CN113584011A - 一种聚多巴胺包覆的塑料填料固定化酶的方法 - Google Patents
一种聚多巴胺包覆的塑料填料固定化酶的方法 Download PDFInfo
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Abstract
本发明为一种聚多巴胺包覆的塑料填料固定化酶的方法。该方法包括以下步骤:该方法将塑料填料放入适合浓度的多巴胺溶液中恒温震荡反应,进行表面修饰改性,使其拥有更好的粘附性、润湿性和表面粗糙度,再将含有固定化酶液的溶胶负载在改性填料上。本发明成本低、效果好、操作简便,得到的塑料载酶填料具有更高的酸碱稳定性、有机溶剂稳定性、储存稳定性和重复使用性。
Description
技术领域:
本发明涉及的固定化酶方法,是一种将塑料填料表面改性并将其应用于涂层和固定化酶领域的方法。
背景技术:
塑料填料因其重量轻、价格便宜、耐高温、耐腐蚀、损坏率低的优势,已被广泛应用于各类填料塔中。但是塑料填料表面能较低,难于被涂层附着,限制了它在涂层及固定化酶等领域的应用,因此需要对塑料填料进行表面改性,使其拥有各种所需的性能。
目前,绝大多数表面改性方法(如表面粗糙化、化学处理法、辐射法、喷涂处理法和塑料改性法等)均有其自身局限性,比如工艺复杂、效果不长久、对基体有特定要求以及对环境不友好等。一直以来人们致力于找寻一种适用于修饰各种基体材料,且操作简便、条件温和、经济高效的表面改性方法。
发明内容:
本发明的目的是针对当前技术中存在的不足,提供一种聚多巴胺包覆的塑料填料固定化酶的方法及应用。该方法将聚丙烯塑料填料放入适合浓度的多巴胺溶液中恒温震荡反应,进行表面修饰改性。在有氧弱碱性溶液中多巴胺会发生氧化自聚合反应,生成具有强粘附作用的聚多巴胺,可以粘附到任何固体基体表面形成原位涂层,并且与材料表面形成牢固且持久的键,使填料拥有更好的粘附性、润湿性和表面粗糙度,更有利于进行固定化酶操作。本发明成本低、效果好、操作简便。
本发明的技术方案为:
一种聚多巴胺包覆的塑料填料固定化酶的方法,该方法包括以下步骤:
(1)塑料填料表面改性:
将清洗并干燥后的塑料材质填料浸入多巴胺溶液中,在室温下于水浴恒温震荡器中连续反应1~2h时间,取出填料,依次用乙醇和去离子水彻底清洗至滤液无色透明,放入55~65℃真空烘箱中隔夜干燥,得到多巴胺改性的聚丙烯填料;
其中,多巴胺溶液的浓度为1.5~2.2mg/mL,其溶剂为pH为8~9的0.05~0.20M的三羟甲基胺基甲烷(Tris)缓冲溶液;
所述的塑料材质填料的清洗方法为:将塑料填料放入异丙醇溶液中超声洗涤20~40min,再用蒸馏水超声洗涤15~20min,取出放入50~60℃真空干燥箱。
所述的塑料材质为聚氯乙烯、聚乙烯或聚丙烯;
(2)脂肪酶的固定化:
将A液和B液分别在0℃冰水浴中搅拌冷却0.5~2min,然后将A液分2~5次倒入B液中,混合液继续在冰水浴中搅拌冷却2~4min,得到溶胶;
其中,A液由甲基三甲氧基硅烷、甲醇、正硅酸甲酯混合而成;B液由氟化钠、聚乙二醇400、去离子水、脂肪酶液混合而成;
体积比为A液:B液=3:1.5~2.5;
所述的A液中,甲基三甲氧基硅烷与正硅酸甲酯的质量比为2.0:1~4.0:1;甲醇添加量为A液总质量的50~60%;
所述的B液中,酶液添加量为B液总质量的50~60%;聚乙二醇400添加量为B液总质量的3.0~4.0%;水添加量为B液总质量的25~35%;氟化钠添加量为B液总质量的10~15%。
所述的脂肪酶液中的脂肪酶为南极假丝酵母脂肪酶A(CaLA)、南极假丝酵母脂肪酶B(CaLB)或褶皱假丝酵母脂肪酶(CRL);
优选为为CaLB酶时,脂肪酶液的组成和质量百分比浓度如下:
