本文主要研究内容
作者袁红梅（2019）在《腺苷发酵高产菌株的选育及其工艺优化》一文中研究指出：腺苷是一种具有重要生理生化作用的内源性嘌呤类核苷,其主要合成方法有化学合成法和生物合成法。在生物合成法中,微生物发酵法因具有成本低、副产物少、对环境无毒无害等特点,已成为了当今社会腺苷生产的主要方式。目前腺苷的市场需求量比较大,但腺苷产量有限,因此如何快速提高腺苷的产量具有很大的研究意义。本论文首先对产腺苷菌株枯草芽孢杆菌BS-0的发酵性能进行了初步研究,然后通过理化因子诱变并结合微孔板发酵初筛、摇瓶复筛,选育出高产且稳定遗传的腺苷生产菌株。再通过单因素、PB实验、最陡爬坡实验和BB实验系统性优化摇瓶生产工艺,提高腺苷产量。最后通过3L发酵罐放大,优化了腺苷发酵工艺。主要研究内容和结果如下:(1)出发菌株BS-0发酵性能的初步研究在出发菌株BS-0的腺苷合成能力分析基础上,对其生长曲线和发酵曲线进行测定,并利用紫外-可见分光光度计法对腺苷标准曲线进行测定,明确了腺苷的标准曲线表达式为Y=0.0063X,其相关性系数R2约为0.9993。对发酵中的摇瓶接种量、摇床转速、摇瓶装液量和纱布层数进行优化,确定了最佳接种量为6%,250 mL摇瓶装液量为30 mL,转速240 r/min,4层纱布层数,发酵72 h时腺苷产量达18.072 g/L。(2)腺苷高产菌株的高通量诱变选育对出发菌株枯草芽孢杆菌BS-0进行常压室温等离子体(ARTP)、UV-LiCL、硫酸二乙酯(DES)和5-溴尿嘧啶(5-BU)诱变处理后,涂布于含结构类似物6-巯基嘌呤(6-MP)的培养基中,获得抗性正突变株,然后通过24微孔板发酵初筛、250 mL摇瓶发酵复筛,选育腺苷高产菌株。以BS-0为出发菌株进行ARTP诱变,筛选到菌株A-2-12腺苷产量达19.654 g/L,较出发菌株BS-0产量提高了6.78%。实验中建立了24微孔板(装液量为0.96 mL)和250 mL摇瓶(装液量为30 mL)发酵腺苷产量的的一致性,相关性系数R2=0.8996,说明两者具有比较好的一致性,可以采用24微孔板代替传统的摇瓶发酵。快速从大量的突变株中筛选出高产突变株,提高筛选效率。在此基础上采用UV-LiCL诱变处理菌株A-2-12,微孔板发酵初筛、摇瓶复筛,得到菌株Z-30,腺苷产量为19.654 g/L,比菌株BS-0产量提高了8.75%。使用烷化剂DES处理菌株Z-30,获得菌株D-21,产苷量达20.895 g/L,比菌株BS-0产量提高了 15.62%。采用碱基类似物5-BU处理菌株D-21,筛选到菌株5-36,其腺苷产量高达22.817g/L,较菌株BS-0产量提高了26.26%。对高产菌株D-21、5-3、5-5、5-6、5-36进行传代培养,发现传代4次后各菌株产苷能力均有所下降,其中菌株5-36的稳定性较好,腺苷产量为22.432 g/L,比出发菌株BS-0产量提高24.13%。(3)高产菌株5-36摇瓶发酵工艺的优化对最终获得的高产菌株5-36进行摇瓶发酵工艺优化,确定了花生饼粉、装液量、次黄嘌呤、发酵时间、pH最适水平分别为2.0%、25 mL、0.12%、72 h、6.75,显著因子为葡萄糖、玉米浆和CTAB。根据显著因子设计最陡爬坡试验确定了响应面的中心点,响应面实验模拟出的葡萄糖、玉米浆、CTAB的最佳浓度分别为13.874%、2.625%和0.056%,此时腺苷产量达23.794 g/L,经验证,腺苷产量为23.789 g/L,较优化前提高了6.05%,较菌株BS-0产量提高了31.63%。(4)高产菌株5-36的放大发酵优化对腺苷高产菌株5-36进行3 L发酵罐放大培养,检测菌浓、还原糖含量、氨基氮、pH以及腺苷产量,未调控时腺苷产量为16.440 g/L。通过优化接种量,确定了最佳接种量为4%,腺苷产量为19.539 g/L,较优化前提高了 18.85%。随后对葡萄糖的补加进行了研究,结果表明腺苷发酵32 h时补加8%葡萄糖,发酵56 h后腺苷产量达23.449 g/L,较补料前提高了20.01%,较未调控前提高了42.63%。本论文首先对出发菌株BS-0发酵工艺进行初步摸索,为后续研究内容的开展提供了实验基础。实验中诱变后的菌体经结构类似物6-MP抗性平板分选后,通过建立微孔板和摇瓶的缩微一致性和高通量发酵,提高了筛选效率,也节省了原料。通过单因素、PB实验、最陡爬坡实验和BB实验由点到面,系统的优化了腺苷的生产工艺,提高了腺苷产量。实验中通过3L发酵罐调控接种量、补加葡萄糖,腺苷产量较未调控前有明显提高。研究结果对于腺苷的生产具有重要的理论与参考价值。
Abstract
xian gan shi yi chong ju you chong yao sheng li sheng hua zuo yong de nei yuan xing piao ling lei he gan ,ji zhu yao ge cheng fang fa you hua xue ge cheng fa he sheng wu ge cheng fa 。zai sheng wu ge cheng fa zhong ,wei sheng wu fa jiao fa yin ju you cheng ben di 、fu chan wu shao 、dui huan jing mo du mo hai deng te dian ,yi cheng wei le dang jin she hui xian gan sheng chan de zhu yao fang shi 。mu qian xian gan de shi chang xu qiu liang bi jiao da ,dan xian gan chan liang you xian ,yin ci ru he kuai su di gao xian gan de chan liang ju you hen da de yan jiu yi yi 。ben lun wen shou xian dui chan xian gan jun zhu ku cao ya bao gan jun BS-0de fa jiao xing neng jin hang le chu bu yan jiu ,ran hou tong guo li hua yin zi you bian bing jie ge wei kong ban fa jiao chu shai 、yao ping fu shai ,shua yo chu gao chan ju wen ding wei chuan de xian gan sheng chan jun zhu 。