绿色木霉纤维素酶系中C1酶的提纯与性质

从绿色木霉(Trichoderma viride) X2-85的麸曲抽提液中,分离出纤维素酶系中的C1酶,经纯化后,用聚丙烯酰胺凝腔电泳和超速离心鉴定,都为均一蛋白。在我们的实验条件下,它对羧甲基纤维素、3-葡萄糖苷及纤维二糖,都不表现活性。该酶能降解徽晶纤维紊、磷酸膨胀纤维素和脱脂棉,主要产物是纤维二糖。用Sephadex G一100凝胶过滤和SDS一聚丙烯酰胺凝胶电泳测定其分子量,分别为54,000和55,000。其沉降系数为4.18。     

花椰菜花叶病毒的酶联免疫吸附分析

我们用酶联免疫吸附分析(ELISA)的双抗体夹心法,研究了花椰菜花叶病毒(CaMV)的定量测定。以提纯的CaMV免疫家兔,得到教价为1：2000的抗血清。经硫酸铵沉淀、DEAE-纤维素(DE-32)离子交换层析纯化了IgG抗体。用于标记的酶为辣根过氧化物酶。该酶经硫酸铵沉淀、DEAE-纤维素(DE一32)离子交换层析、刀豆球蛋白A-琼脂糖4B(ConcanavalinA-Sepharose 4B)亲和层析纯化,使RZ值达2.6。标记抗体制备用过碘酸盐氧化法。     

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。     

洗涤剂用碱性纤维素酶的研究进展

迄今报道的绝大多数纤维素酶的最适pH都在酸性和中性范围 ,当添加到洗涤剂中 ,由于处于碱性环境而无活力 ,不能发挥作用。近年来 ,国内外对由碱性芽孢杆菌 (Bacillussp )产生的碱性纤维素酶 (CMCase,endo β1 ,4 glucanase,EC 3 2 1 4)进行了广泛的研究。对该酶产生菌株的筛选和培养条件、酶学性质 ,以及该酶基因的克隆和表达等方面的研究进行综述 ;并对我国目前未能实现该酶工业化原因进行了初步分析 ,并提出解决途径。     

斜卧青霉纤维素酶系的酶学研究

使用包括疏水相互作用色谱在内的多种色谱分离技术,对斜卧青霉的纤维素酶进行了系统的分离纯化。共分得一种β一葡萄糖苷酶,六种β一葡聚糖纤维二糖水解酶和八种内切β一葡聚糖酶组分。其中六种分别进一步分离为二到六个亚组分。多数达到电泳纯。系统地研究了各组分的分子组成和酶学特征。并用计算机对各酶组分及其它来源的纤维素酶组分的氨基酸组成进行了绕计分析,推论了它们之间的相互关系。据以提出转译后修饰的假说,以解释纤维素酶系多组分性和微异质性产生的原因。     

木耳属种间杂交技术研究*I．木耳菌丝原生质体的形成与再生

本文比较了不同酶液、渗透压稳定剂、酶解媪度及菌丝培养基成份等因素对木耳属(Auricularia)中木耳(Auricularia auricula)和毛木耳(Auricularia polytricha)菌丝释放原生质体的作用及影响。用0.5％纤维素酶加0.5％蜗牛酶的混鸯酶液,以0.6M的MgSO4为稳定剂,在34℃下可自两种菌丝体获得大量原生质体。对原生质体再生条件的研究表明,纤维二糖和菌丝体培养物浸提物对再生有明显促进作用,再生率达20％左右。本文还用VBL型荧光增白剂观察了菌丝脱壁以及原生质体细胞壁再生的过程。     

绿色木霉葡聚糖内切酶EGⅢ基因在大肠杆菌中的表达、定点突变及其动力学特性的研究

将绿色木霉葡聚糖内切酶EGⅢ基因亚克隆到表达载体pET-22b（＋）,构建重组质粒pET-egl3,转化到大肠杆菌BL21（DE3）。利用金属亲和层析对重组EGⅢ进行纯化,纯化后酶比活力达到6u／mg蛋白,最适反应温度为60℃,最适pH为4．0。同时对EGⅢ催化区的氨基酸残基R130和E218进行定点饱和突变,各筛选到一株酶活有提高的突变子R130P和E218F,其比活力为野生型EGⅢ的2．8倍和3．45倍。突变酶E218F的Km提高了一倍,催化效率Kcat提高了5．4倍;而R130P的Km和Kcat没有明显变化。两个突变酶的最适酶解温度和pH分别都提高至65℃和4．4。     

