CN114525269A - 一种基于聚乙二醇和超顺磁性氧化铁纳米颗粒共价结合固定化漆酶的方法 - Google Patents
一种基于聚乙二醇和超顺磁性氧化铁纳米颗粒共价结合固定化漆酶的方法 Download PDFInfo
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Abstract
本发明公开了一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,具体步骤如下:在室温下,将3‑氨基丙基‑三甲氧基硅烷(APTE)溶液缓慢加入氨基聚乙二醇醛基(NH2‑PEG‑CHO)溶液后高速搅拌一段时间后可生成产物AP‑PEG,再缓慢加入谷氨酸水溶液继续高速搅拌可生成产物AP‑PEG‑CHO;接着继续缓慢加入用缓冲液溶解的漆酶搅拌一段时间后可生成产物LA@PEG,最终缓慢加入SPION溶液密封搅拌一段时间可制备终产物LA@PEG@SPIONs,即固定化漆酶,经有机混合溶剂多次洗涤,直到未检测到漆酶,真空干燥后获得灰色粉末LA@PEG@SPION。所获固定化漆酶较游离漆酶具有更高的热稳定催化活性,可磁性回收重复利用,降低使用成本,提高生产经济效益等特点。
Description
技术领域
本发明属于漆酶固定化技术领域,涉及一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法。
背景技术
漆酶是一种含四个铜离子的多酚氧化酶(EC 1.10.3.2)是一种天然的绿色催化剂,广泛存在于真菌(白腐菌、彩绒革盖菌等)、细菌及漆树中,能够催化芳香胺、酚类等化合物。由于具有催化效率高、底物特异性广、对辅因子和特定环境条件要求少、无毒等优点,在制浆造纸、生物转化、脱印漂白和生物传感器制造等领域有巨大的应用潜力。但游离漆酶成本高且稳定性低导致其工业化应用受限,固定化是解决该问题的有效手段之一,可增强酶的热稳定性和使酶易与产物分离以提高酶回收率,显著具有更高经济环保效益。漆酶以单体糖蛋白的形式存在,可用于修饰纳米材料的活性位点包括氨基、羧基和巯基。通过共价键合将漆酶固定在纳米颗粒、微囊、多孔和胶束上,可避免漆酶在工程应用中的解吸,确保可靠、可持续的回收和再利用。
目前,将漆酶固定在各种载体上包括微球、纳米粒子、纳米纤维和膜,可通过增加稳定性、耐久性和实现连续操作来减少游离漆酶的应用限制。其中,磁性Fe3O4纳米颗粒(超顺磁性氧化铁纳米颗粒,SPION)被广泛应用于酶固定化,与过滤或离心相比,它可以在外部磁场中轻松快速地从反应介质中分离出来。Xia等人(Chemical Engineering Journal,2016, 295, 201-206.)制备了吸附容量较高的Fe3O4-NH2-PEI-漆酶胶束,其中酶的活性、稳定性和利用率,以及对酸的适应性、热稳定性和储存稳定性都得到了显著提高。然而,阳离子聚合物聚乙烯亚胺(PEI)的高细胞毒性和环境细胞毒性限制了Fe3O4-NH2-PEI-漆酶胶束的工业化 (Beyth et al., Proceedings of the National Academy of Sciences,2010, 107, 22038)。
传统酶固定化技术主要包括吸附、包埋、结合或化学交联等技术,固定化酶稳定性、牢固性、回收利用便利程度及其催化效率与工业实际应用仍具有较大差距。以纳米磁性材料为代表的新型固定载体以及定向化学修饰为代表的固定化技术取得了显著进展。磁性纳米颗粒是一类利用磁力和磁场的改变来控制粒子运动轨迹的新材料,这一特性不仅便于游离酶与载体结合和分离,同时也便于固定化酶的分离与回收。最常用磁性载体材料为磁性高分子微球,该微球是将磁性粒子和有机高分子相结合,并在其表面连接多种活性基团,如羟基(OH)、羧基(COOH)、醛基(CHO)和巯基(SH)等,从而实现酶的固定化。研究人员将辣根过氧化物酶固定在一种聚乙二醇磁性复合微球上。固定化辣根过氧化物酶负载量高达139.82 mg/g,在稳定性、贮存能力和可重复性利用等方面均有良好的改善,并可用于苯酚降解(Xie et al., Enzyme Microb Technol, 2019, 122: 26-35)。专利号201510872957.X漂珠固定化漆酶及其制备方法,利用的是漂珠与胺基硅烷反应形成氨基改性漂珠,再经1-(3-二甲胺基丙基)-3-乙基碳二亚胺为交联剂得到固体漂珠固定化酶。对废水中活性蓝脱色率达90%,游离漆酶催化活性对比不得而知。专利号201511025976.5一种碳纳米管固定化漆酶及其用途;使用的是琥珀酰亚胺基和氨基三乙酸功能化接枝。固定化漆酶及其制备方法(专利申请号201710757634.5)以经酸碱刻蚀获得的介孔化膨润土为载体,对漆酶进行固定化,但存在温度高(80~100℃)、能耗高等缺点。总之,以铁基SPION磁性微球对漆酶进行固定化技术鲜有报道。
