CN112707977B - 聚苯乙烯型树脂的氨基化方法、氨基化树脂固定酶的方法 - Google Patents
聚苯乙烯型树脂的氨基化方法、氨基化树脂固定酶的方法 Download PDFInfo
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- CN112707977B CN112707977B CN202110329787.6A CN202110329787A CN112707977B CN 112707977 B CN112707977 B CN 112707977B CN 202110329787 A CN202110329787 A CN 202110329787A CN 112707977 B CN112707977 B CN 112707977B
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Abstract
本发明提供了一种聚苯乙烯型树脂的氨基化方法、氨基化聚苯乙烯型树脂固定酶的方法。该氨基化方法包括:在溶剂中,利用催化剂催化聚苯乙烯型树脂和烯胺盐进行傅克烷基化反应,得到氨基化的聚苯乙烯型树脂,其中,催化剂为路易斯酸催化剂。利用傅克烷基化反应将烯胺盐接枝至聚苯乙烯型树脂上,对聚苯乙烯型树脂完成氨基化,傅克烷基化反应的条件易于控制,后处理流程简单,只需要通过洗涤将催化剂和未反应的烯胺盐去除即可,因此本申请的上述氨基化方法步骤少、简单易操作,与此同时,该氨基化方法还避免了贵金属催化剂的使用,减少了生产成本。所得聚苯乙烯树脂作为酶固定化载体时有较好的固定化效果和重复使用性,大大扩充了酶固定化载体库。
Description
技术领域
本发明涉及酶的固定化技术领域,具体而言,涉及一种聚苯乙烯型树脂的氨基化方法、氨基化聚苯乙烯型树脂固定酶的方法。
背景技术
相较与化学催化剂,生物催化剂—酶因其高活性、选择性和底物的专一性好而被广泛应用于工业生产过程中。生物催化正在成为化学品、中间体、精细化学品和最终药物分子制造计划的重要组成部分。虽然酶具有高的区域选择性,高的催化效率和温和的反应条件,但由于游离酶的催化活性受到温度、pH值、溶剂等因素影响,很容易失活。随着工艺需求的不断扩大,对酶应用的效率和经济性的要求也就越来越高。因此,这不仅需要增强酶活性、特异性和生产力,还需要提高酶的可回收性和重复利用性。
通过将酶固定到固体载体上来实现固定化,由此获得非均相固定化酶系统为上述问题提供了一个很好的解决方法。固定化酶在保持了酶特有的催化性能的同时,还呈现出稳定性高,分离回收容易,可重复操作使用,工艺简便等一系列优点。固定化酶的性能主要取决于固定化方法和所用的载体材料。按照酶与载体不同的结合方式,可以分为吸附法、共价结合法、交联法以及包埋法等。其中吸附法是酶与载体通物理吸附作用、范德华力、疏水作用等结合,因操作简单方便、吸附容量大、工业成本低等优点,是固定化酶最常用的方法。但是由于载体和酶之间是用过物理吸附作用结合,结合力较弱,所以存在着酶在重复使用过程中从载体上脱落的问题,而导致转化率下降。共价结合法是将酶和载体通过共价键连接起来。因此载体上必须带有相应的可以和酶反应生成共价键的官能团。相较与吸附法,酶与载体的链接更加牢固,不会存在使用过程中酶掉落的问题,稳定性和循环性更好。
