CN110075809B - 一种提高粉粒几丁质对酶蛋白的吸附能力的方法及其应用 - Google Patents
一种提高粉粒几丁质对酶蛋白的吸附能力的方法及其应用 Download PDFInfo
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Abstract
本发明公开一种提高粉粒几丁质对酶蛋白的吸附能力的方法及其应用。将粉粒几丁质通过高压均质机处理后得到纤维直径为纳米级的几丁质纤维,用于吸附带有几丁质结合域蛋白(ChBD)的融合酶,经过一系列的条件优化后发现,预处理后的几丁质纤维对酶蛋白的吸附效果相较未处理几丁质粉末有了较大提升。本发明提供一种改进常规粉粒几丁质对酶蛋白吸附能力的方法,该方法绿色环保不涉及强酸强碱的使用,制备得到的几丁质纤维具有直径小、比较面积大、结合蛋白能力强的特点,在酶的固定化材料领域具有较好的应用与发展前景。
Description
技术领域
本发明涉及几丁质纤维制备技术领域,具体涉及一种提高粉粒几丁质对酶蛋白的吸附能力的方法及其应用。
背景技术
几丁质(C8H13O5N)n,又称甲壳质或壳多糖,是一类自然界中含量仅次于纤维素的重要多糖,它广泛存在于虾、蟹、昆虫外壳以及一些海洋软体动物的外骨骼中,来源广泛。几丁质是由N-乙酰氨基葡萄糖通过α-1,4糖苷键连接的聚合物,具有独特的分子结构,赋予了其无毒、生物可相容、生物可降解等突出特性。可被制作成各类材料,如膜制品、材料、凝胶、支架、纳米颗粒及纳米纤维等,从而能应用于医疗缝线、抑菌、保健品添加剂等诸多领域。其中,几丁质纳米纤维在已有的无毒、生物兼容性好的基础上,又兼备纳米材料的高表面积、多孔网状结构、优良的机械强度等性质而受到广泛关注。
近年来,用几丁质及其预处理后的几丁质纳米纤维作为固定化材料收到广泛关注,通过在蛋白中表达几丁质结合域(ChBD)基因,酶分子能够特异性亲和吸附在几丁质材料表面,结合力强且不会损失酶活。这类固定化方法省去了传统固定化方法中蛋白纯化步骤,在降低成本的基础上,其固定化载体几丁质本身就是来源广泛的可再生生物基材料,具有良好的应用前景。但几丁质天然结构紧致,比表面积较小,难以高负载量吸附蛋白。因此,需要对几丁质进行预处理以提高其固载量。
如文献“嗜热菌几丁质结合域及其在酶固定化中应用的研究[D]. 南京师范大学,2011.”中报道,用经过预处理得到的胶体几丁质及溶胀几丁质吸附酶蛋白,结合率可在70%以上;而用未处理的壳聚糖吸附酶蛋白时,由于材料比表面积小、纤维直径大等缺点,吸附效果与经处理的胶体几丁质等相差较大。
专利CN106520741A公开了一种利用酸水解几丁质制备几丁质悬液,再用所得几丁质悬液经脱乙酰基酶的作用,得到几丁质纳米纤维的方法。该方法处理几丁质时,涉及强酸的使用,易造成环境污染,且后期酶的使用也使处理成本大大增加,不利于大规模的放大生产。
专利CN108774289A也公开了利用各类溶剂、机械预处理方法与TEMPO氧化体系相结合的方法处理得到平均直径和长度分别为8nm和340nm的几丁质纳米纤维,提高几丁质的比表面积。尽管该方法的处理效果显著,但制备过程设计的溶剂与氧化剂的后期处理较为困难,也易增添环境的负担,同时,制备过程繁琐的步骤也为延长了纳米纤维的生产周期。
