球毛壳菌(Chaetomium globosum)NK102 纤维素酶基因及其表达条件

[目的]生物质的利用是当前生物技术研究的一个热点.本小组分离到一株高效降解纤维素球毛壳菌(Chaetomium globosum)NK102,本文拟探索研究此菌的纤维素酶表达系统并寻找影响酶基因表达的关键因素.[方法]通过对NK102测序,本文界定了球毛壳菌NK102的主要纤维素酶编码基因,使用数字基因表达谱升级版(RNA-Seq)的方法得到纤维素酶基因的表达差异,然后观察了营养、物理条件下纤维素酶基因表达和酶活性变化的情况.[结果]发现随着培养时间的延长,纤维素酶基因整体上表达量升高.在所选基因中,外切葡聚糖酶、纤维二糖脱氢酶和内切葡聚糖酶基因(cbh1,cdh和egl1)的表达量最高.糖代谢的负调控因子ACE I和CreA的随时间表达量均降低,而Hap2/3/5复合体的表达量反而升高.之后检测了不同碳源培养基对纤维素酶基因表达量和酶活性的影响,发现葡萄糖为强阻遏因子,纤维二糖为其诱导物,而山梨醇没有影响.特别是,我们发现光照也影响纤维素酶基因的表达,黑暗条件明显抑制酶基因的表达.[结论]转录组学的方法可以初步探索纤维素酶表达的规律,酶基因的表达受到营养、物理条件的影响.本研究为揭示球毛壳菌降解纤维素分子机理和阐释生物质糖代谢途径提供了有用参考.     

斜卧青霉114-2 cbh1基因的TAIL-PCR克隆及与抗阻遏突变株JU-A10的比较

[目的]研究斜卧青霉(Penicillium decumbens)114-2与其抗阻遏突变株JU-A10外切酶基因序列的差异.[方法]用热不对称交错PCR(TAIL-PCR)和RT-PCR扩增得到斜卧青霉114-2外切葡聚糖酶Ⅰ(cbh1)基因全长和cDNA全长.[结果]cbh1基因全长为1500 bp,含有两个内含子,编码453个氨基酸(GenBank,EF397602).克隆并分析了1.9 kb的cbh1基因上游序列,分别发现了葡萄糖代谢抑制因子CRE Ⅰ与纤维素酶转录调控蛋白ACE Ⅰ的两个的潜在结合位点.[结论]在相同的培养条件下,其抗阻遏突变株JU-A10的外切酶活明显高于野生株114-2.两菌株的cbh1基因序列完全一致,说明外切酶活明显提高不是由于cbh1基因发生突变引起的.     

真菌和细菌纤维素酶的差别及内、外切葡聚糖苷酶的底物专一性

通常认为纤维素的降解过程,首先是纤维素酶（纤酶）分子吸附到纤维素表面,然后,内切葡聚糖苷酶（内切酶）在葡聚糖链的随机位点水解底物,产生寡聚糖;外切葡聚糖苷酶（外切酶）,从葡聚糖链的非还原端进行水解,主要产物为纤维二糖,需要两类酶的"协同"才能完成对纤维素的降解。纤维素酶分子由催化结构域（ｃａｔａｌｙｔｉｃｄｏｍａｉｎ,ＣＤ）、纤维素结合结构域（ｃｅｌｌｕｌｏｓｅ－ｂｉｎｄｉｎｇｄｏｍａｉｎ,ＣＢＤ）和一个连接桥（ｌｉｎｋｅｒ）三部分组成。近年来,纤维素酶分子结构与功能的研究取得了实质性的进展。不同…     

产外切葡聚糖酶真菌的筛选鉴定及毕赤酵母中的表达

【目的】外切葡聚糖酶是纤维素酶组分中一类对结晶纤维素有降解作用的酶类,如何提高外切葡聚糖酶活力是研究的关键问题。【方法】从筛选鉴定得到的一株产外切葡聚糖酶酶活较高的黑曲霉Asp-524菌株出发,通过PCR技术克隆得到外切葡聚糖酶基因序列,生物学信息分析后,构建了毕赤酵母诱导型表达载体,实现了该基因在毕赤酵母中的成功表达。【结果】抗性筛选得到的阳性转化子,用终浓度为1%甲醇诱导5 d后,酶活达到4.74 U/mL。酶学性质分析显示重组外切葡聚糖酶最适pH为5.0,pH稳定性分析显示在pH为4.0-6.0范围内相对稳定,酶活能保持在最高酶活力的80%以上,最适反应温度为50°C,经60°C保温1 h后,酶活仍能保持80%以上。【结论】结果说明该外切葡聚糖酶具有较好的热稳定性和pH稳定性,这一研究为纤维素酶的实际应用奠定了一定基础。     

