CN114940776A - 一种多孔壳聚糖微球及其固定碱性蛋白酶的方法 - Google Patents
一种多孔壳聚糖微球及其固定碱性蛋白酶的方法 Download PDFInfo
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Abstract
本发明公开了一种多孔壳聚糖微球及其固定碱性蛋白酶的方法,由以下步骤制成:(1)将壳聚糖粉末溶于乙酸‑乙酸铵溶液中,再加入纳米二氧化硅粉末,制得改性壳聚糖复合凝胶;(2)将氢氧化钠或氢氧化钾与碳酸钠混合溶解,然后与无水乙醇混合,得到复合碱性成型剂;(3)将步骤(1)所得改性壳聚糖复合凝胶,缓慢加入到复合碱性成型剂中,搅拌成型、静置硬化后用纯净水洗至中性,冷冻干燥后制得多孔壳聚糖微球。本发明所制备的多孔壳聚糖微球,孔隙率与比表面积大、机械强度高,微球内部通道发达且相互贯通,易于酶的吸附、活化与交联,可显著提高碱性蛋白酶的固载率和稳定性,适用于工业推广应用。
Description
技术领域
本发明涉及碱性蛋白酶的固定化技术领域,具体地说,涉及一种多孔壳聚糖微球及其固定碱性蛋白酶的方法。
背景技术
蛋白酶是一种温和的高效催化剂,而碱性蛋白酶是蛋白酶家族中最重要的一类蛋白酶,它能够在极端的碱性环境下发挥作用,被广泛应用于食品、纺织、医药、洗涤等行业。碱性蛋白酶在工业生产中的应用环境较为苛刻,与合成碱性蛋白酶的天然生物细胞环境有着较大的差异,其在体外环境的性能发挥受到不同程度的抑制。如何有效地在体外或是在特定蛋白酶所处的应用环境中稳定蛋白酶的性能是该领域亟待解决的难题之一。
酶的固定化是一种有效的稳定酶的策略,它不仅能够使酶的稳定性得到提高,还能将酶循环利用。可溶性的酶通常可以通过与不溶性载体的表面发生如吸附、交联等相互作用来实现酶的固定化。酶的固定化效果与载体材料的性能直接相关。壳聚糖具有廉价易得、易改性、天然无毒等优良性能,且壳聚糖表面还有着丰富的活泼基团如-NH2、-OH等,利于酶的固定和进一步改性,目前已成功用于多种酶的固定化。钟方旭等直接采用商业壳聚糖固载碱性蛋白酶,获得了一定固定化效果(食品科技,2008,3,49)。梁玉杰等制备了Fe3O4磁性壳聚糖微球,并将其用于蛋白酶的固定化和回收利用(食品与发酵工业,2014,40(8),66)。然而,目前所采用的壳聚糖微球普遍存在酶的固定效率不高的问题,限制了其推广应用。
发明内容
本发明的目的是克服现有技术的不足,提供一种多孔壳聚糖微球及其固定碱性蛋白酶的方法,通过新型多孔壳聚糖微球的设计,丰富并改善载体内部的孔隙结构,为酶的固定提供更多和更稳定的结合位点,提升碱性蛋白酶的固载率和稳定性。
为实现上述目的,本发明采用如下技术方案:
一种多孔壳聚糖微球,由以下步骤制成:
(1)将壳聚糖粉末溶于乙酸-乙酸铵溶液中,再加入纳米二氧化硅粉末,制得改性壳聚糖复合凝胶;
(2)将氢氧化钠或氢氧化钾与碳酸钠混合溶解,然后与无水乙醇混合,得到复合碱性成型剂;
(3)将步骤(1)所得改性壳聚糖复合凝胶,缓慢加入到复合碱性成型剂中,搅拌成型、静置硬化后用纯净水洗至中性,冷冻干燥后制得多孔壳聚糖微球。
