白中目的蛋白亮度，即认为生牛乳中乳过氧化物酶经一步疏水层析纯化纯度大于90％。蛋白溶解于pH 7.0磷酸盐缓冲液中，无需去除硫酸铵即可用于后续实验。
GE HiTrap Octyl FF 1mL预装柱纯化LPO参数设置见表2。
表2：GE HiTrap Octyl FF 1ml预装柱纯化LPO参数设置
Figure PCTCN2018100375-appb-000002
上述实施例的实验结果表明可以通过预处理生牛乳，用50％硫酸铵沉淀杂蛋白，然后经Octyl Sepharose FF预装柱一步疏水层析纯化乳过氧化 物酶，得到的蛋白纯度大于90％，且无需去除硫酸铵即可用于后续实验。本发明建立的乳过氧化物酶纯化方法快速、高效、低成本，为乳过氧化物酶的纯化技术及各领域的应用提供了基础依据。
以上的实施例仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通工程技术对本发明的技术方案作出的各种变型和改进，均应落入本发明的权利要求书确定的保护范围内。

Claims (10)

  1. 一种液态乳中乳过氧化物酶的纯化方法，其特征在于，使用琼脂糖凝胶疏水层析柱对硫酸铵乳清溶液进行疏水层析，分离纯化得到乳过氧化物酶，非必要地，可进一步纯化得到的乳过氧化物酶；优选，所述液态乳为人乳、羊乳、牛乳或其它动物乳，特别优选为牛乳。
  2. 如权利要求1所述纯化方法，其中，琼脂糖凝胶疏水层析柱选自丁基-琼脂糖凝胶疏水层析柱、苯基-琼脂糖凝胶疏水层析柱、辛基-琼脂糖凝胶疏水层析柱，特别优选为辛基-琼脂糖凝胶疏水层析柱。
  3. 如权利要求1-2任一所述纯化方法，其中，所述硫酸铵乳清溶液为含有硫酸铵的乳清溶液，其中所述硫酸铵的浓度为硫酸铵的饱和度为30-70％，优选60-40％，最优选45-55％。
  4. 如权利要求1-3之任一所述纯化方法，其中，所述方法还包括硫酸铵乳清制备步骤；优选进一步还包括制备乳清溶液的步骤；优选进一步还包括液态乳脱脂步骤。
  5. 如权利要求1-4之任一所述纯化方法，其中所述硫酸铵乳清溶液制备步骤包括：
    2-1.将饱和硫酸铵溶液加入乳清溶液混合，制备30-70％，优选40-60％，最优选45-55％的硫酸铵浓度的乳清溶液，搅拌，离心或过滤，分离得到清液，即为硫酸铵乳清溶液；非必要地，所述硫酸铵乳清制备步骤还包括：
    2-2.通过超滤膜或超滤管，处理分离得到的硫酸铵乳清溶液，去除粒径大于0.2μm的大颗粒杂质；
    2-3.如果需要，进一步使用30kDa孔径的超滤膜或超滤管处理2-2步骤得到的硫酸铵乳清溶液，分离去除小于30kDa的杂蛋白，同时浓缩硫酸铵乳清溶液。
  6. 如权利要求1-4之任一所述纯化方法，所述制备乳清溶液的步骤，包括将凝乳酶溶液和脱脂乳混合，加热，离心，制备得到乳清溶液；
    优选，凝乳酶溶液的浓度为0.05-5g/ml，优选0.1-0.5g/mL，特别优选0.2g/mL；
    优选，凝乳酶与脱脂乳的质量体积比为(0.01-2):1(g/L)，优选(0.05-0.2):1(g/L)，特别优选0.08:1(g/L)。
  7. 如权利要求1-4之任一所述纯化方法，所述液态乳脱脂步骤包括：将全脂乳离心分离，去除油脂，制备得到脱脂乳。
  8. 如权利要求1-7之任一的纯化方法，所述层析分离包括如下步骤：
    a.使用超纯水清洗层析柱；
    b.使用100％起始缓冲液平衡层析柱；
    c.将澄清的硫酸铵乳清溶液加到层析柱；
    d.使用20％起始缓冲液和80％洗脱缓冲液清洗层析柱至紫外吸收为0mAU，所述比例为体积百分比；
    e.使用100％洗脱缓冲液洗脱并收集乳过氧化物酶；
    其中，所述起始缓冲液组成及含量为：50mM NaH 2PO 4,50％(NH 4) 2SO 4,2mM CaCl 2,pH 7.0；所述洗脱缓冲液组成及含量为：50mM NaH 2PO 4,2mM CaCl 2,pH 7.0。
  9. 如权利要求1-8任一所述的纯化方法，其特征在于还可以如下步骤：
    h.使用100％洗脱缓冲液平衡层析柱；
    i.使用20％乙醇水溶液保存层析柱。
  10. 如权利要求8或9所述纯化方法，其中，使用1-50个柱体积，优选3-10个柱体积的纯水清洗层析柱；使用1-50个柱体积，优选3-10个柱体积的缓冲液平衡层析柱。
PCT/CN2018/100375 2018-06-06 2018-08-14 一种牛乳中乳过氧化物酶的纯化方法 WO2019232929A1 (zh)

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