微生物资源及开发利用

nullnull微生物资源及开发利用 山东省农业科学院 岳寿松 2011.6.12 威海第三届特种医学暨三省省际联合（山东-河南-湖北）微生态学学术会议提 要提 要肠道微生物资源 从极端微生物到太空微生物 废弃资源的微生物转化 肠 道 微 生 物肠 道 微 生 物null 人类肠道细菌超1000万亿 细菌数量是人体细胞的10倍 PLoS biology. November 2008(11)6:280 Les Dethlefsen David A. Relman et al. 斯坦福大学医学院的戴维·雷尔曼(David Relman)和同事一起，利用一种新型技术——焦磷酸测序法(pyrosequencing)，得到了关于人体肠道内菌落数量更为准确的数据。PLoS biology 人类肠道内的细菌群落数量是原有认识的10倍以上。 nullScience 328, 228 (2010) Matam Vijay-Kumar et al. ……mice genetically deficient in Toll-like receptor 5 (TLR5), a component of the innate immune system that is expressed in the gut mucosa and that helps defend against infection, exhibit hyperphagia and develop hallmark features of metabolic syndrome, including hyperlipidemia, hypertension, insulin resistance, and increased adiposity……. Metabolic Syndrome and Altered Gut Microbiota in Mice Lacking Toll-Like Receptor 5肠道微生物系变化导致代谢综合症。肠道微生物可能影响人类的健康，如胖瘦、糖尿病、胃病、甚至癌症 。缺乏先天性免疫系统中的某种重要成分（TLR5蛋白）的小鼠会产生代谢综合症的标志性特征，如伴有肠道微生物系变化的脂肪积聚的增加及胰岛素抵抗。 将突变小鼠的肠道内微生物转移到无菌小鼠的肠道之中会使接受这些微生物的小鼠出现代谢综合症的几种特征 。 null人肠道微生物菌落基因目录Junjie Qin,Ruiqiang Li et al A human gut microbial gene catalogue established by metagenomic sequencing. Nature| Vol 464.4 March 2010……the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals…. The gene set, ~150 times larger than the human gene complement, …Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, … 124位健康的、超重的和肥胖的成年人以及炎症患者提取的人肠道微生物菌落的基因目录。 比人类基因组大150倍。 超过99%为细菌。 这些基因大部分是在不同个体之间共享的。 nullnature Volume: 473, Pages: 174–180 Date published: (12 May 2011) DOI: doi:10.1038/nature09944 Enterotypes of the human gut microbiome …identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific …This indicates further the existence of a limited number of well-balanced host–microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species highlighting the importance of a functional analysis to understand microbial communities. …individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes …人类肠道微生物系统可拥有特定的分类—肠型 肠型分三类：Enterotype 1, Enterotype 2,Enterotype 3. 分别为Bacteroides型、Prevotella型和Ruminococcus型未来医师或可依据肠型的区划，量身打造病患饮食清单以及开处方，甚至至为抗生素寻求替代品。 肠型与种族、性别、体重、健康或年龄等均无关联。 nullnullThink Twice: How the Gut's "Second Brain" Influences Mood and Well-Being Adam Hadhazy  | February 12, 2010 | A complex, independent nervous system lines the gastrointestinal tract that has been dubbed the "second brain" 心理过程与消化系统紧密相联的程度超出人们的想象。 长期压力过大，过度紧张、焦虑、抑郁、易怒等不良情绪，均可使胃肠道生理功能发生紊乱。 患老年性痴呆症及帕金森氏病的病人中，常在头部和腹部发现同样的组织坏死现象。 疯牛病病人通常是大脑受损而出现精神错乱，与此同时肠器官也经常遭到极度损害。 null nature Vol 467|2 September 2010| doi:10.1038 /nature 09354Bacterial charity work leads to population-wide resistance Henry H. Lee, Michael N. Molla, Charles R. Cantor & James J. CollinsA population-based antibiotic-resistance mechanism a, In the absence of antibiotic stress, wild-type cells naturally produce indole. b, Under antibiotic stress, wild-type cells stop producing indole and eventually die. c, When a drug-resistant mutant emerges, it is able to produce indole even under antibiotic stress. This indole allows the more vulnerable cells in the population to survive the antibiotic stress, by inducing various antibiotic-tolerance mechanisms, thereby boosting the survival capacity of the population. 