CN110643595A - 一种对化学修饰后磁性固定化pla1再活化的方法 - Google Patents
一种对化学修饰后磁性固定化pla1再活化的方法 Download PDFInfo
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Abstract
本发明利用Fe3O4纳米粒子和SiOx复合，得到磁性固定化磷脂酶A1，再用葡聚糖‑醛‑甘氨酸对磁性固定化酶化学修饰，得到Fe3O4/SiOx‑g‑P(GMA)‑PLA1‑Dx‑Gly，通过固定化和化学修饰后的磷脂酶A1的稳定性增加。以修饰后的磷脂酶A1为研究对象，将其应用在大豆油脱胶体系中，修饰后的磁性固定化磷脂酶A1重复使用6次后，相对酶活仍80%，提高了磷脂酶A1的重复使用次数。分离取出，在盐酸胍溶液中温育，处理后的磁性固定化酶悬浮于Tris‑HCl缓冲溶液中，以相对酶活作为评价指标，得出磁性固定化PLA1再活化的最佳条件为溶液pH为7，活化温度为25℃，活化时间为20 h，效应物浓度为0.20 mol/L，此时磁性固定化PLA1的酶活最高，回收酶活为初始酶活的99%，提高了磷脂酶A1的利用率，为工业化生产带来巨大的经济效益。
Description
技术领域
本发明涉及一种对化学修饰后磁性固定化PLA1再活化的方法。
背景技术
磷脂酶A1是油脂酶法脱胶研究和应用中常见的酶，相对于游离酶而言，固定化磷脂酶A1在稳定性上更胜一筹，而磁性固定化磷脂酶A1的出现推动了大豆油连续酶法脱胶可行性的进程。
磁性固定化酶具有独特的应用优势，即在磁性反应器中，可以控制磁场变化来实现固定化酶的分散，不仅提高了酶和底物的接触面积，还可以避免机械搅拌的剪切力对酶的伤害。另外，磁性固定化酶还可以应用到磁流化床中，能非常理想的实现反应后固定化酶和反应产物的分离。磁性固定化酶的载体主要是磁性高分子微球，其制备方法主要包括包埋法、单体聚合法、原位法和可控自由基聚合法。
酶的化学修饰是指为了改变酶的某些性质，用化学方法对酶蛋白质进行改造，方法包括主链切割、剪接和侧链基团的修饰等。化学修饰在酶蛋白表面形成一层覆盖膜，可以防止酶次级键断裂和蛋白质的水解，从而实现对酶的保护。修饰过程向酶分子引入了高亲水性聚合物包衣，这种修饰几乎不会降低酶活，还可以提高酶的稳定性。
随着商品酶的生产技术日益成熟和酶的应用领域逐渐拓展延伸，酶的回收和再活化问题再次引起了更多关注。应用酶学的研究内容包括失活酶的再活化，其目的是提高利用率。在生产应用和技术研究过程中，酶的价格构成了生产成本的主要部分，克服酶再活化的难题，将为社会生产带来巨大的经济效益。
发明内容
本发明首先利用Fe3O4纳米粒子和SiOx复合，采用共价固定的方法制备磁性固定化磷脂酶A1，再用葡聚糖-醛-甘氨酸对磁性固定化酶化学修饰，得到Fe3O4/SiOx-g-P(GMA)-PLA1-Dx-Gly，化学修饰后伯氨基的含量降至47 %，稳定性更高。使其在大豆油脱胶体系中重复使用直至酶活降至80 %，分离取出。
然后在盐酸胍溶液中温育，将处理后的磁性固定化酶悬浮于Tris-HCl缓冲溶液中，当磷酸二氢钠浓度为0.19 mol/L，再活化条件是温度为25 ℃，体系的pH为7.0，再活化时间为20 h， Fe3O4/SiOx-g-P(GMA)-PLA1-Dx-Gly再活化后平均相对酶活为99 %。
具体实施方式
具体实施方式一：
步骤一：本发明首先利用Fe3O4纳米粒子和SiOx复合，采用共价固定的方法制备磁性固定化磷脂酶A1。葡聚糖-醛-甘氨酸的制备。使用了具有50 %氧化度的葡聚糖（MW = 70,000Da），将葡聚糖-醛与等体积的pH=3.5的3 mol/L的甘氨酸混合，加入固体三甲基氨基硼烷至浓度为200 mmol/L。甘氨酸的氨基与葡聚糖中的醛基反应15 h后，通过加入含有100 mg/mL硼氢化钠的pH=10.0的500 mmol/L的碳酸盐缓冲液将反应混合物还原，目的是稳定已经形成的席夫碱，并破坏残留的醛。将该混合物在室温下温育30 min，用盐酸将混合物的pH降至pH=6以破坏硼氢化钠，然后用超纯水透析。再次用50 %高碘酸盐将葡聚糖-甘氨酸氧化，并以1:10的比例用超纯水透析10次，最终得到葡聚糖-醛-甘氨酸。
