CN113930899A - 一种脂肪酶膜及其制备方法和应用 - Google Patents

一种脂肪酶膜及其制备方法和应用 Download PDF

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CN113930899A
CN113930899A CN202111231612.8A CN202111231612A CN113930899A CN 113930899 A CN113930899 A CN 113930899A CN 202111231612 A CN202111231612 A CN 202111231612A CN 113930899 A CN113930899 A CN 113930899A
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lipase
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郦金龙
张倩倩
王子恒
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Beijing Technology and Business University
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Abstract

本发明属于材料化学技术领域,具体涉及一种用于脂肪酶固定的纤维膜,包括亲水纤维‑疏水纤维杂化纤维膜;所述亲水纤维‑疏水纤维杂化纤维膜中亲水纤维和疏水纤维交叉层叠;所述亲水纤维和疏水纤维的质量比为1:5‑5:1;所述亲水纤维直径为0.01μm‑2μm;所述疏水纤维直径为0.1μm‑2μm。上述用于脂肪酶固定的纤维膜可用于食品、制药、化工合成、污水处理中。本发明通过双针对纺的方式完成静电纺疏水纤维和静电纺水凝胶杂化,制备获得了水凝胶‑疏水纤维膜,让水溶性的脂肪酶随着水相吸入膜内部,制备获得了在膜内微小水油界面进行反应的酶膜反应器。不仅高效利用了脂肪酶、提高酶活力,同时能够实现自定义高效组装,且重复利用率高。

Description

一种脂肪酶膜及其制备方法和应用
技术领域
本发明属于材料化学技术领域,具体涉及一种基于水凝胶-疏水纤维杂化结构的脂肪酶高效酶膜反应器的制备方法。
背景技术
脂肪酶(Lipase,EC 3.1.1.3)作为一种重要的工业酶制剂品,在食品、日化、能源、环保、医药、合成等领域己经得到了广泛应用。脂肪酶不同活性的发挥依赖于反应体系的特点,酯水解主要在油水界面进行,脂肪酶对脂质水界面具有高亲和力。“界面催化”是脂肪酶脂水解催化机制的核心特征,脂解的瞬时速率取决于酶和反应物在界面上的二维浓度,而不是它们在体相中的浓度。
脂肪酶可高效地处理石化工业或其他行业的含油废水等,不会造成二次污染,并且能够降低成本。但诸难以回收和重复使用等局限性,严重限制了脂肪酶的工业应用潜力。脂肪酶固定化是一个很有吸引力的策略,可显著提高脂肪酶的实际性能并大大增加了脂肪酶回收的可能性。许多材料如硅藻土、硅胶、天然或合成聚合物、多孔玻璃、活性炭、介孔氧化硅、碳纳米管等都被研究作为固定化载体。但针对于脂肪酶的特殊界面性质设计的载体研究却很少,无法针对进行定向设计,活性不高。其它研究多聚焦于采用乳液界面系统(微乳液、纳米乳液,Pickering乳液等)作为油水催化界面的提供者,以确保脂肪酶的催化具有足够面积的催化界面从而提高脂肪酶的活性,但重复利用率低、资源浪费的问题仍待解决。