综合专利与文献报道看来,几丁质会是一种高效且成本低廉的固定化材料,经过预处理后的几丁质纤维则具备了更好的固定化性能,具有较大的应用潜力。但目前专利所涉及的制备几丁质纳米纤维的过程中,需要涉及化学试剂如强酸强碱,环境污染严重。同时,制备条件苛刻且制备周期长。因此,本领域需要一种高效、清洁、步骤更为简便的几丁质预处理方法。
发明内容
针对现有方法的不足,本发明的目的在于提供一种提高粉粒几丁质对酶蛋白的吸附能力的方法及其应用,本发明方法简便,过程无污染,且固定化酶性能良好,对于几丁质纳米纤维的产业化制备以及新型几丁质固定化材料的发展,具有一定的推动作用。
为解决现有技术问题,本发明采取的技术方案为:
一种提高粉粒几丁质对酶蛋白的吸附能力的方法,包括以下步骤:
步骤1,几丁质粉末的筛选
筛选粒径大小为60-120目的粉粒几丁质;
步骤2,几丁质悬浮液的制备
将粉粒几丁质用纯水洗涤2-5次后,60-85℃下烘干,重悬于蒸馏水中得质量分数为1-4%的几丁质悬浮液;
步骤3,几丁质纳米纤维的制备
将几丁质悬浮液以10-20L/h的进料速率通过高压均质机,压力为200-800 bar,循环1-9次后得几丁质纳米纤维。
作为改进的是,步骤1中几丁质粉末的来源为小龙虾虾壳或蟹壳中一种或两种混合。
上述制备方法所得的几丁质纤维在酶的固定介质中的应用。
上述应用,包括以下步骤:
第一步,含几丁质结合域ChBD的融合蛋白的制备
从Chitinolyticbacter meiyuanensis SYBC-H1中扩增ChBD基因片段,并合成绿色荧光蛋白GFP基因,构建重组质粒pET29a-chbd-gfp,将其导入大肠杆菌BL21(DE3),诱导表达后,收取菌体,裂解后,即得 ChBD-GFP重组蛋白粗酶液;
第二步,将几丁质纳米纤维加入粗酶液至几丁质终浓度为1%-4%后,在温度4-37℃、pH 5.0-8.0条件下进行蛋白的吸附,吸附时间2-30min,即得固定化酶。
作为改进的是,第二步中蛋白吸附的温度为4℃,pH6.8,搅拌速度为200rpm。
作为改进的是,第二步中吸附过程中控制pH所用的缓冲液为磷酸氢二钠-磷酸二氢钠(PBS)缓冲液或柠檬酸-柠檬酸钠缓冲液。
有益效果:
与现有技术相比,本发明的优势在于:
(1)本发明制备方法所得几丁质纳米纤维较未经处理的粉粒几丁质,提高了蛋白吸附性能,从而证明本发明几丁质纳米纤维适合作为固定化材料;
(2)本发明制备方法绿色无污染,步骤简单,易于操作,制备周期短,所得几丁质纤维的直径为纳米级,处理效果好,对几丁质纳米纤维的大规模生产具有一定的推动作用。
附图说明
图1为粉粒几丁质的SEM图;
图2为高压均质法处理得到的几丁质纳米纤维的SEM图;
图3为不同吸附时间下,几丁质纳米纤维对蛋白的吸附情况;
图4为在不同pH的条件下,几丁质纳米纤维对蛋白的吸附情况;
图5为激光共聚焦显微镜下,吸附蛋白的几丁质纳米纤维的荧光图。
具体实施方式
下面将结合实例对本发明做出进一步的详述,实例仅用来解释发明,不构成对本发明保护范围的限定。
实施例1 几丁质粉末的筛选
使用100目的网孔筛,从粒径大小不一的几丁质粉末中筛得直径均匀的100目粉粒几丁质备用,所述的几丁质粉末购自阿拉丁(上海)有限公司。
实施例2 几丁质悬浮液的制备
将实施例1中筛得的100目粉粒几丁质用纯水洗涤3次,置于烘箱中65℃烘干。称取2克烘干的粉粒几丁质,加入蒸馏水至粉粒几丁质终浓度为2%制成几丁质悬浮液。
实施例3 几丁质纳米纤维的制备