纤维素酶滤纸酶活测定方法的改进

微生物的纤维素酶是多组分的酶系。用粘度法测CX酶活(内切β-1,4-葡聚糖酶)和以水杨苷等为底物测定β-葡萄糖苷酶活都能不受其它组分的影响而准确测得～纤酶系中这两个组分的活力。在菌种选育和酶的生产中,主要需了解一纤酶系作用于纤维性材料时产生还原糖的能力,这是C1酶(外切β-1,4-葡聚糖纤维二糖水解酶)与上述     

一株纤维素降解真菌的筛选及鉴定

[目的]分离筛选高效降解纤维素的真菌菌株,并研究其产酶能力.[方法]利用刚果红染色法从甘蔗地土壤中分离纤维素降解真菌,再通过测定滤纸的降解率及发酵酶活复筛.[结果]综合考虑水解圈,水解圈和菌株直径的比值(HC值),滤纸的降解率和复筛酶活,对试验真菌降解纤维素的能力进行综合评价,筛选到具有较强纤维素降解能力的真菌菌株SJ1,经形态学观察及分子生物学鉴定,该菌属于草酸青霉.其滤纸酶活、内切葡聚糖酶酶活(CMC酶活)、β-葡聚糖苷酶酶活和外切葡聚糖酶酶活(CBH酶活)分别为25.15、740.42、58.03和2.442 U/mL.[结论]菌株SJ1是一株十分具有研究开发潜力的纤维素酶生产菌株.     

造纸废水纸浆沉淀物中未培养微生物纤维素酶基因的克隆和鉴定

提取纯化造纸废水纸浆沉淀物的宏基因组DNA并构建16S rDNA文库,系统发育分析显示该环境中存在大量的未培养细菌且具有种类的多样性。以柯斯质粒为载体构建了1个含10000个克隆的宏基因组文库,文库容量为3.53×108bp。筛选文库得到2个表达内切葡聚糖酶活性的克隆、3个表达外切葡聚糖酶活性的克隆和2个表达β-葡萄糖苷酶活性的克隆。从表达不同活性的克隆中分别挑选活性最强的进行鉴定,得到3个新的纤维素酶基因umcel5L、umcel5M和umbgl3D。umcel5L、umcel5M和umbgl3D分别编码产生内切葡聚糖酶、纤维糊精酶和β-葡萄糖苷酶,其编码产物与已报道的纤维素酶一致性最高的分别为43%、48%和46%。这是第一次采用未培养方法对造纸废水纸浆沉淀物中的细菌多样性进行分析并从中克隆纤维素酶基因的报道。     

纤维素酶高产菌株的复合交替诱变选育

以里氏木霉ZM-4为出发菌株,研究了紫外诱变、硫酸二乙酯诱变以及紫外与硫酸二乙酯复合交替诱变等不同诱变方法对其产纤维素酶能力的影响,力求得到高产纤维素酶突变株.结果表明,复合交替诱变的正突变率最高,达45.98%.其中,突变株ZM4-F3具有最高的产酶能力,其滤纸酶活值达11.71U,比出发菌株ZM-4提高了19.75%.对ZM4-F3产纤维素酶酶系进行了详细分析,发现其葡聚糖内切酶、葡聚糖外切酶及β-葡萄糖苷酶酶活均较出发菌株ZM-4有显著提高,其中以β-葡萄糖苷酶酶活增幅最大,达58.3%.利用ZM4-F3降解稻草96h,还原糖产量达2.231g/L,比出发菌株提高了21.7%;利用ZM4-F3降解稻草144h,纤维素分解率和稻草分解率分别达53.01%和68.32%,比出发菌株分别提高了20.2%和14.0%.在对ZM4-F3进行6代连续培养后,仍能保持较高及较稳定的产酶能力,可以应用于工业生产.     

