本文主要研究内容
作者牟颖丽（2019）在《嗜热链球菌β—半乳糖苷酶的转糖基活性研究及表面展示系统的建立》一文中研究指出：嗜热链球菌(Streptococcus thermophilus)是一类重要的食品安全级菌株,常用于酸奶或奶酪等乳制品的生产。嗜热链球菌利用胞内的β-半乳糖苷酶将乳糖分解为葡萄糖和半乳糖,前者通过糖酵解途径转化成乳酸,而后者不能被消耗并全部泵到细胞外,导致乳制品中残留了大量的半乳糖和部分未被利用的乳糖,不利于人类健康。除此之外,β-半乳糖苷酶还能够发挥转糖基活性生成低聚半乳糖(GOS),减少发酵乳中半乳糖的残留。因此,调节其分解活性与转糖基活性有助于生产低糖健康的乳制品,然而对其转糖基活性的相关位点少有报道,限制了其在食品工业的应用。近年来,由于细胞固定化酶有更好的催化活性和稳定性,乳酸菌表面展示系统得到迅速发展,而关于嗜热链球菌表面展示系统的报道较少。本文主要利用生物信息学分析对S.thermophilus&SDMCC050237的β-半乳糖苷酶进行理性改造,从而提高其转糖基活性,同时鉴定并研究了嗜热链球菌的烯醇化酶的表面锚定功能及用于表面展示的潜力。具体的实验结果如下:1.嗜热链球菌中高活性p-半乳糖苷酶的筛选β-半乳糖苷酶对嗜热链球菌的食品工业应用有重要意义。本文使用X-Gal平板检测了嗜热链球菌产β-半乳糖苷酶的能力并以oNPG为底物检测了酶活,发现不同菌株的β-半乳糖苷酶之间存在活性差异。将活性较高的细胞破碎液与乳糖溶液混合反应后利用TLC法初步分析了反应液中糖的成分,结果表明,不同菌株的β-半乳糖苷酶在自然条件下转糖基活性较弱且无明显差距,导致其发酵液中残留了较多的半乳糖。2.β 半乳糖苷酶BgaQ的酶学性质研究及其转糖基活性条件的优化以 S.thermophilus SDMCC050237为材料,克隆其β-半乳糖苷酶基因bgaQ,使用表达质粒pET28a(+)在E.coli DE3中过表达BgaQ蛋白。镍柱亲和层析纯化后获得目标蛋白(113 kDa)。以oNPG和乳糖为底物时BgaQ的酶活分别为4725.3±155.4 U/mg和88.3±1.4U/mg。酶学性质表明,BgaQ的最适温度为50℃,温度耐受范围为37℃～60℃,高温下稳定性下降;最适pH为8.5,pH值稳定性较高。另外,Na-、K-、Mg2-、Ba2+和Co2+促进酶活,而Fe2+、Ni2-和Co2+抑制酶活,该酶对Zn2-和Cu2-不耐受。转糖基活性的条件优化结果表明,该酶生成GOS的最优乳糖浓度为25%,最优温度为50℃,最优pH为6.5。优化后的反应条件为15%的乳糖浓度,pH=6.5,50℃条件下反应5 h,GOS的转化率为45.0±3.4%。3.定点突变提高β-半乳糖苷酶的转糖基活性嗜热链球菌的β-半乳糖苷酶具有分解活性和转糖基活性,其工业应用具有重要意义和广阔前景。为了提高嗜热链球菌β-半乳糖苷酶的转糖基活性,本文对筛选出的β-半乳糖苷酶BgaQ进行同源建模,通过序列叠合找到了两个谷氨酸活性位点,以β-3’-galactosyl-lactose为底物进行Autodocking分析,获得转糖基活性相关位点并通过序列比对分析成功构建了Y801H/P802G突变体蛋白mBgaQ。以oNPG和乳糖为底物测得mBgaQ的酶活力分别为5957.7±209.1 U/mg和81.9±3.6 U/mg,以乳糖为底物时mBgaQ的酶活力基本与野生型相同。酶学性质表明,mBgaQ的最适温度为50℃,在37℃～45℃能发挥活性,对高温的耐受性显著下降;最适pH为8.5,在酸性和中性环境下稳定性较好,在碱性环境下稳定性下降。Na+、K+和Mg2+促进酶活,Zn2+、Ni2++、Mn2+和Co2+抑制酶活,且该酶对Cu2+不耐受。转糖基优化结果显示,mBgaQ生成GOS的最佳乳糖浓度为25%,最佳pH为6.5,最佳反应温度为42℃。优化后的反应条件为:115%乳糖浓度、pH=6.5、50℃下反应5h,最终生成GOS的转化率为58.3±1.4%,与野生型相比提高了 13%。另外,本文检测了低乳糖浓度时mBgaQ与BgaQ生成GOS量以及残留半乳糖量的时间曲线,发现mBgaQ生成GOS的转化率最高为26.7±0.5%,比BgaQ提高了6%,其残留的半乳糖量也低于BgaQ。4.基于烯醉化酶建立嗜热链球菌的表面展示系统烯醇化酶是糖酵解途径的关键酶,不仅能在胞质内发挥活性,还能分泌到细胞表面,是一种间歇蛋白(moonlighting protein)。本文从Sthermophilus CGMCC7.1 79的基因组中找出烯醇化酶编码基因eno M,将EnoM蛋白序列与致病性链球菌的烯醇化酶蛋白序列进行了比对分析,发现具有高度同源性,这表明其烯醇化酶具有表面展示的潜能。随后我们用pET28a(+)质粒分别将GFP蛋白和GFP-EnoM融合蛋白在E.coli DE3中过表达并纯化,结合反应结果显示,与GFP-EnoM融合蛋白孵育的菌体细胞荧光强度显著提高,说明EnoM将GFP蛋白锚定在细胞表面。为了探索EnoM的锚定原理,用不同化学试剂处理菌体细胞,发现经过SDS处理的细胞基本检测不到荧光,表明EnoM与细胞表面的蛋白质结合。对EnoM在乳酸菌中的适用性进行探究,发现其在其它乳酸菌中也具有表面锚定功能。为了进一步证实EnoM的表面展示功能,将分子量较大的β-半乳糖苷酶mBgaQ展示到嗜热链球菌的表面并保持了酶的活性,固定化酶在反复利用8次后仍然维持64%的酶活。为了验证EnoM能否在嗜热链球菌中实现靶向表达,构建了S.thermophilus CGMCC7.179/pLEISS-D2-gfp-enoM重组菌株,提取其胞内蛋白和表面蛋白进行Western Blot检测,发现GFP-EnoM蛋白成功表达但是没有锚定到细胞表面,可能与分泌量不足或者嗜热链球菌致病性的缺失有关。
Abstract
