梭热杆菌纤维小体研究进展

梭热杆菌(Clostridium thermocellum)是一种嗜热厌氧细菌,通过分泌大量纤维素酶高效降解纤维素.根据作用纤维素的不同部位,梭热杆菌分泌的纤维素酶分为内切纤维素酶和外切纤维素酶.纤维小体是由支架蛋白、锚定元件、黏合蛋白、纤维素结合域和催化单位组成的复合体,其独特的结构,使得它可以比真菌纤维素酶更紧密地结合到纤维素表面,这个复合结构结合着多种催化单位,而此特殊的结构是梭热杆菌高效降解纤维素的必要条件.近年来,为更深入透彻地了解纤维小体的结构与功能进行了大量的研究工作,现对相关研究进展进行综述,并给出了未来可能的发展方向.     

极端嗜热古菌——芝田硫化叶菌DNA结合蛋白Ssh12对DNA超螺旋的影响

通过SPSepharose,DNA纤维素和磷酸纤维素等柱层析 ,从极端嗜热古菌———芝田硫化叶菌 (Sulfolobusshibatae)中纯化得到分子量为 11.5ku的DNA结合蛋白Ssh12 .Ssh12约占细胞总蛋白的 4% .该蛋白既能与负超螺旋DNA也能与松弛DNA结合 .利用含单切刻环状DNA进行的切刻闭合分析表明 ,Ssh12在与DNA结合时能够固定负超螺旋 .这种能力在室温 ( 2 2℃ )下很弱 ,而在 3 7℃以上则大大增强 .Ssh12的细胞内含量和固定负超螺旋的能力提示 ,该蛋白对于芝田硫化叶菌染色体DNA的组织以及热稳定性起着重要作用 .     

嗜热毛壳菌纤维素酶（CBHⅡ）cDNA的克隆及在毕赤酵母中的表达

嗜热毛壳菌Chaetomium thermophilum CT2是一种土壤腐生菌,可产生具有重要工业生产价值的纤维素酶类。RACE-PCR获得嗜热毛壳菌纤维二糖水解酶Ⅱ（CBHⅡ）的编码基因(cbh2)。DNA序列分析表明cbh2的开放阅读框由1428个碱基组成,编码476个氨基酸。推断的氨基酸序列包含一个典型真菌纤维素酶的糖结合域（CBD）、催化域（CD）以及二者之间富含脯氨酸和羟基氨基酸的连接桥。根据氨基酸序列推算该酶分子量为53kD,属于糖苷水解酶第六家族,具有该家族催化保守区的典型特征。PCR扩增cbh2的成熟蛋白编码基因,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达纤维二糖水解酶蛋白的重组表达载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,小规模发酵量达1.2 mg/mL。经硫酸铵沉淀、DEAESepharose Fast flow阴离子层析等步骤纯化了该重组表达蛋白。SDS-PAGE得到重组蛋白分子量为67kD,与从嗜热毛壳菌中纯化的该酶分子量一致。该重组纤维二糖水解酶作用的最适合温度50℃,最适pH4.0,在70℃的半衰期为30min,具有较好的热稳定性。     

融合荧光蛋白的纤维素酶表达与性质分析

纤维素酶是能特异性分解纤维素的一系列酶,被广泛应用于食品加工处理、衣物洗涤、农业及造纸等行业。纤维素酶通过不同的水解方式协同作用将纤维素水解为寡糖或可发酵糖。在将纤维素分解成寡糖的过程中,外切葡聚糖酶(CBH)和内切葡聚糖酶(EG)发挥了重要作用。目前关于两种酶协同作用机制和顺序尚未有明确的解释。本研究将嗜热毛壳菌来源的cbh和葡萄穗霉来源的eg分别与绿色荧光蛋白基因gfp和红色荧光蛋白基因mcherry进行融合,并电转入毕赤酵母X33进行异源表达,随后对这两种融合纤维素酶CBH-GFP和EG-MCHERRY进行了性质分析。结果表明：两种融合荧光蛋白的纤维素酶基因在毕赤酵母X33中实现了分泌表达。纯化的CBH-GFP和EG-MCHERRY的蛋白浓度与荧光强度呈正相关关系。对羧甲基纤维素钠(CMC-Na)、磷酸膨胀纤维素(PASC)、滤纸(FP)、微晶纤维素(Avicel)的活性测试结果显示,CBH-GFP对CMC-Na、PASC、Avicel均有催化活性,而EG-MCHERRY对CMC-Na、PASC具有催化活性,CBH-GFP对PASC有最高比酶活(1.1 U/mg);EG-MCHE...     