作为优选的,在上述的多孔壳聚糖微球的制备方法中,步骤(1)所述改性壳聚糖复合凝胶的制备过程为:先向乙酸溶液中加入乙酸铵,使得乙酸铵浓度为0.01-0.1 mol/L,后将壳聚糖粉末溶于乙酸-乙酸铵溶液中制备壳聚糖凝胶,再向其加入与壳聚糖质量比为1:1~1:5的纳米二氧化硅粉末,搅拌得到改性壳聚糖复合凝胶。
作为优选的,在上述的多孔壳聚糖微球的制备方法中,步骤(2)中所述复合碱性成型剂由氢氧化钠或氢氧化钾与碳酸钠混合溶解而成,其摩尔比为1.5-3.5,然后与无水乙醇以5:1 - 2:1的体积比进行混合。
.一种碱性蛋白酶的固定方法,包括以下步骤:
(1) 将权利要求1所制备的多孔壳聚糖微球加入到pH为7.0-9.5的磷酸盐缓冲液中浸润,充分润湿溶胀后加入碱性蛋白酶溶液,在1~10℃下振荡混合得到混合液;
(2) 向混合液中加入交联剂,使碱性蛋白酶负载于多孔壳聚糖微球上,用磷酸盐缓冲液洗涤后,除去多余的交联剂,得到以多孔壳聚糖微球为载体的固定化酶。
作为优选的,在上述的固定方法中,所述多孔壳聚糖微球与碱性蛋白酶的质量比为100:1~20:1,所述交联剂在混合溶液中的体积比浓度为1% - 5%。
作为优选的,在上述的固定方法中,所述磷酸盐缓冲液的pH为7.0 - 9.5。
作为优选的,在上述的固定方法中,所述交联剂为京尼平或戊二醛或EDC/NHS。
与现有技术相比,本发明具有如下有益效果:
本发明所制备的多孔壳聚糖微球,孔隙率与比表面积大、机械强度高,微球内部通道发达且相互贯通,易于酶的吸附、活化与交联,可显著提高碱性蛋白酶的固载率和稳定性,适用于工业推广应用。
具体实施方式
为了更好地解释本发明,以下结合具体实施例进一步阐明本发明的主要内容,但本发明的内容不仅仅局限于以下实例。
实施例1
(1)多孔壳聚糖微球的制备
取乙酸铵溶于乙酸溶液中,使得乙酸铵浓度为0.01 mol/L,将壳聚糖粉末(脱乙酰度>95%)加入到乙酸-乙酸铵溶液中,搅拌制得改性壳聚糖凝胶溶液。以二氧化硅与壳聚糖质量比为1:5的量向上述制得的凝胶中加入纳米二氧化硅,充分搅拌,制备得到改性壳聚糖复合凝胶。将氢氧化钠-碳酸钠混合溶液(两者浓度分别为1.5 mol/L、0.8 mol/L)与无水乙醇以3:1的比例混合,制得成型剂。将所制得改性壳聚糖复合凝胶,缓慢加入到成型剂中,搅拌成型、静置硬化后用纯净水洗至中性,冷冻干燥后制得新型多孔壳聚糖微球。
(2)碱性蛋白酶的固定
将壳聚糖微球加入到pH =7.5为磷酸盐缓冲液中浸润,充分润湿溶胀后,以新型多孔壳聚糖微球与碱性蛋白酶的质量比为25:1,加入碱性蛋白酶溶液,在1~10℃下振荡混合。向载体与蛋白酶的混合液中加入交联剂戊二醛溶液,使得戊二醛在混合液中的浓度为1%,使蛋白酶负载于新型多孔壳聚糖微球上,交联一段时间后,使用pH =7.5为磷酸盐缓冲液充分洗涤,除去多余的交联剂,得到以新型多孔壳聚糖微球为载体的固定化酶。
实施例2
(1)多孔壳聚糖微球的制备
