本文主要研究内容
作者李轲(2019)在《嵌段无序温敏性聚合物载体的合成及其应用研究》一文中研究指出:固定化青霉素G酰化酶(Penicillin G acylase,PGA)是一种被广泛应用的工业催化剂,它可以催化青霉素G钾水解,得到产物6-氨基青霉烷酸(6-APA)。6-APA是半合成青霉素类药物的关键中间体,在抗菌消炎等领域占有很大的市场空间。因此,固定化PGA的一些主要性能,如酶活回收率、响应速率、重复使用性等,直接关联着企业的经济成本与效益。而固定化PGA的性能与载体的结构和性质密切相关,因此,设计合成合适的载体是提高固定化PGA性能的一个关键途径。无机、有机和复合材料作为固定化PGA的主要载体类型,尽管它们在固定化PGA的负载量、响应速率、重复使用性和回收率等方面都得到了不同程度的提高,但相关问题并没有根本改善。而且,研究在如何提高酶活回收率方面的进展却更加缓慢。酶活回收率较低的主要原因在于载体微环境不能适宜酶催化活性最高时所对应的构象需求,导致酶活性中心构象的变形。为探究影响酶活回收率的关键微环境因素,同时兼顾固定化PGA负载量、响应速率和重复使用性,本论文设计合成了两种温敏性嵌段无序聚合物作为PGA的固定化载体。通过利用温敏性聚合物溶胶/凝胶相转变过程提升固定化PGA的响应速率和重复使用性;通过设计载体的多羟基微环境提高载体亲水性,进而维持PGA的高催化活性构象;通过调适靶点间距进一步巩固PGA高催化活性构象,进而提升酶活回收率,研究取得了较好成果,现将研究内容及结果总结如下:1)首先采用可逆加成-断裂链转移(RAFT)聚合方法,以N,N-二乙基丙烯酰胺(PDEA)和甲基丙烯酸β-羟乙酯(HEMA)为功能单体,合成了二嵌段聚合物PDEA-b-PHEMA,并考察了DEA和HEMA比例对PGA酶活性的影响,得出适宜的DEA和HEMA嵌段比例。之后,探究了在载体制备条件下HEMA和甲基丙烯酸缩水甘油酯(GMA)的竞聚率。在上述工作的基础上,合成了具有不同靶点间距的温敏性嵌段无序共聚物载体PDEA-b-PHEMA-b-P(HEMA-co-GMA),并利用核磁氢谱(1HNMR)考察了聚合物的结构及分子量,结果表明,通过1HNMR表征的聚合物中GMA和HEMA摩尔比与通过竞聚率计算结果间的误差范围为1.76-3.47%,凝胶渗透色谱(GPC)表征证明了合成载体的反应为活性/可控自由基聚合。温敏性研究结果表明共聚物载体的低临界相转变温度(LCST)随着无规共聚中单体HEMA的增加而降低;催化性能研究表明固定化PGA响应速率快,且固定化PGA的酶活回收率随靶点间距的增大而增大,当无规共聚物嵌段中HEMA与GMA的摩尔比为8.15:1时(即聚合物PDEA100-b-PHEMA10-b-P(HEMA65-co-GMA8)),固定化PGA的活性回收率可达51.20%,比纯单纯GMA固定化PGA的活性回收率高16.90%。2)改变共聚单体,将无规共聚物中的HEMA改变为甲基丙烯酸甲酯(MMA),采用相似的方法,制备了嵌段无序聚合物载体PDEA-b-PHEMA-b-P(MMA-co-GMA),同样,利用核磁氢谱(1HNMR)考察了聚合物的结构及分子量,结果表明,通过1HNMR表征的聚合物中GMA和HEMA摩尔比与通过竞聚率计算结果间的误差范围为1.32-3.71%,凝胶渗透色谱(GPC)表征证明了合成载体的反应为活性/可控自由基聚合。聚合物的LCST随着聚合物中疏水单体MMA的增加而降低;随着靶点间距的增大,固定化PGA的酶活回收率逐渐增大;催化性能研究表明固定化PGA具有媲美于游离PGA的响应速率,且固定化PGA的酶活回收率随靶点间距的增大而增大,当聚合物中MMA与GMA的摩尔比约为8.75:1时(即聚合物PDEA100-b-PHEMA10-b-P(MMA87-co-GMA10)),固定化PGA的酶活回收率可高达63.51%,比PDEA100-b-PHEMA10-b-P(HEMA65-co-GMA8)高12.31%,且比单纯的GMA固定化PGA高出21.70%。最重要的是,根据HEMA和MMA两种单体作为共聚单体时,固定化PGA酶活回收率随靶点间距增大的变化规律可断定当固定化靶点密度较大时,固定化酶分子因相互挤压导致其活性中心构型变形严重是酶活回收率较低的主要原因,同时多点固定也会影响固定化PGA的酶活回收率。3)基于以上两部分的结果,本部分以最佳MMA和GMA摩尔比的聚合物PDEA100-b-PHEMA10-b-P(MMA87-co-GMA10)为载体,对其固定化工艺条件进行了研究。结果表明:当固定化酶液浓度、时间、pH和温度分别为1.0%、16小时、8.0和37oC时,固定化PGA的酶活回收率最高,其值为68.50%。最后,基于优化条件下制备的固定化PGA,本实验对固定化载体的重复使用性及储存稳定性进行了探究,当载体重复使用10次之后,载体回收率为99%,固定化PGA的相对酶活力为94%;储存250天之后,固定化PGA的相对酶活力为92%,表明固定化PGA有优异的重复使用性和储存稳定性。
Abstract
gu ding hua qing mei su Gxian hua mei (Penicillin G acylase,PGA)shi yi chong bei an fan ying yong de gong ye cui hua ji ,ta ke yi cui hua qing mei su Gjia shui jie ,de dao chan wu 6-an ji qing mei wan suan (6-APA)。6-APAshi ban ge cheng qing mei su lei yao wu de guan jian zhong jian ti ,zai kang jun xiao yan deng ling yu zhan you hen da de shi chang kong jian 。yin ci ,gu ding hua PGAde yi xie zhu yao xing neng ,ru mei huo hui shou lv 、xiang ying su lv 、chong fu shi yong xing deng ,zhi jie guan lian zhao qi ye de jing ji cheng ben yu xiao yi 。er gu ding hua PGAde xing neng yu zai ti de jie gou he xing zhi mi qie xiang guan ,yin ci ,she ji ge cheng ge kuo de zai ti shi di gao gu ding hua PGAxing neng de yi ge guan jian tu jing 。