CN113061601A - 一种基于多点共价作用制备固定化磷脂酶c的方法 - Google Patents
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Abstract
本发明涉及磁性固定化酶领域，公开了一种基于多点共价作用制备固定化磷脂酶C的方法。采用共沉淀法制备了Fe3O4/SiO2磁性纳米粒子，采用超支化缩水甘油进行活化，并对该载体进行羧基修饰，制取一种羧基修饰后的磁性载体；利用多点共价结合的固定方式使载体与磷脂酶C结合，首先，在较低pH下载体上存在的‑COOH与富含正电荷的酶蛋白区域进行阳离子交换，发生静电相互作用。随后提高体系pH，活化后的羧基与酶上的氨基发生酰胺化反应形成共价键。本发明以一种多点共价结合的固定化方式为实际生产中大豆毛油酶法脱胶提供了一种更稳定的固定化磷脂酶，为之后应用到连续化生产中提供依据。
Description
技术领域
本发明涉及一种固定化磷脂酶C的制备方法，尤其涉及一种令载体与酶进行多点反应的共价结合方法。
背景技术
磷脂酶(PLC)广泛存在于动植物及微生物体内，是一类对甘油磷脂具有特异性水解活力的酶，能够水解Sn3上的甘油磷酸键生成磷酸酯和甘油二酯(DAG)。酶法脱胶能够避免磷脂对油脂品质和后续精炼效果的影响；同时，水解产物DAG是中性油，反应后会保留在脱胶油中，提高脱胶得率。
近年来，酶的固定化技术广泛应用到实际生产中，该技术能在一定程度上能够提高酶对外界恶劣环境的抵抗力，降低控制难度，引入磁性纳米粒子做载体，有利于酶与产物的分离、从而降低生产成本，有效的解决游离酶存在的问题。
共价结合法是酶固定化方法的一种，通过载体上的活性基团与酶蛋白表面的氨基酸残基残基共价结合，使酶与载体结合。通过多点共价连接进行酶的固定化能使酶蛋白表面某些区域的刚性化提高，从而起到稳定单体酶的作用。该方法需要载体上易于修饰或存在较多的化学基团。在较低的pH下进行载体和酶蛋白非活性基团的快速静电吸附，吸附后载体上的基团和酶上的基团非常接近，此时增加pH使酶表面的亲核基团与载体的反应，获得额外的共价键。
因此，选择多点共价结合作为固定化方法制备固定化磷脂酶C能有效的改善酶的操作稳定性，对其在生产中的应用显得尤为重要。
发明内容
本发明的目的在于针对现有技术的不足，提供一种基于多点共价作用制备固定化磷脂酶C的方法，制备出一种羧基修饰的磁性载体，所述的磁性固定化磷脂酶C具有较高的稳定性。该酶经重复使用8次仍保留87％的相对活力，具有适宜pH和温度区间不同程度的拓宽，具有更好的pH稳定性和热稳定性，具有较高的应用价值
本发明解决上述技术问题所采用的技术方案为：一种基于多点共价作用制备固定化磷脂酶C的方法，包括以下步骤：
(1)磁性载体的制备
采用共沉淀法制备Fe3O4纳米粒子。取1.00g Fe3O4纳米颗粒，加入10ml TEOS和4ml Tween20，超声分散10分钟。再加入50mL的去离子水，超声10min。将20mL的浓氨水和60ml乙醇与上述溶液混合，35℃水浴并充分搅拌。制得的Fe3O4/SiO2颗粒在40℃真空烘箱中干燥。
(2)载体的活化与修饰
将Fe3O4/SiO2颗粒100g与30μl CH3OK溶液和2.5ml二甲基酰胺置于烧瓶中，在50℃、100rpm下搅拌反应1小时，去除甲醇后加入10ml无水二氧六环，95℃加热。缓慢滴加2.0g缩水甘油。反应2小时，加入甲醇分散，磁分离后用甲醇反复冲洗。烘干12小时后制得HPG活化后的Fe3O4/SiO2载体。三口烧瓶中加入100mg Fe3O4/SiO2/HPG、100mg三乙胺和10ml二甲基甲酰胺，一定量丁二酸酐(SA)，混合液在水浴中加热并在100rpm下搅拌。整个反应在氮气氛围下操作。磁分离后用甲醇冲洗三次。在真空干燥箱中干燥12h，得到Fe3O4/SiO2/HPG-COOH载体，备用。
(3)以多点共价结合法制备固定化磷脂酶C
将l gFe3O4/SiO2/HPG-COOH载体在50mL的磷酸缓冲液中浸泡24小时，磁分离后，调节pH为5.8，缓慢加入50ml 0.1％的EDC交联剂，随后以50rpm的速度在30℃搅拌1h，再加入50ml 0.12％的NHS继续搅拌1h，制备出活性酯修饰的羧基载体，并进行磁分离。随后将载体与50ml 500U/ml的PLC磷酸缓冲溶液在pH为6时反应1h，磁分离。再将载体与酶浸泡在pH为10的碳酸氢盐缓冲溶液中，50℃50rpm搅拌2h，放置24小时进行磁分离，用磷酸盐缓冲溶液对样品洗涤5次以上，得到的固定化酶保存在4℃条件下。
具体实施方式：
具体实施方式一：