(3)载酶填料的制备:采用浸没或喷涂的方式,将上一步得到的溶胶附着在经过改性的塑料材质填料上,将其干燥至恒定质量,即得到载酶填料;
其中,因喷涂过程中会有溶胶喷涂在外造成损失,最终每克填料附着溶胶1~2mL;
所述的喷涂方式具体包括如下步骤:
将经过改性的塑料材质填料平铺到培养皿中,喷枪到填料表面的距离约为10~20cm,喷枪的喷涂流量约为1.5~2.2kg/h,每喷涂3~5秒,进行一次吹风机辅助干燥,待填料表面无流动液体可进行下一次喷涂;经过15~30次“喷涂-吹风机干燥”后,完成喷涂;
所述的步骤(1)中Tris缓冲溶液为加盐酸调至pH为8~9。
与现有技术相比,本发明的有益效果为:
(1)将塑料填料应用于酶催化反应精馏领域,成本更低。
(2)聚多巴胺的修饰能够有效提升塑料填料的表面粗糙度,增加了填料的微观表面积,可以增加凝胶涂层在填料表面的固定点,有利于载酶凝胶与填料间的粘附。利用原子力显微镜(AFM)可以表征的填料改性前后的表面形态,图1为改性前后的AFM图。改性前粗糙度为97.49nm,改性后为232.7nm。
(3)聚多巴胺涂层可以作为多功能的二级反应平台,聚多巴胺以“桥梁”的形式连接填料与酶,可以辅助生物分子共价结合,以共价键和氢键作用将载酶凝胶和部分酶牢牢结合在填料表面,与传统固定化方法或单一的凝胶包埋法相比,具有载酶量更高、固定化酶更稳定和结合力更强的特点。图2为用原子力显微镜在力曲线模式下测量改性前后载酶填料附着力性能测试图,从图中可以看出改性后的粘附力显著增强。
(4)本发明所制的塑料载酶填料具有更高的酸碱稳定性、有机溶剂稳定性、储存稳定性和重复使用性。在pH 10.0的NaOH溶液中孵育12h后,改性前后载酶填料相对酶活分别为75%和81%;在有机溶剂乙酸乙酯中孵育20h后,改性前后载酶填料相对酶活分别为83%和92%;在储存60天后改性前后载酶填料相对酶活分别为86%和91%。
附图说明
图1为改性前后填料表面的原子力显微镜(AFM)图;
图2为用原子力显微镜在力曲线模式下测量改性前后载酶填料附着力性能测试图;其中,图2(a)为改性前的载酶填料附着力性能图,图2(b)为改性后的载酶填料附着力性能图。
图3为多巴胺表面改性前后喷涂溶胶凝胶固定化酶对比图。
图4为实施例5中得到的多巴胺浓度为1.0mg/mL的填料表面图。
具体实施方式
本发明涉及的脂肪酶为南极假丝酵母脂肪酶B(CaLB)为市售公知物质,购于诺维信公司,具体组成和质量百分比浓度如下:
实施例1
取12.114g三羟甲基胺基甲烷(Tris)于1L容量瓶中,加去离子水得到0.1M的Tris缓冲溶液,加盐酸调至pH为8.5。将2g多巴胺溶解在Tris缓冲溶液中,以获得浓度为2.0mg/mL的新鲜多巴胺溶液。将聚丙烯填料放入异丙醇溶液中,超声洗涤30min,再用蒸馏水超声清洗15min,除去表面的油脂及灰尘,取出放入50℃真空干燥箱。将清洗并干燥后的聚丙烯填料浸入多巴胺溶液中,在室温下于水浴恒温震荡器中连续反应1.5h,取出填料,依次用乙醇和去离子水彻底清洗至滤液无色透明,放入60℃真空烘箱中隔夜干燥,得到多巴胺改性的聚丙烯填料。
称取正硅酸甲酯0.548g、甲基三甲氧基硅烷2.056g、甲醇3.39g,将液体混合后作为A液(共6.218mL)。再称取聚乙二醇0.14g、氟化钠0.49g、去离子水0.63g、南极假丝酵母脂肪酶液(脂肪酶含量为6%)2.32g,将液体混合后作为B液(共3.966mL)。A液和B液分别在0℃冰水浴中搅拌冷却1min,再将A液缓慢多次倒入B液中,混合液继续在冰水浴中搅拌冷却3min,然后迅速倒入喷枪中,溶胶在空气泵的气体压力下(1bar)从喷枪的喷嘴中雾化喷射。将多巴胺改性后的聚丙烯填料平铺在一个干净的培养皿中,喷枪到填料的距离约为15cm,调节喷涂流量约为1.82kg/h。因溶胶凝胶在喷涂过程中仍是液体,并未粘附到填料上,为了防止喷枪气流吹掉刚刚涂覆的溶胶溶液,在喷涂过程中每喷涂一层就立刻使用吹风机来辅助干燥(待填料表面无流动液体,进行下一次喷涂)。喷涂量为10g,填料数量为8个,喷涂-干燥组合操作共24次,喷涂时间为8min。喷涂完成后,将其干燥至恒定质量,即得到载酶填料,填料成品负载固定化酶平均1.2g/个,整体约为13.4kg/m3。