将实施例2中所得的几丁质悬浮液,设置高压均质机的进料速率为15L/h,在该进料速率下设置高压均质机的压力参数为600bar,将几丁质悬浮液循环均值处理5次后,制备得到几丁质纳米纤维悬浮液。图1与图2分别为处理前后几丁质的SEM图,图中可清晰对比出,经过高压均质处理的几丁质有更纤细的纳米须。图1中SEM下的几丁质表面表面为粗糙的片层结构,其间夹杂小孔,这种结构不利于酶蛋白的吸附;图2中SEM下经高压均质机处理后的几丁质纤维,直径分布在50nm-100nm,这种纳米纤维结构可吸附更多的酶蛋白。
实施例4 含几丁质结合域ChBD的融合蛋白的制备
为构建含几丁质结合域ChBD的融合蛋白,在pET29a质粒中,以酶切连接的方法,插入来自Chitinolyticbacter meiyuanensis SYBC-H1(江南大学郝之奎赠与)中提取扩增的ChBD基因片段以及扩增得到的绿色荧光蛋白基因gfp片段(绿色荧光蛋白基因由南京擎科生物公司合成),其中,gfp接在chbd基因C端。
将所得重组质粒导入大肠杆菌BL21(DE3)中表达即完成菌株pET29a-chbd-gfp构建。在10mlLB培养基(10g/L蛋白胨,5g/L酵母粉,5g/LNaCl)中,以1%接种量活化菌株,加抗性为20μl卡那霉素,培养条件为37℃,200rpm。活化6h后,以1%的接种量接种于100mlLB培养基中,所加抗性为200μl卡那霉素,培养条件为37℃,200rpm。待菌体密度长OD600至0.6,加入100 μl诱导剂IPTG诱导20h,诱导条件为18℃,200rpm,12h。6000rpm离心收集诱导完的菌体,用蒸馏水洗涤2次,加入10ml柠檬酸-磷酸二氢钠缓冲液(pH6.8)冲悬菌体。使用超声破碎仪破碎细胞,经6000g离心收集上清液即为包含ChBD融合蛋白的粗酶液。该融合蛋白中,ChBD基因用来亲和吸附几丁质,GFP基因用来做吸附后固定化酶的荧光表征。
实施例5 几丁质纳米纤维处理前后的吸附性能研究
分别取实施例2与实施例3中所得的几丁质悬浮液与几丁质纳米纤维悬浮液1ml,6000g离心后去除上清液,沉淀用蒸馏水洗涤2次后,在处理前后的几丁质沉淀中以体积比1:2加入粗酶液进行蛋白吸附实验。吸附条件为4℃,pH6.8,200rpm搅拌。30min的吸附结束后,分别以6000g离心,收集两种几丁质吸附后的上清液,以Bradford法测吸附前的粗酶液中蛋白浓度,以及吸附后两类上清中蛋白的浓度。
(蛋白吸附量=粗酶液中蛋白量-吸附后上清液中蛋白量)测得处理前的几丁质粉末可吸附蛋白量为0.646±0.05mg/ml,而经高压均质处理的几丁质纳米纤维可吸附蛋白量为1.124±0.04mg/ml,吸附效果约为处理前吸附效果的1.74倍,说明处理后的蛋白吸附效果具有显著提升。
实施例6几丁质纳米纤维的最适吸附条件研究
1.最适吸附温度的确定
取1ml实施例3中所得几丁质纳米纤维,6000g离心后沉淀经蒸馏水洗涤2次,按照体积比1:2加入实施例4中所述粗酶液,分别在4℃、20℃、37℃下进行最适吸附温度探究,吸附pH为6.8,搅拌为200rpm,吸附时间为30min。测量蛋白吸附量的方法同实施例4中所述。测得4℃下蛋白吸附量为1.124±0.04mg/ml,20℃下蛋白吸附量为1.434±0.06mg/ml,37℃下蛋白吸附量为0.986±0.05mg/ml,所以选定20℃为最佳吸附温度。
2. 最适吸附时间的确定
