本文主要研究内容
作者张征立（2019）在《固定化酶制备降血糖肽及其生物学活性评价》一文中研究指出：随着生活水平提高,糖尿病的发病率也越来越高,严重威胁人类的生命健康。而生物活性肽作为一种新型降血糖药物,副作用较小,在防治糖尿病方面具有较高的应用价值。传统酶解法制备生物活性肽对自由蛋白酶的需求量较大,且酶活不稳定。目前糙米已成为糖尿病患者的最佳食材,其营养成分丰富,有助于胰脏分泌胰岛素,降低血糖。因此本研究以糙米作为原料,使用固定化酶制备降血糖肽,检测其降血糖活性。以聚偏氟乙烯微孔滤膜（F膜）作为载体,采用共价结合法固定胰蛋白酶,通过响应面试验获得固定化工艺参数,采用扫描电镜和红外光谱对固定化酶进行表征。建立固定化酶制备糙米降血糖肽的水解度动力学模型。构建糙米降血糖肽的分离纯化方法,先后使用超滤、离子交换、凝胶过滤层析与反相高效液相方法,再采用液质联用方法分析降血糖肽氨基酸序列,然后进行分子对接,建立抑制动力学模型,从分子层面研究降血糖肽作用机制与类型。通过体内检测降血糖肽对高血糖小鼠的降血糖作用、生长状况以及肠道菌群平衡的影响,研究肠道菌群与糖尿病之间的关系。试验结果如下:胰蛋白酶固定化最优工艺为:戊二醛浓度2.11wt%,酶浓度6.43mg/mL,温度23.57℃。在最优条件下预测酶活回收率达到63.1947%,经过验证酶活回收率为62.25%。固定化胰蛋白酶的最适温度为55℃,最适pH值为8.5,热稳定性、pH值稳定性均高于自由酶;同时固定化酶在重复使用6次以后,仍然保留初始酶活的67.82%;与自由酶相比,固定化胰蛋白酶的储存稳定性提高了18.00%。根据扫描电镜图可以看出固定化酶载体表面出现大量球状突出点,同时在傅里叶红外光谱图上胰蛋白酶内肽键伸缩振动的酰胺带在11001000cm-1出现特征峰。以水解度作为评价指标,通过单因素试验分析自由酶与固定化酶水解能力,固定化酶最优水解条件为:加酶量520 cm2/g,底物浓度6 mg/mL,温度55℃,pH 8.5,反应时间50min,水解度可以达到24.72%,相比自由酶水解度提高了63.38%。在固定化酶酶解体系最优条件下,固定化酶重复使用5次后,水解度保留初始水解度的72.34%。建立固定化酶可控酶解动力学模型为DH=4.5767ln[1+（5.2121 E0/S0-0.5032）t]。以α-葡萄糖苷酶抑制率作为评价指标,分别检测不同多肽组分的降血糖活性,其中P-II-B-b-2组分活性最大,IC50为0.097mg/mL。根据二级质谱推断降血糖肽的氨基酸组成序列,分子量为362.99的多肽可能是Gly-Met-Arg,分子量为476.11的多肽包括Asn-Trp-Arg、Gly-Asp-Lys-Arg和Asp-Gly-Lys-Arg,分子量为589.12的多肽包括Lys-Arg-Glu-Arg、Pro-Trp-Met-Arg、Val-Trp-Glu-Arg和Cys-Arg-Gly-Pro-Arg。通过分子对接,多肽可以与α-葡萄糖苷酶中心部位紧密结合,抑制α-葡萄糖苷酶的活性。其中Gly-Asp-Lys-Arg的IC50最小,为0.039mg/mL,抑制效果最好。最后通过研究Gly-Asp-Lys-Arg对α-葡萄糖苷酶抑制动力学发现,Km值基本保持不变,而Vm逐渐减少,属于非竞争性抑制作用类型,抑制常数Km=5.405×10-3mol/L。多肽治疗35天后,四氧嘧啶高血糖小鼠的血糖值明显降低,小鼠糖尿病症状,如小鼠毛色、活动能力、反应能力与食欲等各种情况均有明显改善,并提高存活率。通过对小鼠肠道优势菌分离鉴定,肠道菌群包括链球菌、肠杆菌、微球菌、假单胞杆菌与弧菌等。高血糖小鼠肠道内有害菌数量增加,益生菌数量减少。经糙米多肽治疗后,高血糖小鼠血糖降低,肠道微生态平衡得到明显改善恢复。
Abstract
sui zhao sheng huo shui ping di gao ,tang niao bing de fa bing lv ye yue lai yue gao ,yan chong wei xie ren lei de sheng ming jian kang 。er sheng wu huo xing tai zuo wei yi chong xin xing jiang xie tang yao wu ,fu zuo yong jiao xiao ,zai fang zhi tang niao bing fang mian ju you jiao gao de ying yong jia zhi 。chuan tong mei jie fa zhi bei sheng wu huo xing tai dui zi you dan bai mei de xu qiu liang jiao da ,ju mei huo bu wen ding 。mu qian cao mi yi cheng wei tang niao bing huan zhe de zui jia shi cai ,ji ying yang cheng fen feng fu ,you zhu yu yi zang fen bi yi dao su ,jiang di xie tang 。