嗜热毛壳菌基因组密码子偏好性研究

嗜热毛壳菌具有强大的木质纤维素降解能力,将其开发为优异的重组蛋白表达宿主有着广阔的应用前景。蛋白表达宿主的密码子偏好性对重组蛋白的表达水平具有重大影响。为确定嗜热毛壳菌中密码子的使用模式及影响因素,本研究以6 897条CDS序列为对象,对其进行密码子偏好性分析。结果显示,嗜热毛壳菌中GC3的平均含量为66.2%,高于GC1(59.1%)和GC2(45.6%)的平均含量。Effective number of codon(ENC)分析与中性绘图分析结果显示,自然选择是影响嗜热毛壳菌密码子偏好性的主要因素。相关性分析结果显示,芳香族氨基酸比例与GC1含量及蛋白疏水水平呈极显著相关,说明密码子第一位的碱基组成对氨基酸是否具有芳香性影响较大。此外,在嗜热毛壳菌使用频率较高的密码子中,有24个以G/C末端结尾的密码子,进一步确定了23个高表达优越密码子和1个高表达最优密码子(CGC)。通过与其他模式真菌的密码子偏好性进行比较发现：与嗜热毛壳菌在密码子使用频率上差异较小的为嗜热毁丝菌、粗糙脉孢霉,有显著差异的为酿酒酵母。本研究为在嗜热毛壳菌中异源表达重组蛋白提供了目标基因密码子优化的理论依据,为嗜...     

一种嗜热厌氧纤维素降解细菌的分离纯化方法

根据纤维素降解细菌对不溶性纤维素底部的粘附作用,利用Ｈｕｎｇａｔｅ厌氧操作技术直接以不溶性纤维素粉为基质进行滚管,分离和纯化获得嗜热厌氧纤维素降解细菌。     

嗜热子囊菌JCM12803来源的双功能木聚糖/纤维素酶

纤维素和木聚糖的充分利用对于生物燃料的生产是非常重要的。文中利用PCR的方法从嗜热子囊菌Thermoascus crustaceus JCM12803中克隆到一个新颖的双功能木聚糖/纤维素酶基因Tcxyn10a,并将其在毕赤酵母Pichia pastoris GS115中实现高效异源表达。经过蛋白纯化和酶学性质研究分析,TcXyn10A的最适pH值和最适温度分别为5.0和65-70℃,能够在酸性至碱性(pH 3.0-11.0)条件下和60℃下保持稳定;对榉木木聚糖、小麦阿拉伯木聚糖、羧甲基纤维素钠和地衣多糖均有降解活性,比活分别为(1 480±26)U/mg、(2 055±28)U/mg、(7.4±0.2)U/mg和(10.9±0.4)U/mg;同源建模结构以及分子对接试验表明,双功能酶TcXyn10A只含有单一催化结构域,且木聚糖底物与纤维素底物共用一条催化通道。文中为探索双功能酶结构与其功能的关系提供了很好的素材。     