发明内容
本发明提供了一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,解决了游离漆酶成本高且稳定性低导致工业化应用受限等问题,降低了生产应用成本,提高了生产经济效益。
本发明所采用的技术方案如下:一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,步骤为:
1. 本发明公开了一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,具体步骤如下:在室温下,将3-氨基丙基-三甲氧基硅烷(APTE)溶液缓慢加入氨基聚乙二醇醛基(NH2-PEG-CHO)溶液后高速搅拌一段时间后可生成产物AP-PEG,再缓慢加入谷氨酸水溶液继续高速搅拌可生成产物AP-PEG-CHO;接着继续缓慢加入用缓冲液溶解的漆酶搅拌一段时间后可生成产物LA@PEG,最终缓慢加入SPION溶液密封搅拌一段时间可制备终产物LA@PEG@SPIONs,即固定化漆酶,经有机混合溶剂多次洗涤,直到未检测到漆酶,真空干燥后获得灰色粉末LA@PEG@SPION。所获固定化漆酶较游离漆酶具有更高的热稳定催化活性,可磁性回收重复利用,降低使用成本,提高生产经济效益等特点。此外,本专利阐述的漆酶均来自彩绒革盖菌(Coriolus versicolor),购买于美国Sigma-Aldrich试剂有限公司。
第一步,形成AP-PEG的过程:适量的3-氨基丙基-三甲氧基硅烷(APTE)溶液与一定量的氨基聚乙二醇醛基(NH2-PEG-CHO)溶液后在一定的时间和搅拌速率等反应条件下形成AP-PEG。
第二步,形成AP-PEG-CHO的过程:在上述形成的AP-PEG溶液中继续添加适量的谷氨酸水溶液在一定条件下反应生成AP-PEG-CHO,可作为漆酶和SPION载体的支撑基。
第三步,形成LA@PEG的过程:在上述获得的AP-PEG-CHO溶液中继续添加适量的漆酶溶液在一定条件下进行搅拌反应可生成LA@PEG。
第四步,形成LA@PEG@SPIONs:在形成LA@PEG的溶液中适量添加SPIONs溶液在一定条件下进行搅拌反应可生成LA@PEG@SPIONs。
第五步,除杂并干燥处理:用有机混合溶剂多次洗涤,直到未检测到漆酶,并在一定温度下进行真空干燥最终获得灰色粉末LA@PEG@SPION。
2. 如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,适量的3-氨基丙基-三甲氧基硅烷(APTE)溶液是指50~100 mg,用5~10 mL DMSO溶解。适量的氨基聚乙二醇醛基(NH2-PEG-CHO)溶液具体是指1000 ~1500 mg用10~15 mL DMSO溶解后,在一定的时间和搅拌速率条件下是指在600~1000 rpm/min速率下室温搅拌2~4 h形成AP-PEG。
3. 如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,继续添加适量的谷氨酸水溶液在一定条件下反应生成AP-PEG-CHO中谷氨酸质量浓度为50~70%,添加量为2~6 mL;和在600~800rpm/min速率下室温搅拌1~4 h。
4. 如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,继续添加适量的漆酶溶液后在一定条件下进行搅拌反应具体是指用pH 5.0乙酸-乙酸钠缓冲液溶解的漆酶(1.0 mg/mL)5~10 mL的溶液,在一定的时间和搅拌速率条件下是指在600~1000 rpm/min速率下室温搅拌2~4 h。
5. 如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,所述的适量添加SPIONs溶液是指用30~100 mL二甲基亚砜溶解分散SPION(30~100 mg)均匀配置成1~10 mg/mL溶液;所述的在一定条件下进行搅拌反应可生成LA@PEG@SPIONs是指在500~1200 rpm搅拌速率和室温下进行12~72 h的时间反应。
