生孢噬纤维细菌的滤纸纤维素的降解过程研究

生孢噬纤维细菌(Sporocytophaga)是能够降解纤维素的滑动细菌,它可将滤纸和棉花纤维素完全降解;但其可测得的纤维素酶活性极低。为研究其纤维素降解机制,本文采用扫描电子显微镜观察了一株生孢噬纤维细菌(Sporocytophaga sp.) JL01对滤纸纤维素的降解过程,分析了在此过程中滤纸纤维素的降解与菌体形态变化之间的关系。结果表明：生孢噬纤维细菌在纤维素降解过程中,形成长度为2.5μm～4.0μm可弯曲的细长杆状细胞,该细长杆状细胞通过与纤维素分子的吸附或嵌入纤维素中完成对纤维素的降解;在降解后期,杆状细胞转化为直径约为0.6μm的小孢囊休眠细胞。     

改造稀有密码子提高SEA蛋白表达量

利用重叠PCR技术突变了sea基因上一个稀有密码子簇,将此段中稀有密码子全部更换成E.coli最常用密码子,得到sea^m。将sea和sea^m分别克隆于7ZTS表达载体上,并转化JM109（DE3）菌株。结果表明,sea基因的表达十分微弱,而sea^m基因的表达量十分高,约占菌体总蛋白的15 ％。表达产物在体内具有一定的抗肿瘤活性。     

含易降解有机质时对苯二甲酸(TA)厌氧降解动力学

含易降解有机物的对苯二甲酸有机废水在厌氧处理时,其中的易降解有机质通过甲烷发酵被优先利用,其中间代谢物对对苯二甲酸的降解有抑制作用,同时,对苯二甲酸自身的降解也存在底物抑制现象。本文建立了有易降解有机质存在时对苯二甲酸厌氧降解的抑制动力学模型方程：q=q_max SS+Ks[1+(I-0.86)/K_I,s,并利用非线性回归,确定了模型参数q_max=1972.0mgTA/gVSS·d;K_s=20.2844gTA/L;K_I,I=2.041gCOD/L;K_I,s=0.0108 gTA/L,实验数据对该动力学方程能较好拟合。在此基础上提出两段厌氧处理新工艺。     

贝壳状革耳菌和黄孢平革菌固体培养酶系比较

白腐菌黄孢平革菌(Phanerochaete chrysosporium) 与贝壳状革耳菌(Panus conchatus)在类似自然状态的固体培养条件下酶的分泌情况有 较大差异。P.conchatus和P.chrysosporium的主要木素降解酶分别是漆酶和锰过氧化物酶 ;两种菌均产生较高水平的木聚糖酶;P.conchatus在整个培养过程中所产生的内切葡 聚糖酶、微晶纤维素酶和纤维二糖酶活力均比P.chrysosporium相应酶的活力低得多, 尤其是内切葡聚糖酶。研究结果初步揭示了P.conchaus降解木素的主要酶系及选择性降 解木素的原因。     

褐腐真菌产生的羟基自由基HO˙对纤维素作用的研究

由褐腐真菌的典型菌株——密粘褶菌Gloeophyllum trabeum的胞外培养液中分离纯化得到一能还原Fe3 ,产生羟基自由基HO˙的多肽组分(称作Gt因子)。 采用HO˙特异性的抑制剂硫脲,对Gt因子产生的HO˙在纤维素降解中的作用进行了对照研究,结果表明Gt因子及其产生的HO˙在纤维素降解中起着重要的作用,为褐腐菌HO˙氧化降解纤维素机制假说的确立提供了一些依据。     

褐腐真菌产生的羟基自由基HO˙对纤维素作用的研究*

由褐腐真菌的典型菌株——密粘褶菌Gloeophyllum trabeum的胞外培养液中分离纯化得到一能还原Fe3+,产生羟基自由基HO˙的多肽组分(称作Gt因子)。 采用HO˙特异性的抑制剂硫脲,对Gt因子产生的HO˙在纤维素降解中的作用进行了对照研究,结果表明Gt因子及其产生的HO˙在纤维素降解中起着重要的作用,为褐腐菌HO˙氧化降解纤维素机制假说的确立提供了一些依据。     

