本文主要研究内容
作者廖朝勇（2019）在《地衣芽孢杆菌CP-16降解羽毛关键酶高效表达研究》一文中研究指出：我国蛋白质饲料资源供求矛盾突出。每年产生大量羽、毛和蹄角等角蛋白资源,这些角蛋白富含二硫键、氢键及疏水基团,结构稳定不易被动物消化利用。物理、化学方法处理角蛋白存在能耗高、氨基酸破坏严重和环境污染等缺陷。高效生物降解更符合未来发展需求,打开二硫键,破坏角蛋白疏水结构是生物水解角蛋白的关键,实现打开这些化学键关键酶的高效表达对角蛋白生物处理产业化至关重要。本研究拟通过对地衣芽孢杆菌CP-16诱变(物理和化学)育种筛选获得具有高降解角蛋白效率正突变菌,为高效水解角蛋白奠定基础。同时,从CP-16中扩增出二硫键还原酶基因(Trx)、角蛋白酶基因(Ker),构建获得2株枯草芽胞杆菌WB600降解羽毛关键酶重组菌(B.subtilis WB600/pSTOP 1622-pTrx、B.subtilis WB600/pSTOP 1622-pKer)。并分析不同信号肽对目的基因(Trx、Ker)分泌影响,随后对重组菌进行发酵条件优化,实现CP-16降解角蛋白关键酶量产,并应用于角蛋白生物酶解中。运用紫外、硫酸二乙酯对原始菌进行多轮物理和化学诱变,经初筛与复筛得到了羽毛降解关键酶高产正突变菌。经紫外诱变后,突变菌V-8二硫键还原酶酶活、角蛋白酶酶活增幅最大分别为0.15U/mL、15.4U/mL,较出发菌分别提高83.1%、83.3%。硫酸二乙酯诱变后,突变菌D-5二硫键还原酶酶活增幅最大为0.14U/mL,较出发菌提高了67.5%,而突变菌D-2角蛋白酶酶活增幅最大为13.9U/mL,较出发菌提高65.5%。构建了2种角蛋白水解酶的表达质粒(pSTOP 1622-Trx、pSTOP 1622-Ker),电转入WB600并成功表达。在此基础上,通过无缝克隆手段,构建了含不同信号肽的筛选载体,在WB600中实现表达。结果表明,角蛋白酶搭载自身信号肽时胞外分泌角蛋白酶酶活最高。而二硫键还原酶搭载不同信号肽时仍在胞内表达,且酶活有不同程度的降低。通过优化重组菌发酵培养配方,重组菌B.subtilis WB600/pSTOP 1622-pTrx在玉米粉(5.1g/L)、葡萄糖(5.9g/L)、糖蜜(11.9g/L)、甘油(3.0g/L),蛋白胨(20g/L),Nacl(10g/L),发酵液OD600=1时加0.5%木糖诱导发酵48h能明显促进产酶,较其初始培养条件提高80.00%,较正突变菌V-8提高440.00%,较CP-16提高912.50%。重组菌B.subtilis WB600/pSTOP 1622-pKer在玉米粉(5.1g/L)、葡萄糖(5.9g/L)、糖蜜(5.9g/L)、甘油(3.0g/L),蛋白胨(20g/L),Nacl(10g/L),发酵液OD600=1时加0.5%木糖诱导发酵48h能明显促进产酶,较其初始培养条件提高49.74%,较正突变菌V-8提高269.48%,较CP-16提高577.38%。综上结果可得,诱变育种及CP-16降解羽毛关键酶基因枯草芽孢杆菌异源表达均能显著提高CP-16降解羽毛关键酶酶活,且枯草芽孢杆菌异源表达作为CP-16降解羽毛关键酶高效表达手段效果优于诱变育种。
Abstract
wo guo dan bai zhi si liao zi yuan gong qiu mao dun tu chu 。mei nian chan sheng da liang yu 、mao he ti jiao deng jiao dan bai zi yuan ,zhe xie jiao dan bai fu han er liu jian 、qing jian ji shu shui ji tuan ,jie gou wen ding bu yi bei dong wu xiao hua li yong 。wu li 、hua xue fang fa chu li jiao dan bai cun zai neng hao gao 、an ji suan po huai yan chong he huan jing wu ran deng que xian 。gao xiao sheng wu jiang jie geng fu ge wei lai fa zhan xu qiu ,da kai er liu jian ,po huai jiao dan bai shu shui jie gou shi sheng wu shui jie jiao dan bai de guan jian ,shi xian da kai zhe xie hua xue jian guan jian mei de gao xiao biao da dui jiao dan bai sheng wu chu li chan ye hua zhi guan chong yao 。ben yan jiu ni tong guo dui de yi ya bao gan jun CP-16you bian (wu li he hua xue )yo chong shai shua huo de ju you gao jiang jie jiao dan bai xiao lv zheng tu bian jun ,wei gao xiao shui jie jiao dan bai dian ding ji chu 。tong shi ,cong CP-16zhong kuo zeng chu er liu jian hai yuan mei ji yin (Trx)、jiao dan bai mei ji yin (Ker),gou jian huo de 2zhu ku cao ya bao gan jun WB600jiang jie yu mao guan jian mei chong zu jun (B.subtilis WB600/pSTOP 1622-pTrx、B.subtilis WB600/pSTOP 1622-pKer)。bing fen xi bu tong xin hao tai dui mu de ji yin (Trx、Ker)fen bi ying xiang ,sui hou dui chong zu jun jin hang fa jiao tiao jian you hua ,shi xian CP-16jiang jie jiao dan bai guan jian mei liang chan ,bing ying yong yu jiao dan bai sheng wu mei jie zhong 。