因喷涂过程中会有溶胶喷涂在外造成损失,最终每克填料附着溶胶1~2mL;
经考马斯亮蓝法检测未改性的载酶填料蛋白固载率为91.23±2.2%,而改性后的蛋白固载率为97.61±1.6%,酶的利用率有所提高。原填料的粘合强度为48.169MPa,改性后的粘合强度为56.529MPa,表面改性后载酶填料的附着力增强。改性后的填料在储存60天后仍保持约91%的相对活性,高于原填料的86%,表明改性后的载酶填料稳定性在增强。
附图分析:
图1为改性前后填料表面原子力显微镜(AFM)图,(其中,上层标识a的部分为改性前填料表面图,下层标识b的部分为改性后填料表面图。)改性前粗糙度为97.49nm,改性后为232.7nm,表明聚多巴胺的修饰能够有效提升塑料填料的表面粗糙度,除此之外,填料表面形貌也有很大差异,多巴胺处理后填料表面出现了许多由多巴胺组成的规则纳米颗粒,增加了填料的微观表面积,可以增加凝胶涂层在填料表面的固定点,有利于载酶凝胶与填料间的粘附。
图2为用原子力显微镜在力曲线模式下测量改性前后载酶填料附着力性能测试图。当探针接触到表面时都出现了跳触(jump-in)现象,表明涂层与基体间存在吸引力;当探针离开接触表面时均出现跳离(jump-out)现象,表明涂层与基体间存在粘附力,探针彻底从表面分离需要克服此力,所以在向外拉动探针时,最大形变量即表示涂层与基体的粘附力,从图中可以看出,改性后的填料粘附力更强。
图3为塑料填料经多巴胺改性前后分别喷涂溶胶凝胶固定化酶的成品形貌图。图中浅色为未改性的塑料固定化酶填料的形貌,深色的为改性后的塑料固定化酶填料的形貌。两者对比可见改性后的塑料填料表面的酶更多且不易脱落。
实施例2
其他步骤同实施例1,不同之处为MTMS与TMOS的质量比替换为3:1,具体添加量为正硅酸甲酯0.651g、甲基三甲氧基硅烷1.953g。
实验结果显示蛋白固载率为89.62±2.4%;粘合强度为55.734MPa;在pH 10.0中孵育12h后,相对酶活为80%;在有机溶剂乙酸乙酯中孵育20h后,相对酶活为89%;在储存60天后相对酶活为88%
由实验结果可知,硅烷前驱体比例改变后,蛋白固载率和粘合强度变化不大,这是因为蛋白固载率和粘合强度主要取决于填料表面的多巴胺涂层。相对酶活都稍有降低,这是因为脂肪酶CaLB是一种界面活性酶,凝胶基质的疏水性有利于维持酶分子构象并增强酶的活性,而MTMS的减少会降低凝胶基质疏水性,所以酶活都有降低的趋势。
实施例3
其他步骤同实施例1,不同之处为甲醇添加量替换为A液总质量的60%,具体添加量为3.65g。
实验结果显示蛋白固载率为88.42±2.6%;粘合强度为56.762MPa;在pH 10.0中孵育12h后,相对酶活为78%;在有机溶剂乙酸乙酯中孵育20h后,相对酶活为89%;在储存60天后相对酶活为79%。
由实验结果可知,酶活降低较为明显。这是因为甲醇在配方中起到分散剂的作用,可以让酶分散均匀,但甲醇对酶的活性有损伤,会降低酶活,因此相对活性都下降比较明显。
实施例4
其他步骤同实施例1,不同之处为酶液添加量替换为B液总质量的62%,具体添加量为2.48g。
实验结果显示蛋白固载率为80.41±2.1%;粘合强度为57.297Mpa;在pH 10.0中孵育12h后,相对酶活为89%;在有机溶剂乙酸乙酯中孵育20h后,相对酶活为91%;在储存60天后相对酶活为91%。
