Domain structure of cellobiohydrolase II as studied by small angle X-ray scattering: close resemblance to cellobiohydrolase I

Evidence for a domain structure of cellobiohydrolase II (CBH II, 58 kDa) from Trichoderma reesei (Teeri et al., 1987; Tomme et al., 1988) is corroborated by results from SAXS experiments. They indicate a 'tadpole' structure for the intact CBH II in solution (Dmax = 21.5 +/- 0.5 nm; Rg = 5.4 +/- 0.1 nm) and a more isotropic, ellipsoid shape for the core protein (Dmax = 6.0 +/- 0.3 nm; Rg = 2.1 +/- 0.1 nm). The latter was obtained by partial proteolysis with papain which cleaves the native CBH II to give two fragments (Tomme et al., 1988): the core (45 kDa) with the active (hydrolytic) domain and a smaller fragment (11 kDa) coinciding with the tail part of the model and containing the binding domain for unsoluble cellulose. This peptide fragment is conserved in most cellulolytic enzymes from Trichoderma reesei (Teeri et al., 1987). It contains a conserved region (block A) and glycosylated parts (blocks B and B' duplicated and located N-terminally in CBH II). In spite of different domain arrangements in CBH I (blocks B-A at C-terminals) SAXS measurements (Abuja et al., 1988) indicate similar tertiary structures for both cellobiohydrolases although discrete differences in the tail parts exist.     

Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I.

Intact and partially acid hydrolyzed cellulose from Acetobacter xylinum were used as model substrates for cellulose hydrolysis by 1,4-beta-D-glucan-cellobiohydrolase I (CBH I) and 1,4-beta-D-endoglucanase I (EG I) from Trichoderma reesei. A high synergy between CBH I and EG I in simultaneous action was observed with intact bacterial cellulose (BC), but this synergistic effect was rapidly reduced by acid pretreatment of the cellulose. Moreover, a distinct synergistic effect was observed upon sequential endo-exo action on BC, but not on bacterial microcrystalline cellulose (BMCC). A mechanism for endo-exo synergism on crystalline cellulose is proposed where the simultaneous action of the enzymes counteract the decrease of activity caused by undesirable changes in the cellulose surface microstructure.     

The 1,4-beta-D-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei.

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.     

Structural changes in cellobiohydrolase I upon binding of a macromolecular ligand as evident by SAXS investigations

Xylan from Rhodymenia palmata binds to the cellobiohydrolase I from Trichoderma reesei (CBH I) or its core protein, inhibiting their activity. Adsorption onto microcrystalline cellulose (Avicel) is reduced approximately 30% for intact CBH I and nearly 50% for the core, whereas the effects with cellobiose are negligible. Structural changes concomitant with this binding are studied in solution by small angle X-ray scattering. In the "tadpole" structure typical for the CBH I [Abuja et al., 1988] the lengthening of the tail part is the most salient observation when xylan is present which accounts for an increase in Dmax (18.0 to 22.0 nm) and radius of gyration (4.74 to 5.18 nm). When xylan binds to the core the radius of gyration remains nearly unchanged. Here a model can be constructed showing a xylan molecule on the surface of the core protein near the tail part.     

A binding-site-deficient, catalytically active, core protein of endoglucanase III from the culture filtrate of Trichoderma reesei

From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) which was shown to be identical to endoglucanase III (E III, 50 kDa), but lacking the first 61 N-terminal amino acids. This core protein, designated E III core, is fully active against soluble substrates, such as carboxymethylcellulose, whereas both activity against and adsorption to microcrystalline cellulose (Avicel) is markedly decreased. Sedimentation velocity experiments revealed that the intact E III enzyme has much higher asymmetry than the E III core protein, suggesting that the N-terminal region split off constitutes a protruding part of the native enzyme. These results lead to the proposal that native E III consists of two functionally separated domains: a catalytically active core and a protruding N-terminal domain which acts in the binding to insoluble cellulose. The N-terminal peptide missing in E III core corresponds to the heavily glycosylated common structural element found also in the N-terminus of cellobiohydrolase II and in the C-termini of cellobiohydrolase I and endoglucanase I. A similar bifunctional organization could thus be the rule for Trichoderma cellulases, endoglucanases as well as cellobiohydrolases.     

Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.     

Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose

Hydrolysis of microcrystalline cellulose (Avicel) by cellobiohydrolase I and II (CBH I and II) from Trichoderma reesei has been studied. Adsorption and synergism of the enzymes were investigated. Experiments were performed at different temperatures and enzyme/substrate ratios using CBH I and CBH II alone and in reconstituted equimolar mixtures. Fast protein liquid chromatography (FPLC) analysis was found to be an accurate and reproducible method to follow the enzyme adsorption. A linear correlation was found between the conversion and the amount of adsorbed enzyme when Avicel was hydrolyzed by increasing amounts of CBH I and/or CBH II. CBH I had lower specific activity compared to CBH II although, over a wide concentration range, more CBH I was adsorbed than CBH II. Synergism between the cellobiohy-drolases during hydrolysis of the amorphous fraction of Avicel showed a maximum as a function of total enzyme concentration. Synergism measured as a function of bound enzyme showed a continuous increase, which indicates that by decreasing the distance between the two enzymes the synergism is enhanced. The adsorption process for both enzymes was slow. Depending on the enzyme/substrate ratio it took 30-90 min to reach 95% of the equilibrium binding. The amount of bound enzyme decreased with increasing temperature. The two enzymes compete for the adsorption sites but also bind to specific sites. Stronger competition for adsorption sites was shown by CBH I. (c) 1994 John Wiley & Sons, Inc.     

Polyclonal antiserum against noncatalytic part of cellobiohydrolase I from Trichoderma reesei

A specific antiserum to the noncatalytic part of cellobiohydrolase I from Trichoderma reesei was obtained by exhaustion of rabbit antiserum to the native enzyme with its catalytic domain prepared by papain treatment of cellobiohydrolase I tightly adsorbed onto microcrystalline cellulose.     

Enhancement of the affinity of cellobiohydrolase I and its catalytic domain to cellulose in the presence of the reaction product--cellobiose.

The catalytic domain of cellobiohydrolase I from Trichoderma reesei has been obtained by papain treatment of the native enzyme adsorbed onto the surface of microcrystalline cellulose. Both the intact and the truncated enzyme are almost equally active toward soluble fluorogenic derivatives of cellobi-, -tri-, -tetra-, and -pentaose, the fastest and the slowest fluorophore liberation being observed for MUF-cellopenta- and -tetraose, respectively. Titration of the active centers of the intact enzyme and its catalytic domain with MUF-cellotetraose showed their molecular masses to be 49 and 39 kD, respectively, the dissociation constants of the enzyme-soluble ligand complexes being almost equal (65 and 70 nM at 20 degrees C, respectively). In contrast, the intact enzyme and its catalytic core have been shown to significantly (50-60 times) differ in their affinity to insoluble microcrystalline cellulose at low enzyme loading (up to 10 mg per g of the substrate). At 20 degrees C the dissociation constants for the two forms of the enzyme are estimated to be 10 and 500 nM, respectively. Surprisingly, under these conditions the reaction product and inhibitor, cellobiose (Ki = 10 microM), at the concentration 10 mM, increased 3-4-fold the affinity of both the intact cellobiohydrolase and its catalytic domain to cellulose.     

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.     

Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin

The presence of lignin has shown to play an important role in the enzymatic degradation of softwood. The adsorption of enzymes, and their constituent functional domains on the lignocellulosic material is of key importance to fundamental knowledge of enzymatic hydrolysis. In this study, we compared the adsorption of two purified cellulases from Trichoderma reesei, CBH I (Cel7A) and EG II (Cel5A) and their catalytic domains on steam pretreated softwood (SPS) and lignin using tritium labeled enzymes. Both CBH I and its catalytic domain exhibited a higher affinity to SPS than EG II or its catalytic domain. Removal of cellulose binding domain decreased markedly the binding efficiency. Significant amounts of CBH I and EG II also bound to isolated lignin. Surprisingly, the catalytic domains of the two enzymes of T. reesei differed essentially in the adsorption to isolated lignin. The catalytic domain of EG II was able to adsorb to alkaline isolated lignin with a high affinity, whereas the catalytic domain of CBH I did not adsorb to any of the lignins tested. The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.     

The 1,4-beta-D-glucan glucanohydrolases from Phanerochaete chrysosporium. Re-assessment of their significance in cellulose degradation mechanisms.