yun yong zi wai 、liu suan er yi zhi dui yuan shi jun jin hang duo lun wu li he hua xue you bian ,jing chu shai yu fu shai de dao le yu mao jiang jie guan jian mei gao chan zheng tu bian jun 。jing zi wai you bian hou ,tu bian jun V-8er liu jian hai yuan mei mei huo 、jiao dan bai mei mei huo zeng fu zui da fen bie wei 0.15U/mL、15.4U/mL,jiao chu fa jun fen bie di gao 83.1%、83.3%。liu suan er yi zhi you bian hou ,tu bian jun D-5er liu jian hai yuan mei mei huo zeng fu zui da wei 0.14U/mL,jiao chu fa jun di gao le 67.5%,er tu bian jun D-2jiao dan bai mei mei huo zeng fu zui da wei 13.9U/mL,jiao chu fa jun di gao 65.5%。gou jian le 2chong jiao dan bai shui jie mei de biao da zhi li (pSTOP 1622-Trx、pSTOP 1622-Ker),dian zhuai ru WB600bing cheng gong biao da 。zai ci ji chu shang ,tong guo mo feng ke long shou duan ,gou jian le han bu tong xin hao tai de shai shua zai ti ,zai WB600zhong shi xian biao da 。jie guo biao ming ,jiao dan bai mei da zai zi shen xin hao tai shi bao wai fen bi jiao dan bai mei mei huo zui gao 。er er liu jian hai yuan mei da zai bu tong xin hao tai shi reng zai bao nei biao da ,ju mei huo you bu tong cheng du de jiang di 。tong guo you hua chong zu jun fa jiao pei yang pei fang ,chong zu jun B.subtilis WB600/pSTOP 1622-pTrxzai yu mi fen (5.1g/L)、pu tao tang (5.9g/L)、tang mi (11.9g/L)、gan you (3.0g/L),dan bai dong (20g/L),Nacl(10g/L),fa jiao ye OD600=1shi jia 0.5%mu tang you dao fa jiao 48hneng ming xian cu jin chan mei ,jiao ji chu shi pei yang tiao jian di gao 80.00%,jiao zheng tu bian jun V-8di gao 440.00%,jiao CP-16di gao 912.50%。chong zu jun B.subtilis WB600/pSTOP 1622-pKerzai yu mi fen (5.1g/L)、pu tao tang (5.9g/L)、tang mi (5.9g/L)、gan you (3.0g/L),dan bai dong (20g/L),Nacl(10g/L),fa jiao ye OD600=1shi jia 0.5%mu tang you dao fa jiao 48hneng ming xian cu jin chan mei ,jiao ji chu shi pei yang tiao jian di gao 49.74%,jiao zheng tu bian jun V-8di gao 269.48%,jiao CP-16di gao 577.38%。zeng shang jie guo ke de ,you bian yo chong ji CP-16jiang jie yu mao guan jian mei ji yin ku cao ya bao gan jun yi yuan biao da jun neng xian zhe di gao CP-16jiang jie yu mao guan jian mei mei huo ,ju ku cao ya bao gan jun yi yuan biao da zuo wei CP-16jiang jie yu mao guan jian mei gao xiao biao da shou duan xiao guo you yu you bian yo chong 。
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论文详细介绍
论文作者分别是来自中国农业科学院的廖朝勇，发表于刊物中国农业科学院2019-07-05论文，是一篇关于角蛋白酶论文,二硫键还原酶论文,异源表达论文,信号肽论文,发酵条件优化论文,中国农业科学院2019-07-05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，资料来自中国农业科学院2019-07-05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：角蛋白酶论文; 二硫键还原酶论文; 异源表达论文; 信号肽论文; 发酵条件优化论文; 中国农业科学院2019-07-05论文;