6. 如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,所述的用有机混合溶剂进行洗涤是指用乙醇和蒸馏水以体积比1%~99%混合,在一定温度和真空条件下进行真空干燥是指在20~30℃下,真空压力为0.01~0.1 Mpa,真空干燥时间3~9 h。
有益效果:
(1)本发明采用的是室温的绿色反应过程,耗能低、过程环保等特点。
(2)使用的交联剂为聚乙二醇(PEG)具有良好的生物溶解性,不利于细胞粘附,允许其广泛用于食品、药物和生物材料添加剂。PEG的吸收程度取决于其分子量,低分子量(1k以下)的PEG更容易被吸收,并对患者产生过敏反应,而高分子量PEG4k-6k在人体或动物中的吸收更有限,具有无副作用和毒性。
(3)利用PEG可使胶束和纳米粒子的表面光滑,不易损坏。PEG(4k以上)胶束不仅可以通过PEG的静电斥力和空间位阻防止颗粒团聚,还可增加漆酶的负载能力,提高固定化漆酶的柔韧性,减少空间位阻,从而提高酶催化效率。
(4)较传统酶固定化技术如吸附、包埋、结合或化学交联等方法,本专利制备的固定化漆酶稳定性、回收利用便利程度更高,仅通过磁场即可实现漆酶与反应产物快速分离。
(5)本专利的漆酶固定技术具有条件温和、过程简单安全,所需设备简便,重现性好,产品收率高且质量高,工艺稳定,适合工业化生产。
附图说明
附图1 产物(a) SPIONs, (b) NH2-PEG-CHO, (c) LA@PEG5k 和 (d) LA@PEG5k@SPIONs的红外光谱图
附图2 产物SPIONs (a), LA@PEG5K micelles (b), 和 LA@PEG5K@ SPIONs (c)在25℃下记录的广角XRD图谱(A)和磁滞回线(B)
附图3 在水中分散的固定化漆酶LA@PEG@SPIONs微观图:(A-C) TEM电镜图, (D)SEM 电镜图,(E) 粒径分布。
具体实施方式
实施例1
在室温下,将10 mg/mL 的50 mg 3-氨基丙基-三甲氧基硅烷(APTE)溶解于5 mLDMSO溶液缓慢滴入 100 mg/mL的 1000 mg氨基聚乙二醇醛基(NH2-PEG-CHO)溶解于10 mLDMSO溶液后在800 rpm下高速搅拌3 h后形成AP-PEG,再缓慢滴入2 mL的50%谷氨酸水溶液继续在1000 rpm高速搅拌4 h形成AP-PEG-CHO(可作为漆酶和SPION载体的支撑基);缓慢添加10 mL含漆酶(1mg/mL)缓冲液(乙酸-乙酸钠,pH 5.0, 0.2 M)到AP-PEG-CHO溶液后密封在600 rpm速率下搅拌24 h后形成LA@PEG,再缓慢添加到1 mg/mL SPION(30 mg)DMSO (30mL) 溶液密封搅拌制备LA@PEG@SPIONs,最后用纯乙醇、50%乙醇-水和纯水溶剂多次洗涤,直到未检测到漆酶存在,经真空干燥所得灰色粉末LA@PEG@SPION,在25℃下,真空压力为0.01 Mpa,真空干燥时间6 h。将获得的干燥固定化漆酶产物LA@PEG@SPIONs溶解分散于乙酸-乙酸钠缓冲液(0.1M, pH 5.0)中用于测定漆酶活性,以ABTS为底物,在25℃和420 nm波长处,每隔5 s中记录吸光度,得到其经历的时间与吸光度的关系。所测的固定化漆酶LA@PEG@SPION在25℃以底物为ABTS的酶活为115.8 U/mL;在同样条件下游离漆酶所测的酶活为116.74U/mL。此外,漆酶来自彩绒革盖菌,Coriolus versicolor,购买于美国Sigma-Aldrich试剂有限公司具体测定漆酶的步骤如下所述。
漆酶活性的测定
配制0.05 mol/L的HAc-NaAc缓冲液(pH 5.0)和0.5 mmol/L ABTS溶液,在25 ℃预热酶液及ABTS溶液。测定漆酶活性时,取2.9 mL预热的ABTS溶液,加入0.1 mL稀释后的漆酶溶液(酶质量浓度1.8 g/L),迅速摇晃使两者充分混合。用UV-Vis分光光度计记录 420 nm波长处混合液的吸光度从一点(A1)变化到另一点(A2)所经历的时间,A1与A2之间的吸光度差值统一为0.2,测定所用漆酶活性。一个漆酶活力单位(U)是指:漆酶在1 min 转化1 μmolABTS,使产物ABTS+·浓度从a增加至b。漆酶活性计算公式为:
式中:k=0.0492;ABTS+·浓度增加量ΔA为0.2;ΔT为吸光度变化所经历的时间/s;V0为酶活测试底物和酶液的总体积/mL;V 1为漆酶溶液体积/mL;C为漆酶质量浓度/(g/L)。
实施例2