20世纪70年代初,Davankov等通过Friedel-Crafts反应将线型聚苯乙烯交联或将低交联聚苯乙烯再交联合成了一类结构独特、性能优良的多孔聚合物。根据其制备方式,这类反应称为超高交联反应(又称为Davankov超高交联反应)。由超高交联反应制备的多孔聚合物称为超高交联型吸附树脂。超高交联型吸附树脂被认为是继凝胶型、大孔型吸附树脂后的第三代吸附树脂。超高交联吸附树脂通常具有比表面积大、平均孔径小、孔径分布窄、机械强度好等结构特征,目前在有毒有机废水的治理、中草药与抗生素的提取、气体的储存分离等领域显示了广阔的应用前景。
由于氨基具有较好的反应活性,强的受电子性,且易质子化,氨基化的聚苯乙烯树脂在有机药物的分离与钝化、生物医用高分子材料、污水处理吸附、高分子催化剂载体等领域有着不可预估的前景。现在合成胺基化的聚苯乙烯树脂的方法主要有以下几种:
硝基化合物还原法:由硝基化合物经过还原反应得到氨基化合物是合成胺基化聚苯乙烯树脂的一个重要方法。其工艺路线很多,主要有下述几种方式:
催化加氢还原:催化加氢还原法主要是以贵重金属或其合金作为催化剂,例如Ni、Pd、Pt 等,反应条件达到一定的温度和压力时,硝基被还原成氨基。此种方法拥有许多优点,首先可减少人力的投入、机械操作性强;其次,得率较高、产物纯度高、反应步骤可控、绿色无污染。但是其过程中需要贵金属催化,且需要高温高压,对设备的要求较高。这些无疑大大加大了材料的合成成本。水合肼还原法:此方法中需添加催化剂来促进反应的进行,常用的催化剂一种是贵金属类及其合金,通常包括 Pd/C、Pt/C、Raney Ni等。
金属还原法:此种方法应用较早,具体为将金属与酸共存,也有将盐类电解质溶解于水中来实现硝基的还原反应。通过理论分析知,如 Li、Na、K、Mg等电动势在 H之前的金属,在具备一定条件的情况下都能被用作还原剂。但不足之处在于反应结束产生铁泥,会对环境造成严重污染。
溴化胺化法:通过亲电取代反应,使苯环对位接上溴,在钯为催化剂作用下和 LiN(SiMe3)2反应,后处理时选用酸、碱处理,制得聚对氨基苯乙烯。
氯甲基化聚苯乙烯胺基化法:首先将聚苯乙烯微球通过氯甲基化反应在苯环对位接上氯甲基,再与胺化试剂进行胺基化反应,制备得氨基聚苯乙烯。然而这种方法需用到氯甲醚、二氯甲醚,该化学试剂对人体有较大的伤害,具备强烈的致癌风险,现已在国际上大面积被禁止使用。此外,该反应伴随着多取代与后交联等副反应,致使氯甲基树脂结构变得复杂。
氯乙酰化聚苯乙烯胺基化法:通过 F-C 酰基化反应在苯环对位引入酰基,再通过胺基化反应制备胺基化聚苯乙烯,具体反应如下:
发明内容
本发明的主要目的在于提供一种新的聚苯乙烯型树脂的氨基化方法、并用此氨基化聚苯乙烯型树脂用作酶固定化载体,对转氨酶进行固定化。此方法不仅为氨基化聚苯乙烯树脂的合成提供了一种新方法,并且扩充了氨基型酶固定化载体库。
为了实现上述目的,根据本发明的一个方面,提供了一种聚苯乙烯型树脂的氨基化方法,该氨基化方法包括:在溶剂中,利用催化剂催化聚苯乙烯型树脂和烯胺盐进行傅克烷基化反应,得到氨基化的聚苯乙烯型树脂,其中,催化剂为路易斯酸催化剂。
进一步地,上述傅克烷基化反应的温度为40~80℃,优选傅克烷基化反应的时间为12~20h。
进一步地,上述聚苯乙烯型树脂与烯胺盐的质量比2:1~2:3。
进一步地,上述烯胺盐与路易斯酸催化剂的摩尔比为1:3~1:5。
进一步地,上述路易斯酸催化剂为无水三氯化铝、三氯化铁中的任意一种。
进一步地,上述烯胺盐为3-丁烯胺盐酸盐。
进一步地,上述溶剂为1,2-二氯乙烷。
