学位论文-—蜂巢形棉布载体固定化米根霉产果胶酶的半连续化发酵研究.docx
《学位论文-—蜂巢形棉布载体固定化米根霉产果胶酶的半连续化发酵研究.docx》由会员分享,可在线阅读,更多相关《学位论文-—蜂巢形棉布载体固定化米根霉产果胶酶的半连续化发酵研究.docx(65页珍藏版)》请在咨信网上搜索。
1、蜂巢形棉布载体固定化米根霉产果胶酶的半连续化发酵研究摘 要天然纤维素(如橘皮、烟梗)是一种可再生生物质资源,但由于纤维素、半纤维、木质素和果胶等交织在一起形成紧密复杂的结构,造成微生物利用天然纤维素生产乳酸、乙醇等的困难。米根霉在一定培养条件下能产生降解果胶质的酶系。果胶酶被广泛用于食品、纺织、生物技术等领域,其市场需求量日益增长。本文采用一种研究较少但有产果胶酶能力且富有商业价值的安全菌种米根霉,结合棉布载体固定化细胞的技术进行发酵产果胶酶。采用DNS法测定果胶酶(PEC)酶活力和用粘度法测定聚半乳糖醛酸内切酶(Endo-PG)酶活力。首先从纯果胶发酵产果胶酶出发,考察了固定化发酵与游离发酵
2、差别,优化了纯果胶培养基,优化了培养条件并进行半连续发酵试验,研究了发酵液部分果胶酶酶学性质;然后优化烟梗浸液培养基,优化了培养条件并进行半连续试验,还研究了发酵液中纤维素酶活力变化情况。首先,对纯果胶发酵产果胶酶进行研究。在优化前培养基和培养条件下,固定化和游离发酵相比,固定化发酵48h就达到最大产酶量,缩短发酵时间24h,提高产酶量115%。采用单因素和正交试验法优化培养基,结果表明影响产果胶酶的因素依次为:果胶Tween80Zn2+硫酸铵。发酵培养基为:果胶2.5%,硫酸铵1.5%,ZnSO40.6 mmol/L,Tween80 0.15% ,K2HPO4 0.4%,KH2PO4 0.4
3、%;在此基础上采用单因素法优化培养条件,结果为:果胶2.5%,硫酸铵1.5%,ZnSO40.6 mmol/L,Tween80 0.15% ,K2HPO4 0.4%,KH2PO4 0.4%,转速190r/min、装液量50ml/250ml、发酵温度30C、发酵初始pH 5.0、初始孢子浓度0.75106个/mL,培24h,PEC酶活力和Endo-PG酶活力分别为973.47U/mL和165.08U/mL。通过对粗酶液作用pH和温度的研究,得到PEC和Endo-PG为酸性果胶酶,最适pH为4.5,PEC和Endo-PG最适温度分别为45 C和55 C。半连续发酵试验结果表明,在第5批次出现最高PE
4、C酶活力和Endo-PG酶活力,分别为1253.95U/mL和181.94U/mL,分别比首次提高29.8%和18.3%。然后,对烟梗浸液发酵产果胶酶进行研究。采用单因素和正交试验法优化培养基,结果表明影响产果胶酶的因素依次为:烟梗硫酸铵Zn2+Tween80。发酵培养基为:烟梗10%,硫酸铵1.5%,ZnSO40.6 mmol/L,Tween80 0.05%,K2HPO4 0.2%,KH2PO4 0.2%。在此基础上采用单因素法优化培养条件,结果为:烟梗10%,硫酸铵1.5%,ZnSO40.6 mmol/L,Tween80 0.05%,K2HPO4 0.2%,KH2PO4 0.2%,发酵初始
5、pH 5.0,初始孢子浓度0.5106,30C,装液量50mL,转速170r/min,培养48h,PEC酶活力和Endo-PG酶活力最高分别为361.27U/mL和49.22U/mL。烟梗浸液发酵96h,出现纤维素酶活力高峰,滤纸酶活力和羧甲基纤维素钠酶活力分别为33.25U/mL和83.13U/mL。半连续发酵试验结果表明,在第2批次出现最高PEC酶活力和Endo-PG酶活力,分别为430.83U/mL和51.73U/mL,PEC酶活力比首批次提高约22.8%。关键词:米根霉,果胶酶,聚半乳糖醛酸内切酶,固定化,半连续化发酵ABSTRCTNatural cellulose is one of
6、 the renewable resources, such as orange peel and tobacco stem, which are made of cellulose, half fiber, lignin and pectin. The intertwined combination among them leads to form a tight and complex structure, which is hard for microorganisms to digest. Hence, the production of lactic acid or ethanol
7、through using natural cellulose by microorganisms is faced with problems. Rhizopus oryzae has the ability to produce pectinolytic enzymes to degrade pectin.The remove of pectin can make separation of fiber, fiber and lignin easier. There is a growing market demand of pectinolytic enzymes for the fac
8、t that pectinolytic enzymes are widely used in the filed of food, textile, pharmaceutical, paper making, biological technology and so on. Rhizopus oryzae, which owns the ability to degrade pectin and is rich in comercial value, is used to produce pectinolytic enzymes in this research. The immobilize
9、d Rhizopus oryzae with matrix composed of asterisk-configuration fibrous matrices in a honeycomb-shaped, the production of pectinolytic enzymes were carried out from citrus pectin and tobacco stem pectin. Pectinolytic enzymes (PEC) activity was determined by DNS assay and endopolygalacturonase (Endo
