CN113308122A - 一种可降解蛋白塑料及其制备方法 - Google Patents
一种可降解蛋白塑料及其制备方法 Download PDFInfo
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Abstract
本发明涉及材料技术领域，特别涉及一种可降解蛋白塑料及其制备方法。该可降解蛋白塑料由多赖氨酸的工程蛋白交联制成，多赖氨酸的工程蛋白为含有VPGKG重复序列的蛋白。本发明在整个制备过程中，使用水作为载体进行塑性和制备，反应温和，绿色环保，不涉及到有毒的化学物质，同时也不涉及到复杂的制备过程。制备的蛋白塑料具有良好的机械性能，高的拉伸强度和高的韧性，塑料的大小和厚度可以通过制备过程中溶液的浓度及体积来控制。此外，还具有良好的生物相容性和可降解性，可以在蛋白酶的作用下发生降解，制备工艺简单、绿色环保，可开发为新一代环保型蛋白塑料用于进一步应用，具有广泛的应用前景。
Description
技术领域
本发明涉及材料技术领域，特别涉及一种可降解蛋白塑料及其制备方法。
背景技术
石油化工塑料是现在生活中常用的材料之一，广泛应用于包装、建筑、电子等各个领域。常用的石油化工基塑料具有数百甚至更多的稳定的长聚合物链，大多数塑料(例如聚乙烯、聚苯乙烯和聚氯乙烯)需要数千年才能降解。石油化工基塑料不仅污染了全球各地，而且由于其不可生物降解性，也对我们的生态系统造成潜在的不可逆转的破坏。预计至到2025年，将有110亿吨塑料堆积在垃圾填埋场和自然环境中，对生态环境造成严重的污染和破坏。
为解决上述石油化工塑料所带来的环境生态污染问题，对“生物塑料”包括生物基或生物可降解的石化和生物基塑料展开了广泛的研究工作。通过木质化、化学交联和化学改性等方法可以对生物质原料进行处理，制成生物塑料。但在合成过程中有毒化学品的使用以及复杂的加工步骤都限制了生物塑料的进一步广泛应用。而随着生物制造和合成生物学的大力发展，以微生物为基础的生物制造策略已延伸到聚合物，可通过微生物内质粒的设计，合成制备具有不同功能的生物基聚合物，为制造可降解的生物塑料开辟了新的制备途径。然而在制备和使用过程中，生物塑料的机械强度和耐久性仍具有挑战性。
公开号为CN108440974A的“一种高强度明胶蛋白塑料包装膜及其制备方法”通过将长链脂肪酸、明胶和戊二醛三种原料，通过热熔、热压和戊二醛气相交联三步法制备高强度明胶蛋白塑料包装膜。在整个过程中涉及到三个复杂的制备工艺，同时引入了有一定毒性的戊二醛交联剂，一定程度上限制了明胶蛋白塑料包装膜的应用拓展。
发明内容
本发明的目的是为了解决石油化工塑料所带来的环境生态污染问题，克服现有生物塑料合成技术中存在的机械强度的问题，同时避免有毒化学品的使用以及复杂的成型加工步骤，提供一种基于生物工程蛋白的高强高韧生物环保型可降解蛋白塑料及其制备方法。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种可降解蛋白塑料，其由多赖氨酸的工程蛋白交联制成，多赖氨酸的工程蛋白为含有VPGKG重复序列的蛋白。
作为优选，多赖氨酸的工程蛋白中VPGKG重复序列的数量不低于10个。
优选地，多赖氨酸的工程蛋白中VPGKG重复序列的数量为30个或30个以上。
作为优选，多赖氨酸的工程蛋白序列为如下序列中的一种或几种：
a)SEQ ID NO：1至SEQ ID NO：4所示任一序列；或者，
b)与SEQ ID NO：1至SEQ ID NO：4所示任一序列同源性不低于60％的序列。
优选地，多赖氨酸的工程蛋白序列为与SEQ ID NO：1至SEQ ID NO：4所示任一序列同源性不低于80％的序列。
本发明还提供了上述可降解蛋白塑料的制备方法，包括如下步骤：
步骤一、将多赖氨酸的工程蛋白溶于水，得到工程蛋白溶液；