进一步地,上述聚苯乙烯型树脂选自聚苯乙烯树脂、聚苯乙烯-甲基丙烯酸酯树脂、AB8型聚苯乙烯树脂、ECR1090型聚苯乙烯树脂、NKA9型聚苯乙烯树脂、D101型聚苯乙烯树脂、SXD11型聚苯乙烯树脂中的任意一种。
进一步地,上述氨基化方法包括:将聚苯乙烯型树脂和烯胺盐分散在溶剂中,形成待反应体系;在氮气或惰性气氛中,将待反应体系与催化剂混合后加热至傅克烷基化反应的温度进行傅克烷基化反应,得到氨基化的聚苯乙烯型树脂。
根据本发明的另一方面,提供了一种氨基化聚苯乙烯型树脂固定酶的方法,该方法包括:采用上述任一种的氨基化方法制备氨基化的聚苯乙烯型树脂;将氨基化的聚苯乙烯型树脂为载体、以戊二醛为交联剂对酶进行固定化。
进一步地,上述方法包括:采用戊二醛的缓冲溶液对氨基化的聚苯乙烯型树脂进行修饰,得到戊二醛修饰的树脂;将酶液与戊二醛修饰的树脂进行交联反应,实现固定化。
进一步地,上述方法包括:将酶液与氨基化的聚苯乙烯型树脂混合,得到吸附有酶的树脂;将吸附有酶的树脂与戊二醛的缓冲溶液进行交联反应,实现固定化。
进一步地,上述戊二醛的缓冲溶液包括磷酸盐缓冲溶液,戊二醛的缓冲液中戊二醛的质量含量为1~2%,酶为转氨酶。
应用本发明的技术方案,本申请利用傅克烷基化反应一步将烯胺盐接枝至聚苯乙烯型树脂上,对聚苯乙烯型树脂完成氨基化,傅克烷基化反应的条件易于控制,后处理流程简单,只需要通过洗涤将催化剂和未反应的烯胺盐去除即可,因此本申请的上述氨基化方法步骤少、简单易操作,与此同时,该氨基化方法还避免了贵金属催化剂的使用,减少了生产成本。经过氨基化修饰的商业聚苯乙烯树脂可以成功的被用做酶固定化载体,并有较好的固定化效果和重复使用性。这也大大扩充了酶固定化载体库,使在做不同酶的固定化时,无论在载体的孔径大小、比表面积、骨架结构、载体极性等方面,提供了更多的选择。
具体实施方式
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
如本申请背景技术所分析的,现有技术中的氨基化聚苯乙烯型树脂的制备方法或者采用复杂的制备方法或者需要采用多种制剂或者需要复杂的后处理方法,导致其制备方法复杂。为了解决该问题本申请提供了一种聚苯乙烯型树脂的氨基化方法、氨基化聚苯乙烯型树脂固定酶的方法。
在本申请一种典型的实施方式中,提供了一种聚苯乙烯型树脂的氨基化方法,该氨基化方法包括:在溶剂中,利用催化剂催化聚苯乙烯型树脂和烯胺盐进行傅克烷基化反应,得到氨基化的聚苯乙烯型树脂,其中,催化剂为路易斯酸催化剂。
本申请利用傅克烷基化反应将烯胺盐接枝至聚苯乙烯型树脂上,对聚苯乙烯型树脂完成氨基化,傅克烷基化反应的条件易于控制,后处理流程简单,只需要通过洗涤将催化剂和未反应的烯胺盐去除即可,因此本申请的上述氨基化方法步骤少、简单易操作,与此同时,该氨基化方法还避免了贵金属催化剂的使用,减少了生产成本。经过氨基化修饰的商业聚苯乙烯树脂可以成功的被用做酶固定化载体,并有较好的固定化效果和重复使用性。这也大大扩充了酶固定化载体库,使在做不同酶的固定化时,无论在载体的孔径大小、比表面积、骨架结构、载体极性等方面,提供了更多的选择。
为了提高傅克烷基化效率,针对上述反应原料,优选上述傅克烷基化反应的温度为40~80℃,优选傅克烷基化反应的时间为12~20h。
上述傅克烷基化反应实质是在聚苯乙烯的苯环上接枝具有氨基的烷基基团,由于聚苯乙烯型树脂的大分子特点,难以实现各苯环上的均匀接枝,为了尽可能地提高氨基化几率,优选上述聚苯乙烯型树脂与烯胺盐的摩尔比为2:1~2:3。
此外,为了提高催化效率并避免催化剂的浪费,并便于催化剂的去除,优选上述烯胺盐与路易斯酸催化剂的摩尔比为1:3~1:5。
