纤维二糖水解酶I基因的系统进化分析

纤维二糖水解酶I（CBHI）是生物降解纤维素的一种重要的外切酶,它作用于纤维素分子末端,水解β-1,4-糖苷键。纤维二糖水解酶由3个部分组成：具有催化活性的催化结构域,作用为锚定纤维素的纤维素结合域以及连接这两个结构域的一段短肽。已知催化结构域属于糖基水解酶家族7（GH7）,纤维素结合域属于糖类结合模块家族1（CBMl）。为进一步探索CBHI编码基因之间的进化关系,本研究依据CBHI的结构域在GenBank数据库中搜索并鉴定CBHI编码基因并据此构建系统发育树。序列的平均长度为1776bp,平均GC含量为57．64％,平均转换颠换比为0．71,平均遗传距离为0．424。得出结论CBHI编码基因只存在于真菌中,是一个相对活跃的基因,它的进化与物种的进化有着密切的关系。     

Antitumor drugs possessing topoisomerase I inhibition: applicable separation methods

Separation methods for antitumor drugs capable of topoisomerase I inhibition were reviewed in this study. Camptothecin (CPT) its related analogues seemed to be promising anticancer drugs that exhibit topoisomerase I inhibition. This group of compounds contain a closed α-hydroxy-δ-lactone ring (lactone form) that can undergo reversible hydrolysis to form the open-ring form (carboxylate form). In vitro pharmacological study showed that the antitumor activity of the lactone form was higher than that of the carboxylate form. Thus a quantitative method to separate these two forms is important to evaluate the pharmacokinetics and pharmacodynamics of these compounds. Nevertheless, current separation methods are complicated by the pH-dependent instability of the lactone moiety. High-performance liquid chromatography (HPLC) coupled with fluorometric detection has been widely used for the quantitation of the drug as the intact lactone form or as the total lactone carboxylate forms in biological matrices. In this report we reviewed current applicable chromatographic techniques for further bioanalytical studies of CPT derivatives including sample preparations, HPLC columns, mobile phases and additives.     

Computational simulations of the Trichoderma reesei cellobiohydrolase I acting on microcrystalline cellulose Iβ: the enzyme-substrate complex

Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei cellulase system. Although natural cellulases can totally hydrolyze crystalline cellulose to soluble sugars, the current enzyme loadings and long digestion times required render these enzymes less than cost effective for biomass conversion processes. It is clear that cellobiohydrolases must be improved via protein engineering to reduce processing costs. To better understand cellobiohydrolase function, new simulations have been conducted using charmm of cellobiohydrolase I (CBH I) from T.reesei interacting with a model segment (cellodextrin) of a cellulose microfibril in which one chain from the substrate has been placed into the active site tunnel mimicking the hypothesized configuration prior to final substrate docking (i.e., the +1 and +2 sites are unoccupied), which is also the structure following a catalytic bond scission. No tendency was found for the protein to dissociate from or translate along the substrate surface during this initial simulation, nor to align with the direction of the cellulose chains. However, a tendency for the decrystallized cellodextrin to partially re-anneal into the cellulose surface hints that the arbitrary starting configuration selected was not ideal.     

Simulation studies of the insolubility of cellulose

Molecular dynamics simulations have been used to calculate the potentials of mean force for separating short cellooligomers in aqueous solution as a means of estimating the contributions of hydrophobic stacking and hydrogen bonding to the insolubility of crystalline cellulose. A series of four potential of mean force (pmf) calculations for glucose, cellobiose, cellotriose, and cellotetraose in aqueous solution were performed for situations in which the molecules were initially placed with their hydrophobic faces stacked against one another, and another for the cases where the molecules were initially placed adjacent to one another in a co-planar, hydrogen-bonded arrangement, as they would be in cellulose Iβ. From these calculations, it was found that hydrophobic association does indeed favor a crystal-like structure over solution, as might be expected. Somewhat more surprisingly, hydrogen bonding also favored the crystal packing, possibly in part because of the high entropic cost for hydrating glucose hydroxyl groups, which significantly restricts the configurational freedom of the hydrogen-bonded waters. The crystal was also favored by the observation that there was no increase in chain configurational entropy upon dissolution, because the free chain adopts only one conformation, as previously observed, but against intuitive expectations, apparently due to the persistence of the intramolecular O3-O5 hydrogen bond.     