10、-PG) activity was measured by viscosity method. Fistly, the pure pectin was selected as the sbustrate for fermentation. Then, the coparison of enzymes production between immobilized cells and free cells was made. After that, optimization of culture medium and growth conditions was investigated throu
11、gh single factor variable analysis and so on. Also, some properties of the enzyme were under reasearch. Finally, optimization of culture medium made from tobacco stem was investigated through single factor variable analysis and orthogonal experiment. Optimization of growth conditions was investigate
12、d through single factor variable analysis. Six batches of semi-continuous fermentation were carried out in shake flasks.First of all, the study of pure pectin was carried out. Immobilized cells had a higher production of PEC over free cells. Immobilized cells had a max production in 48 hours, which
13、saved 24 hours compared with free cells and improved production by 115%. Results of single factor and orthogonal experimental with immobilized showed the order of factors influencing the production of pectinase was: pectin Tween80 ZnSO4(NH4)2SO4.The suitable culture media was composed of pectin2.5%,
14、 (NH4)2SO4 1.5%, ZnSO40.6 mmol/L ,Tween80 0.15%, K2HPO4 0.4%,KH2PO4 0.4%,rotation speed 190 r/min, liquid volume 50 ml/250 ml, temperature 30C, initial pH 5.0, initial spore concentration 0.75106 /mL. After 24 hours, the PEC activity and Endo-PG activity were 973.47U/mL and 165.08U/mL separately. Ef
15、fects of pH and temperature on crude enzymes fluid were carried out.PEC and Endo-PG might be acidic pectinolytic enzymes. The optimum pH was 4.5 for PEC and Endo-PG, optimum temperature was 45Cand 55C respectively. The highest PEC activity and Endo-PG activity were 1253.95U/mL and 181.94U/mL in the
16、5th batch, about 29.8% and 18.3% higher than the 1st batch. Secondly, optimization of culture medium made from tobacco stem was investigated through single factor variable analysis and orthogonal experiment. Results showed the order of factors influencing the production of pectinase was: tobacco ste
17、m (NH4)2SO4ZnSO4Tween80. The suitable culture media was composed of tobacco stem 10%, (NH4)2SO4 1.5%, ZnSO40.6 mmol/L,Tween80 0.05%, K2HPO4 0.2%,KH2PO4 0.2%,rotation speed 170 r/min, liquid volume 50 ml/250 ml, temperature 30C, initial pH 5.0, initial spore concentration 0.50106 /mL. After 48 hours,
18、 the PEC activity and Endo-PG activity were 361.27U/mL and 49.22U/mL separately. After 96 hours, cellulose enzyme activity reached a peak with filter paper activity of 33.25U/mL and sodium carboxymethyl cellulose activity of 83.13U/mL.The highest PEC activity and Endo-PG activity were 430.83U/mL and