Fusarium oxysporim M1菌株的rDNA ITS鉴定及其纤维素酶活性及纯化研究

以1株分离于北大仓白酒大曲的产纤维素酶真菌M1为材料,对其进行了形态及分子生物学鉴定;纯化并研究了其纤维素酶的酶学性质。以真菌ITS1/ITS4通用引物,扩增真菌M1的rDNA ITS序列,再与GenBank中其他菌株rDNA ITS序列进行比对,使用Mega5.0软件,采用最大似然法进行聚类分析。结果显示,该真菌同已经报道的Fusarium oxysporim strain Bt3聚为一类,一致率达99%,与形态学方法鉴定一致,命名为Fusarium oxysporum M1。该菌具有很高纤维素酶活力,FPA和CMCA分别高达16.84 IU/mL和35.31 IU/mL。经过发酵条件优化酶活性进一步提高。经硫酸铵分级分离、疏水和离子交换层析,纯化了该菌纤维素酶,纯化倍数高达17.97倍,得率为3.676%,SDS-PAGE分析表明,该纤维素酶分子量达60 k Da。本研究为进一步研究该酶高效催化机理及实际应用提供参考。     

纤维素酶在木质纤维素生物质转化中的应用研究

选育得到纤维素酶高产菌株里氏木霉突变菌株(Trichoderma reesei) 813A,优化了其发酵产酶条件。利用该菌株所产纤维素酶对天然木质纤维素的水解糖化过程进行研究,确定了实验条件下最优的糖化条件(温度50℃, pH 4.5,酶浓度6～8 FPU/mL,底物浓度2%)。以玉米叶和杨树叶为天然纤维素原料,水解糖化率分别达到86.2％和56.0%。通过酿酒酵母(Saccharomyces cerevisiae)将糖化液转化为酒精,产乙醇浓度达到 5%～5.8%,转化率为79.4%～92.1%。     

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。     

Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography

Eight chemically modified cellulose supports were tested for their ability to absorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography.     

Screening of cellulases for biofuel production: online monitoring of the enzymatic hydrolysis of insoluble cellulose using high-throughput scattered light detection

A new prospective cellulase assay simultaneously combining high-throughput, online analysis and insoluble cellulosic substrates is described. The hydrolysis of three different insoluble cellulosic substrates, catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast), was monitored using the BioLector - allowing online monitoring of scattered light intensities in a continuously shaken microtiter plate. Cellulase activities could be quantitatively assayed using the BioLector. At low cellulase/cellulose ratios, the Michaelis-Menten parameters of the cellulase mixture were mainly affected by the crystallinity index of the cellulose. Here, the apparent maximum cellulase activities inversely correlated with the crystallinity index of the cellulose. At high cellulase/cellulose ratios the particle size of the cellulose, defining the external surface area accessible to the cellulases, was the key determining factor for cellulase activity. The developed technique was also successfully applied to evaluate the pH optimum of cellulases. Moreover, the non-hydrolytic deagglomeration of cellulose particles was investigated, for the first time, using high-throughput scattered light detection. In conclusion, this cellulase assay ideally links high-throughput, online analysis and realistic insoluble cellulosic substrates in one simple system. It will considerably simplify and accelerate fundamental research on cellulase screening.     

2种纤维素酶水解效果比较及酶蛋白组分分析

比较了自产纤维素酶和商品纤维素酶的水解效果,并采用超滤、层析、SDS-PAGE相结合的方法分析2种纤维素酶蛋白组分的差异。里氏木霉以纸浆为C源合成的自产纤维素酶的水解得率高于商品纤维素酶,自产纤维素酶水解48h的得率为66.24%,商品纤维素酶的得率为52.19%。自产纤维素酶中存在着Cel6A酶组分和XYNⅡ酶组分,而商品纤维素酶中没有检测到这2种酶组分。自产纤维素酶和商品纤维素酶的Cel1A酶组分和Cel7A酶组分间存在着分布和含量上的差异。自产纤维素酶在相对分子质量(2.5～3.5)×104范围内存在着几条蛋白条带,而商品纤维素酶则是在相对分子质量3.5×104附近存在着几条蛋白条带。     

Purification and properties of an endo-cellulase of avicelase type from Irpex lacteus (Polyporus tulipiferae).