嗜热厌氧细菌Caldicellulosiruptor bescii降解木质纤维素研究进展

嗜热厌氧菌Caldicellulosiruptor bescii具有强大的木质纤维素降解能力,能以多种模式植物细胞壁多糖如微晶纤维素Avicel和木聚糖,甚至未经预处理的木质纤维素如柳枝稷作为唯一碳源快速生长,该菌还具有少见的厌氧降解木质素的能力。对基因组注释发现,该菌所编码的蛋白大多为多结构域双功能酶,即在多肽链的N端和C端分别是不同家族的糖苷水解酶,间隔以2-3个碳水化合物结合结构域。该菌降解纤维素相关的酶基因多集中于一个植物细胞壁多糖降解利用的基因簇,例如纤维素酶/木聚糖酶、纤维素酶/甘露聚糖酶和纤维素酶/木葡聚糖酶等。C.bescii的木聚糖酶主要属于GH10家族,该家族的酶底物特异性较为宽泛,氨基酸序列的同源性在18.7%-59.5%间。Caldicellulosiruptor属细菌进化出了一系列的机制使得糖苷水解酶和底物、细菌和木质纤维素能更好的吸附在一起,从而有利于木质纤维素的酶解。C.bescii有12个含SLH结构域的蛋白,以及新发现的黏附蛋白Tāpirin,可能参与了木质纤维素的吸附与利用。综述了近年来对C.bescii降解植物细胞壁的糖苷水解酶的基因资源挖掘方面和降解分子机制方面的研究进展,对高效、多功能高效木质纤维素降解酶的设计和优化具有积极的意义。     

一种嗜热厌氧纤维素降解细菌的分离纯化方法*

根据纤维素降解细菌对不溶性纤维素底物的粘附作用,利用Hungate厌氧操作技术直接以不溶性纤维素粉为基质进行滚管,分离和纯化获得嗜热厌氧纤维素降解细菌。     

解糖热解纤维素菌F32降解未预处理秸秆及产纤维素酶特性分析

【目的】明确极端嗜热厌氧木质纤维素降解菌解糖热解纤维素菌F32代谢特征,并分析其产酶特性。【方法】使用细胞计数法绘制菌株的生长曲线,使用离子色谱及气相色谱进行产物和残糖量分析,以DNS法及对硝基苯酚法检测菌株胞外蛋白的酶活性。【结果】解糖热解纤维素菌F32在以葡萄糖、微晶纤维素和未经预处理小麦秸秆为碳源时生长状况优于解糖热解纤维素菌DSM 8903。在以葡萄糖为碳源进行培养时,与菌株DSM 8903相比,菌株F32具有产乳酸较多,而产氢气较少的特点。在以微晶纤维素和未经预处理小麦秸秆为碳源进行培养时,与菌株DSM 8903相比,菌株F32胞外蛋白具有较高的内切纤维素酶活性和木聚糖酶活性。【结论】解糖热解纤维素菌F32表现出较强的木质纤维素降解能力,其与DSM 8903的产物组成及胞外蛋白的酶活性具有明显差异。     

原生质体融合子代的筛选和鉴定

介绍了绿色木霉N6和黑曲霉856原生质体融合子代的研究。经传代、发酵、筛选,从11株初筛的融合子中得到了3株纤维素酶活高且稳定的菌株AT23、AT16、AT34,其CMC酶活分别为亲本绿色木霉N6的2．2倍、1．4倍、1．2倍。并对其进行制霉菌素抗性试验和可溶性蛋白质凝胶电泳分析鉴定。试验结果证明了AT23、AT16、AT34是基因发生了重组的融合子且具有杂种优势。     

Molecular cloning,expression, and characterization of an endo-beta-1,4-glucanase cDNA from Epidinium caudatum1