将双醛基聚乙二醇溶于水，得到交联剂溶液；
步骤二、将工程蛋白溶液和交联剂溶液混合，震荡，离心去除气泡，得到蛋白交联产物溶液；
步骤三、将蛋白交联产物溶液放入模具，干燥成型。
作为优选，工程蛋白溶液的浓度为100～1000mg/mL。
优选地，工程蛋白溶液的浓度为200～400mg/mL。
作为优选，交联剂溶液的浓度为100～1000mg/mL；
优选地，交联剂溶液的浓度为100～500mg/mL。
更优选地，交联剂溶液的浓度为100～200mg/mL。
作为优选，步骤二中，工程蛋白溶液与交联剂溶液的体积比为(1～5)：(1～5)。
优选地，步骤二中，工程蛋白溶液与交联剂溶液的体积比为1：1。
作为优选，震荡为漩涡震荡，震荡的时间为2～10min。
优选地，震荡为漩涡震荡，震荡的时间为3～5min。
作为优选，离心为：在2000～6000rpm下离心1～5min。
优选地，离心为：在4000rpm下离心2min。
作为优选，干燥为自然风干12～36h。
优选地，干燥为自然风干12～24h。
在本发明中，多赖氨酸的工程蛋白通过将含有目的基因的载体质粒转化进大肠杆菌进行表达、纯化得到。
本发明提供了一种可降解蛋白塑料及其制备方法。该可降解蛋白塑料由多赖氨酸的工程蛋白交联制成，多赖氨酸的工程蛋白为含有VPGKG重复序列的蛋白。本发明相对于现有技术的有益效果是：
本发明提供的一种基于生物工程蛋白的高强高韧生物环保型可降解蛋白塑料，由带多赖氨酸的工程蛋白组成，在水中进行溶解，通过CHO-PEG-CHO进行化学交联，然后经过挥发水分制备而成。在整个过程中，使用水作为载体进行塑性和制备，反应温和，绿色环保，不涉及到有毒的化学物质，同时也不涉及到复杂的制备过程。制备的蛋白塑料具有良好的机械性能，高的拉伸强度和高的韧性，塑料的大小和厚度可以通过制备过程中溶液的浓度及体积来控制。此外，还具有良好的生物相容性和可降解性，可以在蛋白酶的作用下发生降解，制备工艺简单、绿色环保，可开发为新一代环保型蛋白塑料用于进一步应用，具有广泛的应用前景。
附图说明
图1为本专利中具体实施例中所选用的CHO-PEG-CHO化学结构示意图；
图2为实施例1制备得到的蛋白塑料图片；
图3为实施例2制备得到的蛋白塑料图片；
图4为实施例2制备得到的蛋白塑料拉伸性能曲线；
图5为实施例2制备得到的蛋白塑料的TG分析图；
图6为实施例2制备得到的蛋白塑料降解前后图片；
图7为实施例3制备得到的蛋白塑料图片；
图8为实施例4制备得到的蛋白塑料图片。
具体实施方式
本发明公开了一种可降解蛋白塑料及其制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
20种氨基酸英文名、中文名及字母表示：
英文 中文 三字母 单字母
glycine 甘氨酸 Gly G
alanine 丙氨酸 Ala A
valine 缬氨酸 Val V
Leucine 亮氨酸 Leu L
isoleucine 异亮氨酸 Ile I
proline 脯氨酸 Pro P
phenylalanine 苯基丙氨酸 Phe F
tyrosine 酪氨酸 Tyr Y
tryptophan 色氨酸 Trp W
serine 丝氨酸 Ser S
Threonine 苏氨酸 Thr T
cysteine 半胱氨酸 Cys C
methionine 蛋氨酸 Met M
asparagine 天冬氨酸 Asn N
glutamine 谷酰胺 Gln Q
lysine 赖氨酸 Lys K
Arginine 精氨酸 Arg R
Histidine 组氨酸 His H
aspartate 天门冬氨酸 Asp D
glutamate 谷氨酸 Glu E
本发明中所用试剂或仪器均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1：
