CN114082411B - 一种具有类酶催化活性的木质素基碳纳米酶的制备方法 - Google Patents
一种具有类酶催化活性的木质素基碳纳米酶的制备方法 Download PDFInfo
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Abstract
一种具有类酶催化活性的木质素基碳纳米酶的制备方法,它涉及以木质素为原料制备具有类过氧化物酶活性的纳米酶的制备过程。本发明的目的是解决以往的纳米酶制备方法中存在的制备成本高、合成方法复杂等问题。制备方法:以木质素磺酸钠为碳源,氯化钾为掺杂剂,通过水热法合成木质素基碳纳米酶。本发明制备的木质素基碳纳米酶的类过氧化物酶对过氧化氢亲和力高、成本低、环境友好、制备方法简单,且实现了木质素的高价值利用。
Description
技术领域
本发明涉及一种以木质素为原料制备具有类过氧化物酶活性的碳纳米酶的方法。
背景技术
木质素作为植物界继纤维素之后第二大资源的生物质材料,全球每年因制浆造纸和生物炼制产生的木质素达1亿吨以上,来源广且产量大,其中只有约10%得到有效利用。作为自然界可持续利用的芳香资源,木质素对工业农业发展具有重要意义。在工业上,回收的木质素大部分用于能源回收,只有一小部分用于化工生产、增强剂和矿粉粘结剂等;在农业上,木质素可以通过改性、加工等方法合成肥料、土壤疏松剂、农药缓释剂等。目前大部分木质素被作为工业废弃物直接排放或者作为低值燃料进行燃烧,只能制备一些技术层次较低的产品,导致木质素转化价值低。木质素的高价值利用还有待进一步开发。
天然酶是一类生物催化剂,具有种类多样、底物专一等特点,参与生物体内复杂的代谢过程,在食品、农业、制药和化学工业等各个领域中得到积极应用。然而,天然酶也存在一些固有的缺点,如提取工艺复杂、成本较高、稳定性差且容易失活变性。为了克服天然酶的缺陷,具有催化活性的模拟酶被不断地开发出来。
纳米酶是兼具纳米材料的独特性能,又能够模拟酶活性的一类物质。与天然酶相比,纳米酶有许多优点,如催化活性高、成本低、稳定、经济可行和易于大量制备,为实现不同场景的应用提供了可能,因而纳米酶的制备方法也得到了较为广泛的研究。例如中国专利CN 109520946 B公开了一种以纳米金粒子采用硼氢化钠还原氯金酸的方法制备类过氧化物酶的方法。但该方法用到的原料为贵金属,成本较高且硼氢化钠是危险品,易导致实验安全问题。中国专利CN 111517318 B公开了一种通过氮原子对氧化石墨炔进行掺杂制备纳米酶的方法。但该方法采用氨气、氩气等气体为保护气进行高温反应(600~1000℃),操作复杂耗时长成本高,且过程中使用的原料为硫酸、硝酸等强氧化剂,对操作安全存在风险,不够绿色环保。本发明利用产量多且来源广的木质素作为原料,氯化钾为掺杂剂,采用水热法制备了一种具有类酶催化活性的碳纳米酶,解决了纳米酶制备的成本高、合成复杂等问题,同时实现了生物质的高效利用。
发明内容
本发明的目的是要解决纳米酶制备过程中的原料成本高、合成步骤繁琐、实验安全隐患等问题,而提出了一种绿色环保、合成简单、易于操作且可以大规模合成,将低价值的木质素转化成具有类酶催化活性的高价值碳纳米酶的制备方法。
一种具有类酶催化活性的木质素基碳纳米酶的制备方法,具体是按照以下步骤完成:
一、木质素基碳纳米酶的制备过程:在超纯水中加入木质素磺酸钠、氯化钾后,将溶液置于快速混匀器上,使溶液混合均匀,再将混合物转移到内衬为聚四氟乙烯的不锈钢高压反应釜中。将反应釜放入电热恒温鼓风干燥箱中,反应时间设置为11h~13h,温度设定为170℃~190℃;反应结束后将反应釜从烘箱中取出并冷却至室温;步骤一中所述木质素磺酸钠与氯化钾质量比为1g:(0.5g~3g);步骤一中所述木质素磺酸钠质量与超纯水体积比为1g:(50mL~300mL);