土壤来源的五个苏云金芽孢杆菌新亚种的鉴定

从中国土壤中分离的大量苏云金芽孢杆菌菌株中鉴定出H42、H43、H56、H60及H62等5种新H血清型,并进行了形态、培养特征、生化反应、晶体蛋白质成分及毒力特性等项检测鉴定,鉴定了5个苏云金芽孢杆菌新亚种： Bacillus thuringiensis subsp. jinghongiensis (H42), B.thuringiensis subsp. guiyangiensis (H43),B.thuringiensis subsp. rongseni(H56),B.thuringiensis subsp. pingluonsis(H60)及B.thuringiensis subsp. zhaodongensis(H62) 。毒力生物测定证明5个新亚种的代表菌株对棉铃虫(Heliothis armigera)\,小菜蛾(Plutella xylostella)\,柳蓝叶甲(Plagiodera versicolora)幼虫均无毒力。H42、H43、H56、H60对埃及伊蚊(Aedes aegypti)\,斑须按蚊(Anopheles stephensi)及尖音库蚊(Culex pipiens)亦均无毒;H62对埃及伊蚊无毒,但对尖音库蚊与斑须按蚊有低毒。     

滇重楼寄生菌的研究

从滇重楼(Paris polyphylla var.yunnanensis)地下茎中分离和鉴定出两种细菌——蜡状芽孢杆菌(Bacillus cereus)和产碱假单胞菌(Pseudomonas alcaligenes),以及三种真菌——黑团孢霉(Periconia sp.)、白色厚顶孢霉(Pachnocybe albida)和重楼索霉(Hormomyces paridiphilus)。对蜡状芽孢杆菌、产碱假单胞菌和重楼索霉进行了液体培养并测定了胞外多糖含量,结果表明重楼索霉可分泌大量胞外多糖,这可能是导致滇重楼地下茎胶质化和多糖含量增加的原因。     

丝状真菌表达分泌系统中受体菌的构建

黑曲霉糖化酶高产菌株T21经紫外诱变后, 通过酪蛋白平板和蛋白酶活性测定筛选出胞外酸性蛋白酶活力仅为原株076%的菌株A.nigerT21-201,其生长特性和产糖化酶活力与原株基本一致。利用原生质体PEG法将含有报告基因vhb的表达分泌质粒Pgt10-vhb通过与选择标记质粒的共转化导入此蛋白酶部分缺陷株及其原株T21,检测在蛋白酶缺陷株Aspergillus niger T21-201 和原株T21中VHb的分泌表达,结果表明在A.nigerT21-201中VHb表达水平显著高于原株,但Northern blot却显示在两菌株中^vnb基因的转录水平近似,由此证明酸性蛋白酶缺陷对保护外源蛋白产生了显著效果。      

极端嗜热菌海栖热袍菌α-葡萄糖醛酸酶的高效表达和重组酶的纯化

α-葡萄糖醛酸酶作为木聚糖降解的限速酶之一,在木聚糖类半纤维素的生物转化中起着重要的作用。海栖热袍菌Thermotoga maritima是一个嗜极端高温的厌氧细菌,其产生的极耐热性酶类具有非常可观的工业应用前景。但热袍菌属Thermotoga的基因在大肠杆菌中的表达一般较困难。研究了T. maritima中的极耐热性α葡萄糖醛酸酶基因在大肠杆菌不同菌株中的表达水平及纯化技术。结果表明,稀有密码子AGA、AGG和AUA限制了该基因在大肠杆菌中的表达,在大肠杆菌BL21-CodonPlus(DE3)RIL可得到高效表达,重组蛋白表达量达20%,比酶活比野生菌株提高5倍;重组蛋白经热处理和金属Ni²⁺的亲和层析提纯后,达到了电泳纯,提纯倍数为5.1倍,收率为55.1%。对重组菌诱导表达条件的研究表明,营养丰富的TB培养基有助于重组菌的生长, 重组菌生长至OD₆₀₀为0.7～0.8时添加IPTG诱导5h后重组蛋白的表达量最高。     