mo ji 、you ji he fu ge cai liao zuo wei gu ding hua PGAde zhu yao zai ti lei xing ,jin guan ta men zai gu ding hua PGAde fu zai liang 、xiang ying su lv 、chong fu shi yong xing he hui shou lv deng fang mian dou de dao le bu tong cheng du de di gao ,dan xiang guan wen ti bing mei you gen ben gai shan 。er ju ,yan jiu zai ru he di gao mei huo hui shou lv fang mian de jin zhan que geng jia huan man 。mei huo hui shou lv jiao di de zhu yao yuan yin zai yu zai ti wei huan jing bu neng kuo yi mei cui hua huo xing zui gao shi suo dui ying de gou xiang xu qiu ,dao zhi mei huo xing zhong xin gou xiang de bian xing 。wei tan jiu ying xiang mei huo hui shou lv de guan jian wei huan jing yin su ,tong shi jian gu gu ding hua PGAfu zai liang 、xiang ying su lv he chong fu shi yong xing ,ben lun wen she ji ge cheng le liang chong wen min xing qian duan mo xu ju ge wu zuo wei PGAde gu ding hua zai ti 。tong guo li yong wen min xing ju ge wu rong jiao /ning jiao xiang zhuai bian guo cheng di sheng gu ding hua PGAde xiang ying su lv he chong fu shi yong xing ;tong guo she ji zai ti de duo qiang ji wei huan jing di gao zai ti qin shui xing ,jin er wei chi PGAde gao cui hua huo xing gou xiang ;tong guo diao kuo ba dian jian ju jin yi bu gong gu PGAgao cui hua huo xing gou xiang ,jin er di sheng mei huo hui shou lv ,yan jiu qu de le jiao hao cheng guo ,xian jiang yan jiu nei rong ji jie guo zong jie ru xia :1)shou xian cai yong ke ni jia cheng -duan lie lian zhuai yi (RAFT)ju ge fang fa ,yi N,N-er yi ji bing xi xian an (PDEA)he jia ji bing xi suan β-qiang yi zhi (HEMA)wei gong neng chan ti ,ge cheng le er qian duan ju ge wu PDEA-b-PHEMA,bing kao cha le DEAhe HEMAbi li dui PGAmei huo xing de ying xiang ,de chu kuo yi de DEAhe HEMAqian duan bi li 。zhi hou ,tan jiu le zai zai ti zhi bei tiao jian xia HEMAhe jia ji bing xi suan su shui gan you zhi (GMA)de jing ju lv 。zai shang shu gong zuo de ji chu shang ,ge cheng le ju you bu tong ba dian jian ju de wen min xing qian duan mo xu gong ju wu zai ti PDEA-b-PHEMA-b-P(HEMA-co-GMA),bing li yong he ci qing pu (1HNMR)kao cha le ju ge wu de jie gou ji fen zi liang ,jie guo biao ming ,tong guo 1HNMRbiao zheng de ju ge wu zhong GMAhe HEMAma er bi yu tong guo jing ju lv ji suan jie guo jian de wu cha fan wei wei 1.76-3.47%,ning jiao shen tou se pu (GPC)biao zheng zheng ming le ge cheng zai ti de fan ying wei huo xing /ke kong zi you ji ju ge 。wen min xing yan jiu jie guo biao ming gong ju wu zai ti de di lin jie xiang zhuai bian wen du (LCST)sui zhao mo gui gong ju zhong chan ti HEMAde zeng jia er jiang di ;cui hua xing neng yan jiu biao ming gu ding hua PGAxiang ying su lv kuai ,ju gu ding hua PGAde mei huo hui shou lv sui ba dian jian ju de zeng da er zeng da ,dang mo gui gong ju wu qian duan zhong HEMAyu GMAde ma er bi wei 8.15:1shi (ji ju ge wu PDEA100-b-PHEMA10-b-P(HEMA65-co-GMA8)),gu ding hua PGAde huo xing hui shou lv ke da 51.20%,bi chun chan chun GMAgu ding hua PGAde huo xing hui shou lv gao 16.90%。