单细胞组成的群体在种程度上可以像多细胞一样运转。极端环境微生物极端环境微生物太空微生物太空微生物征召细菌进行太空“生物开采”，为将来月球或火星殖民者提炼氧气、营养物质和矿物质。 最有可能的太空“拓荒者”：蓝细菌cyanobacteria,又称为蓝绿藻,适应了地球一些最极端的环境，从极度寒冷干旱的南极麦克多默干燥谷（Antarctic McMurdo Dry Valleys) 到炎热干旱的阿塔卡马沙漠（Atacama Desert)。 意味着其也许有能力在严酷的外太空存活下来。微型矿工将为人类在外星球蓬勃发展助一臂之力Use of cyanobacteria for in-situ resource use in space applications Planetary and Space Science Volume 58, Issue 10, August 2010, 1279-1285 太空微生物太空微生物Planetary and Space Science Volume 58, Issue 10, August 2010, 1279-1285 Use of cyanobacteria for in-situ resource use in space applications Karen Olsson-Francis & CharlesS.Cockell 将几种细菌发射到高度300千米的近地轨道，让它们连续暴露在真空、寒冷、炎热和辐射环境中。接着在有水的情况下把这些细菌接种到不同类型的岩石中，包括类似于月球高地风化层的南非斜长岩（anorthosite)和类似于月球和火星风化层的冰岛火山玄武岩。废弃（角蛋白质）资源 的微生物转化 废弃（角蛋白质）资源 的微生物转化 null 近年来，随着分子生物学技术的发展与应用，角蛋白酶研究进展迅速，特别是角蛋白酶降解疯牛病的致病因子“Prion蛋白”的发现，使之成为研究热点。由于Prion蛋白的β折叠构象与羽毛角蛋白类似，因此以羽毛角蛋白质为材料研究降解机理具有更重要价值和意义。Keratinase has been discovered to degrade the " Prion protein" virulence factor of mad cow disease in recent years. Feather keratin has a protein structure similar to that of the prion. It’s a rich, β-sheet structure, similar to the pathogenic unfolded isoform of prions.降解羽毛微生物分离及多样性研究降解羽毛微生物分离及多样性研究30个样本 746个具有分解蛋白质微生物菌落 130株具降解羽毛的能力 得到了枯草芽孢杆菌（Bacillus sublitis)YYW-1、NKY-12，嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia)YHYJ-1、NKY-3、NKY-4、NKY-5、NKY-6和NKY-7，产气单胞菌属Aeromonas sp.NKY-13，微杆菌属Microbacterium sp. JJD-1、芽孢杆菌属Bacillus sp.JJD-2、金黄杆菌属Chryseobacterium sp.JJD-3等属和种的18株高效菌株. nullnull至少有5株为S. maltophilia（≧40%） 说明S. maltophilia羽毛废弃物分解菌中优势种群。 一个典型样品，有42株菌具有分解羽毛的能力；其中13株分解羽毛能力很强。分属于3个以上的属 。说明降解羽毛微生物系统发育的多样性。 null The native keratinase showed strong degrading capability on feather, wool and hair. Feather and wool were degraded completely at 36 h and 72 h after inculated with the keratinase, respectively; and hair was degraded obviously at 84 h. B1B2C2 野生菌接种于羽毛、羊毛、毛发三种培养基，观察对三种底物的降解情况。36小时能将羽毛完全降解，72小时将羊毛完全降解，84小时后对头发有明显降解。 Studies on feather degradating and keratinase properties of S. maltophilia YHYJ-1 嗜麦芽窄食单胞菌菌YHYJ-1角蛋白酶 KerF 基因克隆、 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 达、纯化及酶学性质分析null Protein content in fermented liquid Protein concentration was determined according to Bradford(1976)，using bovine serum albumin(BSA) as standard substrate． 发酵液蛋白质含量 nullS. maltophilia YHYJ-1发酵产酶曲线 Time course production of keratinase in feather substrate null Optimum temperature and thermal stability of crude keratinase The optimum temperature of crude enzyme was 50℃. And the keratinase was stable at 40～45℃. 