二、木质素基碳纳米酶的纯化过程:将冷却后反应釜中的混合物置于离心机中,设置转速为5500rpm~6000rpm,时间为8~10min,离心之后将所得的上清液经过0.22μm微孔滤头过滤,然后在超纯水中经过透析袋透析20h~28h;取透析袋(3000Da~3500Da)外部溶液通过旋转蒸发仪浓缩,将旋蒸后的液体冷冻干燥,得到木质素基碳纳米酶。
一种对木质素基碳纳米酶的类酶催化活性检测过程,具体按以下步骤完成:
设置50μL不同浓度的木质素基碳纳米酶水溶液、50μL不同浓度的过氧化氢溶液和50μL不同浓度的3`3`5`5`-四甲基联苯胺溶液的体系,并用pH=4的醋酸-醋酸钠缓冲液(0.1mol/L)将体系稀释2mL;同时设置以下两个对照组:用pH=4的醋酸-醋酸钠缓冲液(0.1mol/L)将50μL不同浓度的过氧化氢溶液和50μL不同浓度的3`3`5`5`-四甲基联苯胺溶液稀释至2mL的体系和用pH=4的醋酸-醋酸钠缓冲液(0.1mol/L)将50μL不同浓度的木质素基碳纳米酶水溶液和50μL不同浓度的3`3`5`5`-四甲基联苯胺溶液稀释至2mL的体系。将上述三组溶液在室温下黑暗中孵育10分钟后,通过紫外-可见吸收分光光度计记录溶液的吸光度谱图;步骤中所述木质素基碳纳米酶水溶液的浓度为2mg/mL~5mg/mL;步骤中所述过氧化氢溶液的浓度为0.5mmol/L~5mmol/L;步骤中所述3`3`5`5`-四甲基联苯胺溶液的浓度为9mmol/L~11mmol/L。
本发明优点:1、本发明以木质素为碳源,以氯化钾为掺杂剂制备纳米酶,所合成的纳米酶无需昂贵的原材料,且原料来源广、成本低,实现了木质素的高度有效利用,使木质素的利用技术层次提高;2、本发明采用水热法制备木质素基碳纳米酶,合成方法简单,易于操作,可大规模生产,环境友好;3、本发明制备得到的具有类酶催化活性的木质素基碳纳米酶水溶性强,易于保存,以过氧化氢为底物时的Km=0.19mmol/L,低于辣根过氧化物酶对过氧化氢的Km=3.7mmol/L,即对过氧化氢亲和力较强。
附图说明
图1是实施例1具有类酶催化活性的木质素基碳纳米酶的透射电镜图;由图1可以看出,制备的木质素基碳纳米酶分散性好,为球状结构且直径分布均匀,主要分布在2~5nm范围内;
图2是对实施例1木质素基碳纳米酶类酶催化活性检测的示意图;由图2可得,木质素基碳纳米酶、3`3`5`5`-四甲基联苯胺溶液体系在光谱扫描范围(500~800nm)内未出现吸收峰(曲线a),说明木质素基碳纳米酶不能氧化3`3`5`5`-四甲基联苯胺溶液;与过氧化氢溶液、3`3`5`5`-四甲基联苯胺溶液对应的吸收光谱在652nm处有一个弱吸收峰(曲线b),说明3`3`5`5`-四甲基联苯胺能被过氧化氢微弱氧化;与木质素基碳纳米酶、过氧化氢、3`3`5`5`-四甲基联苯胺体系对应的光谱曲线(曲线c)在652nm处有一个强吸收峰。结果表明,所制备的木质素基碳纳米酶具有类过氧化物酶催化活性,可催化过氧化氢氧化3`3`5`5`-四甲基联苯胺而发生吸光度变化的反应;
图3是实施例1中木质素基碳纳米酶的催化机理检测示意图,采用异丙醇作为羟基自由基清除剂,图3中得到,随着异丙醇体积增加,体系在652nm处的吸光度下降,说明木质素基碳纳米酶催化过氧化氢生成的羟基自由基被异丙醇所清除,即木质素基碳纳米酶有类过氧化物酶活性;
