CN110042095A - 一种固定化纤维素酶的制备方法 - Google Patents
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Abstract
本发明公开了一种固定化纤维素酶的制备方法,以纤维素酶为原料,以UiO‑66MOFs材料为载体,对纤维素酶进行固定化。其具体包括以下步骤:(1)取50‑100mg纤维素酶溶于超纯水中,加入50‑100mgUiO‑66MOFs材料,在20‑40℃恒温水浴中振荡8‑20h,得到振荡产物;(2)将步骤(1)中所述振荡产物离心,得到离心上清液和离心产物;(3)将步骤(2)中所述离心产物干燥,得到固定化纤维素酶。其具有酶载量高,活性回收率高的优点。将本发明固定化纤维素酶应用在再造烟叶原料预处理时可有效降解再造烟叶中的纤维素大分子,增加主要致香成分,降低烟碱,提高再造烟叶香气以及协调性。
Description
技术领域
本发明涉及采用物理吸附法制备固定化纤维素酶,尤其涉及一种固定化纤维素酶的制备方法。
背景技术
随着能源的日益枯竭,人们的注意力越来越多的聚集到可再生能源中,而纤维素是可再生能源也是全世界储量最大的能源。开发利用纤维素可大大减低现在对能源短缺的恐慌,作者Meng C报道了一种利用纤维素的方法(Meng C et al.Activation ofCellulose by Supercritical Tetrafluoroethane and Its Application in Synthesisof Cellulose Acetate.Industrial&Engineering Chemistry Research,2015,54(48))。由于纤维素的特殊结构,纤维素的分解较其他多糖更难,虽然有一些报道关于纤维素的降解,但是条件苛刻如GaoY等人报道的利用高温使纤维素降解。(GaoYet al.Hydrothermaldegradation ofhemicelluloses from triploidpoplar in hot compressed water at180-340C.Polymer Degradation&Stability,2016,126:S014139101630026X)。目前最有效的降解纤维素的方法就是利用纤维素酶进行纤维素的降解。纤维素酶降解纤维素广泛应用于新饲料的开发,洗涤剂的添加剂以及在食品工业、烟草行业等。例如在烟草工业中其可有效降低纤维素的浓度优化烟叶的质量,改变其口感等。目前最有效的纤维素酶的生产方式就是生物发酵方式如Sandhu S K在2013年报道的纤维素酶的发酵生产(Sandhu S K etal.Two-Stage Statistical Medium Optimization for Augmented CellulaseProduction via Solid-State Fermentation by Newly Isolated Aspergillus nigerHN-1and Application of Crude Cellulase Consortium in Hydrolysis of RiceStraw.Journal of Agricultural and Food Chemistry,2013,61(51):12653-12661),纤维素酶作为一种生物催化剂,因其具有高选择性、催化反应条件温和、无污染等特点,广泛应用于食品加工、医药和精细化工等行业。但天然酶稳定性差、易失活、不能重复使用,而且生产成本高、纯化困难,使其难以在工业中更为广泛的应用。
固定化酶的概念和技术得以提出和发展,成为近几年酶工程研究的重点。纤维素酶固定化方法也发展起来,如Chao Yang在2016年报道了相应固定化纤维素酶的方法(ChaoY,et al.Surface functionalized natural inorganic nanorod for highly efficientcellulase immobilization.Rsc Advances,2016,6(80):76855-76860)。