在室温下,将15 mg/mL 的75 mg 3-氨基丙基-三甲氧基硅烷(APTE)溶解于5 mLDMSO溶液缓慢滴入 120 mg/mL的 1200 mg氨基聚乙二醇醛基(NH2-PEG-CHO)溶解于10 mLDMSO溶液后在1000 rpm下高速搅拌2.5 h后形成AP-PEG,再缓慢滴入5 mL的50%谷氨酸水溶液继续在800 rpm高速搅拌4 h形成AP-PEG-CHO(可作为漆酶和SPION载体的支撑基);缓慢添加20 mL含漆酶(2mg/mL)缓冲液(乙酸-乙酸钠,pH 5.0, 0.2 M)到AP-PEG-CHO溶液后密封在600 rpm速率下搅拌36 h后形成LA@PEG,再缓慢添加到2 mg/mL SPION(50 mg)DMSO(25 mL) 溶液密封搅拌制备LA@PEG@SPIONs,最后用纯乙醇、70%乙醇-水和纯水溶剂多次洗涤,直到未检测到漆酶存在,经真空干燥所得灰色粉末LA@PEG@SPION,在25℃下,真空压力为0.01 Mpa,真空干燥时间9 h。以底物为ABTS,在25℃室温下固定化漆酶LA@PEG@SPIONs的酶活为120.6 U/mL。此外,漆酶来自彩绒革盖菌,Coriolus versicolor,购买于美国Sigma-Aldrich试剂有限公司在同样条件下游离漆酶所测的酶活为122.39 U/mL。测定漆酶方法见实施例1。
实施例3
在室温下,将10 mg/mL 的50 mg 3-氨基丙基-三甲氧基硅烷(APTE)溶解于5 mLDMSO溶液缓慢滴入 100 mg/mL的 1000 mg氨基聚乙二醇醛基(NH2-PEG-CHO)溶解于10 mLDMSO溶液后在800 rpm下高速搅拌4 h后形成AP-PEG,再缓慢滴入2 mL的50%谷氨酸水溶液继续在1000 rpm高速搅拌3 h形成AP-PEG-CHO(可作为漆酶和SPION载体的支撑基);缓慢添加10 mL含漆酶(1.0 mg/mL)缓冲液(乙酸-乙酸钠,pH 5.0, 0.2 M)到AP-PEG-CHO溶液后密封在600 rpm速率下搅拌48 h后形成LA@PEG,再缓慢添加到1 mg/mL SPION(30 mg)DMSO(30 mL) 溶液密封搅拌制备LA@PEG@SPIONs,最后用纯乙醇、50%乙醇-水和纯水溶剂多次洗涤,直到未检测到漆酶存在,经真空干燥所得灰色粉末LA@PEG@SPION,在25℃下,真空压力为0.01 Mpa,真空干燥时间8 h。以底物为ABTS,在25℃室温下固定化漆酶LA@PEG@SPIONs的酶活为119.88 U/mL。此外,漆酶来自彩绒革盖菌,Coriolus versicolor,购买于美国Sigma-Aldrich试剂有限公司。在同样条件下游离漆酶所测的酶活为117.59 U/mL。测定漆酶方法见实施例1。
对比例
仅考量不同温度下(30℃、50℃和100℃)游离漆酶和固定化漆酶的酶活对比,其他实验条件一致。参照文献(谢普军等,食品科学,2018,39(16):90-97)中漆酶活性的测定方法,使用游离漆酶在30℃时以ABTS为底物的酶活为115.3 U/mL,本专利获得的固定化漆酶在30℃时以ABTS为底物的酶活为113.8 U/mL;游离漆酶在50℃时以ABTS为底物的酶活为103.68 U/mL,本专利获得的固定化漆酶在50℃时以ABTS为底物的酶活为117.5 U/mL;游离漆酶在100℃时以ABTS为底物的酶活为0.3 U/mL,本专利获得的固定化漆酶在100℃时以ABTS为底物的酶活为28.65 U/mL;因此,本发明制备的固定化漆酶较游离漆酶的热稳定性更高,由于共价键合了超顺磁氧化铁纳米颗粒使其具有磁性可回收重复利用等特点。
Claims (6)
1.本发明公开了一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,具体步骤如下:在室温下,将3-氨基丙基-三甲氧基硅烷(APTE)溶液缓慢加入氨基聚乙二醇醛基(NH2-PEG-CHO)溶液后高速搅拌一段时间后可生成产物AP-PEG,再缓慢加入谷氨酸水溶液继续高速搅拌可生成产物AP-PEG-CHO;接着继续缓慢加入用缓冲液溶解的漆酶搅拌一段时间后可生成产物LA@PEG,最终缓慢加入SPION溶液密封搅拌一段时间可制备终产物LA@PEG@SPIONs,即固定化漆酶,经有机混合溶剂多次洗涤,直到未检测到漆酶,真空干燥后获得灰色粉末LA@PEG@SPION;所获固定化漆酶较游离漆酶具有更高的热稳定催化活性,可磁性回收重复利用,降低使用成本,提高生产经济效益等特点,此外,本专利阐述的漆酶均来自彩绒革盖菌(Coriolus versicolor),购买于美国Sigma-Aldrich试剂有限公司;
