CN113061595A - 一种以环氧基修饰的磁性壳聚糖纳米粒子为载体的固定化磷脂酶c的方法 - Google Patents
一种以环氧基修饰的磁性壳聚糖纳米粒子为载体的固定化磷脂酶c的方法 Download PDFInfo
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Abstract
本发明涉及磁性固定化酶领域,公开了一种以环氧基修饰的磁性壳聚糖纳米粒子为载体的固定化磷脂酶C的方法。采用环氧氯丙烷对共价法制备的磁性壳聚糖纳米粒子进行修饰,再对环氧基载体进行醛基活化,制取了一种具有醛基和氨基的磁性载体,在pH的改变下,利用多点共价结合的方式将磁性壳聚糖纳米载体与磷脂酶C结合,通过氨基与氨基之间的静电吸附和氨基与醛基之间的共价结合,将PLC的非活性中心基团与载体多点共价结合,生产出一种稳定性更好固定化PLC。本发明重点解决的问题不再是为了实现酶与底物的快速分离,更多的集中在优化酶的特性上,制备的固定化酶具有更高的活性和稳定性。之后在工业上的大规模生产提供了理论依据。
Description
技术领域
本发明涉及一种固定化磷脂酶C的制备方法,尤其涉及一种以环氧基修饰的磁性壳聚糖纳米粒子为载体令载体与酶进行多点反应的共价结合方法。
背景技术
壳聚糖是几丁质的去乙酰化衍生物,是一种同时具有羟基和氨基的天然多糖,具有良好的生物相容性,同时具有无毒、易改性、可生物降解、低成本可再生等特点,因其独特的理化性质和生物学特性广泛应用在食品,制药,电化学,生物传感器等领域。
近年来,作为一种可再生的绿色资源,壳聚糖作为载体被广泛的应用在酶固定化的研究中。将大分子壳聚糖纳米化可以起到减少载体与酶、载体与载体之间团聚现象的出现,制备的磁性壳聚糖纳米载体可以起到固定化酶与底物快速分离的作用。选择环氧氯丙烷代替交联剂戊二醛,能够降低交联的不定向进行,避免出现除载体与酶之外的交联发生。将经过环氧基修饰的磁性壳聚糖纳米粒子进行醛基活化,使磁性壳聚糖纳米载体同时具有醛基和氨基。便于进一步进行酶的共价固定。
磷脂酶C(PLC)能够特异性水解磷脂,已被广泛的应用在油脂脱胶以及药物输送中。在PLC对磷脂的水解过程中,主要反应基团为-COOH,而-NH2位于非活性中心可以作为酶的固定化位点。根据壳聚糖及PLC的特性,通过调整pH,使酶上的-COOH与载体上的-NH2进行静电吸附快速结合起来,再使酶的-NH2与载体的醛基进行共价结合,使二者稳定结合。
因此,选择环氧基修饰的壳聚糖磁性纳米粒子进行磷脂酶C的多点共价固定化,能够有效的避免戊二醛带来的过度交联以及非定向共价结合过程中对酶活性位点的破坏,对实际生产有重要的指导意义。
发明内容
本发明的目的在于针对现有技术的不足,提供以环氧基修饰的磁性壳聚糖纳米粒子为载体的固定化磷脂酶C的方法,制备出一种同时带有氨基和醛基的磁性壳聚糖纳米载体,所制备的磁性固定化磷脂酶C具有较高的稳定性。该酶具有适宜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,在储藏60天后仍保留93%的相对活力,60℃水浴180min后相对活力高于80%。经重复使用8次仍保留89%的相对活力,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)Fe3O4磁性纳米粒子的制备
在磁力搅拌下将FeCl3·6H2O(5.11g)和FeCl2·4H2O(1.99g)溶解在纯净水(100mL)中,并将反应溶液在氮气保护下加热至70℃。然后,用氢氧化铵(28%)将反应体系的pH值调节至10,并将温度升高至80℃达1小时。最后,溶液冷却至室温后,Fe3O4用磁体分离纳米颗粒,并用纯净水洗涤直至中性。
(2)载体的活化与修饰
将壳聚糖(0.035g溶于3mL蒸馏水)和7g磁性流体的溶液溶于3.5L蒸馏水。用1MHCl将混合物的pH降低至3.7,并将混合物在60℃的超声浴中处理60分钟。该容器装有机械搅拌器,搅拌12小时以使壳聚糖共价键合到金属氧化物纳米颗粒上。然后,将混合物在磁体上放置24小时,并沉淀壳聚糖官能化的磁赤铁矿纳米颗粒。弃去上清液,将颗粒用蒸馏水洗涤并风干。
(3)环氧基修饰磁性壳聚糖纳米载体的制备
在45℃下将2mL4.8 g·L-1环氧氯丙烷和2.7g·L-1NaOH的水溶液与1g壳聚糖颗粒接触2h。通过在4℃下将1g载体悬浮在6mL含40g·L-1NaOH,37%(v/v)丙酮和3.8g·L-1NaBH4的水溶液中来进行载体活化。然后,加入环氧氯丙烷直至最终浓度为185g·L-1,并将混合物在25℃下搅拌18h。修饰后的载体用20%(v/v)的丙酮水溶液和蒸馏水洗涤。
(4)载体的醛基活化
通过使10mL的0.5M H2SO4溶液与1g载体接触1小时,使残留的环氧基水解。在25℃下,使10mLNaIO4水溶液(10–25mM)与1g载体接触2小时,进行二醇氧化。
(5)PLC的共价固定
将50mL 600U/ml PLC与l g载体在50mLpH为5.8的磷酸盐缓冲溶液中浸泡1h,磁分离后,在50mL pH为10的碳酸氢盐缓冲液中浸泡24小时,磁分离后,在50℃下,50rpm搅拌2h。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。