不同产纤维素酶菌种特定底物培养产酶活力比较

纤维素酶是由几种不同酶组成的复合酶系,由于酶催化反应底物的复杂性,从不同霉菌和不同底物发酵分离得到的纤维素酶组分和酶活力有着很大差别.本论文的主要目的主要是针对不同底物和不同霉菌进行产纤维素酶活力比较评价.实验选用CGMCC3.3002 和CICC13048 两种菌,使用液体发酵法在微晶纤维素和糠醛渣两种底物上进行产酶培养,在特定的时间测定其产的纤维素酶的纤维二糖酶活力、内切葡聚糖酶活力、外切葡聚糖酶活力以及滤纸酶活.产滤纸酶活比较高的菌株CGMCC3.3002,以糠醛渣为特定底物的滤纸酶活要高于微晶纤维素特定底物,且CGMCC3.3002 在微晶纤维素和糠醛渣底物的酶活力差异较大,该菌株可通过紫外诱变使其酶活力更高,有望用于糠醛渣生产燃料乙醇的过程中.     

黑曲霉纤维二糖水解酶基因cbhA在毕赤酵母中异源表达

[目的]对黑曲霉纤维二糖水解酶cbhA基因进行了克隆和在毕赤酵母中的真核表达。[方法]采用PCR方法扩增黑曲霉纤维二糖水解酶A(Cellobiohydrolase A,CBHA)基因,获得的DNA序列与cbhA基因表现出高度相似,推导出的氨基酸序列与真菌CBHA酶也高度相似,属于糖基水解酶第7家族。将扩增得到的cbhA基因克隆到毕赤酵母表达载体pPIC9K上,与α-因子信号肽序列形成融合蛋白,进一步通过电转化方法将线性化质粒p PIC9K-cbhA转化至毕赤酵母GS115菌株进行表达。[结果]在甲醇诱导下,重组菌株CMC比酶活力是对照的2.5倍,SDS-PAGE分析结果也确认了cbhA基因在重组菌株GS115/p PIC9K-cbhA中的表达。对该酶性质的分析表明,重组CBHA酶水解CMC底物最适p H值为5.0,最适温度为55℃。[结论]黑曲霉纤维二糖水解酶基因cbhA的克隆和其真核表达工程菌株的构建,为获得纤维二糖水解酶A高产菌株,实现纤维素酶多组分的人工组装奠定了基础。     

Purification and properties of two exo-cellobiohydrolases from a cellulolytic culture of Fusarium lini

Two distinct exo-cellobiohydrolases (1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) have been isolated from culture filtrates of Fusarium lini by repeated ammonium sulphate fractionation and isoelectric focusing. The purified enzymes were evaluated for physical properties, kinetics and the mechanism of their action. The results of this work were as follows. (1) A two-step enzyme purification procedure was developed, involving isoelectric focusing and ammonium sulphate fractionation. (2) Yields of pure cellobiohydrolases I and II were 45 and 36 mg l^?1 of culture broth, respectively. (3) Both enzymes were found to be homogeneous, as determined by ultracentrifugation, isoelectric focusing, electrophoresis in polyacrylamide gels containing SDS and chromatography on Sephadex. (4) The molecular weights of the two cellobiohydrolases, as determined by gel filtration and SDS gel electrophoresis, were 50 000–57 000. (5) Both cellobiohydrolases had low viscosity-reducing and reducing sugar activity from carboxymethyl cellulose and high activity with Walseth cellulose and Avicel. (6) The enzymes produced only cellobiose as the end product from filter paper and Avicel, indicating that they are true cellobiohydrolases. (7) Cellobiohydrolase I hydrolysed d-xylan whereas cellobiohydrolase II was inactive towards d-xylan. (8) There was a striking synergism in filter paper activity when cellobiohydrolase was supplemented with endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21).     

Purification and characterization of two cellobiohydrolases from Chaetomium thermophile var. coprophile

Cellobiohydrolases I and II were purified to homogeneity from culture filtrates of a thermophilic fungus, Chaetomium thermophile var. coprophile, by using a combination of ion-exchange and gel filtration chromatographic procedures. The molecular weights of cellobiohydrolase I and II were estimated to be 60,000 and 40,000 and the enzymes were found to be glycoproteins containing 17 and 22.8% carbohydrate, respectively. The two forms differed in their amino-acid composition mainly with respect to threonine, alanine, methionine and arginine. Antibodies produced against either form of cellobiohydrolases failed to cross-react with the other. The tryptic maps of the two enzymes were found to be different. The temperature optima for cellobiohydrolase I and II were 75 and 70 degrees C, and they were optimally active at pH 5.8 and 6.4, respectively. Both enzymes were stable at higher temperatures and were able to degrade crystalline cellulosic materials.     