A physico-chemical, functional and structural characterization, including partial sequence data, of three major 1,4-beta-D-glucan glucanohydrolases (EC. 3.2.1.4) isolated from the culture filtrate of the white-rot fungus Phanerochaete chrysosporium, shows that all three enzymes belong to a single family of cellulases. EG44, pI 4.3, (named after its apparent molecular mass in kDa), shows a clear homology with Schizopyllum commune Endoglucanase I (EGI); whereas EG38, pI 4.9, (named in the same manner) is related more closely to Trichoderma reesei (Trichoderma longibrachiatum) Endoglucanase III (EGIII). EG36, pI 5.6-5.7, is probably an EG38 protein lacking its cellulose binding domain. Strong synergistic action is induced by the enzymes acting in concert with cellobiohydrolases (CBHI and CBHII) from the same organism, indicating a highly effective enzymatic system for cellulose degradation. Controlled proteolysis with papain has allowed a so far unique cleavage of endoglucanases EG44 and EG38 into two domains: a core protein, which virtually lacks the capacity to absorb onto microcrystal-line cellulose but retains full catalytic activity against carboxymethyl cellulose and low molecular weight soluble substrates; and a peptide fragment corresponding to the cellulose binding domain. The latter appears to be of paramount significance in the mechanisms involved in the hydrolysis of microcrystalline cellulose.     

Characterization of commercial Trichoderma reesei cellulase preparations by denaturing electrophoresis (SDS-PAGE) and immunostaining using monoclonal antibodies.

Fifteen different cellulase preparations from Trichoderma reesei, obtained either commercially or from pilot plants, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using monoclonal antibodies against two cellobiohydrolases (CBH I, CBH II), an endoglucanase (EG I), and beta-glucosidase. The staining patterns were compared with the activities of the preparations against filter paper (FPU), carboxymethylcellulose (CMC-ase), cellobiose (beta-glucosidase), and azocasein (protease). Variable amounts of proteolytic degradation products of CBH I, CBH II, and EG I were seen in most samples, and only half of them contained intact beta-glucosidase. The degree of proteolysis did not correlate with any significant difference in the respective activities of these preparations against filter paper cellulose or carboxymethylcellulose. In more than 50% of all cases a decreased beta-glucosidase activity and the absence of intact beta-glucosidase protein in Western blots was observed in preparations displaying high proteolytic activity.     

Effect of endoglucanase and cellobiohydrolase from Trichoderma reesei on cellulose microfibril structure

Abstract Cellobiohydrolase (CBH, EC 3.2.91) was purified to homogeneity from Trichoderma reesei culture fluids by means of preparative isoelectric focussing (IEF). Its isoelectric points was 4.2. The degradation product of crystalline cellulose (Avicel and cotton) was predominantly cellobiose. The action of purified endoglucanase (EG) and CBH on cellulose microfibrils was followed by transmission electron microscopy (TEM) observations after Pt-C shadowing of the specimen. EG pretreatment of microfibrils resulted in submicrofibril formation. Addition of CBH induced the conversion of submicrofibrils into heterogeneous cellulose clusters and into homogeneous cellulose plaques. One structural effect of CBH was the increase in accessible cellulose surface area, possibly providing intermolecular entrace of water molecules between adjacent cellulose chains. Plaque formation is interpreted as a visible CBH action on crystalline cellulose to form swollen water-insoluble cellulose intermediates.     

The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its Molecular Size

Degradation of cotton cellulose by Trichoderma reesei endoglucanase I (EGI) and cellobiohydrolase II (CBHII) was investigated by analyzing the insoluble cellulose fragments remaining after enzymatic hydrolysis. Changes in the molecular-size distribution of cellulose after attack by EGI, alone and in combination with CBHII, were determined by size exclusion chromatography of the tricarbanilate derivatives. Cotton cellulose incubated with EGI exhibited a single major peak, which with time shifted to progressively lower degrees of polymerization (DP; number of glucosyl residues per cellulose chain). In the later stages of degradation (8 days), this peak was eventually centered over a DP of 200 to 300 and was accompanied by a second peak (DP, (apprx=)15); a final weight loss of 34% was observed. Although CBHII solubilized approximately 40% of bacterial microcrystalline cellulose, the cellobiohydrolase did not depolymerize or significantly hydrolyze native cotton cellulose. Furthermore, molecular-size distributions of cellulose incubated with EGI together with CBHII did not differ from those attacked solely by EGI. However, a synergistic effect was observed in the reducing-sugar production by the cellulase mixture. From these results we conclude that EGI of T. reesei degrades cotton cellulose by selectively cleaving through the microfibrils at the amorphous sites, whereas CBHII releases soluble sugars from the EGI-degraded cotton cellulose and from the more crystalline bacterial microcrystalline cellulose.     

