CN103756990A - Papain preparation and immobilization method thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种木瓜蛋白酶制剂及固定化方法，包括如下步骤：（1）将纤维素纳米晶基材分散在pH为5-9的缓冲溶液中，配置成基材溶液；（2）将（1）中所得的基材溶液与木瓜蛋白酶溶液或木瓜蛋白酶浆状物混合，在0-35℃振荡混合1-60min，得到混合悬浊液；（3）将（2）所得的混合悬浊液与聚丙烯酰胺溶液混合，震荡1-60min；（4）将（3）中所得的固定化木瓜蛋白酶洗涤后冷冻干燥即得。本发明具有固定化木瓜蛋白酶固定化效率高、酶活回收率高，操作简便，条件温和等优点。

The invention discloses a papain preparation and an immobilization method, which comprises the following steps: (1) dispersing a cellulose nanocrystal substrate in a buffer solution with a pH of 5-9 to form a substrate solution; (2) dissolving the Mix the substrate solution obtained in (1) with papain solution or papain slurry, shake and mix at 0-35°C for 1-60 minutes to obtain a mixed suspension; (3) mix the mixed suspension obtained in (2) solution mixed with polyacrylamide solution, and shaken for 1-60 minutes; (4) The immobilized papain obtained in (3) was washed and then freeze-dried. The invention has the advantages of high immobilization efficiency of the immobilized papain, high recovery rate of enzyme activity, simple and convenient operation, mild conditions and the like.

Description

Translated fromChinese

一种木瓜蛋白酶制剂及固定化方法A kind of papain preparation and immobilization method

技术领域technical field

本发明涉及一种木瓜蛋白酶固定化方法及酶制剂。The invention relates to a method for immobilizing papain and an enzyme preparation.

背景技术Background technique

纤维素是世界上最丰富的天然高分子聚合物之一，对纤维素的高附加值利用是国内外的研究热点。纤维素纳米晶体具有高结晶度，高机械强度，大比表面积等性能，在食品、医药、生物（酶的固定化）、新能源等领域有较好的应用前景。Cellulose is one of the most abundant natural polymers in the world, and the high value-added utilization of cellulose is a research hotspot at home and abroad. Cellulose nanocrystals have high crystallinity, high mechanical strength, large specific surface area and other properties, and have good application prospects in food, medicine, biology (enzyme immobilization), new energy and other fields.

目前酶固定化方法常用的用双功能团试剂对酶和载体进行交联，如戊二醛、甲醛、以及二亚胺类试剂，其中二醛类交联剂成本较低，但反应激烈，酶与载体质量比低(18.8mg（酶）/g（载体）Gulay Bayramoglu,B.Filiz Senkal,MeltemYilmaz,M.Yakup Arica.Immobilization and stabilization of papain on poly(hydroxyethylmethacrylate–ethylenglycol dimethacrylate)beads grafted with epoxy functional polymerchains via surface-initiated-atom transfer radical polymerization(SI-ATRP).BioresourceTechnology.Volume102,Issue21,2011,9833–9837)，酶固定化效率不高，增加了制备成本，另一方面，二亚胺类试剂价格较高，交联时间较长（12-24h活化，2h交联），步骤繁复（Khaled A.Mahmoud,Edmond Lam,Sabahudin Hrapovic,and John H.T.Luong.Preparation of Well-Dispersed Gold/Magnetite Nanoparticles Embedded on CelluloseNanocrystals for Efficient Immobilization of Papain Enzyme.ACS Appl.Mater.Interfaces2013,5,4978-4985）。因此，有必要发明一种操作简单，酶固定化效率高，反应条件温和的反应。At present, enzyme immobilization methods commonly use bifunctional reagents to cross-link the enzyme and the carrier, such as glutaraldehyde, formaldehyde, and diimine reagents. Among them, the cost of dialdehyde cross-linking agents is relatively low, but the reaction is intense. Low mass ratio to carrier (18.8mg (enzyme)/g (carrier) Gulay Bayramoglu, B. Filiz Senkal, MeltemYilmaz, M. Yakup Arica. Immobilization and stabilization of papain on poly(hydroxyethylmethacrylate–ethylenglycol dimethacrylate)beads grafted with cepoxy polymertion via surface-initiated-atom transfer radical polymerization (SI-ATRP).BioresourceTechnology.Volume102, Issue21, 2011, 9833–9837), the enzyme immobilization efficiency is not high, which increases the preparation cost. On the other hand, the price of diimine reagents Higher, longer cross-linking time (12-24h activation, 2h cross-linking), complicated steps (Khaled A. Mahmoud, Edmond Lam, Sabahudin Hrapovic, and John H.T.Luong. Preparation of Well-Dispersed Gold/Magnetite Nanoparticles Embedded on Cellulose Nanocrystals for Efficient Immobilization of Papain Enzyme. ACS Appl. Mater. Interfaces 2013, 5, 4978-4985). Therefore, it is necessary to invent a reaction with simple operation, high enzyme immobilization efficiency and mild reaction conditions.