Exo- and endo-glucanolytic activity of cellulases purified from Trichoderma reesei

Four cellulases, produced by Trichoderma reesei, have been purified by preparative isoelectric focusing (Rotofor), size exclusion (Sephacryl 100 HR), anionic (Mono Q) and cationic (Mono S) chromatography and chromatofocusing (Mono P). Enzymatic activity with a large number of substrates allowed the proteins to be classified as: cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II. The exo- or endo-glucanase character of these enzymes was analysed by using a technique based on the measurement of the Avicel insoluble fibres reducing power. © Rapid Science Ltd. 1998     

Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.     

Purification of the cellulase complex produced byPenicillium camemberti and its partial characterization

Three cellulase components (FP-ase, CMC-ase and cellobiase) were purified by affinity binding on Avicel followed by Sephadex G-25, DEAE-Sepharose, DEAE-cellulose and Sephadex G-100 chromatography from the culture filtrate of the newly isolated strain Penicillium camemberti. The isolated enzymes had the properties of cellobiohydrolase, endo-1,4-beta-D-glucanase and cellobiase and their respective molar masses were 99, 87 and 61 kDa as determined by molecular sieve chromatography on Sephadex G-100. The amino acid composition of each fraction was also determined.     

Cellobiohydrolase from Trichoderma reesei.

A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.     

Comparison of four purified extracellular 1,4-beta-D-glucan cellobiohydrolase enzymes from Trichoderma viride.

Four electrophoretically distinct 1,4-beta-D-glucan cellobiohydrolase enzymes (exo-cellobiohydrolase, EC 3.2.1.91) from Trichoderma viride have been purified to homogeneity. Three enzymes (A, B, and C) were from a commercial T. viride preparation whereas the other (D) was from T. viride QM 9123 grown on cellulose in submerged culture. The enzymes were similar with respect to ultraviolet light absorption, amino acid and amino sugar composition, heat stability, molecular weight, specific activity, and carboxyterminal residues, indicating very nearly identical polypeptide portions. The enzymes also exhibited immunological cross-reactivity. The enzymes differed most in the content and composition of covalently bound neutral carbohydrate.     

Development of a screening method for the indentification of a novelSaccharomyces cerevisiae mutant over-expressingTrichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L^?1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.     

Cellulase from Fusarium solani: Purification and properties of the C1 component

The C₁ component from Fusarium solani cellulase was purified extensively by molecular-sieve chromatography on Ultrogel AcA-54 and ion-exchange chromatography on DEAE-Sephadex. The purified component showed little capacity for hydrolysing highly ordered substrates (e.g., cotton fibre), but poorly ordered substrates (e.g., H₃PO₄-swollen cellulose), and the soluble cello-oligosaccharides cellotetraose and cellohexaose, were readily hydrolysed; cellobiose was the principal product in each case. Attack on O-(carboxymethyl)cellulose, a substrate widely used for measuring the activity of the randomly acting enzymes (C_x enzymes) of the cellulase complex, was minimal, and ceased after the removal of a few unsubstituted residues from the end of the chain. These observations, and the fact that the rate of change of degree of polymerisation of H₃PO₄-swollen cellulose was very slow compared with that effected by the randomly acting endoglucanases (C_x, CM-cellulases), indicate that C₁ is a cellobiohydrolase. Fractionation by a variety of methods gave no evidence for the non-identity of the cellobiohydrolase and the component that acted in synergism with the randomly acting C_x enzyme when solubilizing cotton fibre.     

Purification and partial characterisation of three endo-glucanases from dichomitus squalens

Three endo-glucanases (En I-III) were obtained by chromatofocusing fractionation of a culture supernatant from the white-rot fungus Dichomitus squalens. They were purified further on Phenyl-Sepharose CL-4B, DEAE-Trisacryl, and Ultrogel AcA 54; ∼21-, ∼16-, and ∼9-fold purifications were obtained for En I-III, respectively. The enzymes appeared as homogeneous proteins on disc gel electrophoresis with and without SDS (sodium dodecyl sulphate), and on isoelectric focusing; the respective mol. wts. were 42,000, 56,000, and 47,000, and the isoelectric points 4.8, 4.3, and 4.1. Optimum conditions for the hydrolysis of CM-cellulose were pH 4.8 and 55° for each enzyme, and each was stable over the pH range 4.0–8.5 but inactivated completely within 30 min at 70°. None of the purified enzymes exhibited β-d-glucosidase or cellobiohydrolase activity, but En II was weakly active towards laminaran and xylan. En I and En II acted more randomly on CM-cellulose than did En III. Cellotetraose was degraded by each endo-glucanase, whereas only En III could hydrolyse cellotriose.