在上述方法中,用于傅克烷基化的路易斯酸催化剂可以从现有技术中傅克烷基化常用的路易斯酸催化剂中进行选择,为了使催化剂便于从产物中分离出来,并保证高效催化,优选上述路易斯酸催化剂为无水三氯化铝、三氯化铁中的任意一种或两种的组合。
在一些实施例中,用于上述氨基化方法的烯胺盐为3-丁烯胺盐酸盐,实现高效的氨基化。
用于本申请的上述溶剂主要是为了溶解烯胺盐和路易斯酸催化剂,以便于其与聚苯乙烯型树脂的充分接触,为了提高烯胺盐和路易斯酸催化剂的溶解性,优选上述溶剂为1,2-二氯乙烷。
用于本申请的聚苯乙烯型树脂包括但不限于聚苯乙烯树脂、聚苯乙烯-甲基丙烯酸酯树脂、AB8型聚苯乙烯树脂、ECR1090型聚苯乙烯树脂、NKA9型聚苯乙烯树脂、D101型聚苯乙烯树脂、SXD11型聚苯乙烯树脂中的任意一种。即无论是纯的聚苯乙烯树脂还是改性聚苯乙烯树脂,均可采用上述氨基化方法进行氨基修饰。
在本申请一些实施例中,上述氨基化方法包括:将聚苯乙烯型树脂和烯胺盐分散在溶剂中,形成待反应体系;在氮气或惰性气氛中,将待反应体系与催化剂混合后加热至傅克烷基化反应的温度进行傅克烷基化反应,得到氨基化的聚苯乙烯型树脂。首先将烯胺盐和聚苯乙烯型树脂在溶剂中分散,从而使烯胺盐能够预先进入聚苯乙烯型树脂的孔隙中,然后再加入催化剂进行傅克烷基化反应,提高了反应速度和烷基化的均匀性。
在本申请另一种典型的实施方式中,提供了一种氨基化聚苯乙烯型树脂固定酶的方法,该方法包括:采用上述任一种的氨基化方法制备氨基化的聚苯乙烯型树脂;将氨基化的聚苯乙烯型树脂为载体、以戊二醛为交联剂对酶进行固定化。
采用本申请的氨基化方法制备氨基化的聚苯乙烯型树脂,制备方法简单,成本低,且可以通过该氨基化方法灵活调整氨基化程度,进而对下一步的酶的固定化程度进行灵活调整。
在本申请一些实施例中,上述方法包括:采用戊二醛的缓冲溶液对氨基化的聚苯乙烯型树脂进行修饰,得到戊二醛修饰的树脂;将酶液与戊二醛修饰的树脂进行交联反应,实现固定化。
上述方法先采用戊二醛对树脂进行修饰,以利用聚苯乙烯型树脂上的氨基与戊二醛交联形成交联网络;然后在将酶的氨基残基与戊二醛发生交联,进而实现对酶的交联固化,该过程使得酶的交联固化效率较高,尤其是适用于对戊二醛敏感的酶类。
在本申请另一些实施例中,上述方法包括:将酶液与氨基化的聚苯乙烯型树脂混合,得到吸附有酶的树脂;将吸附有酶的树脂与戊二醛的缓冲溶液进行交联反应,实现固定化。
上述方法首先将聚苯乙烯型树脂通过吸附作用将酶吸附在其内外表面,然后再与戊二醛进行反应,实现利用戊二醛将酶通过交联方式固定在聚苯乙烯型树脂上。上述方法尤其适用于对戊二醛不敏感的酶类。
用于本申请的戊二醛的缓冲溶液可以参考交联固化所用的常规缓冲溶液,比如上述戊二醛的缓冲溶液包括磷酸盐缓冲溶液。另外,为了提高酶交联固定率,优选上述戊二醛的缓冲液中戊二醛的质量含量为1~2%,以提高戊二醛的利用率。在固定酶的过程中,可以通过调整酶相对于载体的量对负载量进行调整,在此不再赘述。
由于上述固定化是利用戊二醛的醛基和酶的氨基酸残基之间的交联作用,因此本申请的方法适用于常规酶类,比如上述酶选自转氨酶
以下将结合实施例和对比例,进一步说明本申请的有益效果。
氨基化实施例
实施例1-1
聚苯乙烯树脂合成
将含0.5% PVA的150 mL水溶液加入到500 mL四口瓶中,并升温至40 ℃。将16 g苯乙烯,5 g二乙烯苯(80%),10 mL甲苯,20 mL正庚烷,100 mg的过氧化苯甲酰混合均匀后,加至四口瓶中。开启搅拌调整液滴至合适的粒径后升温至80 ℃保温2 h,随后升温至88 ℃保温4 h。反应结束后冷却至室温,过滤得到目标珠体,依次用热水,乙醇,去离子水清洗,筛分烘干后得到聚苯乙烯树脂。