zai tong guo chan yin su 、PBshi yan 、zui dou pa po shi yan he BBshi yan ji tong xing you hua yao ping sheng chan gong yi ,di gao xian gan chan liang 。zui hou tong guo 3Lfa jiao guan fang da ,you hua le xian gan fa jiao gong yi 。zhu yao yan jiu nei rong he jie guo ru xia :(1)chu fa jun zhu BS-0fa jiao xing neng de chu bu yan jiu zai chu fa jun zhu BS-0de xian gan ge cheng neng li fen xi ji chu shang ,dui ji sheng chang qu xian he fa jiao qu xian jin hang ce ding ,bing li yong zi wai -ke jian fen guang guang du ji fa dui xian gan biao zhun qu xian jin hang ce ding ,ming que le xian gan de biao zhun qu xian biao da shi wei Y=0.0063X,ji xiang guan xing ji shu R2yao wei 0.9993。dui fa jiao zhong de yao ping jie chong liang 、yao chuang zhuai su 、yao ping zhuang ye liang he sha bu ceng shu jin hang you hua ,que ding le zui jia jie chong liang wei 6%,250 mLyao ping zhuang ye liang wei 30 mL,zhuai su 240 r/min,4ceng sha bu ceng shu ,fa jiao 72 hshi xian gan chan liang da 18.072 g/L。(2)xian gan gao chan jun zhu de gao tong liang you bian shua yo dui chu fa jun zhu ku cao ya bao gan jun BS-0jin hang chang ya shi wen deng li zi ti (ARTP)、UV-LiCL、liu suan er yi zhi (DES)he 5-xiu niao mi ding (5-BU)you bian chu li hou ,tu bu yu han jie gou lei shi wu 6-qiu ji piao ling (6-MP)de pei yang ji zhong ,huo de kang xing zheng tu bian zhu ,ran hou tong guo 24wei kong ban fa jiao chu shai 、250 mLyao ping fa jiao fu shai ,shua yo xian gan gao chan jun zhu 。yi BS-0wei chu fa jun zhu jin hang ARTPyou bian ,shai shua dao jun zhu A-2-12xian gan chan liang da 19.654 g/L,jiao chu fa jun zhu BS-0chan liang di gao le 6.78%。shi yan zhong jian li le 24wei kong ban (zhuang ye liang wei 0.96 mL)he 250 mLyao ping (zhuang ye liang wei 30 mL)fa jiao xian gan chan liang de de yi zhi xing ,xiang guan xing ji shu R2=0.8996,shui ming liang zhe ju you bi jiao hao de yi zhi xing ,ke yi cai yong 24wei kong ban dai ti chuan tong de yao ping fa jiao 。kuai su cong da liang de tu bian zhu zhong shai shua chu gao chan tu bian zhu ,di gao shai shua xiao lv 。zai ci ji chu shang cai yong UV-LiCLyou bian chu li jun zhu A-2-12,wei kong ban fa jiao chu shai 、yao ping fu shai ,de dao jun zhu Z-30,xian gan chan liang wei 19.654 g/L,bi jun zhu BS-0chan liang di gao le 8.75%。shi yong wan hua ji DESchu li jun zhu Z-30,huo de jun zhu D-21,chan gan liang da 20.895 g/L,bi jun zhu BS-0chan liang di gao le 15.62%。cai yong jian ji lei shi wu 5-BUchu li jun zhu D-21,shai shua dao jun zhu 5-36,ji xian gan chan liang gao da 22.817g/L,jiao jun zhu BS-0chan liang di gao le 26.26%。