将1g Fe3O4/SiOx-g-P(GMA)-PLA1与10 mL含有30 mg/mL葡聚糖-醛-甘氨酸的pH=7.0的0.2 mol/L的磷酸二氢钠溶液在4 ℃一起温育12 h。达到交联反应的终点，将悬浮液过滤并重悬浮于4 ℃含有1 mg/mL硼氢化钠的pH=10.0的100 mmol/L的碳酸氢钠中，该制备物被称为Fe3O4/SiOx-g-P(GMA)-PLA1-Dx-Gly。
步骤二：将经过化学修饰的磁性固定化PLA1应用于大豆油的脱胶中，收集大豆油脱胶实验中失活的化学修饰磁性固定化PLA1进行前处理。按照0.25 g/ml的比例，将一定量的灭活的修饰磁性固定化PLA1置于适量pH=7.0的含有8 mol/L盐酸胍的Tris-HCl缓冲溶中温育30 min。然后，在1 min内将pH值迅速调节至8.5，并向悬浮液中补充含有10 mg二硫苏糖醇的pH=7.0的0.1 mmol/L的Tris-HCl缓冲液。
将前处理后的化学修饰磁性固定化PLA1从上述盐酸胍溶液中过滤出来，用去离子水彻底洗涤以去除二硫苏糖醇和变性剂。将失活的化学修饰磁性固定化PLA1重新悬浮于一定pH的50 mmol/L的Tris-HCl缓冲溶液中，加入硫代二硫化物交换催化剂，，加入一定浓度的磷酸二氢钾溶液作为效应物，再活化的温度为15、20、25、35、45 ℃，再活化的pH分别为6、6.5、7、7.5、8，再活化的时间为10、15、20、25、30 h，效应物浓度为0.10、0.15、0.20、0.25、0.30 mol/L。
具体实施方式二：本实施方式与具体实施方式一不同点在于步骤二再活化温度的不同，分别为20、25、35 ℃，其它步骤与具体实施方式一相同。
具体实施方式三：本实施方式与具体实施方式一不同点在于步骤二再活化的pH不同，分别为6.5、7、7.5，其它步骤与具体实施方式一相同。
具体实施方式四：本实施方式与具体实施方式一不同点在于步骤二再活化的时间不同，分别为15、20、25 h，其它步骤与具体实施方式一相同。
具体实施方式五：本实施方式与具体实施方式一不同点在于步骤二效应物浓度不同，分别为0.15、0.20、0.25 mol/L，其它步骤与具体实施方式一相同。
Claims (5)
1.一种对化学修饰后磁性固定化PLA1再活化的方法，其特征步骤如下：
步骤一：本发明首先利用Fe3O4纳米粒子和SiOx复合，采用共价固定的方法制备磁性固定化磷脂酶A1，葡聚糖-醛-甘氨酸的制备，使用了具有50 %氧化度的葡聚糖（MW = 70,000Da），将葡聚糖-醛与等体积的pH=3.5的3 mol/L的甘氨酸混合，加入固体三甲基氨基硼烷至浓度为200 mmol/L，甘氨酸的氨基与葡聚糖中的醛基反应15 h后，通过加入含有100 mg/mL硼氢化钠的pH=10.0的500 mmol/L的碳酸盐缓冲液将反应混合物还原，目的是稳定已经形成的席夫碱，并破坏残留的醛，将该混合物在室温下温育30 min，用盐酸将混合物的pH降至pH=6以破坏硼氢化钠，然后用超纯水透析，再次用50 %高碘酸盐将葡聚糖-甘氨酸氧化，并以1:10的比例用超纯水透析10次，最终得到葡聚糖-醛-甘氨酸，将1g Fe3O4/SiOx-g-P(GMA)-PLA1与10 mL含有30 mg/mL葡聚糖-醛-甘氨酸的pH=7.0的0.2 mol/L的磷酸二氢钠溶液在4 ℃一起温育12 h，达到交联反应的终点，将悬浮液过滤并重悬浮于4 ℃含有1 mg/mL硼氢化钠的pH=10.0的100 mmol/L的碳酸氢钠中，该制备物被称为Fe3O4/SiOx-g-P(GMA)-PLA1-Dx-Gly；
步骤二：将经过化学修饰的磁性固定化PLA1应用于大豆油的脱胶实验中，收集大豆油脱胶实验中失活的化学修饰磁性固定化PLA1进行前处理，按照0.25 g/ml的比例，将一定量的灭活的修饰磁性固定化PLA1置于适量pH=7.0的含有8 mol/L盐酸胍的Tris-HCl缓冲溶中温育30 min，然后，在1 min内将pH值迅速调节至8.5，并向悬浮液中补充含有10 mg二硫苏糖醇的pH=7.0的0.1 mmol/L的Tris-HCl缓冲液，将前处理后的化学修饰磁性固定化PLA1从上述盐酸胍溶液中过滤出来，用去离子水彻底洗涤以去除二硫苏糖醇和变性剂，将失活的化学修饰磁性固定化PLA1重新悬浮于一定pH的50 mmol/L的Tris-HCl缓冲溶液中，加入硫代二硫化物交换催化剂，加入一定浓度的磷酸二氢钾溶液作为效应物，再活化的温度为15、20、25、35、45 ℃，再活化的pH分别为6、6.5、7、7.5、8，再活化的时间为10、15、20、25、30 h，效应物浓度为0.10、0.15、0.20、0.25、0.30 mol/L。