Functional display of fungal cellulases from <Emphasis Type="Italic">Trichoderma </Emphasis><Emphasis Type="Italic">reesei</Emphasis> on phage M13

To test whether the phage display technology could be applied in cellulase engineering, phagemids harboring the genes encoding the mature forms of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from filamentous fungus Trichoderma reesei were constructed, respectively. CBH I and EG I fused to the phage coat protein encoded by the g3 gene were expressed and displayed on phage M13. The phage-bound cellulases retained their activities as determined by hydrolysis of the corresponding substrates, Also, their binding abilities to insoluble cellulose substrate were confirmed by an ELISA method. Overall, these results demonstrate that cellulases can be displayed on phage surface while maintaining their biological function, thus providing an alternative for directed evolution and high-throughput screening for improved cellulases.     

Structural and functional domains of cellobiohydrolase I from trichoderma reesei

Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, D_max=6.5 nm) and for the intact enzyme (Rg=4.27 nm, D_max=18 nm) indicate very different shapes. The resulting models show a tadpole-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.Abbreviations SAXS small angle X-ray scattering - SDS-PAGE SDS-polyacrylamide gel electrophoresis - IEF-PAG polyacrylamide gel isoelectric focusing; cellobiohydrolase (CBH, 1,4--glucan cellobio hydrolase (E.C.3.2.1.91)) - D_max maximum diameter - Rg radius of gyration     

微紫青霉CBHI酶的CBD蛋白编码区在大肠杆菌中的亚克隆及表达

对插入质粒pUC18－181上的微紫青霉（Penicilliumjanthinellum）CBHI酶的cDNA基因进行一系列DNA体外操作,包括进行序列定向缺失,最后将两末端修饰为平端后进行连接使质粒环化。用得到的产生序列定向缺失的重组质粒转化大肠杆菌JM109。利用CBD能吸附到结晶纤维素上的特性,从随机选取的24个缺失转化子中筛选到一株含CBD编码区的转化子JM109（pUC18C）,所表达的CBD融合蛋白分子量为21kD．JM109（pUC18C）所产生的LacZ-CBD融合蛋白可通过对纤维素的吸附-解吸附过程一步纯化。其IPTG诱导的pNPC酶活力为零,表明该菌已不再具有CBHI酶活力。     

The enzymatic hydrolysis rate of cellulose decreases with irreversible adsorption of cellobiohydrolase I

Protein adsorption onto solid substrates usually takes place in an irreversible fashion and this irreversible adsorption also occurs in some enzymatic reactions. In this work the adsorption behavior of intact β-1, 4-glucan-cellobiohydrolase (CBH I) from Trichoderma reesei onto microcrystalline cellulose was monitored by surface plasmon resonance and UV-spectral method. It was found that there existed reversible binding and irreversible binding of CBH I during its interaction with cellulose substrate. To evaluate the influence of adsorption on cellulose enzymatic hydrolysis, the reaction dynamics on pure cellulose were determined. A plot of the hydrolysis rate against the surface density of irreversibly adsorbed CBH I, revealed an inverse relationship in which an apparent decrease in the hydrolysis rate was observed with increasing surface density. Taken together, results presented here should be useful for modifying the binding characteristics of CBH I and making them more effective in cellulose hydrolysis.     