角蛋白酶的最适反应温度及热稳定性 Effect of temperature on activity of crude keratinase Temperature stability of crude keratinase nullOptimum pH and pH stability of crude keratinase The optimum pH of crude enzyme was 9.0 . The activity of keratinase was stable in the pH 6.6 and 8.4 indicated that it is a combination enzyme. 角蛋白酶最适pH值及pH值稳定性 嗜麦芽窄食单胞菌角蛋白酶粗酶最适pH值为9.0；在pH 8.4时比较稳定，同时在pH6.6时也相对稳定。由这两个峰可以初步推断此角蛋白酶可能为一种复合酶。Effect of pH on activity of crude keratinase Effect of pH on stability of crude keratinase Cloning , Expression ,Purification and Functional Analysis of S. maltophilia YHYJ-1 keratinase gene KerF Cloning , Expression ,Purification and Functional Analysis of S. maltophilia YHYJ-1 keratinase gene KerF Expression and PurificationCloning of gene KerF Functional analysis of keratinaseKerF基因克隆表达纯化酶活性质分析嗜麦芽窄食单胞菌菌YHYJ-1角蛋白酶 KerF 基因克隆、表达、纯化及酶学性质分析nullPrimer design P1：5'-GAACGAACATTTCATCTTGCT-3'； P2：5‘-GGTCTGAGGAACAGCGTG-3’ . Design the P3, P4 without signal peptide according to expression vector pET28a（+）: P3：5'-AAACCATGGCTCAGGTAACGCAACC-3'； NcoⅠ P4：5'-AAAAAGCTTGTACTGGGCGTTGAGGGTC-3' Hind III嗜麦芽窄食单胞菌R551-3全基因组已测序，基因组序列和注释已登陆GenBank，根据推定的嗜麦芽窄食单胞菌R551-3的角蛋白酶基因序列 设计 领导形象设计圆作业设计ao工艺污水处理厂设计附属工程施工组织设计清扫机器人结构设计 引物P1、P2。测序后针对pET28a（+）表达载体设计去信号肽引物P3、P4。null Extraction of genomic DNA Extract the DNA of Stenotrophomonas maltophilia as the template for further PCR.Agarose gel eletrophoresis of genomic DNA of S.maltophilia YHYJ-1 嗜麦芽窄食单胞菌总DNA提取 提取嗜麦芽窄食单胞菌的染色体DNA ，作为下一步PCR扩增的 模板 个人简介word模板免费下载关于员工迟到处罚通告模板康奈尔office模板下载康奈尔 笔记本 模板 下载软件方案模板免费下载 。 M:Marker (1Kb DNA Ladder); 1: Genomic DNA of S.maltophilia YHYJ-1nullPCR amplification of keratinase gene and connection of the target gene and T-vector . 角蛋白酶基因KerF的扩增及与T载体的连接 以S.maltophilia YHYJ-1的基因组DNA为模板，进行PCR扩增，得到一条大小为1980bp左右的特异性条带。连接后的重组质粒pMD18-KerF经EcoR I和Hind III双酶切后出现2条带。 PCR amplification of Keratinase gene KerF M:Marker (DL2000 Plus) ;1:Keratinase gene KerFIdentification of recombinant plasmid pMD18-KerF by digestion(EcoR I,Hind III)nullSequencing and signal peptide analysis of the target gene The keratinase gene(KerF), include complete sequence of 1 981 bp and a 1 734 bp open reading frame and encode 580 amino acid, was cloned from S.maltophilia(GenBank Accession No.HM590650).Using SignalP3.0 to assay the signal peptide of KerF gene.KerF基因序列测定与信号肽分析 利用DNAman软件和NCBI的BLAST进行序列同源性分析，结果显示，该基因全长1981 bp，含有一个1743 bp的开放阅读框，编码580个氨基酸。将该基因提交GenBank，获得登录号HM590650。利用SignalP3.0软件分析推导氨基酸的信号肽nullConstruction of pET28-kerF recombinant plasmid pET28-kerF重组质粒构建null PCR amplification of KerF gene without signal peptide and identification of recombinant plasmid pET28-kerF by digestion (Nco I, Hind III). 去信号肽的KerF片段PCR扩增及与pET28表达载体的连接 设计引物P3、P4在PCR时将KerF片段的信号肽切除，电泳检测后得到一段与预计结果大小一致的条带（1756bp）。将KerF片段与pET28表达载体连接，提取转化子质粒，进行Nco I和Hind III双酶切鉴定。PCR amplification of gene KerF without signal peptide Identification of recombinant plasmid pET28-kerF by digestion(Nco I,Hind III) null Expression of S. maltophilia Keratinase Gene （KerF） in Escherichia coli 重组角蛋白酶的表达 以重组质粒pET28-kerF转化大肠杆菌BL21（DE3）， 37℃振荡培养1h至OD600约为0.1，25℃ ，0.1 mmol/L IPTG诱导表达，重组质粒pET28-kerF在相对分子量50kD处出现一条特异蛋白带。 