2.根据权利要求1所述的一种提高磁性固定化PLA1再活化的方法，其特征在于步骤二中选取再活化温度分别为20、25、35 ℃。
3.根据权利要求1所述的一种提高磁性固定化PLA1再活化的方法，其特征在于步骤二中选取再活化的pH分别为6.5、7、7.5。
4.根据权利要求1所述的一种提高磁性固定化PLA1再活化的方法，其特征在于步骤二中选取再活化的时间分别为15、20、25 h。
5.根据权利要求1所述的一种提高磁性固定化PLA1再活化的方法，其特征在于步骤二中选取效应物浓度分别为0.15、0.20、0.25 mol/L。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4599197A (en) * | 1982-12-22 | 1986-07-08 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| US5077392A (en) * | 1988-10-17 | 1991-12-31 | Boehringer Mannheim Gmbh | Process for activation of recombinant protein produced by prokaryotes |
| JP2003102494A (ja) * | 2001-07-26 | 2003-04-08 | Sangaku Renkei Kiko Kyushu:Kk | 蛋白質のリフォールディング方法及び連続リフォールディング方法並びに連続リフォールディング装置 |
| CN102649954A (zh) * | 2012-05-17 | 2012-08-29 | 兰州大学 | 磁性纳米粘土载体固定化酶及其再生的方法 |
-
2019
- 2019-10-21 CN CN201911002105.XA patent/CN110643595A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4599197A (en) * | 1982-12-22 | 1986-07-08 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| US5077392A (en) * | 1988-10-17 | 1991-12-31 | Boehringer Mannheim Gmbh | Process for activation of recombinant protein produced by prokaryotes |
| JP2003102494A (ja) * | 2001-07-26 | 2003-04-08 | Sangaku Renkei Kiko Kyushu:Kk | 蛋白質のリフォールディング方法及び連続リフォールディング方法並びに連続リフォールディング装置 |
| CN102649954A (zh) * | 2012-05-17 | 2012-08-29 | 兰州大学 | 磁性纳米粘土载体固定化酶及其再生的方法 |
Non-Patent Citations (2)
| Title |
|---|
| RAFAEL ET AL.: "Improved reactivation of immobilized-stabilized lipase from Thermomyces lanuginosus by its coating with highly hydrophilic polymers", 《JOURNAL OF BIOTECHNOLOGY》 * |
| YU ET AL.: "Characterization of immobilized phospholipase A1 on magnetic nanoparticles for oil degumming application", 《LWT-FOOD SCIENCE AND TECHNOLOGY》 * |
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