Function and mechanism of a low-molecular-weight peptide produced by Gloeophyllum trabeum in biodegradation of cellulose

A special low-molecular-weight peptide named Gt factor, was isolated and purified via HPLC from the culture extract of the brown-rot fungus Gloeophyllum trabeum. It had high-affinity Fe(3+)-chelating ability and could reduce Fe(3+) to Fe(2+). In the presence of O(2), it could produce hydroxyl radicals HO*. The effects of Gt factor on cellulose degradation suggested that Gt factor could disrupt inter- and intra- hydrogen bonds in cellulose chains by a HO*-involved mechanism. This resulted in depolymerization of cellulose chains, which produced more reducing and non-reducing ends, thus making cellulose accessible for further degradation. This pathway was quite different from the hydrolytic processes driven by cellulases, and Gt factor might play an important role in the early stage of cellulose depolymerization by brown-rot fungi.     

密粘褶菌Gloeophyllum trabeum胞外低分子量多肽的分离纯化及其对纤维素生物降解作用

通过HPLC高效液相层析由褐腐真菌中能强烈降解木质纤维素的代表菌株密粘褶菌Gloeo phyllumtrabeum的胞外培养液 ,分离纯化得到一低分子量的活性多肽组分 (Gt因子 ) .Gt因子具有较好热稳定性 ,在pH 2 5～ 6 5范围内保持稳定 .Gt因子分子量在 4 0 0 0左右 ,等电点pI 6 6 .Gt因子具有络合Fe3 + 的能力 ,且能够将Fe3 + 还原为Fe2 + .在O2 存在时 ,能以纤维素物质为电子供体形成羟基自由基HO·.利用循环伏安法 ,观察到Gt因子与纤维素底物之间的氧化还原过程 .研究表明 ,Gt因子极有可能在褐腐菌的纤维素降解初期起着重要的作用 .     

褐腐真菌纤维素降解初期产生羟基自由基的普遍性研究*

本文通过对11株褐腐菌的研究,发现其普遍具有产生HO的特性;而且于褐腐菌的胞外培养液中均得到了部分分离纯化的、非酶的III组分,III组分普遍具有还原Fe3+为Fe2+的能力。III组分对纤维素底物的作用实验表明,HO的氧化机制在褐腐菌对纤维素降解的初期普遍存在,HO氧化断裂纤维素链内或链间的氢键,继而断裂糖苷键,使纤维素暴露出更多的还原性和非还原性末端,为纤维素酶的进一步降解提供有利条件。     

A peptide-mediated and hydroxyl radical HO*-involved oxidative degradation of cellulose by brown-rot fungi

A special low-molecular-weight peptide named Gt factor, was isolated and purified from the extracellular culture of brown-rot fungi Gloeophyllum trabeum via gel filtration chromatography and HPLC. It has been shown to reduce Fe³⁺ to Fe²⁺. Electron paramagnetic resonance (EPR) spectroscopy revealed Gt factor was able to drive H₂O₂ generation via a superoxide anion O₂ ^.- intermediate and mediate the formation of hydroxyl radical HO^. in the presence of O₂. All the results indicated that Gt factor could oxidize the cellulose, disrupt the inter- and intrahydrogen bonds in cellulose chains by a HO^. -involved mechanism. This resulted in depolymerization of the cellulose, which made it accessible for further enzymatic hydrolysis.     