yin ci ben yan jiu yi cao mi zuo wei yuan liao ,shi yong gu ding hua mei zhi bei jiang xie tang tai ,jian ce ji jiang xie tang huo xing 。yi ju pian fu yi xi wei kong lv mo （Fmo ）zuo wei zai ti ,cai yong gong jia jie ge fa gu ding yi dan bai mei ,tong guo xiang ying mian shi yan huo de gu ding hua gong yi can shu ,cai yong sao miao dian jing he gong wai guang pu dui gu ding hua mei jin hang biao zheng 。jian li gu ding hua mei zhi bei cao mi jiang xie tang tai de shui jie du dong li xue mo xing 。gou jian cao mi jiang xie tang tai de fen li chun hua fang fa ,xian hou shi yong chao lv 、li zi jiao huan 、ning jiao guo lv ceng xi yu fan xiang gao xiao ye xiang fang fa ,zai cai yong ye zhi lian yong fang fa fen xi jiang xie tang tai an ji suan xu lie ,ran hou jin hang fen zi dui jie ,jian li yi zhi dong li xue mo xing ,cong fen zi ceng mian yan jiu jiang xie tang tai zuo yong ji zhi yu lei xing 。tong guo ti nei jian ce jiang xie tang tai dui gao xie tang xiao shu de jiang xie tang zuo yong 、sheng chang zhuang kuang yi ji chang dao jun qun ping heng de ying xiang ,yan jiu chang dao jun qun yu tang niao bing zhi jian de guan ji 。shi yan jie guo ru xia :yi dan bai mei gu ding hua zui you gong yi wei :wu er quan nong du 2.11wt%,mei nong du 6.43mg/mL,wen du 23.57℃。zai zui you tiao jian xia yu ce mei huo hui shou lv da dao 63.1947%,jing guo yan zheng mei huo hui shou lv wei 62.25%。gu ding hua yi dan bai mei de zui kuo wen du wei 55℃,zui kuo pHzhi wei 8.5,re wen ding xing 、pHzhi wen ding xing jun gao yu zi you mei ;tong shi gu ding hua mei zai chong fu shi yong 6ci yi hou ,reng ran bao liu chu shi mei huo de 67.82%;yu zi you mei xiang bi ,gu ding hua yi dan bai mei de chu cun wen ding xing di gao le 18.00%。gen ju sao miao dian jing tu ke yi kan chu gu ding hua mei zai ti biao mian chu xian da liang qiu zhuang tu chu dian ,tong shi zai fu li xie gong wai guang pu tu shang yi dan bai mei nei tai jian shen su zhen dong de xian an dai zai 11001000cm-1chu xian te zheng feng 。yi shui jie du zuo wei ping jia zhi biao ,tong guo chan yin su shi yan fen xi zi you mei yu gu ding hua mei shui jie neng li ,gu ding hua mei zui you shui jie tiao jian wei :jia mei liang 520 cm2/g,de wu nong du 6 mg/mL,wen du 55℃,pH 8.5,fan ying shi jian 50min,shui jie du ke yi da dao 24.72%,xiang bi zi you mei shui jie du di gao le 63.38%。