虽然相继出现了很多固定化纤维素酶的制备方法,但是大都应用了交联剂或其他会与酶反应的体系,导致在交联过程中难免会出现酶结构的改变等,如(Pei J et al.ANovel Layered Anchoring Structure Immobilized Cellulase via Covalent Bindingof Cellulase on MNPs Anchored by LDHs.Journal of Inorganic and OrganometallicPolymers and Materials,2018),因此,开发一种简便快捷的固定化纤维素酶技术极其重要。
为了解决以上问题,提出本发明。
发明内容
本发明提供一种固定化纤维素酶的制备方法,以纤维素酶为原料,以UiO-66MOFs材料为载体,对纤维素酶进行固定化。
其中,金属有机骨架(metal-organic frameworks,MOFs),是由金属离子或金属簇单元与含有N、O等元素的有机配体分子通过配位作用自组装形成的一类具有周期性多维立体结构的多孔晶态材料,也被称为多孔配位聚合物(porous coordination polymers,PCPs)或有机-无机杂化材料(Organic-Inorganic hybrid materials)。由于金属和有机配体种类的种类繁多,且金属与配体间的连接方式各异,使得MOFs呈现出多样性结构。MOFs还具有高度有序性、孔径可调、易功能化和具有极高的比表面积和孔隙率等特点,使其在催化、分离、气体储存、传感、生物医学成像以及药物传输等方面具有良好的应用前景。UiO-66是以四价金属锆的正八面体[Zr6O4(OH)4]次级结构单元作为无机节点与12个对苯二甲酸有机配体自组装形成的三维骨架结构,具有超高的化学和热稳定性,因此UiO-66材料作为固定化酶的载体具有优良的特性。
优选地,固定化纤维素酶的制备方法包括以下步骤:
(1)吸附:取50-100mg纤维素酶溶于超纯水中,加入50-100mgUiO-66 MOFs材料,在20-40℃恒温水浴中振荡8-20h,得到振荡产物。
(2)离心:将步骤(1)中所述振荡产物离心,得到离心上清液和离心产物。
(3)干燥:将步骤(2)中所述离心产物干燥,得到固定化纤维素酶。
其中,步骤(1)中UiO-66 MOFs的BET比表面积大于1000m2/g,郎格缪尔比表面积大于1400m2/g。
优选地,步骤(2)中离心次数为两次,离心机的转速为6000-8000r/min,且第二次离心转速不超过8000r/min,经两次离心后上层上清液合并为离心上清液,下层沉淀通过超纯水再洗涤三次得到离心产物。
其中,步骤(2)中离心上清液用Bradford法(考马斯亮蓝法)测酶负载量,取所述离心上清液1mL,水1mL分别置于两只试管,再各加5mLG-250染色液,在595nm测OD值。
优选地,步骤(3)中干燥温度为30-40℃,干燥条件为真空干燥。
其中,步骤(3)中所述固定化纤维素酶用DNS法(3,5-二硝基水杨酸比色法)测酶活,DNS法测酶活使用的缓冲液为pH=7.0的磷酸氢二钠-柠檬酸缓冲液。取三只试管依次加入pH为7.0的磷酸氢二钠-柠檬酸缓冲液。再分别取离心上清液2mL,水2mL,负载酶50mg+2mL水置于装缓冲液的试管中,将试管置于50℃水浴锅10min,加1mL2%羧甲基纤维素钠(CMC)溶液于每根试管中再水浴10min,取出加入已配置的DNS溶液(3,5-二硝基水杨酸溶液)3mL,将试管置于沸水中煮沸6min,取出急冷,稀释至25mL在540nm下测OD值。其中,DNS溶液的配置方法为:3.15gDNS溶于150ml H2O中,3.15gNaOH溶于50ml H2O中,将NaOH缓慢加到DNS中,再加入溶于200mlH2O的92.5g酒石酸钾钠,再依次加入2.5g苯酚和2.5g亚硫酸氢钠,整个配制过程保持在45℃。
相对于现有技术,本发明具有以下有益效果:
1、本发明以纤维素酶为原料,以UiO-66 MOFs材料为载体,对纤维素酶进行固定化,该方法UiO-66 MOFs材料可直接通过物理吸附对纤维素酶进行负载,不会破坏纤维素酶的结构,避免固化过程中改变纤维素酶的结构,从而使其保持了良好的酶活性,同时UiO-66MOFs材料性质稳定,进一步保证固定化纤维素酶中纤维素酶的结构不被改变。
2、本发明制备的固定化纤维素酶中纤维素酶的负载率为60.5%,酶活为1342U/μg,相对于纤维素酶原料的酶活率为70%,同时在相同条件下固定化纤维素酶循环使用4次后相对于制备的固定化纤维素酶仍然具有21.4%的酶活,因此本发明制备的固定化纤维素酶具有酶载量高,酶活性回收率高的优点,这可能是由于本发明制备的固定化纤维素酶中UiO-66 MOFs材料与纤维素酶之间存在氢键化学键作用,这种作用力使UiO-66 MOFs材料与纤维素酶之间更加稳定,从而进一步发挥UiO-66 MOFs材料与纤维素酶之间的协同催化能力。