第一步,形成AP-PEG的过程:适量的3-氨基丙基-三甲氧基硅烷(APTE)溶液与一定量的氨基聚乙二醇醛基(NH2-PEG-CHO)溶液后在一定的时间和搅拌速率等反应条件下形成AP-PEG;
第二步,形成AP-PEG-CHO的过程:在上述形成的AP-PEG溶液中继续添加适量的谷氨酸水溶液在一定条件下反应生成AP-PEG-CHO,可作为漆酶和SPION载体的支撑基;
第三步,形成LA@PEG的过程:在上述获得的AP-PEG-CHO溶液中继续添加适量的漆酶溶液在一定条件下进行搅拌反应可生成LA@PEG;
第四步,形成LA@PEG@SPIONs:在形成LA@PEG的溶液中适量添加SPIONs溶液在一定条件下进行搅拌反应可生成LA@PEG@SPIONs;
第五步,除杂并干燥处理:用有机混合溶剂多次洗涤,直到未检测到漆酶,并在一定温度下进行真空干燥最终获得灰色粉末LA@PEG@SPION。
2.如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,适量的3-氨基丙基-三甲氧基硅烷(APTE)溶液是指50~100 mg,用5~10 mL DMSO溶解;
适量的氨基聚乙二醇醛基(NH2-PEG-CHO)溶液具体是指1000 ~1500 mg用10~15 mLDMSO溶解后,在一定的时间和搅拌速率条件下是指在600~1000 rpm/min速率下室温搅拌2~4 h形成AP-PEG。
3.如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,继续添加适量的谷氨酸水溶液在一定条件下反应生成AP-PEG-CHO中谷氨酸质量浓度为50~70%,添加量为2~6 mL;和在600~800 rpm/min速率下室温搅拌1~4 h。
4.如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,继续添加适量的漆酶溶液后在一定条件下进行搅拌反应具体是指用pH 5.0乙酸-乙酸钠缓冲液溶解的漆酶(1.0 mg/mL)5~10 mL的溶液,在一定的时间和搅拌速率条件下是指在600~1000 rpm/min速率下室温搅拌2~4 h。
5.如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,所述的适量添加SPIONs溶液是指用30~100 mL二甲基亚砜溶解分散SPION(30~100 mg)均匀配置成1~10 mg/mL溶液;所述的在一定条件下进行搅拌反应可生成LA@PEG@SPIONs是指在500~1200 rpm搅拌速率和室温下进行12~72 h的时间反应。
6.如权利要求1所述的一种基于聚乙二醇(PEG)和超顺磁性氧化铁纳米颗粒(SPION)共价结合固定化漆酶的方法,其特征在于,所述的用有机混合溶剂进行洗涤是指用乙醇和蒸馏水以体积比1%~99%混合,在一定温度和真空条件下进行真空干燥是指在20~30℃下,真空压力为0.01~0.1 Mpa,真空干燥时间3~9 h。
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CN104911175A (zh) * | 2015-07-02 | 2015-09-16 | 南京工业大学 | 氨基硅烷化磁性纳米粒子共固定化漆酶和介体系统及其制备方法 |
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CN104478068A (zh) * | 2014-10-30 | 2015-04-01 | 华中科技大学 | 固定化漆酶及木质素类介体处理抗生素污染水体的方法 |
CN104911175A (zh) * | 2015-07-02 | 2015-09-16 | 南京工业大学 | 氨基硅烷化磁性纳米粒子共固定化漆酶和介体系统及其制备方法 |
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CN117085147A (zh) * | 2023-10-17 | 2023-11-21 | 潍坊医学院 | 一种负载阿霉素的碳基给药系统及其制备方法和应用 |
CN117085147B (zh) * | 2023-10-17 | 2023-12-22 | 潍坊医学院 | 一种负载阿霉素的碳基给药系统及其制备方法和应用 |
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