一种高强高韧生物环保型可降解蛋白塑料的制备方法，所述方法具体步骤如下：
步骤一：配制质量分数为200mg/mL的蛋白的水溶液，充分震荡搅拌均匀；所用蛋白的序列如下所示(SEQ ID NO：1)：
MGAGPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGWPHHHHHH(36个VPGKG重复序列)
步骤二：配制质量分数为100mg/mL的CHO-PEG-CHO水溶液，充分震荡搅拌均匀；
步骤三：分别取步骤一以及步骤二得到的溶液，进行等体积混合。上述液体混合后立即漩涡震荡5min；
步骤四：将步骤三中的混合液体在4000rpm下进行离心2min，去除溶液中悬浮的气泡；
步骤五：将步骤四所得的溶液倾倒在四氟乙烯模具表面，在空气中自然风干12h，即可得到高强高韧生物环保型可降解蛋白塑料(图2)。
实施例2：
一种高强高韧生物环保型可降解蛋白塑料的制备方法，所述方法具体步骤如下：
步骤一：配制质量分数为200mg/mL的蛋白的水溶液，充分震荡搅拌均匀；所用蛋白的序列如下所示(SEQ ID NO：2)：
MGAGPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGWPHHHHHH(72个VPGKG重复序列)
步骤二：配制质量分数为200mg/mL的CHO-PEG-CHO水溶液，充分震荡搅拌均匀；
步骤三：分别取步骤一以及步骤二得到的溶液，进行等体积混合。上述液体混合后立即漩涡震荡3min；
步骤四：将步骤三中的混合液体在4000rpm下进行离心2min，去除溶液中悬浮的气泡；
步骤五：将步骤四所得的溶液倾倒在四氟乙烯模具表面，在空气中自然风干12h，即可得到高强高韧生物环保型可降解蛋白塑料(图3)。将所得的蛋白塑料进行力学性能拉伸测试，测试结果如图4所示，证明所制备的蛋白塑料具有良好的力学性能，包括高的强度以及良好的韧性，TG分析见图5。
为了验证蛋白塑料的可降解性，在制备蛋白塑料的过程中加入极少量的食用性素色增强对比度，将所得的蛋白塑料放入弹性蛋白酶缓冲溶液中(0.2mg/mL弹性蛋白酶溶解在100μM碳酸氢铵溶液中)，放置在37℃条件下震荡24h，整个溶液变成黄色，如图6所示，证明蛋白塑料已经完全降解。
实施例3：
一种高强高韧生物环保型可降解蛋白塑料的制备方法，所述方法具体步骤如下：
步骤一：配制质量分数为200mg/mL的蛋白的水溶液，充分震荡搅拌均匀；所用蛋白的序列如下所示(SEQ ID NO：3)：
MGAGPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGWPHHHHHH(144个VPGKG重复序列)
步骤二：配制质量分数为150mg/mL的CHO-PEG-CHO水溶液，充分震荡搅拌均匀；
步骤三：分别取步骤一以及步骤二得到的溶液，进行等体积混合。上述液体混合后立即漩涡震荡3min；
步骤四：将步骤三中的混合液体在4000rpm下进行离心2min，去除溶液中悬浮的气泡；
步骤五：将步骤四所得的溶液倾倒在四氟乙烯模具表面，在空气中自然风干12h，即可得到高强高韧生物环保型可降解蛋白塑料(图7)。
实施例4：
一种高强高韧生物环保型可降解蛋白塑料的制备方法，所述方法具体步骤如下：
步骤一：配制质量分数为200mg/mL的蛋白的水溶液，充分震荡搅拌均匀；所用蛋白的序列如下所示(SEQ ID NO：4)：
MPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSHTTHHAPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGPAATAVSDIWPHHHHHH(60个VPGKG重复序列)