Cloning, sequencing, and expression of the cellulase genes of Humicola grisea var. thermoidea

We have cloned an endoglucanase (EGI) gene and a cellobiohydrolase (CBHI) gene of Humicola grisea var. thermoidea using a portion of the Trichoderma reesei endoglucanase I gene as a probe, and determined their nucleotide sequences. The deduced amino acid sequence of EGI was 435 amino acids in length and the coding region was interrupted by an intron. The EGI lacks a hinge region and a cellulose-binding domain. The deduced amino acid sequence of CBHI was identical to the H. grisea CBHI previously reported, with the exception of three amino acids. The H. grisea EGI and CBHI show 39.8% and 37.7% identity with the T. Reesei EGI, respectively. In addition to TATA box and CAAT motifs, putative CREA binding sites were observed in the 5′ upstream regions of both genes. The cloned cellulase genes were expressed in Aspergillus oryzae and the gene products were purified. The optimal temperatures of CBHI and EGI were 60 °C and 55–60 °C, respectively. The optimal pHs of these enzymes were 5.0. CBHI and EGI had distinct substrate specificities: CBHI showed high activity toward Avicel, whereas EGI showed high activity toward carboxymethyl cellulose (CMC).     

The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride

TheP-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HC1) that did not affect the tryptophan fluorescence of this enzyme. The K_m of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combine reversibly with I molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.     

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters.     

Pretreatment of microcrystalline cellulose flakes with CaCl2 increases the surface area, and thus improves enzymatic saccharification

Glucose production from cellulose flakes with cellulases was improved after pretreatment with saturated CaCl2 at room temperature. When pretreated microcrystalline cellulose flakes (Funacel II, Funakoshi Co., Ltd, Tokyo, Japan) were saccharified with the cellulases, 76.8% of the substrate was converted into glucose within 5 h, whereas the corresponding conversion rate of water-pretreated cellulose flakes was 33.8%. To clarify the mechanism of the promotion, cellobiohydrolase I purified from Trichoderma longibrachiatum was used as the model cellulase, which degraded CaCl2-pretreated cellulose more quickly than the water-pretreated cellulose under tested conditions. The maximum amount of the enzyme bound to CaCl2-pretreated cellulose at 37 degrees C was estimated as 1.14 nmol/mg of cellulose, whereas that to water-pretreated cellulose was 0.527 nmol/mg of cellulose. The specific activity of the bound enzyme greatly decreased with the increase of the surface density (rho) of the bound enzyme, and no significant positive effects of the CaCl2-pretreatment on the specific activity could be observed at the same rho value, suggesting that the promotion was attributed mainly to the increase of the surface area of cellulose. The effect was also observed with dewaxed cotton or filter paper, but not with nata de coco cellulose or bagasse cellulose as the substrates. This suggests that the CaCl2-pretreatment serves to increase the surface area of cellulose flakes via liberation of cellulose particles which were artificially aggregated during harsh drying processes of the flakes.     

Purification of cellobiohydrolase I fromTrichoderma reesei using monoclonal antibodies in an immunomatrix

Summary The several components of the fungal cellulase system present practical problems in devising facile and efficient schemes for their purification. We report on a new single-step affinity chromatographic method for purification of cellobiohydrolase I ofTrichoderma reesei based on its selective absorption and elution using an immunomatrix constructed with CnBr-activated Sepharose 4B and monoclonal antibody specific for the enzyme. Isoenzymes of cellobiohydrolase I were purified directly from crude culture filtrate. The method is fast, simple, and of high resolution.     

嗜热毛壳菌外切葡聚糖纤维二糖水解酶的纯化和部分性质研究

研究液体发酵嗜热毛壳菌(Chaetomium thermophilum)产生的一种外切葡聚糖纤维二糖水解酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Sephacryl S-100分子筛层析、Q Sepharose Fast Flow强阴离子层析等步骤后获得凝胶电泳均一的外切葡聚糖纤维二糖水解酶。经12.5%SDS-PAGE和凝胶过滤层析方法测得该酶的分子量大小约为66.3kDa和67.1kDa。该酶反应的最适温度和pH值分别为65℃和5.0。在60℃以下酶比较稳定,在70℃酶的半衰期为1h,在80℃下保温20min仍具有20%的活性,该酶的热稳定性较中温真菌的同类酶高,与国外报道的嗜热真菌的同类酶热稳定性接近。以pNPC为底物的Km值为0.956mmol/L。