19、 51.73U/mL in the 2nd batch. PEC activity was about 22.8% higher than the 1st batch.Keywords: Rhizopus oryzae, pectinolytic enzymes, endopolygalacturonase, immobilization, semi-continuous fermentation目 录摘 要IABSTRCTII1绪论11.1问题的提出及研究意义11.1.1问题的提出11.1.2研究意义21.2国内外研究现状21.2.1果胶分布、化学结构和分类21.2.2果胶酶分类和作用方式3
20、1.2.3果胶酶生产菌种41.2.4真菌果胶酶的表达调控61.2.5 微生物果胶酶条件优化研究现状71.2.6米根霉产果胶酶研究进展71.2.7固定化细胞产果胶酶的进展81.2.8果胶酶生产过程中的影响因素91.2.9果胶酶的应用111.2.10果胶质原料的研究121.3本课题研究的目的及主要内容121.3.1研究目的121.3.2本课题研究的主要内容121.3.3创新点132.棉布载体固定化发酵产果胶酶的研究142.1前言142.2材料和方法142.2.1菌种142.2.2主要仪器和试剂142.2.3培养基152.2.4载体的制备152.2.5发酵培养方法152.2.6分析方法162.3结果
21、分析182.3.1酶液稀释倍数的确定182.3.2游离发酵与固定化发酵的比较192.4讨论192.4.1酶液稀释倍数对酶活力测定准确性的影响192.4.2游离发酵与固定化发酵192.5小结193固定化发酵果胶酶纯果胶培养基的优化203.1前言203.2材料和方法203.2.1菌种203.2.2主要仪器和试剂203.2.3培养基213.2.4发酵培养方法213.2.5分析方法213.3结果分析223.3.1碳源种类的选择223.3.2果胶浓度选择233.3.3氮源种类选择233.3.4硫酸铵浓度选择243.3.5 Tween80对产酶的影响253.3.6金属离子的对产酶的影响253.3.7发酵培
22、养基的正交实验263.4讨论273.4.1碳源种类及果胶浓度对产酶的影响273.4.2氮源种类及硫酸铵对产酶的影响283.4.3Tween80对产酶的影响283.4.4金属离子对产酶的影响283.4.5正交实验结果分析293.5小结294纯果胶培养基半连续发酵果胶酶304.1前言304.2材料和方法304.2.1菌种、主要仪器和试剂304.2.2培养基304.2.4发酵培养方法314.2.5分析方法314.3结果分析314.3.1转速的选择314.3.2装液量选择324.3.3温度选择324.3.4初始pH的选择334.3.5初始孢子浓度的选择334.3.6生长和产酶的时间曲线344.3.7半
23、连续发酵试验354.3.8粗酶液部分酶学性质364.4讨论374.4.1转速和装液量对产酶的影响374.4.2 pH对产酶的影响374.4.3初始孢子浓度对产酶的影响384.4.4产酶与pH变化的关系384.4.5半连续发酵的稳定性384.4.6粗酶液部分酶学性质384.5小结395. 固定化发酵果胶酶烟梗浸液培养基的优化405.1前言405.2材料和方法405.2.1试剂和设备405.2.2烟梗浸液的制备415.2.3培养基及发酵培养方法415.2.4分析方法415.3结果分析415.3.1两种制备烟梗浸液方法的比较415.3.2烟梗浓度的选择425.3.3硫酸铵浓度的选择425.3.4 Z
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- 学位 论文 蜂巢 棉布 载体 固定 化米根霉产 果胶酶 连续 发酵 研究
1、咨信平台为文档C2C交易模式,即用户上传的文档直接被用户下载,收益归上传人(含作者)所有;本站仅是提供信息存储空间和展示预览,仅对用户上传内容的表现方式做保护处理,对上载内容不做任何修改或编辑。所展示的作品文档包括内容和图片全部来源于网络用户和作者上传投稿,我们不确定上传用户享有完全著作权,根据《信息网络传播权保护条例》,如果侵犯了您的版权、权益或隐私,请联系我们,核实后会尽快下架及时删除,并可随时和客服了解处理情况,尊重保护知识产权我们共同努力。
2、文档的总页数、文档格式和文档大小以系统显示为准(内容中显示的页数不一定正确),网站客服只以系统显示的页数、文件格式、文档大小作为仲裁依据,平台无法对文档的真实性、完整性、权威性、准确性、专业性及其观点立场做任何保证或承诺,下载前须认真查看,确认无误后再购买,务必慎重购买;若有违法违纪将进行移交司法处理,若涉侵权平台将进行基本处罚并下架。
3、本站所有内容均由用户上传,付费前请自行鉴别,如您付费,意味着您已接受本站规则且自行承担风险,本站不进行额外附加服务,虚拟产品一经售出概不退款(未进行购买下载可退充值款),文档一经付费(服务费)、不意味着购买了该文档的版权,仅供个人/单位学习、研究之用,不得用于商业用途,未经授权,严禁复制、发行、汇编、翻译或者网络传播等,侵权必究。
4、如你看到网页展示的文档有www.zixin.com.cn水印,是因预览和防盗链等技术需要对页面进行转换压缩成图而已,我们并不对上传的文档进行任何编辑或修改,文档下载后都不会有水印标识(原文档上传前个别存留的除外),下载后原文更清晰;试题试卷类文档,如果标题没有明确说明有答案则都视为没有答案,请知晓;PPT和DOC文档可被视为“模板”,允许上传人保留章节、目录结构的情况下删减部份的内容;PDF文档不管是原文档转换或图片扫描而得,本站不作要求视为允许,下载前自行私信或留言给上传者【胜****】。
5、本文档所展示的图片、画像、字体、音乐的版权可能需版权方额外授权,请谨慎使用;网站提供的党政主题相关内容(国旗、国徽、党徽--等)目的在于配合国家政策宣传,仅限个人学习分享使用,禁止用于任何广告和商用目的。
6、文档遇到问题,请及时私信或留言给本站上传会员【胜****】,需本站解决可联系【 微信客服】、【 QQ客服】,若有其他问题请点击或扫码反馈【 服务填表】;文档侵犯商业秘密、侵犯著作权、侵犯人身权等,请点击“【 版权申诉】”(推荐),意见反馈和侵权处理邮箱:1219186828@qq.com;也可以拔打客服电话:4008-655-100;投诉/维权电话:4009-655-100。