脂肪酶的固定化是提升脂肪酶催化效能的重要途径,相关领域涉及的固定化材料与固定化方法非常丰富,显著提升酶催化活性的同时,固定化酶的温各种物理性能和重复使用性也得到改善。静电纺丝技术已经应用在了多种酶的载用过程,纺出的纤维尺寸均一、组成多样,具有高孔隙率和比表面积,能够有效降低传质阻力,作为酶载体的应用有巨大潜力。但在凝胶技术以及静电纺丝与脂肪酶催化结合研究上,已有的国内外研究基本仍侧重于利用水凝胶或静电纺丝纤维膜实现脂肪酶的包埋或固定化,针对脂肪酶界面催化机制进行定点设计的高度界面化结构的杂化酶膜反应器并无较多研究。
发明内容
针对目前脂肪酶酶膜反应器中存在的问题,本发明提供一种脂肪酶膜,有更高的催化活力;不会造成酶的大量浪费和污染,可多次重复利用;有利于产物的收集。
为实现上述目的,本发明采用如下技术方案。
一种用于脂肪酶固定的纤维膜,包括亲水纤维-疏水纤维杂化纤维膜;所述亲水纤维-疏水纤维杂化纤维膜中亲水纤维和疏水纤维交叉层叠;所述亲水纤维和疏水纤维的质量比为1:5-5:1;所述亲水纤维直径为0.01μm-2μm;所述疏水纤维直径为0.1μm-2μm。
优选地,所述纤维膜的厚度为0.1-0.45mm;单位面积重量为0.008-0.15g/cm2。更优选地,所述纤维膜的厚度为0.1-0.3mm;平均单位面积重量为0.008-0.065g/cm2
优选地,所述亲水纤维的材料选自聚丙烯酸(PAA),甲基丙烯酸酐化明胶(GelMA),聚乙烯醇(PVA),壳聚糖(CS)和海藻酸钠(SA)中的至少一种;所述疏水纤维的材料选自聚氨酯(PU),聚乳酸(PLA),乙基纤维素(EC),聚偏氟乙烯(PVDF)和聚苯乙烯(PS)中的至少一种。
更优选地,所述亲水纤维的材料为聚丙烯酸;所述疏水纤维的材料为聚氨酯。
一种上述纤维膜的制备方法,包括以下步骤:通过双针对纺的静电纺丝方法制备获得用于脂肪酶固定的纤维膜。
优选地,所述双针对纺的参数设置为0.08:0.05-0.05:0.08的流速比。
一种脂肪酶膜的制备方法,包括以下步骤:
(1)将用于脂肪酶固定的纤维膜凝胶化,使酶膜的亲水纤维转化为水凝胶纤维,获得凝胶化纤维膜;
(2)将脂肪酶固定于凝胶化酶膜上,获得脂肪酶膜。
优选地,所述凝胶化的方法可采用物理交联或化学交联。如,加热,紫外,冷冻,交联剂交联。
优选地,固定的方法为吸附法、包埋法、交联法或共价结合法。
一种上述制备方法获得的脂肪酶膜。
一种上述脂肪酶膜制备的脂肪酶膜反应器。
一种上述用于脂肪酶固定的纤维膜,脂肪酶膜或脂肪酶膜反应器在食品、制药、化工合成、污水处理中的应用。如,生物柴油的制备、手性化合物拆分、工业皮革处理、食品脱脂加工。
本发明具有以下优点:
本发明通过双针对纺的方式完成静电纺疏水纤维和静电纺水凝胶杂化,制备获得了水凝胶-疏水纤维膜,让水溶性的脂肪酶随着水相吸入膜内部,制备获得了在膜内微小水油界面进行反应的酶膜反应器。在本发明的亲水纤维和疏水纤维的质量比下,随着疏水纤维的比例增加,纤维膜的单位面积重量增加,且吸附的酶量增加;在本发明的亲水纤维和疏水纤维的质量比下,随着疏水纤维的增加,单位酶活降低,随着亲水纤维的增加,单位酶活增加。当超出上述亲水纤维和疏水纤维的质量比和单位面积重量范围时,单位酶活出现下降。本发明的脂肪酶膜不仅高效利用了脂肪酶、提高酶活力,同时能够实现自定义高效组装,且重复利用率高。为未来设计多样高效智能的酶膜反应器提供了一条有效的设计思路。
附图说明
图1为用于脂肪酶固定的纤维膜中纤维结构的激光共聚焦显微图,其中:1-亲水纤维网络,2-疏水纤维网络;
图2为凝胶化纤维膜的电镜图;