聚苯乙烯树脂氨基化
取1 g烘干的聚苯乙烯树脂及1 g 3-丁烯胺盐酸盐分散到100 mL 1,2-二氯乙烷中,室温下搅拌1 h,形成待反应体系。随后称取2 g 无水三氯化铝加入到上述待反应体系中,继续搅拌10 min,升温至80 ℃,于N2保护下反应20 h。反应结束后冷却至室温,依次用甲醇、去离子水清洗得到氨基化苯乙烯树脂:PS-NH2。
实施例1- 2
聚苯乙烯-甲基丙烯酸酯树脂合成
将含0.5% PVA和10% NaCl的240 mL水溶液加入到500 mL四口瓶中,并升温至40℃。将6 g甲基丙烯酸甲酯,15 g二乙烯苯(80%),20 mL 1,2-二氯乙烷,40 mL甲基环己烷,100 mg 偶氮二异丁腈混合均匀后,加至四口瓶中。开启搅拌调整液滴至合适的粒径后升温至70 ℃保温2 h,随后升温至80 ℃保温4 h,85 ℃保温1 h。反应结束后冷却至室温,过滤得到目标珠体,依次用热水,乙醇,去离子水清洗,筛分烘干得到聚苯乙烯-甲基丙烯酸酯树脂(PDMA)。
PDMA树脂修饰
取1 g烘干的PDMA树脂及0.7 g 3-丁烯胺盐酸盐分散到100 mL 1,2-二氯乙烷中,室温下搅拌1 h,形成待反应体系。随后称取2 g无水三氯化铝加入到上述待反应体系中,继续搅拌10 min后升温至80 ℃,于N2保护下反应20 h。反应结束后冷却至室温,依次用甲醇,去离子水清洗得到氨基化树脂:PDMA-NH2。
实施例1- 3
取1 g 烘干的ECR1090型聚苯乙烯树脂(购买自Purlite)及1 g 3-丁烯胺盐酸盐分散到100 mL 1,2-二氯乙烷中,室温下搅拌1 h,形成待反应体系。随后称取2 g无水三氯化铝加入到上述待反应体系中,继续搅拌10 min,升温至80 ℃,于N2保护下反应20 h。反应结束后冷却至室温,依次用甲醇,去离子水清洗得到氨基化ECR1090树脂:ECR1090-NH2。
实施例1- 4
取1 g 烘干的AB8树脂(购买自南开合成有限公司)及1 g 3-丁烯胺盐酸盐分散到100 mL 1,2-二氯乙烷中,室温下搅拌1 h,形成待反应体系。随后称取2 g无水三氯化铝加入到上述待反应体系中,继续搅拌10 min,升温至80 ℃,于N2保护下反应20 h。反应结束后冷却至室温,依次用甲醇,去离子水清洗得到氨基化AB8树脂:AB8-NH2。
实施例1- 5
取1 g 烘干的NKA9树脂(购买自南开合成有限公司)及1g 3-丁烯胺盐酸盐分散到100 mL 1,2-二氯乙烷中,室温下搅拌1 h,形成待反应体系。随后称取2 g无水三氯化铝加入到上述待反应体系中,继续搅拌10 min,升温至80 ℃,于N2保护下反应20 h。反应结束后冷却至室温,依次用甲醇,去离子水清洗得到氨基化NKA9树脂:NKA9-NH2。
实施例1- 6
取1 g 烘干的SXD-11树脂(购买自安徽三星有限公司)及1 g 3-丁烯胺盐酸盐分散到100 mL 1,2-二氯乙烷中,室温下搅拌1 h,形成待反应体系。随后称取2 g无水三氯化铝加入到上述待反应体系中,继续搅拌10 min,升温至80 ℃,于N2保护下反应20 h。反应结束后冷却至室温,依次用甲醇,去离子水清洗得到氨基化SXD-11树脂:SXD-11-NH2。
实施例1-7