Lignin degradation by a novel peptide, Gt factor, from brown rot fungus Gloeophyllum trabeum

Gt factor, a pure component isolated from Gloeophyllum trabeum, was used to decompose lignin materials. The radical intermediates, degradation products and structural changes of treated materials were analyzed by infrared spectrum, NMR and electron paramagnetic resonance, etc. The results indicate that Gt factor makes an oxidative attack on lignin via HO., which might be the initial step in lignocellulose degradation by brown rot fungus.     

Mechanisms of wood degradation by brown-rot fungi: chelator-mediated cellulose degradation and binding of iron by cellulose

Iron, hydrogen peroxide, biochelators and oxalate are believed to play important roles in cellulose degradation by brown-rot fungi. The effect of these compounds in an 'enhanced' Fenton system on alpha-cellulose degradation was investigated specifically in regard to molecular weight distribution and cellulose-iron affinity. This study shows that the degradative ability of an ultrafiltered low molecular weight preparation of chelating compounds isolated from the brown-rot fungus Gloeophyllum trabeum (termed 'Gt chelator') increased with increasing Gt chelator concentration when the FeIII to Gt chelator ratio was greater than about 30:1. When this ratio was less than 30:1, increasing Gt chelator concentration did not accelerate cellulose degradation. In excess hydrogen peroxide, cellulose degradation increased and then decreased with increasing iron concentration when FeIII was present in excess of the Gt chelator. The critical ratio of FeIII to Gt chelator varied depending on the concentration of hydrogen peroxide in the system. Increasing iron concentration above a critical iron:chelator ratio inhibited cellulose degradation. The optimum pH for cellulose degradation mediated by Gt chelator was around 4.0. A comparison of the effects of 2,3-DHBA (a chelator that reduces iron similarly to Gt chelator) and Gt chelator with respect to cellulose degradation demonstrated the same pattern of cellulose degradation. Cellulose-iron affinity studies were conducted at three pH levels (3.6, 3.8, 4.1), and the binding constants for cellulose-FeIII, cellulose-FeII and cellulose-FeIII in the presence of Gt chelator were calculated. The binding constants for cellulose-FeIII at all three pH levels were much higher than those for cellulose-FeII, and the binding constants for cellulose-FeIII in the presence of Gt chelator were very close to those for cellulose-FeII. This is probably the result of FeIII reduction to FeII by Gt chelator and suggests that chelators from the fungus may be able to sequester iron from cellulose and reduce it in near proximity to the cellulose and thereby better promote depolymerization. The free radical generating system described has potential for use in a variety of industrial processing and pollution control applications.     

粉红黏帚霉67-1菌株寄生核盘菌菌核的酶学动态研究

对粉红黏帚霉67-1菌株侵染核盘菌菌核过程的多种细胞壁降解酶活性进行了连续测定,以研究几丁质酶等在这一寄生互作体系中的可能作用。结果表明:葡聚糖酶活性变化表现活跃,且随寄生过程呈增加趋势,配对法T检验结果表明,第10d的处理与对照酶活性差异达到最大;几丁质酶、蛋白酶活性变化表现较低,而纤维素酶未检测得到。酶学动态变化与之前石蜡切片显微观察的结果在时间上表现一致;认为葡聚糖酶可能是粉红黏帚霉67-1菌株寄生核盘菌菌核的关键酶。     

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.     

Design of a fungal cellulose-binding domain for PCR amplification and expression in E. coli

A new fungal cellulose binding domain (CBD) from Stachybotris sp. has been cloned. Multiple sequence alignment of the CBD from 34 fungi shows highest sequence identity at the ends of the domains. The two primers from these regions were amplified by PCR giving a 120-bp product. Two of these, from Trichoderma sp. and Stachybotris sp. were subsequently cloned, sequenced and confirmed to be of the CBD family. The CBD from Stachybotris sp. was expressed in E. coli fused to g3p of the M13 phage and with a c-myc tag. The secreted fusion protein adsorbed on acid-swollen cellulose thereby confirming its functionality.