图3为单层和多层酶膜反应器结构示意图;
图4为不同加热凝胶化时间对单位酶活力的影响;
图5为脂肪酶膜不同循环次数后单位酶活剩余率;
图6为不同纺丝比例对单位酶活力的影响;
图7为酶膜反应器内部油水分布的激光共聚焦显微图,其中:1-水相;2-油相;
图8为不同层数酶膜反应器的单位酶活力变化。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 用于脂肪酶固定的纤维膜的制备
(1)称取PAA加入含有0.1%(w/w)氯化钠的95%乙醇中,获得8%(w/w)的PAA溶液,即亲水纤维纺丝液;
称取PU加入N,N-二甲基甲酰胺和四氢呋喃等体积混合的溶剂中,获得22%(w/w)PU溶液,即疏水纤维纺丝液;
(2)PAA溶液和PU溶液分别装入5mL平推注射器,采用0.4mm的射流针头,针头水平放置且距接收器12-15cm;设置PAA溶液的流速设定为0.08-0.05cm/min,纺丝电压为正压16kV,负压-3kV;设置PU溶液的流速设定为0.05-0.08cm/min,纺丝电压为正压13kV,负压-3kV;
两个射流针头在300rpm/min金属滚筒接收器两侧对称放置,且在纺丝过程中都进行1000mm/min的轴向往复运动,接收器表面裹上硅油纸进行纤维接收;
(3)在30-35℃,30-40%空气湿度下纺丝6h得到厚度为0.15mm左右的纤维膜,得到的PAA纤维直径为0.01μm-2μm,PU纤维直径为0.1μm-2μm,平均单位面积重量为0.008-0.065g/cm2
将获得的纤维膜分别用荧光染料尼罗蓝和尼罗红,然后进行激光共聚焦纤维观察。荧光染料尼罗蓝和尼罗红能分别对亲水纤维和疏水纤维进行染色,染色后的杂化膜的在640nm、488nm波长激光下发射出不同颜色,如图1所示:其中红色纤维代表亲水纤维PAA,绿色纤维代表疏水纤维PU。由图1可知,获得的纤维膜是亲水纤维和疏水纤维是交叉层叠的。
实施例2 脂肪酶膜和酶膜反应器的制备
(1)将实施例1中的纤维膜150-160℃加热30min、60min、90min凝胶化,使酶膜的亲水纤维转化为水凝胶纤维,获得凝胶化纤维膜,加热60min凝胶化的电镜图如图2所示;然后切割成0.6mm直径的圆片,备用;
(2)天野脂肪酶(Lipase PS Amano SD,来自Burkholderia cepacia,≥30000 U/g)加入pH=7.0的磷酸盐缓冲液中,制备得到2mg/mL的脂肪酶溶液;
(3)将凝胶化纤维膜原片浸泡在脂肪酶溶液中12h,取出拭干表面残留酶液得到脂肪酶膜;
(4)将多层脂肪酶膜用长针从圆心处固定成图3所示的脂肪酶酶膜反应器,各脂肪酶膜之间间距为0.1-1.2cm。
实施例3 脂肪酶膜的制备
按照实施例1中的方法制备纤维膜,不同在于步骤(3)中纺丝18h得到厚度为0.45mm左右的纤维膜,得到的PAA纤维直径为0.01μm-2μm,PU纤维直径为0.1μm-2μm,单位面积重量为0.10-0.12g/cm2
按照实施例2中的方法制备脂肪酶膜,不同在于步骤(1)中,150-160℃加热60min凝胶化,获得脂肪酶膜的制备。
对比例1 亲水脂肪酶膜的制备
(1)称取PAA加入含有0.1%(w/w)氯化钠的95%乙醇中,获得8%(w/w)的PAA溶液,即亲水纤维纺丝液;
(2)PAA溶液装入5mL平推注射器,采用0.4mm的射流针头,针头水平放置且距接收器12-15cm,设置PAA溶液的流速设定为0.08cm/min,纺丝电压为正压16kV,负压-3kV;