An endo-beta-1,4-glucanase gene (epi3) from the rumen ciliated protozoan Epidinium caudatum was cloned from a cDNA library constructed by using the lambda ZAP II vector. The enzymatic activity of the gene product was detected by the Congo red assay, using carboxymethyl cellulose (CMC) as substrate. The nucleotide sequence of epi3 revealed 1,253 nucleotides with an open reading frame for a protein (Epi3) of 356 amino acids (Mr -41,014). Epi3 shows high homology with family 5 endoglucanase genes and with genes from protozoa isolated from sources other than the rumen. The specific activity of Epi3 produced in Escherichia coli was 5.544, 2.754, and 0.295 mmol of glucose min(-1) mg(-1) protein when the substrates used were CMC, beta-glucan, and xylan, respectively. A beta-1,4-linked trisaccharide of glucose was the preferred substrate of Epi3, as determined by analysis with the p-nitrophenyl form of the substrate. To our knowledge, this is the first report of the isolation of an endoglucanase gene from a rumen protozoan.     

Differential expression of protein S genes during Myxococcus xanthus development

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.     

登革病毒衣壳蛋白靶向核酸酶表达系统的建立及应用

根据登革 2型病毒衣壳蛋白C基因和葡萄球菌核酸酶SN基因序列设计引物 ,从构建的原核表达载体pLEX D2C SN中扩增获得编码登革病毒衣壳蛋白和葡萄球菌核酸酶的融合基因D2C SN ,将其插入到真核表达载体pcDNA6 V5 His中 ,筛选获得重组质粒pcDNA D2C SN .电穿孔转染BHK细胞后 ,5mg Lblasticidin压力筛选 ,通过RT PCR、间接免疫荧光和免疫印迹鉴定表达的蛋白 ,体外DNA消化试验检测核酸酶活性 .结果表明 ,融合蛋白D2C SN在BHK细胞中获得了稳定表达 ,表达的融合蛋白能够被抗登革病毒衣壳蛋白的单克隆抗体特异识别 ,并具有良好的核酸酶活性 ,能够对DNA进行切割 .同时 ,BHK细胞中稳定表达的融合蛋白D2C SN能够有效抑制登革病毒的增殖 ,使其感染性降低 10 3 ～ 10 4倍 .这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于人类抗登革病毒感染奠定了基础     

Expression of the cry11A gene of Bacillus thuringiensis ssp. israelensis in Saccharomyces cerevisiae

The complete cry11A region gene of Bacillus thuringiensis ssp. israelensis was fused in frame to the 3' end of the GST gene under the control of the Saccharomyces cerevisiae HXK1 promoter. The fusion protein GST-cry11A was expressed in S. cerevisiae strain AMW13C+. The fusion gene GST-cry11A was expressed when yeast cells were grown on galactose and a nonfermentable medium containing ethanol as carbon and energy source. When the cells were grown in glucose, mannose, fructose, or glycerol as carbon sources, the GST-cry11A gene was repressed. Thus, a regulated expression in accordance with the regulatory activity of the HXK1 gene promoter has been detected. The GST-cry11A fusion protein was detected in the transformed yeasts as a soluble protein. The fusion protein was purified by affinity chromatography using glutathione-Sepharose beads. Cell-free extracts from transformed yeasts grown in ethanol-containing culture media showed insecticidal activity against third-instar Aedes aegypti larvae. This insecticidal activity was increased about 4-fold when the purified fusion protein was assayed.     

人源Cdc25C蛋白的融合表达及纯化

目的:表达并纯化有活性的GST-Cdc25C融合蛋白,以用于Cdc25C功能研究。方法:利用RT-PCR克隆MCF-7细胞的cdc25c全长基因;在大肠杆菌中表达GST-Cdc25C融合蛋白;利用GSH交联的琼脂糖珠纯化GST-Cdc25C融合蛋白;通过体外磷酸酶活性分析检测GST-Cdc25C融合蛋白的磷酸酶活性。结果:克隆获得1465 bp的人源cdc25c全长基因,并克隆至pGEX-4T-1原核表达载体;在原核系统中可溶性表达了相对分子质量约87×103的GST-Cdc25C融合蛋白;通过亲和纯化获得的GST-Cdc25C融合蛋白具有较好的磷酸酶活性。结论:得到了有磷酸酶活性的GST-Cdc25C融合蛋白,可用于后续的Cdc25C功能研究。     