以Fe3O4/SiO2/HPG-COOH作为载体，在反应温度40℃，PLC添加量400U/ml，PLC与EDC摩尔比分别为1:1、1:10、1:20、1:50、1:100的条件下，反应4h。考察PLC与EDC摩尔比对固定化酶的活力的影响。
具体实施方式二：
本实施方式与具体实施方式一的不同点在于PLC添加量分别为200、300、400、500、600、700和800U/ml，考察PLC添加量对固定化酶的活力的影响。
具体实施方式三：
本实施方式与具体实施方式一的不同点在于反应温度分别为35、40、45、50、55和60℃，考察反应温度对固定化酶的活力的影响。
具体实施方式四：
本实施方式与具体实施方式一的不同点在于反应时间分别为2、3、4、5、6、7和8h，考察反应时间对固定化酶的活力的影响。
具体实施方式五：
分别选用磁性固定化PLC和游离PLC，对100g毛油进行脱胶反应2h，在60℃条件下，在pH 4～8的范围分别进行试验，测定游离酶和固定化酶的酶活力。
具体实施方式六：
本实施方式与具体实施方式五的不同点在于在pH7.0，40～80℃的范围进行试验，测定游离酶和固定化酶的酶活力。
具体实施方式七：
在最适pH和最适温度的条件下研究酶的重复使用效果。
Claims (4)
1.一种基于多点共价作用制备固定化磷脂酶C的方法，其特征通过以下步骤实现：
步骤一：采用共沉淀法制备Fe3O4纳米粒子。取1.00g Fe3O4纳米颗粒，加入10ml TEOS和4ml Tween20，超声分散10分钟。再加入50mL的去离子水，超声10min。将20mL的浓氨水和60ml乙醇与上述溶液混合，35℃水浴并充分搅拌。制得的Fe3O4/SiO2颗粒在40℃真空烘箱中干燥；
步骤二：将Fe3O4/SiO2颗粒100g与30μl CH3OK溶液和2.5ml二甲基酰胺置于烧瓶中，在50℃、100rpm下搅拌反应1小时，去除甲醇后加入10ml无水二氧六环，95℃加热。缓慢滴加2.0g缩水甘油。反应2小时，加入甲醇分散，磁分离后用甲醇反复冲洗。烘干12小时后制得HPG活化后的Fe3O4/SiO2载体。三口烧瓶中加入100mg Fe3O4/SiO2/HPG、100mg三乙胺和10ml二甲基甲酰胺，一定量丁二酸酐(SA)，混合液在水浴中加热并在100rpm下搅拌。整个反应在氮气氛围下操作。磁分离后用甲醇冲洗三次。在真空干燥箱中干燥12h，得到Fe3O4/SiO2/HPG-COOH载体，备用；
步骤三：将lg Fe3O4/SiO2/HPG-COOH载体在50mlpH为5.8的磷酸盐缓冲溶液中浸泡1h，磁分离后，在50mLpH为10的碳酸氢盐缓冲液中浸泡24小时，磁分离后，缓慢加入一定量的EDC活化剂，50℃下，50rpm搅拌2h。反应一段时间后进行磁分离，同时使用磷酸盐缓冲溶液对样品进行冲洗，得到的固定化酶保存在4℃条件下；
步骤四：以Fe3O4/SiO2/HPG-COOH作为载体，在反应温度40℃，PLC添加量400U/ml，PLC与EDC摩尔比分别为1:1、1:10、1:20、1:50、1:100的条件下，反应4h。考察PLC与EDC摩尔比对固定化酶的活力的影响。保证其他条件不变调整PLC添加量为200、300、400、500、600、700和800U/ml，考察PLC添加量对固定化酶的活力的影响。保证其他条件不变调整反应温度分别为35、40、45、50、55和60℃，考察反应温度对固定化酶的活力的影响。保证其他条件不变调整反应时间分别为2、3、4、5、6、7和8h，考察反应时间对固定化酶的活力的影响；
步骤五：分别选用磁性固定化PLC和游离PLC，对100g毛油进行脱胶反应2h，在60℃条件下，pH 4～8的范围分别进行试验，测定游离酶和固定化酶的酶活力。在pH7.0、40～80℃的范围进行试验，测定游离酶和固定化酶的酶活力；
步骤六：在最适pH和最适温度的条件下，将固定化磷脂酶C应用在大豆毛油脱胶中，重复使用8次并测定固定化酶的酶活力重复使用效果。
2.根据权利要求1所述的一种基于多点共价作用制备固定化磷脂酶C的方法，其特征在于步骤四中最佳固定化条件为PLC与EDC的摩尔比1：20，PLC添加量500U/ml，反应温度45℃，反应时间5h。
3.根据权利要求1所述的一种基于多点共价作用制备固定化磷脂酶C的方法，其特征在于步骤五中最适pH和最适温度分别为7.5和55℃。
4.根据权利要求1所述的一种基于多点共价作用制备固定化磷脂酶C的方法，其特征在于步骤六中固定化酶重复使用8次仍保留87％的相对活力。
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