CN100387711C - 一种用于溶菌酶亲和层析的硅胶负载表面大孔甲壳素基质 - Google Patents
一种用于溶菌酶亲和层析的硅胶负载表面大孔甲壳素基质 Download PDFInfo
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Abstract
本发明公开了一种用于溶菌酶亲和层析的新型硅胶负载表面大孔甲壳素基质。本发明首先将壳聚糖负载在硅胶上,采用含有环氧基团、水溶性的无机硅烷试剂γ-环氧丙氧丙基三甲氧基硅烷作为交联剂制备硅胶负载交联壳聚糖微珠,乙酰化后得到硅胶负载甲壳素基质。基质粒径可通过改变所用硅胶支载材料的粒径方便调节。本发明壳聚糖微珠的交联是在水溶液中进行的,过程简单,无需任何有机溶剂。采用水溶性的聚乙二醇20000作为致孔剂,所得基质具有优良的表面大孔结构,适于蛋白质亲和层析。本发明采用氧化法脱去壳聚糖上未乙酰化的游离氨基,基质的非特异性吸附小。本发明制备的硅胶负载表面大孔甲壳素基质可用于鸡蛋清中溶菌酶的亲和纯化,一步纯化可得到高比活力的溶菌酶。
Description
【技术领域】
本发明涉及一种溶菌酶亲和层析基质,特别是适用于溶菌酶亲和层析的硅胶负载具有表面大孔结构的甲壳素基质。
【背景技术】
溶菌酶(Lysozyme,EC 3.2.1.17),又称胞壁质酶,或N-乙酰胞壁质聚糖水解酶,是一种专门作用于微生物细胞壁的水解酶,广泛存在于动物、植物和微生物中,具有杀菌和破壁的作用。溶菌酶具有稳定的化学性质和良好的生物相容性,对人体安全无毒、无副作用,已在食品行业的杀菌、防腐和保鲜,发酵工业的菌体破裂,及抗菌、抗病毒、促进血液凝固、分解脓液和组织修复等临床治疗和诊断方面取得广泛应用。随着生物工程的发展,溶菌酶已成为基因工程及细胞工程中质粒、包涵体和细胞破壁必不可少的工具酶。目前,溶菌酶是一种国内外很紧俏的生化物质,国内的溶菌酶生产未能满足国内市场需要,部分溶菌酶及其制剂需要进口。现有溶菌酶的制备多以蛋清为原料,提取方法包括直接结晶法、盐析法及乙醇沉淀法、超滤纯化浓缩法及树脂层析纯化技术。然而,结晶及沉淀法费时且生产率较低、超滤法需要特殊的设备。因此,离子交换和亲和层析两种树脂层析纯化溶菌酶技术应用较为广泛。离子交换法除梯度或分段洗脱及树脂的再生和平衡带来的设备复杂、操作繁琐等问题外,还存在纯化倍数较低的缺点。而具有高度特异性和专一选择性的亲和层析法,操作工艺简单、周期短、得率高,广泛应用于蛋白质和多肽等生物分子的分离纯化。制备性能优良的基质材料是亲和层析技术的关键所在。亲和层析基质材料应具有高选择性吸附目标蛋白的能力、稳定的机械和化学性能、且易于制备,价格低廉。此外,用于蛋白质纯化的层析基质还必须有利于蛋白分子构像和活性的保持,因此基质必须具有大的比表面积、大的孔网络和高的孔隙度。目前用于制备溶菌酶的亲和层析基质主要有N-乙酰基-D-葡萄糖胺-琼脂糖、甲壳素粉。然而,N-乙酰基-D-葡萄糖胺-琼脂糖价格十分昂贵,制备过程需使用剧毒试剂溴化氰作为活化试剂,操作复杂。