发明内容Contents of the invention

本发明的目的在于克服现有技术的缺陷，提供一种简单的基于静电自组装技术的固定化方法，形成了纤维素基材-酶-丙烯酰胺三层结构的固定化酶。The purpose of the present invention is to overcome the defects of the prior art, provide a simple immobilization method based on electrostatic self-assembly technology, and form an immobilized enzyme with a three-layer structure of cellulose substrate-enzyme-acrylamide.

一种木瓜蛋白酶固定化方法，包括如下步骤：A method for immobilizing papain, comprising the steps of:

（1）将纤维素纳米晶基材分散在pH为5-9的缓冲溶液中，配置成基材溶液；(1) Dispersing the cellulose nanocrystal substrate in a buffer solution with a pH of 5-9 to form a substrate solution;

（2）将（1）中所得的基材溶液与木瓜蛋白酶溶液或木瓜蛋白酶浆状物混合，在0-35℃振荡混合1-60min，得到混合悬浊液；(2) Mix the substrate solution obtained in (1) with the papain solution or papain slurry, shake and mix at 0-35°C for 1-60 minutes to obtain a mixed suspension;

（3）将（2）所得的混合悬浊液与聚丙烯酰胺溶液混合，震荡1-60min；(3) Mix the mixed suspension obtained in (2) with the polyacrylamide solution, and shake for 1-60 minutes;

（4）将（3）中所得的固定化木瓜蛋白酶洗涤后冷冻干燥即得。(4) The immobilized papain obtained in (3) is washed and then freeze-dried.

所述步骤（1）中的纤维素纳米晶基材为将纤维素材料通过硫酸水解所得到纤维素纳米晶体。The cellulose nanocrystal substrate in the step (1) is the cellulose nanocrystal obtained by hydrolyzing the cellulose material with sulfuric acid.

所述聚丙烯酰胺与木瓜蛋白酶的质量比为0.11～2。The mass ratio of the polyacrylamide to papain is 0.11-2.

所述木瓜蛋白酶与基材的质量比为1:（10-100）。The mass ratio of the papain to the substrate is 1: (10-100).

所述步骤（2）、（3）中震荡速率为100-500rpm。The vibration rate in the steps (2) and (3) is 100-500 rpm.

所述步骤（2）中木瓜蛋白酶包括木瓜蛋白酶酶粉、胞外产木瓜蛋白酶或细胞破壁后含木瓜蛋白酶的发酵液。The papain in the step (2) includes papain enzyme powder, extracellular papain or fermentation broth containing papain after cell wall breaking.

所述纤维素材料为微晶纤维素、纤维素纤维和棉花中的一种或两种以上。The cellulose material is one or more of microcrystalline cellulose, cellulose fiber and cotton.