取1 g 烘干的D101树脂(购买自安徽三星有限公司)及1 g 3-丁烯胺盐酸盐分散到100 mL 1,2-二氯乙烷中,室温下搅拌1 h,形成待反应体系。随后称取2 g无水三氯化铝加入到上述待反应体系中,继续搅拌10 min,升温至80 ℃,于N2保护下反应20 h。反应结束后冷却至室温,依次用甲醇,去离子水清洗得到氨基化D101树脂:D101-NH2。
对上述各实施例得到的氨基化苯乙烯树脂采用茚三酮显色法和酸碱滴定法检测其中的氨基含量,使用氮气吸脱附法测其比表面积与孔径,检测结果见表1中
表1
酶固定化实施例
以转氨酶为模型,转氨酶来源于Chromobacterium violaceum DSM30191(CVTA)为模型物,其序列SEQ ID NO.1为:
MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFFGTTHPPVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGMKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPLGAVFVGDRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMMLAFTLVKNKAKRELFPDFGEIGTLCEDIFFRNNLIMTAQGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA。
实施例 2-1
用20 mM pH为7.0的磷酸盐缓冲液配制1%戊二醛溶液,分别称取1 g实施例1-1的氨基化苯乙烯树脂和苯乙烯树脂分散于上述溶液中。于30 ℃下孵化1 h,随后用去离子水洗涤三次,得到红色的戊二醛修饰的氨基化苯乙烯树脂和戊二醛修饰的苯乙烯树脂,树脂经戊二醛修饰后,会变成红棕色,所以根据颜色变化判断戊二醛是否实现成功修饰。
取4 mL配制好的酶液(1V酶液加3V磷酸盐缓冲液)加入到戊二醛修饰的两种树脂(1 g) 中进行固定化,制得氨基化苯乙烯固定化酶及苯乙烯树脂固定化酶。
测体系固定前后蛋白含量差值计算得到酶的负载量,其中氨基化苯乙烯固定化酶的固定化酶负载量为26.44 mg/g;苯乙烯树脂固定化酶的固定化酶负载量为11.09 mg/g。
实施例 2-2
用20 mM pH为7.0的磷酸盐缓冲液配制1%戊二醛溶液,分别称取1 g PDMA树脂、PDMA-NH2树脂分散于上述溶液中。于30 ℃下孵化1 h,随后用去离子水洗涤三次,得到红色的戊二醛修饰的PDMA树脂和戊二醛修饰的PDMA-NH2树脂。
取4 mL配制好的酶液(1V酶液加3V磷酸盐缓冲液)加入到戊二醛活化后的两种树脂(1 g)中进行固定化,制得PDMA-NH2固定化酶及PDMA固定化酶。
所制得的PDMA-NH2固定化酶的固定化酶负载量为28.35 mg/g;所制得的PDMA固定化酶的固定化酶负载量为9.17 mg/g。
实施例 2-3
用20 mM pH为7.0的磷酸盐缓冲液配制1%戊二醛溶液,分别称取1 g ECR1090,ECR1090-NH2分散于上述溶液中。于30 ℃下孵化1 h,随后用去离子水洗涤三次,得到红色的戊二醛修饰的ECR1090树脂和戊二醛修饰的ECR1090-NH2树脂。
取4 mL配制好的酶液(1V酶液加3V磷酸盐缓冲液)加入到戊二醛活化后的两种树脂(1 g)中进行固定化,制得ECR1090-NH2固定化酶及ECR1090固定化酶。