(3)在30-35℃,30-40%空气湿度下纺丝得到厚度为0.15mm左右的亲水纤维膜;其中亲水纤维的直径为0.01µm-2μm;
(4)将上述亲水纤维膜150-160℃加热60min凝胶化,使酶膜的亲水纤维转化为水凝胶纤维,获得凝胶化纤维膜;然后切割成0.6mm直径的圆片,备用;
(5)天野脂肪酶(Lipase PS Amano SD,来自Burkholderia cepacia,≥30000 U/g)加入pH=7.0的磷酸盐缓冲液中,制备得到2mg/mL的脂肪酶溶液;
(6)将凝胶化纤维膜原片浸泡在脂肪酶溶液中12h,取出拭干表面残留酶液得到亲水脂肪酶膜。
对比例2 硅藻土固定化脂肪酶的制备
参照林海蛟,等(2019)的方法(林海蛟,张云,孙爱君,等.基于无机载体的先交联后吸附固定化脂肪酶的方法[J].暨南大学学报:自然科学与医学版,2019(3):195-205.),使用双功能环氧交联剂新戊二醇二缩水甘油醚和硅藻土载体固定化相同量的天野脂肪酶,采用先交联后吸附的方法;交联条件:pH5.5,温度35℃,交联剂质量分数0.5%,交联时间2h;吸附条件:温度40℃,载体质量1.5g,吸附时间5h。交联结束后加入硅藻土载体吸附,再继续放在恒温摇床中固定化。吸附结束后将固定化酶抽滤、常温烘干,即得硅藻土固定化脂肪酶。
应用例1 脂肪酶膜在降解p-NPP中的应用
以磷酸对硝基苯酯(p-NPP)为底物检测不同脂肪酶膜的催化性能:
(1)将p-NPP配制为浓度为2mg/mL的异辛烷溶液;
(2)将单片实施例2中制备的不同加热时间的脂肪酶膜浸入2mL的p-NPP溶液,1500r/min的搅拌条件下进行酶促反应,15min后加入2mL无水乙醇终止反应,再加入1mL磷酸盐缓冲液(pH 7.0)冲出产物,水相振荡均匀后,410nm波长检测产物浓度,BCA法测出膜内吸附酶量,计算酶活。单位脂肪酶酶活力=
Figure DEST_PATH_IMAGE002
(3)按照步骤(2)的方法再循环4次,计算单位酶活力;
同时,以酶液(同膜内吸附酶量、水量相同)和对比例1中的亲水脂肪酶膜为对照,加入2mL的p-NPP溶液组成传统二维界面,得到单位酶活力。
从图4可知,不同凝胶化时间得到的脂肪酶膜中的单位脂肪酶活力都高于酶液对照,将酶液中单位酶活力最低的设为100%,凝胶化时间设定为60min时,脂肪酶膜的单位酶活力最高,为传统二维界面对照组的2.56倍。
从图5可知,单片脂肪酶膜在不另加酶的条件下,循环使用4次,单位酶活仍保持在较高水平,为初始酶活的63%。
从图6可知,不同比例亲水/疏水纤维得到的脂肪酶膜中的单位脂肪酶活力都高于亲水脂肪酶膜,将亲水脂肪酶膜的酶活力设为100%,得到在纺丝比例为0.08/0.05(亲水/疏水)时,脂肪酶膜的单位酶活最高,较亲水脂肪酶膜,酶活提高10.63倍。
将实施例1和2中纺丝比例为0.08/0.05(亲水/疏水),凝胶时间为60min,纺丝时间为6h的脂肪酶膜(平均单位面积重量为0.05g/cm2)与实施例3中的脂肪酶膜进行相同条件下的p-NPP酶促降解,结果显示:与酶液相比,实施例获得的脂肪酶膜的酶活提高2.56倍,实施例3获得的脂肪酶膜与酶液相比酶活提高2.03倍,实施例中比实施例3中的酶活提高倍数更高。说明,随着纤维膜厚度的增加,即单位面积重量的增加,酶活呈现出先增加后降低的趋势。过大的单位面积重量会因传质阻力过大而降低单位酶活。
应用例2 脂肪酶膜反应器在降解p-NPP中的应用
以磷酸对硝基苯酯(p-NPP)为底物检测不同脂肪酶膜反应器的催化性能:
(1)将p-NPP配制为浓度为2mg/mL的异辛烷溶液;