本发明原理：纤维素材料经过硫酸水解生成纤维素纳米晶后，其表面糖链上的活性羟基部分被酸（优选硫酸）酯化，因酸根带有负电荷，因此，酸水解纤维素纳米晶表面带有负电荷。木瓜蛋白酶在ph<7时，酶蛋白表面带有部分正电荷，在水溶液中，能与带负电荷的纤维素纳米晶发生静电自组装，形成表面带部分正电荷的酶-纤维素纳米晶复合物。聚丙烯酰胺在pH5-9范围内带有负电荷，能与上述带部分正电荷的酶-纤维素纳米晶复合物再一次发生静电自组装，从而实现酶的固定化。The principle of the invention: after the cellulose material is hydrolyzed by sulfuric acid to generate cellulose nanocrystals, the active hydroxyl groups on the sugar chains on the surface are esterified by acid (preferably sulfuric acid), because the acid radicals have negative charges, therefore, acid hydrolysis of cellulose nanocrystals The surface is negatively charged. When papain is ph<7, the surface of the enzyme protein is partially positively charged. In aqueous solution, it can electrostatically self-assemble with negatively charged cellulose nanocrystals to form a partially positively charged enzyme-cellulose nanocrystal complex. things. The polyacrylamide has a negative charge in the pH range of 5-9, and can undergo electrostatic self-assembly with the above-mentioned partially positively charged enzyme-cellulose nanocrystal complex again, thereby realizing the immobilization of the enzyme.

本发明相对于具有以下优点：The present invention has the following advantages relative to:

（1）发明使用简单的静电自组装技术，在固定化酶过程中不使用戊二醛、甲醛等双功能试剂，成本较低，反应温和，能够较大程度保持酶活（60%～99%），提高酶活回收率，固定化效果好，操作简便。(1) Invented and used simple electrostatic self-assembly technology, no bifunctional reagents such as glutaraldehyde and formaldehyde were used in the process of immobilizing enzymes, the cost was low, the reaction was mild, and the enzyme activity could be maintained to a large extent (60%-99% ), improving the recovery rate of enzyme activity, good immobilization effect, and easy operation.

（2）本发明的固定化木瓜蛋白酶方法，能够用于木瓜蛋白酶、过氧化物酶等一系列在pH2-10下zeta电位为正的酶，并具有作为蛋白质药物载体的潜质。(2) The immobilized papain method of the present invention can be used for a series of enzymes with positive zeta potential at pH 2-10, such as papain and peroxidase, and has the potential as a protein drug carrier.

附图说明Description of drawings

图1为实施例2中所得到的酶制剂在不同pH之下的相对酶活。Fig. 1 is the relative enzyme activity of the enzyme preparation obtained in Example 2 under different pH.

图2为实施例2中所得到的酶制剂在不同温度下的相对酶活。Fig. 2 is the relative enzyme activity of the enzyme preparation obtained in Example 2 at different temperatures.

具体实施方式Detailed ways

下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

实施例1Example 1

将80mg纤维素纳米晶分散在4ml pH5的磷酸缓冲溶液中，加入4ml含有3.298mg木瓜蛋白酶溶液，在30℃、速率200rpm下震荡60min，加入0.5ml0.5%聚丙烯酰胺水溶液，在30℃、速率200rpm下震荡30min，洗涤后冷冻干燥得到固定化酶产品。固定化酶效率为98.40%，产品中木瓜蛋白酶与纤维素纳米晶基材比例为41.22mg/g。Disperse 80mg of cellulose nanocrystals in 4ml of pH5 phosphate buffer solution, add 4ml of papain solution containing 3.298mg, shake at 30°C for 60min at a rate of 200rpm, add 0.5ml of 0.5% polyacrylamide aqueous solution, and incubate at 30°C, Shake at a rate of 200 rpm for 30 min, wash and then freeze-dry to obtain an immobilized enzyme product. The immobilized enzyme efficiency is 98.40%, and the ratio of papain to cellulose nanocrystal substrate in the product is 41.22mg/g.