具体实施方式:
具体实施方式一:
以环氧基修饰的磁性壳聚糖纳米载体作为载体,在反应温度50℃,反应时间为3h,PLC添加量为200、300、400、500、600、700和800U/ml的条件下时,考察PLC添加量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。
具体实施方式三:
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。
具体实施方式四:
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。
具体实施方式五:
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。
具体实施方式六:
在最适pH和最适温度的条件下研究酶的重复使用效果。
具体实施方式七:
将游离酶与磁性固定化酶,在4℃条件下储藏60d,每隔10天对酶活力进行一次测定,并计算相对酶活力,以此考察酶的储藏稳定性。
具体实施方式八:
用磷酸盐缓冲液调节体系的pH为6.0,保持在60℃水浴下270min,并每隔30min取一定样品,测定相对活力,研究长时间加热对PLC相对活力的影响。
Claims (4)
1.一种以环氧基修饰的磁性壳聚糖纳米粒子为载体的固定化磷脂酶C的方法,其特征通过以下步骤实现:
步骤一:在磁力搅拌下将FeCl3·6H2O(5.11g)和FeCl2·4H2O(1.99g)溶解在纯净水(100mL)中,并将反应溶液在氮气保护下加热至70℃。然后,用氢氧化铵(28%)将反应体系的pH值调节至10,并将温度升高至80℃达1小时。最后,溶液冷却至室温后,Fe3O4用磁体分离纳米颗粒,并用纯净水洗涤直至中性;
步骤二:将壳聚糖(0.035g溶于3mL蒸馏水)和7g磁性流体的溶液溶于3.5L蒸馏水。用1MHCl将混合物的pH降低至3.7,并将混合物在60℃的超声浴中处理60分钟。该容器装有机械搅拌器,搅拌12小时以使壳聚糖共价键合到金属氧化物纳米颗粒上。然后,将混合物在磁体上放置24小时,并沉淀壳聚糖官能化的磁赤铁矿纳米颗粒。弃去上清液,将颗粒用蒸馏水洗涤并风干;
步骤三:在45℃下将2mL4.8 g·L-1环氧氯丙烷和2.7g·L-1NaOH的水溶液与1g壳聚糖颗粒接触2h。通过在4℃下将1g载体悬浮在6mL含40g·L-1NaOH,37%(v/v)丙酮和3.8g·L-1NaBH4的水溶液中来进行载体活化。然后,加入环氧氯丙烷直至最终浓度为185g·L-1,并将混合物在25℃下搅拌18小时。修饰后的载体用20%(v/v)的丙酮水溶液和蒸馏水洗涤;
步骤四:通过使10mL的0.5M H2SO4溶液与1g载体接触1小时,使残留的环氧基水解。在25℃下,使10mL NaIO4水溶液(10–25mM)与1g载体接触2小时,进行二醇氧化;
步骤五:将g PLC与lg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,在50℃下,50rpm搅拌2h。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;
步骤六:以环氧基修饰的磁性壳聚糖纳米载体作为载体,在反应温度50℃,反应时间为3h,PLC添加量为200、300、400、500、600、700和800U/ml的条件下时,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;
步骤七: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;
步骤八: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。将游离酶与磁性固定化酶,在4℃条件下储藏60d,每隔10天对酶活力进行一次测定,并计算相对酶活力,以此考察酶的储藏稳定性。用磷酸盐缓冲液调节体系的pH为6.0,保持在60℃水浴下270min,并每隔30min取一定样品,测定相对活力,研究长时间加热对PLC相对活力的影响。
2.根据权利要求1所述的一种以环氧基修饰的磁性壳聚糖纳米粒子为载体的固定化磷脂酶C的方法,其特征在于步骤六中最佳固定化条件为PLC添加量600U/ml,反应温度45℃,反应时间5h。
3.根据权利要求1所述的一种以环氧基修饰的磁性壳聚糖纳米粒子为载体的固定化磷脂酶C的方法,其特征在于步骤七中最适pH和最适温度分别为8.0和60℃。
4.根据权利要求1所述的一种以环氧基修饰的磁性壳聚糖纳米粒子为载体的固定化磷脂酶C的方法,其特征在于步骤八中固定化酶重复使用8次仍保留89%的相对活力。固定化酶在储藏60天后仍保留93%的相对活力,60℃水浴180min后相对活力高于80%。
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