取乙酸铵溶于乙酸溶液中,使得乙酸铵浓度为0.05 mol/L,将壳聚糖粉末(脱乙酰度>95%)加入到乙酸-乙酸铵溶液中,搅拌制得改性壳聚糖凝胶溶液。以二氧化硅与壳聚糖质量比为1:5的量向上述制得的凝胶中加入纳米二氧化硅,充分搅拌,制备得到改性壳聚糖复合凝胶。将氢氧化钠-碳酸钠混合溶液(两者浓度分别为1.5 mol/L、0.6 mol/L)与无水乙醇以4:1的比例混合,制得成型剂。将所制得改性壳聚糖复合凝胶,缓慢加入到成型剂中,搅拌成型、静置硬化后用纯净水洗至中性,冷冻干燥后制得新型多孔壳聚糖微球。
(2)碱性蛋白酶的固定
将壳聚糖微球加入到pH =8.0为磷酸盐缓冲液中浸润,充分润湿溶胀后,以新型多孔壳聚糖微球与碱性蛋白酶的质量比为50:1,加入碱性蛋白酶溶液,在1~10℃下振荡混合。向载体与蛋白酶的混合液中加入交联剂戊二醛溶液,使得戊二醛在混合液中的浓度为2%,使蛋白酶负载于新型多孔壳聚糖微球上,交联一段时间后,使用pH =8.0为磷酸盐缓冲液充分洗涤,除去多余的交联剂,得到以新型多孔壳聚糖微球为载体的固定化酶。
实施例3
(1)多孔壳聚糖微球的制备
取乙酸铵溶于乙酸溶液中,使得乙酸铵浓度为0.025 mol/L,将壳聚糖粉末(脱乙酰度>95%)加入到乙酸-乙酸铵溶液中,搅拌制得改性壳聚糖凝胶溶液。以二氧化硅与壳聚糖质量比为1:4的量向上述制得的凝胶中加入纳米二氧化硅,充分搅拌,制备得到改性壳聚糖复合凝胶。将氢氧化钠-碳酸钠混合溶液(两者浓度分别为2 mol/L、0.75 mol/L)与无水乙醇以3:1的比例混合,制得成型剂。将所制得改性壳聚糖复合凝胶,缓慢加入到成型剂中,搅拌成型、静置硬化后用纯净水洗至中性,冷冻干燥后制得新型多孔壳聚糖微球。
(2)碱性蛋白酶的固定
将壳聚糖微球加入到pH =7.0为磷酸盐缓冲液中浸润,充分润湿溶胀后,以新型多孔壳聚糖微球与碱性蛋白酶的质量比为30 : 1,加入碱性蛋白酶溶液,在1~10℃下振荡混合。向载体与蛋白酶的混合液中加入交联剂戊二醛溶液,使得戊二醛在混合液中的浓度为3.5%,使蛋白酶负载于新型多孔壳聚糖微球上,交联一段时间后,使用pH =7.0为磷酸盐缓冲液充分洗涤,除去多余的交联剂,得到以新型多孔壳聚糖微球为载体的固定化酶。
实施例4
(1)多孔壳聚糖微球的制备
取乙酸铵溶于乙酸溶液中,使得乙酸铵浓度为0.06 mol/L,将壳聚糖粉末(脱乙酰度>95%)加入到乙酸-乙酸铵溶液中,搅拌制得改性壳聚糖凝胶溶液。以二氧化硅与壳聚糖质量比为1:4的量向上述制得的凝胶中加入纳米二氧化硅,充分搅拌,制备得到改性壳聚糖复合凝胶。将氢氧化钾-碳酸钠混合溶液(两者浓度分别为2 mol/L、0.8 mol/L)与无水乙醇以5:1的比例混合,制得成型剂。将所制得改性壳聚糖复合凝胶,缓慢加入到成型剂中,搅拌成型、静置硬化后用纯净水洗至中性,冷冻干燥后制得新型多孔壳聚糖微球。
(2)碱性蛋白酶的固定
将壳聚糖微球加入到pH =8.5为磷酸盐缓冲液中浸润,充分润湿溶胀后,以新型多孔壳聚糖微球与碱性蛋白酶的质量比为40 : 1,加入碱性蛋白酶溶液,在1~10℃下振荡混合。向载体与蛋白酶的混合液中加入交联剂戊二醛溶液,使得戊二醛在混合液中的浓度为1.5%,使蛋白酶负载于新型多孔壳聚糖微球上,交联一段时间后,使用pH =8.5为磷酸盐缓冲液充分洗涤,除去多余的交联剂,得到以新型多孔壳聚糖微球为载体的固定化酶。
对比例1
