本文主要研究内容
作者黄婷婷（2019）在《磷脂酶D的重组优化表达及生物转化制备磷脂酰丝氨酸》一文中研究指出：磷脂酰丝氨酸（phosphatidylserine;PS）作为大脑中的主要酸性磷脂,具有提高认知力、抗压抑等功效,可用于医药、食品等领域。由于自然界中含量较少,且提取困难,近年来,人们开始关注酶转化法制备PS。即通过磷脂酶D（phospholipase D;EC 3.1.4.4;PLD）催化底物磷脂酰胆碱和L-丝氨酸合成。目前虽然取得了一定研究成果,但仍存在一些不足之处:（1）野生菌PLD分泌量远低于工业生产标准;（2）现有研究中PS多采用有机相和水相两相催化体系生成。基于此,本研究以Bacillus subtilis WB600为宿主,研究了PLD的异源高效表达,并优化了PS在纯水相中的生成条件。主要研究结果如下:（1）PLD的异源表达及信号肽优化。将Streptomyces racemochromogenes来源的PLD基因按照枯草芽孢杆菌密码子优化性合成,并根据N端带电荷数和H端疏水性选择了7条来源于枯草芽孢杆菌的信号肽,将其融在PLD基因N端,将这7段融合片段克隆到表达质粒pSTOP并转入B.subtilis WB600中表达,比较了不同信号肽牵引下的PLD分泌效率。结果显示,PLD在B.subtilis WB600中成功实现胞外异源分泌表达,且在信号肽amyE介导下获得最高胞外酶活11.3 U×mL-1,是内源性信号肽介导下胞外酶活分泌量的5.27倍,ywbN信号肽介导的胞外PLD酶活最低(3.2 U×mL-1)。（2）表达载体优化。分别构建了3个重组表达质粒pSTOP-PLD-amyE-his、pMA0911-PLD-amyE-his和pP43-PLD-amyE-his,包含这3个质粒的重组菌分别命名为STT1、ST2和ST3。结果显示,ST2和ST3的PLD酶活高于STT1,ST2菌株获得最高酶活19.1 U×mL-1,比对照提高约69.03%,通过对3株菌的胞外上清液进行Western Blot检测,在约53 kDa处均可见明显单一条带,与报道条带大小相符。（3）RBS及spacer区优化。通过RBS Calculator v2.0软件对重组质粒pMA0911-PLD-amyE-his的RBS及spacer区进行优化,选择了翻译效率较原始序列分别提高3、6、9、12倍的四种组合,分别得到RS1、RS2、RS3和RS4这4株重组菌。结果显示RS1菌株获得最高胞外酶活24.2 U×mL-1,较对照提高约26.7%,而RS4菌株胞外酶活反而较对照降低约11.8%。我们发现随着RBS强度的增加,PLD胞外分泌量呈现先升高后降低的趋势,这表明RBS的翻译强度并不明显与PLD的分泌量呈正相关。（4）15 L发酵罐实验及酶学性质研究。15 L发酵罐实验,酶活提高约5倍,达到116.2U×mL-1。对RS1菌株进行遗传稳定性实验,重组质粒经过5代后的保留率仍为100%。纯化RS1菌株的胞外上清液,测定PLD酶学性质。结果表明,PLD蛋白条带大小约为53 kDa,与已报道条带大小相符,最适反应温度45℃,最适反应pH 5.5,4℃下可长期保藏。（5）优化纯水相中生产PS工艺,避免了有机相的使用。最佳反应条件下:PC60:L-丝氨酸1:10(g×g-1)、酶添加量500 U、反应温度45℃、反应pH 5.5、反应时间6 h、氯化钙添加量10%。反应6 h后,以PC60为底物,PS转化率达到96.7%,PC剩余2.3%,副产物PA仅生成0.2%,底物向产物实现高效转化。
Abstract
lin zhi xian si an suan （phosphatidylserine;PS）zuo wei da nao zhong de zhu yao suan xing lin zhi ,ju you di gao ren zhi li 、kang ya yi deng gong xiao ,ke yong yu yi yao 、shi pin deng ling yu 。you yu zi ran jie zhong han liang jiao shao ,ju di qu kun nan ,jin nian lai ,ren men kai shi guan zhu mei zhuai hua fa zhi bei PS。ji tong guo lin zhi mei D（phospholipase D;EC 3.1.4.4;PLD）cui hua de wu lin zhi xian dan jian he L-si an suan ge cheng 。mu qian sui ran qu de le yi ding yan jiu cheng guo ,dan reng cun zai yi xie bu zu zhi chu :（1）ye sheng jun PLDfen bi liang yuan di yu gong ye sheng chan biao zhun ;（2）xian you yan jiu zhong PSduo cai yong you ji xiang he shui xiang liang xiang cui hua ti ji sheng cheng 。ji yu ci ,ben yan jiu yi Bacillus subtilis WB600wei su zhu ,yan jiu le PLDde yi yuan gao xiao biao da ,bing you hua le PSzai chun shui xiang zhong de sheng cheng tiao jian 。zhu yao yan jiu jie guo ru xia :（1）PLDde yi yuan biao da ji xin hao tai you hua 。jiang Streptomyces racemochromogeneslai yuan de PLDji yin an zhao ku cao ya bao gan jun mi ma zi you hua xing ge cheng ,bing gen ju Nduan dai dian he shu he Hduan shu shui xing shua ze le 7tiao lai yuan yu ku cao ya bao gan jun de xin hao tai ,jiang ji rong zai PLDji yin Nduan ,jiang zhe 7duan rong ge pian duan ke long dao biao da zhi li pSTOPbing zhuai ru B.subtilis WB600zhong biao da ,bi jiao le bu tong xin hao tai qian yin xia de PLDfen bi xiao lv 。