3、将本发明固定化纤维素酶应用在再造烟叶生产中的原料预处理时可有效降解再造烟叶中的纤维素大分子,通过调节其化学成分组成,增加主要致香成分,降低烟碱,从而改善再造烟叶的内在质量,降低再造烟叶的杂气,提高再造烟叶香气以及协调性。
4、本发明以UiO-66 MOFs材料为载体,UiO-66 MOFs材料具有超高的比表面积,且UiO-66 MOFs材料合成简便。同时固定化纤维素酶的制备过程中,无需高温高压,有机溶剂或昂贵的设备,在工业化应用时可降低生产成本,可应用与多种领域如食品,发酵等工业。本发明建立了一种简单、快速、高负载率的固定化纤维素酶的制备方法。
附图说明
图1为本发明UiO-66 MOFs材料N2吸附脱附图;
图2为本发明牛血清白蛋白蛋白质标准曲线;
图3为本发明葡萄糖标准曲线;
其中,牛血清白蛋白蛋白质标准曲线中运用Bradford法测酶浓度,葡萄糖标准曲线中运用DNS法测葡萄糖浓度。
具体实施方式
以下结合实施例对本发明做进一步描述。需要说明的是,实施例不能作为对本发明保护范围的限制。本领域的技术人员应当理解,在本发明基础上所作的任何改进和变化都在本发明的保护范围之内。
以下实施例所用化学试剂都是常规试剂,均可商购获得。
对比例1
利用UiO-66 MOFs材料负载中性蛋白酶水解络蛋白,利用folin试剂测酶活。称取100mgUiO-66 MOFs材料于试管中,加入100mg中性蛋白酶酶粉及10mLpH7.5的缓冲溶液,充分混匀后将其置于30℃水浴振荡吸附12h,得到振荡产物。将振荡产物离心两次,收集合并上清液得到离心上清液,对离心沉淀用超纯水洗涤三次,得到离心产物,将离心产物置于30℃烘箱干燥,得到固定化中性蛋白酶。
酶活测试方法:将1mL(10mg/ml)酶液和1mL(5mg/ml)酪蛋白底物分别预热2min后,混合均匀并在40℃恒温反应10min。加入0.4mol/L三氯乙酸2mL,继续水浴保温10min。离心取1mL上清液,加入0.4mol/L碳酸钠溶液5ml和1mL稀释后的福林试剂(folin:H2O=1:2),混合均匀,在40℃恒温水浴中显色10min,在660nm下测吸光度。
对比例2
利用UiO-66 MOFs材料负载果胶酶,利用DNS试剂测酶活。称取100mgUiO-66 MOFs材料于试管中,加入100mg果胶酶酶粉及10mLpH7.5的缓冲溶液,充分混匀后将其置于30℃水浴振荡吸附12h,得到振荡产物。将振荡产物离心两次,收集合并上清液得到离心上清液,对离心沉淀用超纯水洗涤三次,得到离心产物,将离心产物置于30℃烘箱干燥,得到固定化果胶酶。
酶活测试方法:DNS法测酶活用缓冲液为pH=7.0的磷酸氢二钠-柠檬酸缓冲液。取三只试管依次加入pH为7.0的磷酸氢二钠-柠檬酸缓冲液。再分别取离心上清液2mL,水2mL,负载酶50mg+2mL水置于装缓冲液的试管中,将试管置于50℃水浴锅10min,加1mL2%羧甲基纤维素钠(CMC)溶液于每根试管中再水浴10min,取出加入已配置的DNS溶液(3,5-二硝基水杨酸溶液)3mL,将试管置于沸水中煮沸6min,取出急冷,稀释至25mL在540nm下测OD值。
实施例1
固定化纤维素酶的制备:称取100mgUiO-66 MOFs材料于试管中,加入100mg纤维素酶及10mL水,充分混匀后将其置于30℃水浴振荡吸附5h,得到振荡产物。将振荡产物离心两次,收集合并上清液得到离心上清液,对离心沉淀用超纯水洗涤三次,得到离心产物,将离心产物置于30℃烘箱干燥,得到固定化纤维素酶样品1。
其中离心上清液留存用于测纤维素酶负载率,固定化纤维素酶样品1用于测纤维素酶的酶活。
固定化纤维素酶的负载率测定:将1mL离心上清液,1mL水分别加到两支试管中,再分别加入5mL染色液于试管中,充分混匀,等待5min在595nm测OD值。(根据蛋白质标准方程:Y=0.7875X+0.02278可计算酶液浓度,负载率=原酶酶液浓度(10mg/ml)-计算出的酶液浓度/原酶酶液浓度)