SDS-PAGE analysis of the expression product at different induced times. 1: Marker; 1-7: recombinant strain induced by IPTG at 1～7h, respectively; 8-14:recombinant strain lysate induced by IPTG at 1～7h, respectively.nullPurification of keratinase KerF Recombinant KerF was purified to electrophoretical homogeneity after nickel affinity chromatographyand exhibited a high specific activity of 726U/mg. 重组角蛋白酶的纯化 重组角角蛋白酶末端含有His标签，可采用镍亲和层析法快速纯化。对纯化的角蛋白酶进行蛋白含量测定和酶活测定，得出重组角蛋白酶比酶活力达到26U/mg。Analysis of recombinant expressed keratinase by SDS-PAGE. M, Marker; 1, purified keratinase; 2, recombinant strain pET28-kerF lysate induced by IPTG null Optimum temperature and thermal stability of keratinase Ker Effect of temperature on enzyme activity Effect of temperature on enzyme stability KerF角蛋白酶的最适作用温度和温度稳定性 在pH 7.4，反应30 min的条件下，于25～75 ℃范围内，每隔5℃，分别测定酶活，以酶活最高为100%计算相对酶活，由此确定酶的最适作用温度。将酶液于30、35、40、45、50、55℃下保温处理不同时间，测定残余酶活性。以未保温（4℃放置）的酶样活性为100%，由此确定酶的热稳定。null Optimum pH and pH stability of keratinas KerF The optimum pH was determined at 30℃using the following buffers：Acetic acid/sodium acetate buffer (pH 4.2 ～ 5.4), Tris-HC1 buffer(pH 6.0～8.4), and Glycine/NaOH buffer (pH 9.0～9.6)． The enzyme solution was preincubated for 30min in different pH buffer .Then the residual activity was measured． Effect of pH on enzyme activity Effect of pH on enzyme activity Effect of pH on enzyme activity KerF角蛋白酶的最适pH值及pH稳定性 Effect of pH on enzyme activity Effect of pH on enzyme activity Effects of pH on enzyme activityEffect of pH on enzyme stability Effect of pH on enzyme stabilitynullEffect of metal ions on keratinase KerF activity 金属离子对KerF角蛋白酶活性的影响nullEffect of proteinase inhibitors and organic solvent on keratinase activity 有机溶剂、蛋白酶抑制剂等对KerF角蛋白酶活性的影响nullEffect of sulfur compounds on keratinase activity含硫化合物对角蛋白酶的影响 nullSubstrate specificity of keratinase Recombinant KerF showed degrading capability on various kind of proteins such as soluble keratin, feather, wool, hair and bovine serum albumin. KerF角蛋白酶的底物利用特性 重组角蛋白酶以羊毛、头发、羽毛粉和牛血清白蛋白为底物对可溶性角蛋白酶的相对酶活分别为103.7%、99.3%、76.0%和94.1%。说明该角蛋白酶不仅可利用β折叠结构角蛋白质羽毛作为底物，而且可以利用α螺旋结构角蛋白质的毛发类底物。 Keratinase activity on various protein substrates nullThe keratinase from Stenotrophomonas maltophilia YHYJ-1 hydrolysed a broad range of protein substrates inclues the protein chain in α-helix (α-keratin, wool and hair) and β-sheet (β-keratin, feather) structures and showed different properties to keratinases from other genera micoorganisms reported. The enzyme seems to be a novel keratinase and could be used to improve nutritional values of animal feeds containing feather, or hard fibers treatment in the textile industry. 目前正研究KerF 与S. maltophilia YHYJ-1降解羽毛的相关性，并构建KerF高表达分泌型真菌表达系统，最终实现产业化。null微生物资源是最丰富、最重要而且曾一度被忽视的生物资源。 微生物资源的开发利用，必将为人类健康、自然资源高效利用和可持续发展起重要作用。小结敬请批评指正敬请批评指正谢谢！