zai gu ding hua mei mei jie ti ji zui you tiao jian xia ,gu ding hua mei chong fu shi yong 5ci hou ,shui jie du bao liu chu shi shui jie du de 72.34%。jian li gu ding hua mei ke kong mei jie dong li xue mo xing wei DH=4.5767ln[1+（5.2121 E0/S0-0.5032）t]。yi α-pu tao tang gan mei yi zhi lv zuo wei ping jia zhi biao ,fen bie jian ce bu tong duo tai zu fen de jiang xie tang huo xing ,ji zhong P-II-B-b-2zu fen huo xing zui da ,IC50wei 0.097mg/mL。gen ju er ji zhi pu tui duan jiang xie tang tai de an ji suan zu cheng xu lie ,fen zi liang wei 362.99de duo tai ke neng shi Gly-Met-Arg,fen zi liang wei 476.11de duo tai bao gua Asn-Trp-Arg、Gly-Asp-Lys-Arghe Asp-Gly-Lys-Arg,fen zi liang wei 589.12de duo tai bao gua Lys-Arg-Glu-Arg、Pro-Trp-Met-Arg、Val-Trp-Glu-Arghe Cys-Arg-Gly-Pro-Arg。tong guo fen zi dui jie ,duo tai ke yi yu α-pu tao tang gan mei zhong xin bu wei jin mi jie ge ,yi zhi α-pu tao tang gan mei de huo xing 。ji zhong Gly-Asp-Lys-Argde IC50zui xiao ,wei 0.039mg/mL,yi zhi xiao guo zui hao 。zui hou tong guo yan jiu Gly-Asp-Lys-Argdui α-pu tao tang gan mei yi zhi dong li xue fa xian ,Kmzhi ji ben bao chi bu bian ,er Vmzhu jian jian shao ,shu yu fei jing zheng xing yi zhi zuo yong lei xing ,yi zhi chang shu Km=5.405×10-3mol/L。duo tai zhi liao 35tian hou ,si yang mi ding gao xie tang xiao shu de xie tang zhi ming xian jiang di ,xiao shu tang niao bing zheng zhuang ,ru xiao shu mao se 、huo dong neng li 、fan ying neng li yu shi yu deng ge chong qing kuang jun you ming xian gai shan ,bing di gao cun huo lv 。tong guo dui xiao shu chang dao you shi jun fen li jian ding ,chang dao jun qun bao gua lian qiu jun 、chang gan jun 、wei qiu jun 、jia chan bao gan jun yu hu jun deng 。gao xie tang xiao shu chang dao nei you hai jun shu liang zeng jia ,yi sheng jun shu liang jian shao 。jing cao mi duo tai zhi liao hou ,gao xie tang xiao shu xie tang jiang di ,chang dao wei sheng tai ping heng de dao ming xian gai shan hui fu 。
论文参考文献
论文详细介绍
论文作者分别是来自江苏科技大学的张征立，发表于刊物江苏科技大学2019-07-10论文，是一篇关于固定化酶论文,酶解动力学论文,糙米降血糖肽论文,分子对接论文,抑制动力学论文,小鼠肠道优势菌分离与鉴定论文,江苏科技大学2019-07-10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，资料来自江苏科技大学2019-07-10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：固定化酶论文; 酶解动力学论文; 糙米降血糖肽论文; 分子对接论文; 抑制动力学论文; 小鼠肠道优势菌分离与鉴定论文; 江苏科技大学2019-07-10论文;