甲壳素(Chitin)广泛存在于虾、蟹、蛹及昆虫等动物外壳以及菌类、藻类植物的细胞壁中。作为一种天然碱性多糖,甲壳素分子中含有丰富的羟基、氨基。同时,因其资源丰富,具有无毒、无害、良好的生物相容性、生物可降解性、可再生性等环境友好特征而受到人们的极大关注。甲壳素分子中含有可与溶菌酶特异性结合的N-乙酰基-D-葡萄糖胺结构,可直接用于溶菌酶的亲和纯化。然而,甲壳素分子中有较强的分子间和分子内氢键,不溶于水、稀酸、稀碱、浓碱、一般有机溶剂,可溶于浓的盐酸、硫酸、磷酸和无水甲酸,但同时主链发生降解,极大地制约了甲壳素的广泛应用。通常将甲壳素粉碎后直接用于溶菌酶的亲和纯化。这样得到的甲壳素微珠内部孔径较小,可用于蛋白结合的表面积小,蛋白吸附量低,层析操作时传质阻力较大、柱压较高。
壳聚糖(Chitosan)是甲壳素脱乙酰化后的产物,又名甲壳胺或可溶性甲壳质,是最为重要的甲壳素衍生物。相对于甲壳素,壳聚糖的溶解性大大改善,化学性质也更为活泼。壳聚糖分子中的氨基易于参加化学反应,衍生容易。将壳聚糖进行交联制得多孔微珠后,再对其N-乙酰化可得到甲壳素多孔微珠。此法与甲壳素直接粉碎法相比,可以得到粒径及孔径可控的甲壳素多孔微珠。壳聚糖微珠的制备通常采用微乳液聚合法。壳聚糖在含表面活性剂、致孔剂的微乳液体系中,在交联剂如戊二醛、甲醛、环氧氯丙烷、甘油双环氧丙基醚等存在下交联成球。然而,此方法通常需使用大量的液体石蜡等有机溶剂,微珠中包含的表面活性剂及致孔剂抽提较为困难。
本发明的目的是针对当前甲壳素微珠存在的问题,提供一种性能优良、适用于溶菌酶亲和层析的大孔甲壳素基质。本发明首先制备硅胶负载交联壳聚糖,经乙酰化得到硅胶负载甲壳素基质。本发明将壳聚糖负载在硅胶上,基质粒径可通过改变所用硅胶支载材料的粒径方便调节,且所得基质机械性能好。采用的交联试剂为含有环氧基团、水溶性的无机硅烷试剂γ-环氧丙氧丙基三甲氧基硅烷,壳聚糖微珠的制备是在水溶液中进行,过程简单,无需任何有机溶剂。本发明采用水溶性的聚乙二醇20000作为致孔剂,经简单的热抽提即可完全除去,所得基质具有优良的表面大孔,适于蛋白质亲和层析。本发明采用氧化法脱去未酰化的游离氨基,基质的非特异性吸附小。本发明制备的硅胶负载表面大孔甲壳素基质可用于鸡蛋清中溶菌酶的亲和纯化,一步纯化可得高比活力的溶菌酶。
【发明内容】
本发明提供一种性能优良、适用于溶菌酶亲和层析的大孔甲壳素基质。本发明硅胶负载表面大孔甲壳素基质是通过以下步骤完成的:
——a在1mol/L 100mL醋酸溶液中加入壳聚糖2-4g,致孔剂聚乙二醇5-15g,磁力搅拌30min,过滤;
——b在滤液中加入无机硅烷试剂γ-环氧丙氧丙基三甲氧基硅烷2-8mL,磁力搅拌30mini
——c向溶液中加入60-80目硅胶50g,搅拌均匀,超声40min后倾倒到培养皿中,搅拌均匀,室温静置48h后,40℃烘干;
——d将所得固体置于0.5mol/L 100mL氨水溶液中,80℃抽提,每2h更换氨水溶液,8h后将固体取出,蒸馏水洗至中性,40℃烘干;
——e将所得固体置于95mL甲醇中,加入5mL醋酸酐,搅拌2h,所得固体蒸馏水洗至中性;
——f固体置于0.1mol/L盐酸溶液中,搅拌使其悬浮,慢慢加入亚硝酸钠溶液,搅拌2h后用碱将溶液调至中性,蒸馏水彻底清洗所得固体,40℃烘干,得硅胶负载表面大孔甲壳素基质。