Evidence that the xylanase activity from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> Oα is encoded by the endoglucanase precursor gene (<Emphasis Type="Italic">sso1354</Emphasis>) and characterization of the associated cellulase activity

Sulfolobus solfataricus strain Oalpha was previously isolated for its ability to grow on minimal medium supplemented with xylan as a carbon source. The strain exhibited thermostable xylanase activity but several attempts to identify the gene encoding for the activity failed. Further studies showed that the xylanase displayed activity on carboxymethylcellulose (CMC) and the new activity was characterized. It exhibited an optimal temperature and pH of 95 degrees C and 3.5, respectively, and a half-life of 53 min at 95 degrees C. The enzyme, which was demonstrated to be glycosylated, hydrolyzed CMC in an endo-manner releasing cellobiose and other cello-oligomers. Analysis of the tryptic fragments by tandem mass spectrometry led to identification of the endoglucanase precursor, encoded by the sso1354 gene, as the protein possessing dual activity. The efficiency of the SSO1354 protein in degrading cellulosic and hemicellulosic fractions contained in agronomic residues was tested at low pH and high temperature. Cellulose and xylan were degraded to glucose and xylose at 90 degrees C, pH 4 by an enzyme mix consisting of SSO1354 and additional glycosyl hydrolases from S. solfataricus Oalpha. Given its role in saccharification processes requiring high temperatures and acidic environments, SSO1354 represents an interesting candidate for the utilization of agro-industrial waste for fuel production.     

Heterologous expression of endo-beta-1,4-D-glucanase from Clostridium cellulovorans in Clostridium acetobutylicum ATCC 824 following transformation of the engB gene.

Heterologous expression of the Clostridium cellulovorans engB gene by Clostridium acetobutylicum BKW-1 was detected as zones of hydrolysis on carboxymethyl cellulose (CMC) Trypticase glucose yeast plates stained with Congo red. The extracellular cellulase preparation from C. acetobutylicum BKW-1 has a specific activity towards CMC which is more than fourfold that present in C. acetobutylicum ATCC 824. Western blot (immunoblot) analysis using the C. cellulovorans anti-EngB primary antibody demonstrated that an additional 44-kDa protein band was present in the supernatant derived from C. acetobutylicum BKW-1 but was not present in ATCC 824 or ATCC 824(pMTL500E).     

人组织型纤溶酶原激活剂-水蛭素融合基因的构建及其在毕赤酵母中的表达

构建并表达兼有溶栓和抗凝活性、减少出血副作用的人组织型纤溶酶原激活剂(t-PA)和水蛭素(HV2)的融合蛋白。通过提取总RNA和RT-PCR获得t-PA基因,与HV2基因通过活化凝血因子X(Fxa)识别序列（IEGR）的对应碱基序列连接构成融合蛋白基因,融合蛋白基因经pGEM-T、pIC9克隆至表达载体pIC9K上,电转导入毕赤酵母（Pichia pastoris）GS115。转化子摇瓶内甲醇诱导表达。纤维蛋白平板溶圈法和纤维蛋白凝块法分别检测溶栓和抗凝活性。琼脂糖凝胶电泳结果显示克隆的t-PA基因片段大小为1700bp,序列测定结果表明其35位氨基酸由文献报道的精氨酸突变为色氨酸。限制性酶切和PCR鉴定结果均表明融合蛋白基因已克隆入表达载体和宿主菌。甲醇利用实验、G418抗性筛选获得多拷贝甲醇利用快型克隆。甲醇诱导表达产物具有纤溶活性并可被抗t-PA抗体抑制。完整融合蛋白无抗凝活性,但以Fxa裂解后可释放抗凝活性。同时,融合蛋白以单链和双链两种形式存在。融合蛋白在血栓部位特有的Fxa作用下靶向释放抗凝活性,具有溶栓抗凝双功能,有望降低临床出血副作用。