ECR1090-NH2固定化酶的固定化酶负载量为21.82 mg/g;ECR1090固定化酶的固定化酶负载量为21.09 mg/g。
实施例 2-4
用20 mM pH为7.0的磷酸盐缓冲液配制1%戊二醛溶液,分别称取1 g AB8,AB8-NH2分散于上述溶液中。于30℃下孵化1 h,随后用去离子水洗涤三次,得到红色的戊二醛修饰的AB8树脂和戊二醛修饰的AB8-NH2树脂。
取4 mL配制好的酶液(1V酶液加3V磷酸盐缓冲液)加入到戊二醛活化后的两种树脂(1 g)中进行固定化,制得AB8-NH2固定化酶及AB8固定化酶。
AB8-NH2固定化酶的固定化酶负载量为28.37 mg/g、AB8固定化酶的固定化酶负载量为12.91 mg/g。
实施例 2-5
用20 mM pH为7.0的磷酸盐缓冲液配制1%戊二醛溶液,分别称取1 g NKA9,NKA9-NH2分散于上述溶液中。于30 ℃下孵化1 h,随后用去离子水洗涤三次,得到红色的戊二醛修饰的NKA9树脂和戊二醛修饰的NKA9-NH2树脂。
取4 mL配制好的酶液(1V酶液加3V磷酸盐缓冲液)加入到戊二醛活化后的两种树脂(1 g)中进行固定化,制得NKA9-NH2固定化酶及NKA9固定化酶。
NKA9-NH2固定化酶的固定化酶负载量为44.56 mg/g、及NKA9固定化酶的固定化酶负载量为42.31 mg/g。
实施例 2-6
用20 mM pH为7.0的磷酸盐缓冲液配制1%戊二醛溶液,分别称取1 g SXD-11,SXD-11-NH2分散于上述溶液中。于30℃下孵化1 h,随后用去离子水洗涤三次,得到红色的戊二醛修饰的SXD-11树脂和戊二醛修饰的SXD-11-NH2树脂。
取4 mL配制好的酶液(1V酶液加3V磷酸盐缓冲液)加入到戊二醛活化后的两种树脂(1 g)中进行固定化,制得SXD-11-NH2固定化酶及SXD-11固定化酶。
SXD-11-NH2固定化酶的固定化酶负载量为13.31 mg/g、及SXD-11固定化酶的固定化酶负载量为13.41 mg/g。
酶催化反应路线如下:
在20 mL的反应瓶中,加入0.5 mL 甲醇,溶解0.1 g羰基底物,并加入15 eq 异丙胺盐酸盐和25.0 mg PLP(5’-磷酸吡哆醛),补加0.1 M 磷酸盐缓冲液(pH 8.0)至反应液终体积为5 mL,形成待反应体系;
分别独立地向待反应体系中加入0.1 g转氨酶酶粉、或由0.1 g转氨酶酶粉制备的固定化酶,在47 ℃搅拌20 h。体系经HPLC检测转化率,每一轮反应结束后将固定化酶分离,投入下一轮反应中重复使用,考察重复使用次数,结果记录在表2中。
表2
从以上的描述中,可以看出,本发明上述的实施例实现了如下技术效果:
本申请利用傅克烷基化反应将烯胺盐接枝至聚苯乙烯型树脂上,对聚苯乙烯型树脂完成氨基化,傅克烷基化反应的条件易于控制,后处理流程简单,只需要通过洗涤将催化剂和未反应的烯胺盐去除即可,因此本申请的上述氨基化方法步骤少、简单易操作,与此同时,该氨基化方法还避免了贵金属催化剂的使用,减少了生产成本。经过氨基化修饰的商业聚苯乙烯树脂可以成功的被用做酶固定化载体,并有较好的固定化效果和重复使用性。这也大大扩充了酶固定化载体库,使在做不同酶的固定化时,无论在载体的孔径大小、比表面积、骨架结构、载体极性等方面,提供了更多的选择。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 凯莱英医药集团(天津)股份有限公司