本发明制备的硅胶负载表面大孔甲壳素基质,是由硅胶负载交联壳聚糖微珠乙酰化得到的。将壳聚糖涂覆在硅胶上,通过交联制得硅胶负载壳聚糖微珠,避免了纯壳聚糖造粒难的缺点。同时,硅胶具有大的比表面积和优良的机械强度,制备的载体材料机械强度高。
本发明壳聚糖的交联是通过交联试剂实现的。水溶性的γ-环氧丙氧丙基三甲氧基硅烷交联剂含有双官能团:硅甲氧基和环氧基团。硅甲氧基可以通过在溶液中的水解、缩合形成无机氧化物网络。同时,壳聚糖分子中的氨基与环氧基团的反应,可以实现壳聚糖链之间的交联并将壳聚糖成功引入到无机网络中。壳聚糖的交联在水溶液中进行,过程简单,无需任何有机溶剂。壳聚糖分子中丰富的羟基、氨基及无机硅烷组分产生的硅醇基团可提供大量的氢键形成位点,有利于在硅胶表面制备得到结构均匀、性能优良的材料。
本发明亲和层析载体,采用一种高分子聚合物-聚乙二醇20000作为致孔剂。聚乙二醇分子中丰富的醚键使其具有优良的水溶性。由于聚乙二醇分子中具有大量的氢键形成位点(氧原子),与硅胶、交联壳聚糖存在大量的氢键而具有良好的相容性。抽提过程中,温度的升高导致氢键的断裂和聚乙二醇的溶解,使得载体材料具有表面多孔结构。红外光谱谱图证明表面抽提过的载体中不含聚乙二醇。SIRION扫描电子显微镜(FEI,USA)用于观测固体的表面形貌,固体均经镀金后进行测试。图1为制备过程中使用的硅胶、硅胶负载交联壳聚糖微珠及本专利制得的硅胶负载甲壳素基质的扫描电子显微镜图。硅胶表面为平整的无孔结构(图1A,B),硅胶负载交联壳聚糖微珠中(图1C,D)硅胶表面的壳聚糖层十分明显,并且具有与硅胶表面无孔形貌具有显著性差异的多孔结构。交联壳聚糖微珠经乙酰化后制得的甲壳素基质(图1E,F),较之前者,形貌无明显改变,为微米级贯穿式表面大孔。这主要是由于上述壳聚糖的交联及有机-无机杂化作用使得硅胶负载交联壳聚糖具有一定的硬度和强度,在乙酰化过程中形貌不发生明显变化。
采用GENENIS4000能量色散X-射线微分析仪(EDAX,USA)测定硅胶、硅胶负载甲壳素基质的X-射线能量色散光谱,如图2所示。本发明的硅胶负载甲壳素基质的X-射线能量色散光谱(图1B)相对于硅胶的X-射线能量色散光谱(图1A)有显著差异,证明了甲壳素的涂覆。
以鸡蛋清中溶菌酶的提取为例,检测本专利所得层析基质亲和纯化溶菌酶的效果。
溶菌酶活性测定是将Micrococcus lysodeikticus菌体悬浮于0.067mol/L的pH=6.3磷酸盐缓冲液中。取此菌液2.97mL置于石英比色皿中,加入30μL酶溶液,混合均匀后室温下测定450nm波长下的吸光度变化值。单位溶菌酶活性定义为上述条件下,每分钟水解菌体使吸光度下降0.001单位的酶量。
总蛋白含量测定采用考马斯亮兰染色法,以牛血清蛋白为标准蛋白。
鸡蛋清中亲和纯化溶菌酶过程如下:将硅胶负载大孔甲壳素基质以匀浆法装柱(10cm×2.6cm i.d.),以平衡缓冲液(0.1mol/L的磷酸盐缓冲液中,pH=8.0)平衡柱子。将10mL鸡蛋清加入0.1mol/L 30mL磷酸盐缓冲液(pH=8.0)中,匀浆10min,8000转冷冻离心20min。将所得上清液以1mL/min的流速上柱,然后用40mL平衡缓冲液以1.5mL/min的流速冲洗柱子,除去柱中未结合的蛋白。最后,用0.1mol/L醋酸溶液以1.5mL/min流速洗脱溶菌酶。收集活性部分,即为纯化的溶菌酶。经总蛋白、酶活性测定,每毫克总蛋白中溶菌酶活力为48000U。