取1ml实施例3中所得几丁质纳米纤维,6000g离心后沉淀经蒸馏水洗涤2次,按照体积比1:2加入实施例4中所述粗酶液,在20℃、pH6.8、200rpm的搅拌下分别吸附2min、5min、10min、30min,进行最适吸附时间的探究。测量蛋白吸附量的方法同实施例4中所述。吸附时间结果如图3,吸附10min时蛋白量趋于饱和,确定10min为最适吸附时间。
3.最适吸附pH的确定
取1ml实施例3中所得几丁质纳米纤维,6000g离心后沉淀经蒸馏水洗涤2次,按照体积比1:2加入实施例4中所述粗酶液,在20℃、200rpm的搅拌下分别分别在pH值为5.0、5.6、6.2、6.8、7.4、8.0的体系下吸附10min ,进行最适吸附pH的探究。测量蛋白吸附量的方法同实施例4中所述,且吸附结果如图4,确定6.8为最适吸附pH。
综合下来,最优吸附条件为吸附温度20℃,吸附时间10min,吸附pH6.8。经过高压均质法处理得到的几丁质纳米纤维,相较粉粒几丁质,吸附能力约为未处理时的2.2倍。(最适条件下几丁质纳米纤维吸附蛋白量:1.421±0.08mg/ml;粉粒几丁质吸附蛋白量:0.646±0.05mg/ml)。图5为高压均质法处理过的几丁质纤维在激光共聚焦显微镜下的荧光图。
用高压均质法预处理粉粒几丁质具有良好的吸附性能,可显著提高粉粒几丁质对酶蛋白的吸附能力,是一种高效的处理方法。同时制备几丁质纳米纤维的过程,也具有步骤简便、绿色清洁、制备周期短的优势,利于工业化的放大作用。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (4)
1.一种几丁质纳米纤维在酶的固定介质中的应用,其特征在于,包括以下步骤:
第一步,含几丁质结合域ChBD的融合蛋白的制备
从Chitinolyticbacter meiyuanensis SYBC-H1中扩增ChBD基因片段,并合成绿色荧光蛋白GFP基因,构建重组质粒pET29a-chbd-gfp,将其导入大肠杆菌BL21(DE3),诱导表达后,收取菌体,裂解后,即为ChBD-GFP重组蛋白粗酶液;
第二步,将几丁质纳米纤维加入粗酶液至几丁质终浓度为1%-4%后,在温度4-37℃、pH 5.0-8.0条件下进行蛋白的吸附,吸附时间2-30min,即得固定化酶;其中,所述几丁质纳米纤维的制备方法,具体步骤如下:
步骤1,几丁质粉末的筛选
筛选粒径大小为60-120目的粉粒几丁质;
步骤2,几丁质悬浮液的制备
将粉粒几丁质用纯水洗涤2-5次后,60-85℃下烘干,重悬于蒸馏水中得质量分数为1-4%的几丁质悬浮液;
步骤3,几丁质纳米纤维的制备
将几丁质悬浮液以10-20L/h的进料速率通过高压均质机,压力为200-800bar,循环1-9次后得几丁质纳米纤维。
2.根据权利要求1所述的几丁质纳米纤维在酶的固定介质中的应用,其特征在于,步骤1中几丁质粉末的来源为小龙虾虾壳或蟹壳中一种或两种混合。
3.根据权利要求1所述的几丁质纳米纤维在酶的固定介质中的应用,其特征在于,包括以下步骤:第二步中蛋白吸附的温度为4℃,pH6.8, 搅拌速度为200rpm。
4.根据权利要求1所述的几丁质纳米纤维在酶的固定介质中的应用,其特征在于,包括以下步骤:第二步中吸附过程中控制pH所用的缓冲液为磷酸氢二钠-磷酸二氢钠缓冲液或柠檬酸-柠檬酸钠缓冲液。
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