(2)将实施例2中制备的加热60min的脂肪酶膜反应器(脂肪酶膜片数分别为2片、4片、8片)浸入2mL的p-NPP溶液,1500r/min的搅拌条件下进行酶促反应,15min后加入2mL无水乙醇终止反应,再加入1mL磷酸盐缓冲液(pH 7.0)冲出产物,水相振荡均匀后,410nm波长检测产物浓度,BCA法测出膜内吸附酶量,计算酶活。单位脂肪酶酶活力=
Figure DEST_PATH_IMAGE002A
;同时,以酶液(同膜内吸附酶量、水量相同)和对比例2中的固定化脂肪酶为对照。
图7为酶膜反应器内每片脂肪酶膜中油水分布的激光共聚焦显微图,证实了其能够实现微观上的油水交叉,即三维界面的效果。
根据图8可知,随着脂肪酶膜片数的增加,脂肪酶膜反应器中的脂肪酶活先增加后下降,但与对照相比,其酶活提高倍数逐渐增加,分别为2.56、4.54、6.15、6.71倍。
与对比例2中的固定化脂肪酶相比,实施例2中的脂肪酶膜的单位脂肪酶活力了至少1.46倍。
采用不同亲水材料,如,PAA、GelMA、PVA、CS、SA、HA中任意一种;和疏水材料,如,PU、PLA、EC、PVDF、PS中任意一种采用适当的双针对纺参数,以亲水纤维:疏水纤维=1:5-5:1(w/w)获得纤维直径0.01-2μm的用于脂肪酶固定的纤维膜,纤维膜的密度为0.008-0.065g/cm2,然后以加热或化学交联的方式凝胶化、以吸附法、包埋法、化学法、交联法和共价结合法固定化脂肪酶。在磷酸对硝基苯酯酶促降解过程中,相比单纯酶液反应,脂肪酶膜的单位酶活力提高1.2-15倍。

Claims (10)

1.一种用于脂肪酶固定的纤维膜,其特征在于,包括亲水纤维-疏水纤维杂化纤维膜;所述亲水纤维-疏水纤维杂化纤维膜中亲水纤维和疏水纤维交叉层叠;所述亲水纤维和疏水纤维的质量比为1:5-5:1;所述亲水纤维直径为0.01μm-2μm;所述疏水纤维直径为0.1μm-2μm。
2.根据权利要求1所述的纤维膜,其特征在于,所述纤维膜的厚度为0.1-0.45mm;平均单位面积重量值为0.008-0.15g/cm2
3.根据权利要求1所述的纤维膜,其特征在于,所述亲水纤维的材料选自聚丙烯酸,甲基丙烯酸酐化明胶,聚乙烯醇,壳聚糖和海藻酸钠中的至少一种;所述疏水纤维的材料选自聚氨酯,聚乳酸,乙基纤维素,聚偏氟乙烯和聚苯乙烯中的至少一种;优选地,所述亲水纤维的材料为聚丙烯酸;所述疏水纤维的材料为聚氨酯。
4.一种如权利要求1-3任一所述纤维膜的制备方法,其特征在于,包括以下步骤:通过双针对纺的静电纺丝方法制备获得用于脂肪酶固定的纤维膜。
5.根据权利要求4所述的制备方法,其特征在于,所述双针对纺的参数设置为0.08:0.05-0.05:0.08的流速比。
6.一种脂肪酶膜的制备方法,其特征在于,包括以下步骤:
(1)将用于脂肪酶固定的纤维膜凝胶化,使酶膜的亲水纤维转化为水凝胶纤维,获得凝胶化纤维膜;
(2)将脂肪酶固定于凝胶化酶膜上,获得脂肪酶膜。
7.根据要求6所述的制备方法,其特征在于,所述凝胶化的方法可采用物理交联或化学交联;所述固定的方法为吸附法、包埋法、交联法或共价结合法。
8.一种如权利要求6或7任一制备方法获得的脂肪酶膜。
9.一种如权利要求8所述的脂肪酶膜制备的脂肪酶膜反应器。
10.一种如权利要求1-3任一所述用于脂肪酶固定的纤维膜,如权利要求8所述的脂肪酶膜或如权利要求9所述的脂肪酶膜反应器在食品、制药、化工合成、污水处理中的应用。
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