Cellobiohydrolase hydrolyzes crystalline cellulose on hydrophobic faces

Biodegradation of plant biomass is a slow process in nature, and hydrolysis of cellulose is also widely considered to be a rate-limiting step in the proposed industrial process of converting lignocellulosic materials to biofuels. It is generally known that a team of enzymes including endo- and exocellulases as well as cellobiases are required to act synergistically to hydrolyze cellulose to glucose. The detailed molecular mechanisms of these enzymes have yet to be convincingly elucidated. In this report, atomic force microscopy (AFM) is used to image in real-time the structural changes in Valonia cellulose crystals acted upon by the exocellulase cellobiohydrolase I (CBH I) from Trichoderma reesei. Under AFM, single enzyme molecules could be observed binding only to one face of the cellulose crystal, apparently the hydrophobic face. The surface roughness of cellulose began increasing after adding CBH I, and the overall size of cellulose crystals decreased during an 11-h period. Interestingly, this size reduction apparently occurred only in the width of the crystal, whereas the height remained relatively constant. In addition, the measured cross-section shape of cellulose crystal changed from asymmetric to nearly symmetric. These observed changes brought about by CBH I action may constitute the first direct visualization supporting the idea that the exocellulase selectively hydrolyzes the hydrophobic faces of cellulose. The limited accessibility of the hydrophobic faces in native cellulose may contribute significantly to the rate-limiting slowness of cellulose hydrolysis.     

Synergism of Trichoderma reesei cellulases while degrading different celluloses

Summary The synergistic action of purified cellulases from Trichoderma reesei in hydrolysis of cellulose decreased with increasing substrate concentration, depended strongly on the the type of cellulose used, and was maximal on crystalline cellulose. Contrarily, the activity of the individual cellulases was highest on amorphous cellulose. The binary combinations CBH I/EG III and CBH I/CBH II exhibited the greatest degree of synergism on crystalline cellulose.     

微紫青霉CBHⅠ酶纤维素结合结构域在大肠杆菌中的分泌型表达及性质研究

为研究纤维素酶纤维素结合结构域的结构与功能 ,进而深入了解天然纤维素的生物降解机制和提高纤维素酶的生物工艺学价值 ,采用 PCR技术体外扩增了携带微紫青霉外切葡聚糖纤维二糖水解酶 ( CBH ) CBD编码区的 DNA片段 ,将 CBD编码区 DNA片段插入带有 Erwiniacarotovora pe1 B前导肽序列的大肠杆菌质粒 p KK- tac- new上进行了表达 .携带微紫青霉 CBDCBH 编码区的大肠杆菌重组菌株 DH5α( p KK- tac- new- 8)产生有活性的分泌型 CBDCBH 蛋白 .SDS-PAGE检测显示所产生的 CBDCBH 蛋白分子量约 1 0 .8k D.在 IPTG诱导下 ,该菌株所产生的CBDCBH 蛋白含量达 45.2 mg/L,且 90 %以上的 CBD蛋白分泌到培养物上清液中 .结晶纤维素 CF-1 1溶液经 CBDCBH 处理后 ,浊度比对照提高了 1 2 8.9% ,天然棉花纤维结构经 CBDCBH 处理后产生一定程度的非水解性降解作用 ,表明微紫青霉 CBDCBH 具有解聚天然结晶纤维素的作用 .     

Pilot scale production of a heterologous Trichoderma reesei cellulase by Saccharomyces cerevisiae.

Cellobiohydrolase II of Trichoderma reesei was produced in laboratory and pilot scale using a transformant strain of Saccharomyces cerevisiae harbouring a multicopy expression plasmid. Different strategies were compared for concentration and partial purification of the enzyme produced in a 200 1 pilot cultivation. After efficient separation of biomass and sub-cellular particulate matter, a combination of ultrafiltration and adsorbent treatment for removal of protein impurities was used to provide a concentrate for chromatographic purification. Effective purification of the CBH II protein was obtained by passing the concentrate through a column of DEAE Sepharose, on which almost all the yeast proteins were adsorbed. The purified enzyme reacted with antibodies prepared against T. reesei CBH II and catalyzed partial solubilization of crystalline cellulose to soluble sugars.