本发明的优点及效果:
1、本发明硅胶负载表面大孔甲壳素基质的制备,先制得硅胶负载交联壳聚糖微珠,乙酰化后得到硅胶负载甲壳素基质。所得基质粒径可通过改变所用硅胶支载材料的粒径方便调节。
2、本发明采用的壳聚糖交联剂为水溶性的无机硅烷试剂γ-环氧丙氧丙基三甲氧基硅烷,壳聚糖交联过程条件温和,无需任何有机溶剂。
3、本发明制得的溶菌酶亲和层析载体,具有优良的表面多孔结构,适于蛋白质亲和层析。
4、本发明采用氧化法脱去未酰化的游离氨基,基质的非特异性吸附小。
【附图说明】
图1、硅胶、硅胶负载交联壳聚糖微珠、硅胶负载甲壳素基质的扫描电子显微镜图(A-F的放大倍数分别为100、20000、5000、40000、5000和40000倍)
图2、硅胶、硅胶负载甲壳素基质的X-射线能量色散光谱
【具体实施方式】
在1mol/L 100mL醋酸溶液中加入精制壳聚糖2g、致孔剂聚乙二醇10g,磁力搅拌30min,滤布过滤后除去不溶残渣;向滤液中加入无机硅烷试剂γ-环氧丙氧丙基三甲氧基硅烷2mL,磁力搅拌30min后边搅拌边在溶液中加入60-80目硅胶50g,搅拌均匀,超声40min;将悬浮液倾倒到培养皿中,搅拌均匀,室温静置48h,40℃烘干。将所得固体置于0.5mol/L100mL氨水溶液中,80℃抽提,每2h更换氨水溶液,8h后将固体取出,蒸馏水洗至中性,40℃烘干后置于95mL甲醇中,加入5mL醋酸酐,搅拌2h,固体用蒸馏水洗至中性后置于0.1mol/L盐酸中,搅拌使其悬浮,慢慢加入亚硝酸钠溶液,搅拌2h后用碱将溶液调至中性,蒸馏水彻底清洗所得固体,40℃烘干,即可得到亲和层析纯化溶菌酶的硅胶负载表面大孔甲壳素基质。
Claims (1)
1.一种用于溶菌酶亲和层析的硅胶负载表面大孔甲壳素基质,其特征是该亲和层析基质是通过以下步骤制备的:
——a在1mol/L 100mL醋酸溶液中加入壳聚糖2-4g,致孔剂聚乙二醇5-15g,磁力搅拌30min,过滤;
——b在滤液中加入无机硅烷试剂γ-环氧丙氧丙基三甲氧基硅烷2-8mL,磁力搅拌30min;
——c向完成b步骤所得溶液中加入60-80目硅胶50g,搅拌均匀,超声40min后倾倒到培养皿中,搅拌均匀,室温静置48h后,40℃烘干;
——d将完成c步骤所得固体置于0.5mol/L 100mL氨水溶液中,80℃抽提,每2h更换氨水溶液,8h后将固体取出,蒸馏水洗至中性,40℃烘干;
——e将完成d步骤所得固体置于95mL甲醇中,加入5mL醋酸酐,搅拌2h,所得固体用蒸馏水洗至中性;
——f将完成e步骤所得固体置于0.1mol/L盐酸溶液中,搅拌使其悬浮,慢慢加入亚硝酸钠溶液,搅拌2h后用碱将溶液调至中性,蒸馏水彻底清洗所得固体,40℃烘干,得硅胶负载表面大孔甲壳素基质。
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