步骤二：配制质量分数为100mg/mL的CHO-PEG-CHO水溶液，充分震荡搅拌均匀；
步骤三：分别取步骤一以及步骤二得到的溶液，进行等体积混合。上述液体混合后立即漩涡震荡5min；
步骤四：将步骤三中的混合液体在4000rpm下进行离心2min，去除溶液中悬浮的气泡；
步骤五：将步骤四所得的溶液倾倒在四氟乙烯模具表面，在空气中自然风干12h，即可得到高强高韧生物环保型可降解蛋白塑料。蛋白塑料的形状可通过改变膜具的形貌进行调控，所得蛋白塑料的照片如图8所示。
本发明通过制备带多赖氨酸的工程蛋白，利用CHO-PEG-CHO进行化学交联，通过水溶及挥发水的方式制备基于生物工程蛋白的蛋白塑料。制备的蛋白塑料具有良好的机械性能，同时可以在蛋白酶的作用下发生降解。整个制备过程，工艺简单，绿色环保，不涉及到有毒化学物质，使用水作为载体进行塑性和制备。制备的蛋白塑料具有良好的拉伸强度和韧性，膜的大小和厚度可控，可开发为新一代环保型蛋白塑料用于进一步应用。
对比例1
制备方法同实施例2，所用蛋白序列如下所示(SEQ ID NO：5)：MGAGPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGWPHHHHHH
但该制备方法并没有形成蛋白塑料，因此当蛋白序列中无K(赖氨酸)时，无法通过本发明制备方法形成蛋白塑料。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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Claims (10)
1.一种可降解蛋白塑料，其特征在于，其由多赖氨酸的工程蛋白交联制成，所述多赖氨酸的工程蛋白为含有VPGKG重复序列的蛋白。
2.根据权利要求1所述的可降解蛋白塑料，其特征在于，所述多赖氨酸的工程蛋白中VPGKG重复序列的数量不低于10个。
3.根据权利要求1所述的可降解蛋白塑料，其特征在于，所述多赖氨酸的工程蛋白中VPGKG重复序列的数量为30个或30个以上。
4.根据权利要求1至3中任一项所述的可降解蛋白塑料，其特征在于，所述多赖氨酸的工程蛋白序列为如下序列中的一种或几种：
a)SEQ ID NO：1至SEQ ID NO：4所示任一序列；或者，
b)与SEQ ID NO：1至SEQ ID NO：4所示任一序列同源性不低于60％的序列。
5.根据权利要求4所述的可降解蛋白塑料，其特征在于，所述多赖氨酸的工程蛋白序列为与SEQ ID NO：1至SEQ ID NO：4所示任一序列同源性不低于80％的序列。
6.权利要求1至5中任一项所述可降解蛋白塑料的制备方法，其特征在于，包括如下步骤：
步骤一、将多赖氨酸的工程蛋白溶于水，得到工程蛋白溶液；
将双醛基聚乙二醇溶于水，得到交联剂溶液；
步骤二、将工程蛋白溶液和交联剂溶液混合，震荡，离心去除气泡，得到蛋白交联产物溶液；
步骤三、将蛋白交联产物溶液放入模具，干燥成型。
7.根据权利要求6所述的制备方法，其特征在于，所述工程蛋白溶液的浓度为100～1000mg/mL；所述交联剂溶液的浓度为100～1000mg/mL；
步骤二中，所述工程蛋白溶液与所述交联剂溶液的体积比为(1～5)：(1～5)。
8.根据权利要求6所述的制备方法，其特征在于，所述震荡为漩涡震荡，震荡的时间为2～10min；
所述离心为：在2000～6000rpm下离心1～5min。
9.根据权利要求6所述的制备方法，其特征在于，所述干燥为自然风干12～36h。
10.根据权利要求6至9中任一项所述的制备方法，其特征在于，所述多赖氨酸的工程蛋白通过将含有目的基因的载体质粒转化进大肠杆菌进行表达、纯化得到。
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