shi re lian qiu jun (Streptococcus thermophilus)shi yi lei chong yao de shi pin an quan ji jun zhu ,chang yong yu suan nai huo nai lao deng ru zhi pin de sheng chan 。shi re lian qiu jun li yong bao nei de β-ban ru tang gan mei jiang ru tang fen jie wei pu tao tang he ban ru tang ,qian zhe tong guo tang jiao jie tu jing zhuai hua cheng ru suan ,er hou zhe bu neng bei xiao hao bing quan bu beng dao xi bao wai ,dao zhi ru zhi pin zhong can liu le da liang de ban ru tang he bu fen wei bei li yong de ru tang ,bu li yu ren lei jian kang 。chu ci zhi wai ,β-ban ru tang gan mei hai neng gou fa hui zhuai tang ji huo xing sheng cheng di ju ban ru tang (GOS),jian shao fa jiao ru zhong ban ru tang de can liu 。yin ci ,diao jie ji fen jie huo xing yu zhuai tang ji huo xing you zhu yu sheng chan di tang jian kang de ru zhi pin ,ran er dui ji zhuai tang ji huo xing de xiang guan wei dian shao you bao dao ,xian zhi le ji zai shi pin gong ye de ying yong 。jin nian lai ,you yu xi bao gu ding hua mei you geng hao de cui hua huo xing he wen ding xing ,ru suan jun biao mian zhan shi ji tong de dao xun su fa zhan ,er guan yu shi re lian qiu jun biao mian zhan shi ji tong de bao dao jiao shao 。ben wen zhu yao li yong sheng wu xin xi xue fen xi dui S.thermophilus&SDMCC050237de β-ban ru tang gan mei jin hang li xing gai zao ,cong er di gao ji zhuai tang ji huo xing ,tong shi jian ding bing yan jiu le shi re lian qiu jun de xi chun hua mei de biao mian mao ding gong neng ji yong yu biao mian zhan shi de qian li 。ju ti de shi yan jie guo ru xia :1.shi re lian qiu jun zhong gao huo xing p-ban ru tang gan mei de shai shua β-ban ru tang gan mei dui shi re lian qiu jun de shi pin gong ye ying yong you chong yao yi yi 。ben wen shi yong X-Galping ban jian ce le shi re lian qiu jun chan β-ban ru tang gan mei de neng li bing yi oNPGwei de wu jian ce le mei huo ,fa xian bu tong jun zhu de β-ban ru tang gan mei zhi jian cun zai huo xing cha yi 。jiang huo xing jiao gao de xi bao po sui ye yu ru tang rong ye hun ge fan ying hou li yong TLCfa chu bu fen xi le fan ying ye zhong tang de cheng fen ,jie guo biao ming ,bu tong jun zhu de β-ban ru tang gan mei zai zi ran tiao jian xia zhuai tang ji huo xing jiao ruo ju mo ming xian cha ju ,dao zhi ji fa jiao ye zhong can liu le jiao duo de ban ru tang 。2.β ban ru tang gan mei BgaQde mei xue xing zhi yan jiu ji ji zhuai tang ji huo xing tiao jian de you hua yi S.thermophilus SDMCC050237wei cai liao ,ke long ji β-ban ru tang gan mei ji yin bgaQ,shi yong biao da zhi li pET28a(+)zai E.coli DE3zhong guo biao da BgaQdan bai 。