A culture filtrate of Irpex lacteus (Polyporus tulipiferae) was fractionated initially by salting out with ammonium sulfate, and a cellulase [EC 3.2.1.4.] fraction with high Avicel-hydrolyzing activity (formerly called Avicelase) was extensively purified by a series of column chromatography procedures. This purified endo-cellulase showed a less random hydrolytic mechanism, and was obtained in a yield of 0.04% with respect to the starting material. Its specific activity was enhanced approximately 30 times over that of the starting material. The cellulase component showed a single peak on both ultracentrifugal and acrylamide disc electrophoretic analyses. Its molecular weight was estimated to be 56,000. It contained 12.2% carbohydrate; the major sugar constituents were glucose and mannose. Regarding the amino acid composition, the contents of aspartic acid and glycine were highest, followed by those of glutamic acid, serine, and theonine. The cellulase component was not markedly inhibited by most metal ions tested excepted for Hg2+. This purified endo-cellulase attacked a series of cellooligosaccharides, beta-cellobioside, CM-cellulose, and insoluble, cellulosic substrates. In the digests from insoluble substrates, glucose, cellobiose, cellotriose, and cellotetraose were detectable, but the amount of cellobiose was the largest by far. In constrast, cellobiose and glucose were produced in almost equal amounts from beta-cellobioside.     

Purification and characterization of cellulolytic enzymes fromTrichoderma harzianum

Two forms of filter paper activity (filter paper activity; cellulose 1,4-β-cellobiosidase, EC 3.2.1.91) and single forms of CM-cellulase (carboxymethyl cellulase; endo-l,4-β-glucanase, EC 3.2.1.4) and β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) from the culture filtrate ofTrichoderma harzianum were separated and partially purified by (NH₄)₂SO₄ precipitation, ion-exchange chromatography and gel filtration. The final preparation was purified about 12-, 20- and 27-fold for FP-activity, CM-oellulase and β-glueosidase, respectively. The pH and temperature optima, stability, kinetic parameters, effeet of metal ions and molar mass of each was determined. A distinct type of synergistic action between cellulase components was observed for efficientin vitro saccharification of dewaxed cotton.     

The determination of cellulase activity by gas--liquid chromatography.

A new procedure for the determination of cellulase activity is described. The cellulases are incubated for 30 min at 39°C with finely divided cellulose at pH 6.9, and the glucose and cellobiose produced during the incubation are silylated and measured by gas-liquid chromatography. The precision of various steps in the procedure are determined and the optimum conditions for the enzyme assay are established. The coefficient of variation for the assay was 2.4 to 4.5%, depending on the substrate used. Although the method is specifically developed for the measurement of cellulase activity in mixed enzyme preparations from sheep rumen contents, it is applicable for the determination of other cellulases.     

A New Technique for Localization of Cellulase in situ Using Silver Nitrate

A new staining technique has been developed for the histochemical localization of cellulase in plant tissues by light microscopy. The products of cellulolysis are reducing sugars which can reduce the salts of heavy metals under appropriate conditions. The present technique relies on the deposition of black silver oxide due to reduction of alkaline silver nitrate to detect cellulase in tissues.     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Multicomplex cellulase-xylanase system of Clostridium papyrosolvens C7.

The cellulase system of Clostridium papyrosolvens C7 was fractionated by means of ion-exchange chromatography into at least seven high-molecular-weight multiprotein complexes, each with different enzymatic and structural properties. The molecular weights of the complexes, as determined by gel filtration chromatography, ranged from 500,000 to 660,000, and the isoelectric points ranged from 4.40 to 4.85. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the complexes showed that each complex had a distinct polypeptide composition. Avicelase, carboxymethyl cellulase, and xylanase activity profiles differed from protein complex to protein complex. Three of the complexes hydrolyzed crystalline cellulose (Avicel). Activity zymograms of gels (following electrophoresis under mildly denaturing conditions) revealed different carboxymethyl cellulase-active proteins in all complexes but xylanase-active proteins in only two of the complexes. The xylanase specific activity of these two complexes was more than eightfold higher than that of the unfractionated cellulase preparation. A 125,000-M(r) glycoprotein with no apparent enzyme activity was the only polypeptide present in all seven complexes. Experiments involving recombination of samples eluted from the ion-exchange chromatography column indicated that synergistic interactions occurred in the hydrolysis of crystalline cellulose by the cellulase system. We propose that the C. papyrosolvens enzyme system responsible for the hydrolysis of crystalline cellulose and xylan is a multicomplex system comprising at least seven diverse protein complexes.     

Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate

With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.