固定化纤维素酶的酶活测试:取三只试管依次加入pH为7.0的磷酸氢二钠-柠檬酸缓冲液。再分别取离心上清液2mL,水2mL,负载酶50mg+2mL水,分别置于装缓冲液的三根试管中,将三根试管置于50℃水浴锅10min,加1mL2%羧甲基纤维素钠(CMC)溶液于每根试管中再水浴10min,取出分别加入已配置的DNS溶液3mL,将三根试管置于沸水中煮沸6min,取出急冷,稀释至25mL在540nm下测OD值。由OD值可计算水解生成的葡萄糖浓度(图3的标准方程为:Y=0.78845X+0.01158)。再根据酶活定义:每一分钟从2%CMC溶液中水解生成1μg葡萄糖为一个酶活单位。酶活计算:(计算出的葡萄糖浓度×25×1000)÷10
实施例2
固定化纤维素酶的制备方法与实施例1相同,但是改变吸附时间,将实施例1中的吸附5h改为吸附7h,得到固定化纤维素酶样品2。其他不变在相同条件下测固定化纤维素酶的负载率及固定化纤维素酶的酶活。
实施例3
固定化纤维素酶的制备方法与实施例1相同只是改变吸附时间,将实施例1中的吸附5h改为吸附9h,得到固定化纤维素酶样品3。其他不变在相同条件下测固定化纤维素酶的负载率固定化纤维素酶的酶活。
实施例4
固定化纤维素酶的制备方法与实施例1相同只是改变吸附时间,将实施例1中的吸附5h改为吸附12h,得到固定化纤维素酶样品4。其他不变在相同条件下测固定化纤维素酶的负载率及固定化纤维素酶的酶活。
将对比例1中的固定化中性蛋白酶、对比例2中的固定化果胶酶和实施例4中的固定化纤维素酶样品4中三个样品的酶活分别与固定化之前的中性蛋白酶、果胶酶和纤维素酶对比,得到固定化中性蛋白酶、固定化果胶酶和固定化纤维素酶相对原酶酶活率,结果见表1。通过表1可以看出固定化纤维素酶相对原酶酶活率比固定化中性蛋白酶和固定化果胶酶相对原酶酶活率均高,即利用UiO-66 MOFs材料进行负载纤维素酶效果比负载中性蛋白酶和果胶酶好,这可能是由于UiO-66 MOFs材料与纤维素酶之间存在氢键化学键作用,这种作用力使UiO-66 MOFs材料与纤维素酶之间更加稳定,从而进一步发挥UiO-66 MOFs材料与纤维素酶之间的协同催化能力。
表1不同固定化酶相对于原酶的酶活率
将固定化纤维素酶样品4在相同条件下(将样品450mg加入pH为7.0的磷酸氢二钠-柠檬酸缓冲液。再加水2mL,置于试管中,将试管置于50℃水浴锅10min,加1mL2%羧甲基纤维素钠(CMC)溶液于试管中再水浴10min,取出加入已配置的DNS溶液3mL,将试管置于沸水中煮沸6min,取出急冷,稀释至25mL在540nm下测OD值。),经不同循环的次数后其相对于第一次使用的固定化纤维素酶酶活见表2。
表2固定化纤维素酶样品4经不同循环次数后其相对酶活
由表2可以看出,固定化纤维素酶样品4在循环使用4次后,其酶活相对于初始酶活仍能达到21.4%,本发明制备的固定化纤维素酶具有活性回收率高的优点。
实施例5
固定化纤维素酶的制备方法与实施例1相同只是改变吸附时间,将实施例1中的吸附5h改为吸附15h,得到固定化纤维素酶样品5。其他不变在相同条件下测固定化纤维素酶的负载率及固定化纤维素酶的酶活。
其中固定化纤维素酶样品1、固定化纤维素酶样品2、固定化纤维素酶样品3、固定化纤维素酶样品4和固定化纤维素酶样品5的负载率及酶活,见表3。
表3不同固定化纤维素酶的负载率及酶活
从表3中可以看出,固定化纤维素酶的制备过程中吸附时间越长,固定化纤维素酶的负载率越大,但是在吸附时间为12h时,固定化纤维素酶的酶活取的最大值,即本发明固定化纤维素酶的制备过程吸附时间为12h,得到的固定化纤维素酶酶活性最高。
Claims (5)
1.一种固定化纤维素酶的制备方法,其特征在于,以纤维素酶为原料,以UiO-66 MOFs材料为载体,对纤维素酶进行固定化。
2.根据权利要求1所述的制备方法,其特征在于,其包括以下步骤:
(1)吸附:取50-100mg纤维素酶溶于超纯水中,加入50-100mgUiO-66 MOFs材料,在20-40℃恒温水浴中振荡8-20h,得到振荡产物;
(2)离心:将步骤(1)中所述振荡产物离心,得到离心上清液和离心产物;
(3)干燥:将步骤(2)中所述离心产物干燥,得到固定化纤维素酶。
3.根据权利要求2所述的制备方法,其特征在于,步骤(1)中UiO-66 MOFs的BET比表面积大于1000m2/g,郎格缪尔比表面积大于1400m2/g。
4.根据权利要求2所述的制备方法,其特征在于,步骤(2)中离心次数为两次,离心机的转速大于6000r/min,且第二次离心转速不超过8000r/min。
5.根据权利要求2所述的制备方法,其特征在于,步骤(3)中干燥温度为30-40℃,干燥条件为真空干燥。
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