nie zhu qin he ceng xi chun hua hou huo de mu biao dan bai (113 kDa)。yi oNPGhe ru tang wei de wu shi BgaQde mei huo fen bie wei 4725.3±155.4 U/mghe 88.3±1.4U/mg。mei xue xing zhi biao ming ,BgaQde zui kuo wen du wei 50℃,wen du nai shou fan wei wei 37℃～60℃,gao wen xia wen ding xing xia jiang ;zui kuo pHwei 8.5,pHzhi wen ding xing jiao gao 。ling wai ,Na-、K-、Mg2-、Ba2+he Co2+cu jin mei huo ,er Fe2+、Ni2-he Co2+yi zhi mei huo ,gai mei dui Zn2-he Cu2-bu nai shou 。zhuai tang ji huo xing de tiao jian you hua jie guo biao ming ,gai mei sheng cheng GOSde zui you ru tang nong du wei 25%,zui you wen du wei 50℃,zui you pHwei 6.5。you hua hou de fan ying tiao jian wei 15%de ru tang nong du ,pH=6.5,50℃tiao jian xia fan ying 5 h,GOSde zhuai hua lv wei 45.0±3.4%。3.ding dian tu bian di gao β-ban ru tang gan mei de zhuai tang ji huo xing shi re lian qiu jun de β-ban ru tang gan mei ju you fen jie huo xing he zhuai tang ji huo xing ,ji gong ye ying yong ju you chong yao yi yi he an kuo qian jing 。wei le di gao shi re lian qiu jun β-ban ru tang gan mei de zhuai tang ji huo xing ,ben wen dui shai shua chu de β-ban ru tang gan mei BgaQjin hang tong yuan jian mo ,tong guo xu lie die ge zhao dao le liang ge gu an suan huo xing wei dian ,yi β-3’-galactosyl-lactosewei de wu jin hang Autodockingfen xi ,huo de zhuai tang ji huo xing xiang guan wei dian bing tong guo xu lie bi dui fen xi cheng gong gou jian le Y801H/P802Gtu bian ti dan bai mBgaQ。yi oNPGhe ru tang wei de wu ce de mBgaQde mei huo li fen bie wei 5957.7±209.1 U/mghe 81.9±3.6 U/mg,yi ru tang wei de wu shi mBgaQde mei huo li ji ben yu ye sheng xing xiang tong 。mei xue xing zhi biao ming ,mBgaQde zui kuo wen du wei 50℃,zai 37℃～45℃neng fa hui huo xing ,dui gao wen de nai shou xing xian zhe xia jiang ;zui kuo pHwei 8.5,zai suan xing he zhong xing huan jing xia wen ding xing jiao hao ,zai jian xing huan jing xia wen ding xing xia jiang 。Na+、K+he Mg2+cu jin mei huo ,Zn2+、Ni2++、Mn2+he Co2+yi zhi mei huo ,ju gai mei dui Cu2+bu nai shou 。zhuai tang ji you hua jie guo xian shi ,mBgaQsheng cheng GOSde zui jia ru tang nong du wei 25%,zui jia pHwei 6.5,zui jia fan ying wen du wei 42℃。you hua hou de fan ying tiao jian wei :115%ru tang nong du 、pH=6.5、50℃xia fan ying 5h,zui zhong sheng cheng GOSde zhuai hua lv wei 58.3±1.4%,yu ye sheng xing xiang bi di gao le 13%。