实施例2Example 2

将563.2mg纤维素纳米晶分散在25ml pH5.5缓冲溶液中，加入20ml含有57.994mg木瓜蛋白酶溶液，在30℃、速率200rpm下震荡30min，加入6ml0.5%聚丙烯酰胺水溶液，在30℃、速率200rpm下震荡30min，洗涤后冷冻干燥得到固定化酶产品，酶活回收率65.3%。Disperse 563.2mg of cellulose nanocrystals in 25ml of pH5.5 buffer solution, add 20ml of papain solution containing 57.994mg, shake at 30°C for 30min at a speed of 200rpm, add 6ml of 0.5% polyacrylamide aqueous solution, at 30°C, Shake at a rate of 200 rpm for 30 minutes, freeze-dry after washing to obtain the immobilized enzyme product, and the recovery rate of the enzyme activity is 65.3%.

实施例3Example 3

将80mg纤维素纳米晶分散在4ml pH6.0缓冲溶液中，加入4ml含有3.297mg木瓜蛋白酶溶液，在30℃、速率200rpm下震荡30min，加入0.3ml0.5%聚丙烯酰胺水溶液，在35℃、速率200rpm下震荡30min，洗涤后冷冻干燥得到固定化酶产品。固定化酶效率为97.60%，酶与纤维素纳米晶基材比例为40.23mg/g。Disperse 80mg of cellulose nanocrystals in 4ml of pH6.0 buffer solution, add 4ml of papain solution containing 3.297mg, shake at 30°C at a rate of 200rpm for 30min, add 0.3ml of 0.5% polyacrylamide aqueous solution, at 35°C, Shake at a rate of 200 rpm for 30 min, wash and then freeze-dry to obtain an immobilized enzyme product. The efficiency of immobilized enzyme was 97.60%, and the ratio of enzyme to cellulose nanocrystal substrate was 40.23mg/g.

实施例4Example 4

将80mg纤维素纳米晶分散在4ml pH5的磷酸缓冲溶液中，加入4ml含有2.268mg木瓜蛋白酶溶液，在30℃、速率100rpm下震荡10min，加入0.1ml0.5%聚丙烯酰胺水溶液，在30℃、速率100rpm下震荡50min，洗涤后冷冻干燥得到固定化酶产品。固定化酶效率为75.21%，产品中木瓜蛋白酶与纤维素纳米晶基材比例为21.32mg/g。Disperse 80mg of cellulose nanocrystals in 4ml of pH5 phosphate buffer solution, add 4ml of papain solution containing 2.268mg, shake at 30°C for 10min at a rate of 100rpm, add 0.1ml of 0.5% polyacrylamide aqueous solution, and incubate at 30°C, Shake at a rate of 100 rpm for 50 min, wash and freeze-dry to obtain the immobilized enzyme product. The immobilized enzyme efficiency was 75.21%, and the ratio of papain to cellulose nanocrystal substrate in the product was 21.32mg/g.

实施例5Example 5

将80mg纤维素纳米晶分散在4ml pH5.5缓冲溶液中，加入4ml含有2.268mg木瓜蛋白酶溶液，在30℃、速率200rpm下震荡20min，加入0.1ml0.5%聚丙烯酰胺水溶液，在20℃、速率200rpm下震荡40min，洗涤后冷冻干燥得到固定化酶产品。固定化酶效率为67.22%，酶与纤维素纳米晶基材比例为19.06mg/g。Disperse 80mg of cellulose nanocrystals in 4ml pH5.5 buffer solution, add 4ml solution containing 2.268mg papain, shake at 30°C for 20min at a rate of 200rpm, add 0.1ml0.5% polyacrylamide aqueous solution, Shake at a speed of 200 rpm for 40 min, wash and then freeze-dry to obtain the immobilized enzyme product. The efficiency of immobilized enzyme was 67.22%, and the ratio of enzyme to cellulose nanocrystal substrate was 19.06mg/g.