<120> 聚苯乙烯型树脂的氨基化方法、氨基化树脂固定酶的方法
<130> PN147915
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 459
<212> PRT
<213> 紫色色杆菌DSM30191(Chromobacterium violaceum DSM30191)
<400> 1
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Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
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Claims (14)
1.一种聚苯乙烯型树脂的氨基化方法,其特征在于,所述氨基化方法包括:
在溶剂中,利用催化剂催化聚苯乙烯型树脂和烯胺盐进行傅克烷基化反应,得到氨基化的聚苯乙烯型树脂,其中,所述催化剂为路易斯酸催化剂,所述烯胺盐为3-丁烯胺盐酸盐。
2.根据权利要求1所述的氨基化方法,其特征在于,所述傅克烷基化反应的温度为40~80℃。
3.根据权利要求2所述的氨基化方法,其特征在于,所述傅克烷基化反应的时间为12~20h。
4.根据权利要求1所述的氨基化方法,其特征在于,所述聚苯乙烯型树脂与所述烯胺盐的质量比2:1~2:3。
5.根据权利要求1所述的氨基化方法,其特征在于,所述烯胺盐与所述路易斯酸催化剂的摩尔比为1:3~1:5。
6.根据权利要求1所述的氨基化方法,其特征在于,所述路易斯酸催化剂为无水三氯化铝、三氯化铁中的任意一种或两种的组合。
7.根据权利要求1所述的氨基化方法,其特征在于,所述溶剂为1,2-二氯乙烷。
8.根据权利要求1所述的氨基化方法,其特征在于,所述聚苯乙烯型树脂选自聚苯乙烯树脂、聚苯乙烯-甲基丙烯酸酯树脂中的任意一种。
9.根据权利要求1所述的氨基化方法,其特征在于,所述聚苯乙烯型树脂选自AB8型聚苯乙烯树脂、ECR1090型聚苯乙烯树脂、NKA9型聚苯乙烯树脂、D101型聚苯乙烯树脂、SXD11型聚苯乙烯树脂中的任意一种。
10.根据权利要求1至6中任一项所述的氨基化方法,其特征在于,所述氨基化方法包括:
将所述聚苯乙烯型树脂和所述烯胺盐分散在所述溶剂中,形成待反应体系;
在氮气或惰性气氛中,将所述待反应体系与所述催化剂混合后加热至傅克烷基化反应的温度进行傅克烷基化反应,得到所述氨基化的聚苯乙烯型树脂。
11.一种氨基化聚苯乙烯型树脂固定酶的方法,其特征在于,所述方法包括:
采用权利要求1至10中任一项所述的氨基化方法制备氨基化的聚苯乙烯型树脂;
将所述氨基化的聚苯乙烯型树脂为载体、以戊二醛为交联剂对酶进行固定化。
12.根据权利要求11所述的方法,其特征在于,所述方法包括:
采用戊二醛的缓冲溶液对所述氨基化的聚苯乙烯型树脂进行修饰,得到戊二醛修饰的树脂;
将酶液与所述戊二醛修饰的树脂进行交联反应,实现所述固定化。
13.根据权利要求11所述的方法,其特征在于,所述方法包括:
将所述酶液与所述氨基化的聚苯乙烯型树脂混合,得到吸附有酶的树脂;
将所述吸附有酶的树脂与戊二醛的缓冲溶液进行交联反应,实现所述固定化。
14.根据权利要求12或13所述的方法,其特征在于,所述戊二醛的缓冲溶液包括磷酸盐缓冲溶液,所述戊二醛的缓冲液中戊二醛的质量含量为1~2%,所述酶为转氨酶。
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