(1)传统壳聚糖微球的制备
将壳聚糖粉末(脱乙酰度>95%)加入到乙酸溶液中,搅拌制得壳聚糖的凝胶溶液。将1.5 mol/L的氢氧化钠溶液与无水乙醇以3:1的比例混合,制得成型剂。将壳聚糖凝胶缓慢加入到成型剂中,搅拌成型、静置硬化后用纯净水洗至中性,冷冻干燥后制得传统壳聚糖微球。
(2)碱性蛋白酶的固定
将壳聚糖微球加入到pH =7.5为磷酸盐缓冲液中浸润,充分润湿溶胀后,以壳聚糖微球与碱性蛋白酶的质量比为25:1,加入碱性蛋白酶溶液,在1~10℃下振荡混合。向载体与蛋白酶的混合液中加入交联剂戊二醛溶液,使得戊二醛在混合液中的浓度为1%,使蛋白酶负载于新型多孔壳聚糖微球上,交联一段时间后,使用pH =7.5为磷酸盐缓冲液充分洗涤,除去多余的交联剂,得到以传统壳聚糖微球为载体的固定化酶。
实施例5 酶负载量的测试
采用考马斯亮蓝法对碱性蛋白酶的固载量进行测试,首先配制考马斯亮蓝染液,后以牛血清蛋白为标准蛋白质配置梯度浓度标准液并根据蛋白质显色原理绘制浓度标准曲线。
接着对样品溶液中蛋白质浓度进行测定,取待测酶溶液1 mL或取适量待测酶样品溶液稀释到1 mL,加入5 mL的考马斯亮蓝染液,摇匀后静置5-10 min,在595 nm处测其吸光度,与标准曲线进行对照,求得酶的浓度。由此可测得固定前碱性蛋白酶溶液与固定化后上层清液中的蛋白质浓度,计算得到固定化酶负载量。实施例1-4与对比例1测试结果如表1所示。
实施例6 酶活力测试方法
采用国标法对碱性蛋白酶的活力进行测试,即取1 mL的蛋白酶溶液或等量的固定化酶于40 ℃预热5 min,加入1 mL提前预热的pH 7.5 的1%酪蛋白溶液,混匀后于40 ℃下反应10 min,加入2 mL的10% TCA溶液终止反应,过滤或离心,取1 mL上清液依次加入5 mL0.4 M碳酸钠溶液与1 mL福林酚显色剂,40℃下显色20 min,使用分光光度计于680 nm处测试吸光度,以灭活的酶液为空白对照,平行测定三次。酶活单位定义为在一定条件下(本实验为25 ℃,pH 7.5),每分钟催化酪蛋白水解产生1μg酪氨酸所需的碱性蛋白酶量。实施例1-4与对比例1测试结果如表1所示。
表1多孔壳聚糖微球对碱性蛋白酶的负载率及固定酶活性
对比例 | 实施例1 | 实施例2 | 实施例3 | 实施例4 | |
负载率(%) | 38.29 | 78.35 | 70.55 | 72.84 | 76.52 |
酶活性(U/mg) | 120.14 | 316.32 | 266.48 | 278.53 | 302.39 |
Claims (7)
1.一种多孔壳聚糖微球,其特征在于由以下步骤制成:
(1)将壳聚糖粉末溶于乙酸-乙酸铵溶液中,再加入纳米二氧化硅粉末,制得改性壳聚糖复合凝胶;
(2)将氢氧化钠或氢氧化钾与碳酸钠混合溶解,然后与无水乙醇混合,得到复合碱性成型剂;
(3)将步骤(1)所得改性壳聚糖复合凝胶,缓慢加入到复合碱性成型剂中,搅拌成型、静置硬化后用纯净水洗至中性,冷冻干燥后制得多孔壳聚糖微球。