图4是实施例1中木质素基碳纳米酶的稳态动力学研究示意图。采用米氏动力学方程分别研究了木质素基碳纳米酶对于3`3`5`5`-四甲基联苯胺和过氧化氢的亲和力,即。其中,米氏常数(Km)和最大反应速率(Vmax)为米氏方程的两个参数,分别用于描述酶与底物的亲和程度和在最佳反应条件下酶催化反应的最大速率。Km的数值只与酶自身有关,且Km的值越小说明底物与酶的亲和力越大。Vmax表示酶被底物饱和时的反应速度,其数值越大表明酶在该条件下的催化活性越高。经过对此方程的斜率和截距的计算,得到以过氧化氢为底物时的Km=0.19mmol/L,Vmax=11.63×10-8M s-1;以3`3`5`5`-四甲基联苯胺为底物时的Km=0.85mmol/L,Vmax=2.51×10-6M s-1。
具体实施方式
实施例1:
本实施方式是一种具有类酶催化活性的木质素基碳纳米酶的制备方法,具体是按以下步骤完成的:
一、木质素基碳纳米酶的制备过程:在20mL超纯水中加入0.1g木质素磺酸钠、0.1g氯化钾后,将溶液置于快速混匀器上混合均匀,再将混合物转移到内衬为聚四氟乙烯的不锈钢高压反应釜中。将反应釜放入电热恒温鼓风干燥箱中,反应时间设置为12h,温度设定为180℃;反应结束后将反应釜从烘箱中取出并冷却至室温;
二、木质素基碳纳米酶的纯化过程:将冷却后反应釜中的混合物置于离心机中,设置转速为6000rpm,时间为10min,离心之后将所得的上清液经过0.22μm微孔滤头过滤,然后在超纯水中经过透析袋(3500Da)透析24h;取透析袋外部溶液通过旋转蒸发仪浓缩,将旋蒸后的液体冷冻干燥,得到木质素基碳纳米酶。
实施例2:
本实施方式是一种对木质素基碳纳米酶的类酶催化活性检测过程,具体按以下步骤完成的:
设置50μL3mg/mL的木质素基碳纳米酶水溶液、50μL 5mmol/L的过氧化氢溶液和50μL10mmol/L的3`3`5`5`-四甲基联苯胺溶液的体系并用pH=4的醋酸-醋酸钠缓冲液(0.1mol/L)将体系稀释2mL;同时设置以下两个对照组:用pH=4的醋酸-醋酸钠缓冲液(0.1mol/L)将50μL 5mmol/L的过氧化氢溶液、50μL 10mmol/L的3`3`5`5`-四甲基联苯胺溶液稀释至2mL的体系和用pH=4的醋酸-醋酸钠缓冲液(0.1mol/L)将50μL 3mg/mL的木质素基碳纳米酶水溶液、50μL 10mmol/L的3`3`5`5`-四甲基联苯胺溶液稀释至2mL的体系。将上述三组溶液在室温下黑暗中孵育10分钟后,通过紫外-可见吸收分光光度计记录溶液的吸光度谱图。
Claims (2)
1.一种具有类酶催化活性的木质素基碳纳米酶的制备方法,其特征在于是按照以下步骤制备的:
一、木质素基碳纳米酶的制备过程:在超纯水中加入木质素磺酸钠、氯化钾后,将溶液置于快速混匀器上,使溶液混合均匀,再将混合物转移到内衬为聚四氟乙烯的不锈钢高压反应釜中,将反应釜放入电热恒温鼓风干燥箱中,反应时间设置为11h ~ 13h,温度设定为170℃ ~ 190℃;反应结束后将反应釜从烘箱中取出并冷却至室温;步骤一中所述木质素磺酸钠与氯化钾质量比为1g:0.5g ~ 3g;步骤一中所述木质素磺酸钠质量与超纯水体积比为1g:50mL ~ 300mL;
二、木质素基碳纳米酶的纯化过程:将冷却后反应釜中的混合物置于离心机中,设置转速为5500rpm ~ 6000rpm,时间为8 ~ 10min,离心之后将所得的上清液经过0.22μm微孔滤头过滤,然后在超纯水中经过透析袋透析20h ~ 28h;取截留分子量3000 ~ 3500Da的透析袋外部溶液通过旋转蒸发仪浓缩,将旋蒸后的液体冷冻干燥,得到木质素基碳纳米酶。
2. 根据权利要求1所述的一种木质素基碳纳米酶的制备方法,其特征在于步骤二中所述经透析袋透析,在55℃ ~ 65℃下旋转蒸发浓缩。
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