ling wai ,ben wen jian ce le di ru tang nong du shi mBgaQyu BgaQsheng cheng GOSliang yi ji can liu ban ru tang liang de shi jian qu xian ,fa xian mBgaQsheng cheng GOSde zhuai hua lv zui gao wei 26.7±0.5%,bi BgaQdi gao le 6%,ji can liu de ban ru tang liang ye di yu BgaQ。4.ji yu xi zui hua mei jian li shi re lian qiu jun de biao mian zhan shi ji tong xi chun hua mei shi tang jiao jie tu jing de guan jian mei ,bu jin neng zai bao zhi nei fa hui huo xing ,hai neng fen bi dao xi bao biao mian ,shi yi chong jian xie dan bai (moonlighting protein)。ben wen cong Sthermophilus CGMCC7.1 79de ji yin zu zhong zhao chu xi chun hua mei bian ma ji yin eno M,jiang EnoMdan bai xu lie yu zhi bing xing lian qiu jun de xi chun hua mei dan bai xu lie jin hang le bi dui fen xi ,fa xian ju you gao du tong yuan xing ,zhe biao ming ji xi chun hua mei ju you biao mian zhan shi de qian neng 。sui hou wo men yong pET28a(+)zhi li fen bie jiang GFPdan bai he GFP-EnoMrong ge dan bai zai E.coli DE3zhong guo biao da bing chun hua ,jie ge fan ying jie guo xian shi ,yu GFP-EnoMrong ge dan bai fu yo de jun ti xi bao ying guang jiang du xian zhe di gao ,shui ming EnoMjiang GFPdan bai mao ding zai xi bao biao mian 。wei le tan suo EnoMde mao ding yuan li ,yong bu tong hua xue shi ji chu li jun ti xi bao ,fa xian jing guo SDSchu li de xi bao ji ben jian ce bu dao ying guang ,biao ming EnoMyu xi bao biao mian de dan bai zhi jie ge 。dui EnoMzai ru suan jun zhong de kuo yong xing jin hang tan jiu ,fa xian ji zai ji ta ru suan jun zhong ye ju you biao mian mao ding gong neng 。wei le jin yi bu zheng shi EnoMde biao mian zhan shi gong neng ,jiang fen zi liang jiao da de β-ban ru tang gan mei mBgaQzhan shi dao shi re lian qiu jun de biao mian bing bao chi le mei de huo xing ,gu ding hua mei zai fan fu li yong 8ci hou reng ran wei chi 64%de mei huo 。wei le yan zheng EnoMneng fou zai shi re lian qiu jun zhong shi xian ba xiang biao da ,gou jian le S.thermophilus CGMCC7.179/pLEISS-D2-gfp-enoMchong zu jun zhu ,di qu ji bao nei dan bai he biao mian dan bai jin hang Western Blotjian ce ,fa xian GFP-EnoMdan bai cheng gong biao da dan shi mei you mao ding dao xi bao biao mian ,ke neng yu fen bi liang bu zu huo zhe shi re lian qiu jun zhi bing xing de que shi you guan 。
论文参考文献
论文详细介绍
论文作者分别是来自山东大学的牟颖丽，发表于刊物山东大学2019-07-16论文，是一篇关于嗜热链球菌论文,半乳糖苷酶论文,低聚半乳糖论文,表面展示系统论文,烯醇化酶论文,山东大学2019-07-16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，资料来自山东大学2019-07-16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：嗜热链球菌论文; 半乳糖苷酶论文; 低聚半乳糖论文; 表面展示系统论文; 烯醇化酶论文; 山东大学2019-07-16论文;