2)gai bian gong ju chan ti ,jiang mo gui gong ju wu zhong de HEMAgai bian wei jia ji bing xi suan jia zhi (MMA),cai yong xiang shi de fang fa ,zhi bei le qian duan mo xu ju ge wu zai ti PDEA-b-PHEMA-b-P(MMA-co-GMA),tong yang ,li yong he ci qing pu (1HNMR)kao cha le ju ge wu de jie gou ji fen zi liang ,jie guo biao ming ,tong guo 1HNMRbiao zheng de ju ge wu zhong GMAhe HEMAma er bi yu tong guo jing ju lv ji suan jie guo jian de wu cha fan wei wei 1.32-3.71%,ning jiao shen tou se pu (GPC)biao zheng zheng ming le ge cheng zai ti de fan ying wei huo xing /ke kong zi you ji ju ge 。ju ge wu de LCSTsui zhao ju ge wu zhong shu shui chan ti MMAde zeng jia er jiang di ;sui zhao ba dian jian ju de zeng da ,gu ding hua PGAde mei huo hui shou lv zhu jian zeng da ;cui hua xing neng yan jiu biao ming gu ding hua PGAju you pi mei yu you li PGAde xiang ying su lv ,ju gu ding hua PGAde mei huo hui shou lv sui ba dian jian ju de zeng da er zeng da ,dang ju ge wu zhong MMAyu GMAde ma er bi yao wei 8.75:1shi (ji ju ge wu PDEA100-b-PHEMA10-b-P(MMA87-co-GMA10)),gu ding hua PGAde mei huo hui shou lv ke gao da 63.51%,bi PDEA100-b-PHEMA10-b-P(HEMA65-co-GMA8)gao 12.31%,ju bi chan chun de GMAgu ding hua PGAgao chu 21.70%。zui chong yao de shi ,gen ju HEMAhe MMAliang chong chan ti zuo wei gong ju chan ti shi ,gu ding hua PGAmei huo hui shou lv sui ba dian jian ju zeng da de bian hua gui lv ke duan ding dang gu ding hua ba dian mi du jiao da shi ,gu ding hua mei fen zi yin xiang hu ji ya dao zhi ji huo xing zhong xin gou xing bian xing yan chong shi mei huo hui shou lv jiao di de zhu yao yuan yin ,tong shi duo dian gu ding ye hui ying xiang gu ding hua PGAde mei huo hui shou lv 。3)ji yu yi shang liang bu fen de jie guo ,ben bu fen yi zui jia MMAhe GMAma er bi de ju ge wu PDEA100-b-PHEMA10-b-P(MMA87-co-GMA10)wei zai ti ,dui ji gu ding hua gong yi tiao jian jin hang le yan jiu 。jie guo biao ming :dang gu ding hua mei ye nong du 、shi jian 、pHhe wen du fen bie wei 1.0%、16xiao shi 、8.0he 37oCshi ,gu ding hua PGAde mei huo hui shou lv zui gao ,ji zhi wei 68.50%。zui hou ,ji yu you hua tiao jian xia zhi bei de gu ding hua PGA,ben shi yan dui gu ding hua zai ti de chong fu shi yong xing ji chu cun wen ding xing jin hang le tan jiu ,dang zai ti chong fu shi yong 10ci zhi hou ,zai ti hui shou lv wei 99%,gu ding hua PGAde xiang dui mei huo li wei 94%;chu cun 250tian zhi hou ,gu ding hua PGAde xiang dui mei huo li wei 92%,biao ming gu ding hua PGAyou you yi de chong fu shi yong xing he chu cun wen ding xing 。
论文参考文献
论文详细介绍
论文作者分别是来自兰州理工大学的李轲,发表于刊物兰州理工大学2019-07-18论文,是一篇关于载体论文,青霉素酰化酶论文,靶点间距论文,酶活回收率论文,微环境论文,兰州理工大学2019-07-18论文的文章。本文可供学术参考使用,各位学者可以免费参考阅读下载,文章观点不代表本站观点,资料来自兰州理工大学2019-07-18论文网站,若本站收录的文献无意侵犯了您的著作版权,请联系我们删除。
标签:载体论文; 青霉素酰化酶论文; 靶点间距论文; 酶活回收率论文; 微环境论文; 兰州理工大学2019-07-18论文;