2.如权利要求1所述的多孔壳聚糖微球的制备方法,其特征在于,步骤(1)所述改性壳聚糖复合凝胶的制备过程为:先向乙酸溶液中加入乙酸铵,使得乙酸铵浓度为0.01-0.1mol/L,后将壳聚糖粉末溶于乙酸-乙酸铵溶液中制备壳聚糖凝胶,再向其加入与壳聚糖质量比为1:1~1:5的纳米二氧化硅粉末,搅拌得到改性壳聚糖复合凝胶。
3.如权利要求1所述的多孔壳聚糖微球的制备方法,其特征在于,步骤(2)中所述复合碱性成型剂由氢氧化钠或氢氧化钾与碳酸钠混合溶解而成,其摩尔比为1.5-3.5,然后与无水乙醇以5:1 - 2:1的体积比进行混合。
4.一种碱性蛋白酶的固定方法,其特征在于包括以下步骤:
(1) 将权利要求1所制备的多孔壳聚糖微球加入到pH为7.0-9.5的磷酸盐缓冲液中浸润,充分润湿溶胀后加入碱性蛋白酶溶液,在1~10℃下振荡混合得到混合液;
(2) 向混合液中加入交联剂,使碱性蛋白酶负载于多孔壳聚糖微球上,用磷酸盐缓冲液洗涤后,除去多余的交联剂,得到以多孔壳聚糖微球为载体的固定化酶。
5.如权利要求4所述的固定方法,其特征在于,所述多孔壳聚糖微球与碱性蛋白酶的质量比为100:1~20:1,所述交联剂在混合溶液中的体积比浓度为1% - 5%。
6.如权利要求4所述的固定方法,其特征在于,所述磷酸盐缓冲液的pH为7.0 - 9.5。
7.如权利要求4所述的固定方法,其特征在于,所述交联剂为京尼平或戊二醛或EDC/NHS。
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JPH0994090A (ja) * | 1995-09-29 | 1997-04-08 | Fuji Spinning Co Ltd | 酵素固定化用担体の製造方法 |
CN101892217A (zh) * | 2010-06-02 | 2010-11-24 | 中国水产科学研究院黄海水产研究所 | 磁性壳聚糖复合微球固定化海洋碱性蛋白酶的制备方法 |
CN106754861A (zh) * | 2016-12-26 | 2017-05-31 | 浙江工商大学 | 一种多孔磁性铜离子金属螯合载体及其制备方法、利用载体固定化木瓜酶的方法及其应用 |
CN108740997A (zh) * | 2018-07-25 | 2018-11-06 | 华中农业大学 | 一种蛋白酶壳聚糖微球的制备方法 |
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JPH0994090A (ja) * | 1995-09-29 | 1997-04-08 | Fuji Spinning Co Ltd | 酵素固定化用担体の製造方法 |
CN101892217A (zh) * | 2010-06-02 | 2010-11-24 | 中国水产科学研究院黄海水产研究所 | 磁性壳聚糖复合微球固定化海洋碱性蛋白酶的制备方法 |
CN106754861A (zh) * | 2016-12-26 | 2017-05-31 | 浙江工商大学 | 一种多孔磁性铜离子金属螯合载体及其制备方法、利用载体固定化木瓜酶的方法及其应用 |
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