实施例6Example 6

将80mg纤维素纳米晶分散在4ml pH6.5缓冲溶液中，加入4ml含有3.297mg木瓜蛋白酶溶液，在30℃、速率200rpm下震荡60min，加入0.3ml0.5%聚丙烯酰胺水溶液，在35℃、速率200rpm下震荡30min，洗涤后冷冻干燥得到固定化酶产品。固定化酶效率为98.47%，酶与纤维素纳米晶基材比例为40.59mg/g。Disperse 80mg of cellulose nanocrystals in 4ml of pH6.5 buffer solution, add 4ml of papain solution containing 3.297mg, shake at 30°C for 60min at a rate of 200rpm, add 0.3ml of 0.5% polyacrylamide aqueous solution, and incubate at 35°C, Shake at a rate of 200 rpm for 30 min, wash and then freeze-dry to obtain an immobilized enzyme product. The efficiency of immobilized enzyme was 98.47%, and the ratio of enzyme to cellulose nanocrystal substrate was 40.59mg/g.

Claims (8)

Translated fromChinese

1.一种木瓜蛋白酶固定化方法，其特征在于，包括如下步骤：1. a papain immobilization method, is characterized in that, comprises the steps:（1）将纤维素纳米晶基材分散在pH为5-9的缓冲溶液中，配置成基材溶液；(1) Dispersing the cellulose nanocrystal substrate in a buffer solution with a pH of 5-9 to form a substrate solution;（2）将（1）中所得的基材溶液与木瓜蛋白酶溶液或木瓜蛋白酶浆状物混合，在0-35℃振荡混合1-60min，得到混合悬浊液；(2) Mix the substrate solution obtained in (1) with the papain solution or papain slurry, shake and mix at 0-35°C for 1-60 minutes to obtain a mixed suspension;（3）将（2）所得的混合悬浊液与聚丙烯酰胺溶液混合，震荡1-60min；(3) Mix the mixed suspension obtained in (2) with the polyacrylamide solution, and shake for 1-60 minutes;（4）将（3）中所得的固定化木瓜蛋白酶洗涤后冷冻干燥即可。(4) The immobilized papain obtained in (3) may be washed and freeze-dried.2.根据权利要求1所述的方法，其特征在于，所述步骤（1）中的纤维素纳米晶基材为将纤维素材料通过硫酸水解所得到纤维素纳米晶体。2 . The method according to claim 1 , wherein the cellulose nanocrystal substrate in the step (1) is cellulose nanocrystals obtained by hydrolyzing cellulose materials with sulfuric acid. 3 .3.根据权利要求2所述的方法，其特征在于，所述聚丙烯酰胺与木瓜蛋白酶的质量比为0.11～2。3. The method according to claim 2, characterized in that the mass ratio of the polyacrylamide to papain is 0.11-2.4.根据权利要求1或2或3所述的方法，其特征在于，所述木瓜蛋白酶与基材的质量比为1:（10-100）。4. The method according to claim 1, 2 or 3, characterized in that the mass ratio of papain to substrate is 1:(10-100).5.根据权利要求1或2或3所述的方法，其特征在于，所述步骤（2）、（3）中震荡速率为100-500rpm。5. The method according to claim 1, 2 or 3, characterized in that, in the steps (2) and (3), the shaking speed is 100-500 rpm.6.根据权利要求1或2或3所述的方法，其特征在于，所述步骤（2）中木瓜蛋白酶包括木瓜蛋白酶酶粉、胞外产木瓜蛋白酶或细胞破壁后含木瓜蛋白酶的发酵液。6. The method according to claim 1, 2 or 3, wherein the papain in the step (2) includes papain enzyme powder, extracellular papain or fermentation broth containing papain after cell wall breaking .7.根据权利要求1或2或3所述的方法，其特征在于，所述纤维素材料为微晶纤维素、纤维素纤维和棉花中的一种或两种以上。7. The method according to claim 1, 2 or 3, characterized in that the cellulose material is one or more of microcrystalline cellulose, cellulose fiber and cotton.8.根据权利要求1～7任意一项方法制备的木瓜蛋白酶制剂。8. The papain preparation prepared according to any one method of claims 1-7.

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