dui gao chan jun zhu D-21、5-3、5-5、5-6、5-36jin hang chuan dai pei yang ,fa xian chuan dai 4ci hou ge jun zhu chan gan neng li jun you suo xia jiang ,ji zhong jun zhu 5-36de wen ding xing jiao hao ,xian gan chan liang wei 22.432 g/L,bi chu fa jun zhu BS-0chan liang di gao 24.13%。(3)gao chan jun zhu 5-36yao ping fa jiao gong yi de you hua dui zui zhong huo de de gao chan jun zhu 5-36jin hang yao ping fa jiao gong yi you hua ,que ding le hua sheng bing fen 、zhuang ye liang 、ci huang piao ling 、fa jiao shi jian 、pHzui kuo shui ping fen bie wei 2.0%、25 mL、0.12%、72 h、6.75,xian zhe yin zi wei pu tao tang 、yu mi jiang he CTAB。gen ju xian zhe yin zi she ji zui dou pa po shi yan que ding le xiang ying mian de zhong xin dian ,xiang ying mian shi yan mo ni chu de pu tao tang 、yu mi jiang 、CTABde zui jia nong du fen bie wei 13.874%、2.625%he 0.056%,ci shi xian gan chan liang da 23.794 g/L,jing yan zheng ,xian gan chan liang wei 23.789 g/L,jiao you hua qian di gao le 6.05%,jiao jun zhu BS-0chan liang di gao le 31.63%。(4)gao chan jun zhu 5-36de fang da fa jiao you hua dui xian gan gao chan jun zhu 5-36jin hang 3 Lfa jiao guan fang da pei yang ,jian ce jun nong 、hai yuan tang han liang 、an ji dan 、pHyi ji xian gan chan liang ,wei diao kong shi xian gan chan liang wei 16.440 g/L。tong guo you hua jie chong liang ,que ding le zui jia jie chong liang wei 4%,xian gan chan liang wei 19.539 g/L,jiao you hua qian di gao le 18.85%。sui hou dui pu tao tang de bu jia jin hang le yan jiu ,jie guo biao ming xian gan fa jiao 32 hshi bu jia 8%pu tao tang ,fa jiao 56 hhou xian gan chan liang da 23.449 g/L,jiao bu liao qian di gao le 20.01%,jiao wei diao kong qian di gao le 42.63%。ben lun wen shou xian dui chu fa jun zhu BS-0fa jiao gong yi jin hang chu bu mo suo ,wei hou xu yan jiu nei rong de kai zhan di gong le shi yan ji chu 。shi yan zhong you bian hou de jun ti jing jie gou lei shi wu 6-MPkang xing ping ban fen shua hou ,tong guo jian li wei kong ban he yao ping de su wei yi zhi xing he gao tong liang fa jiao ,di gao le shai shua xiao lv ,ye jie sheng le yuan liao 。tong guo chan yin su 、PBshi yan 、zui dou pa po shi yan he BBshi yan you dian dao mian ,ji tong de you hua le xian gan de sheng chan gong yi ,di gao le xian gan chan liang 。shi yan zhong tong guo 3Lfa jiao guan diao kong jie chong liang 、bu jia pu tao tang ,xian gan chan liang jiao wei diao kong qian you ming xian di gao 。yan jiu jie guo dui yu xian gan de sheng chan ju you chong yao de li lun yu can kao jia zhi 。
论文参考文献
论文详细介绍
论文作者分别是来自安徽工程大学的袁红梅，发表于刊物安徽工程大学2019-07-18论文，是一篇关于腺苷论文,诱变论文,菌株选育论文,优化论文,补料论文,安徽工程大学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，资料来自安徽工程大学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。