由实验结果可知,蛋白固载率下降较明显。通常,随着酶质量分数增加,相应的结合位点增加,酶与底物的结合效果更好,但是酶过多时,酶分子并不能被完全包埋,会产生团聚现象,因此使得蛋白固载率趋于下降。
实施例5
其他步骤同实施例1,不同之处为多巴胺溶液由2.0mg/mL替换为1.0mg/mL。
实验结果显示蛋白固载率为84.54±1.2%;粘合强度为53.864MPa;在pH 10.0中孵育12h后,相对酶活为82%;在有机溶剂乙酸乙酯中孵育20h后,相对酶活为85%;在储存60天后相对酶活为86%;填料表面存在开裂现象。
图4为多巴胺浓度为1.0mg/mL的样品表面电镜图,黑色方框线区域为出现裂缝的区域。填料表面出现较为明显的裂纹,这是因为多巴胺在配方中起到中间桥梁作用,可以帮助酶与塑料填料更好地结合,多巴胺浓度降低导致塑料填料表面的粘附性降低,因此酶粘合强度不够易脱落导致开裂。
本发明未尽事宜为公知技术。
Claims (7)
1.一种聚多巴胺包覆的塑料填料固定化酶的方法,其特征为该方法包括以下步骤:
(1)塑料填料表面改性:
将清洗并干燥后的塑料材质填料浸入多巴胺溶液中,在室温下于水浴恒温震荡器中连续反应1~2h时间,取出填料,依次用乙醇和去离子水清洗,放入55~65℃真空烘箱中隔夜干燥,得到多巴胺改性的聚丙烯填料;
(2)脂肪酶的固定化:
将A液和B液分别在0℃冰水浴中搅拌冷却0.5~2min,然后将A液分2~5次倒入B液中,混合液继续在冰水浴中搅拌冷却2~4min,得到溶胶;
其中,A液由甲基三甲氧基硅烷、甲醇、正硅酸甲酯混合而成;B液由氟化钠、聚乙二醇400、去离子水、脂肪酶液混合而成;
体积比为A液:B液=3:1.5~2.5;
所述的A液中,甲基三甲氧基硅烷与正硅酸甲酯的质量比为2.0:1~4.0:1;甲醇添加量为A液总质量的50~60%;
所述的B液中,酶液添加量为B液总质量的50~60%;聚乙二醇400添加量为B液总质量的3.0~4.0%;水添加量为B液总质量的25~35%;氟化钠添加量为B液总质量的10~15%;
(3)载酶填料的制备:采用浸没或喷涂的方式,将上一步得到的溶胶附着在经过改性的塑料材质填料上,将其干燥至恒定质量,即得到载酶填料;
其中,每克填料附着溶胶1~2mL。
2.如权利要求1所述的聚多巴胺包覆的塑料填料固定化酶的方法,其特征为所述的塑料材质填料的清洗方法为:将塑料填料放入异丙醇溶液中,超声洗涤20~40min,再用蒸馏水超声清洗15~20min,取出放入50~60℃真空干燥箱。
3.如权利要求1所述的聚多巴胺包覆的塑料填料固定化酶的方法,其特征为所述的塑料材质为聚氯乙烯、聚乙烯或聚丙烯。
4.如权利要求1所述的聚多巴胺包覆的塑料填料固定化酶的方法,其特征为所述的多巴胺溶液的浓度为1.5~2.2mg/mL,其溶剂为pH为8~9的0.05~0.20M的三羟甲基胺基甲烷(Tris)缓冲溶液。
5.如权利要求1所述的聚多巴胺包覆的塑料填料固定化酶的方法,其特征为所述的脂肪酶液中的脂肪酶为南极假丝酵母脂肪酶A(CaLA)、南极假丝酵母脂肪酶B(CaLB)或褶皱假丝酵母脂肪酶(CRL)。
7.如权利要求1所述的聚多巴胺包覆的塑料填料固定化酶的方法,其特征为所述的喷涂方式具体包括如下步骤:
将经过改性的塑料材质填料平铺到培养皿中,喷枪到填料表面的距离约为10~20cm,喷枪的喷涂流量约为1.5~2.2kg/h,每喷涂3~5秒,进行一次吹风机辅助干燥,待填料表面无流动液体,进行下一次喷涂;经过15~30次“喷涂-吹风机干燥”后,完成喷涂。
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