jie guo xian shi ,PLDzai B.subtilis WB600zhong cheng gong shi xian bao wai yi yuan fen bi biao da ,ju zai xin hao tai amyEjie dao xia huo de zui gao bao wai mei huo 11.3 U×mL-1,shi nei yuan xing xin hao tai jie dao xia bao wai mei huo fen bi liang de 5.27bei ,ywbNxin hao tai jie dao de bao wai PLDmei huo zui di (3.2 U×mL-1)。（2）biao da zai ti you hua 。fen bie gou jian le 3ge chong zu biao da zhi li pSTOP-PLD-amyE-his、pMA0911-PLD-amyE-hishe pP43-PLD-amyE-his,bao han zhe 3ge zhi li de chong zu jun fen bie ming ming wei STT1、ST2he ST3。jie guo xian shi ,ST2he ST3de PLDmei huo gao yu STT1,ST2jun zhu huo de zui gao mei huo 19.1 U×mL-1,bi dui zhao di gao yao 69.03%,tong guo dui 3zhu jun de bao wai shang qing ye jin hang Western Blotjian ce ,zai yao 53 kDachu jun ke jian ming xian chan yi tiao dai ,yu bao dao tiao dai da xiao xiang fu 。（3）RBSji spacerou you hua 。tong guo RBS Calculator v2.0ruan jian dui chong zu zhi li pMA0911-PLD-amyE-hisde RBSji spacerou jin hang you hua ,shua ze le fan yi xiao lv jiao yuan shi xu lie fen bie di gao 3、6、9、12bei de si chong zu ge ,fen bie de dao RS1、RS2、RS3he RS4zhe 4zhu chong zu jun 。jie guo xian shi RS1jun zhu huo de zui gao bao wai mei huo 24.2 U×mL-1,jiao dui zhao di gao yao 26.7%,er RS4jun zhu bao wai mei huo fan er jiao dui zhao jiang di yao 11.8%。wo men fa xian sui zhao RBSjiang du de zeng jia ,PLDbao wai fen bi liang cheng xian xian sheng gao hou jiang di de qu shi ,zhe biao ming RBSde fan yi jiang du bing bu ming xian yu PLDde fen bi liang cheng zheng xiang guan 。（4）15 Lfa jiao guan shi yan ji mei xue xing zhi yan jiu 。15 Lfa jiao guan shi yan ,mei huo di gao yao 5bei ,da dao 116.2U×mL-1。dui RS1jun zhu jin hang wei chuan wen ding xing shi yan ,chong zu zhi li jing guo 5dai hou de bao liu lv reng wei 100%。chun hua RS1jun zhu de bao wai shang qing ye ,ce ding PLDmei xue xing zhi 。jie guo biao ming ,PLDdan bai tiao dai da xiao yao wei 53 kDa,yu yi bao dao tiao dai da xiao xiang fu ,zui kuo fan ying wen du 45℃,zui kuo fan ying pH 5.5,4℃xia ke chang ji bao cang 。（5）you hua chun shui xiang zhong sheng chan PSgong yi ,bi mian le you ji xiang de shi yong 。zui jia fan ying tiao jian xia :PC60:L-si an suan 1:10(g×g-1)、mei tian jia liang 500 U、fan ying wen du 45℃、fan ying pH 5.5、fan ying shi jian 6 h、lv hua gai tian jia liang 10%。fan ying 6 hhou ,yi PC60wei de wu ,PSzhuai hua lv da dao 96.7%,PCsheng yu 2.3%,fu chan wu PAjin sheng cheng 0.2%,de wu xiang chan wu shi xian gao xiao zhuai hua 。
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论文详细介绍
论文作者分别是来自江南大学的黄婷婷，发表于刊物江南大学2019-10-21论文，是一篇关于磷脂酶论文,磷脂酰丝氨酸论文,枯草芽孢杆菌论文,分泌表达论文,江南大学2019-10-21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，资料来自江南大学2019-10-21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：磷脂酶论文; 磷脂酰丝氨酸论文; 枯草芽孢杆菌论文; 分泌表达论文; 江南大学2019-10-21论文;