Estimation of Cellobiohydrolase Ⅰ Activity by Numerical Differentiation of Dynamic Ultraviolet Spectroscopy

1,4-β-D-glucan cellobiohydrolase Ⅰ （CBH Ⅰ）, p-nitrophenyl β-D-cellobioside, p-nitrophenol and cellobiose show distinct ultraviolet spectra, allowing the design of an assay to track the dynamic process of p-nitrophenyl β-D-cellobioside hydrolysis by CBH Ⅰ. Based on the linear relationship between p-nitrophenol formation in the hydrolysate and its first derivative absorption curve of AUC340-400 m （area under the curve）, a new sensitive assay for the determination of CBH Ⅰ activity was developed. The dynamic parameters of catalysis reaction, such as Vm and kcat, can all be derived from this result. The influence of β-glucosidase and endoglucanase in crude enzyme sample on the assay was discussed in detail. This approach is useful for accurate determination of the activity of CBHs.     

Production and Characteristics of Avicel-Digesting and Non-Avicel-Digesting Cellobiohydrolases from Aspergillus ficum

Two immunologically related cellobiohydrolases, cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II), were purified from Aspergillus ficum. The Avicel-adsorbable CBH I (molecular weight, 128,000) digested Avicel, cotton, and cellulose powder to cellobiose, but the Avicel-unadsorbable CBH II (molecular weight, 50,000) could not digest those substrates. Both enzymes hydrolyzed insoluble cellooligosaccharides ( 25) to cellobiose. High-pressure liquid chromatographic analysis of soluble cellooligosaccharide hydrolysates revealed that both enzymes split off strictly cellobiose units from the nonreducing end of the cellulose chain with an exowise mechanism. CBH I showed glucosyltransferase activity, but CBH II did not. The N-bromosuccinimideoxidized CBH I was completely inactive but retained the ability to adsorb to Avicel. This suggested that CBH I has separate sites for binding to cellulose and for catalyzing cleavage of glycosidic linkages. Cellobiohydrolases were of two types, CBH I and CBH II. The former can adsorb to and digest Avicel, while the latter can do neither.     

Kinetics of the hydrolysis of cellulose by beta-1,4-glucan cellobiohydrolase of Trichoderma viride

The cellulolytic enzyme beta-1,4-glucan cellobiohydrolase (CBH) has been isolated from the crude mixture of cellulase enzymes of Trichoderma viride by gel filtration and ion-exchange methods, and some aspects of its kinetic behaviour have been examined. Studies of the initial rates of the CBH-catalyzed production of cellobiose from fibrous alpha-cellulose show that (i) the dissociation constant for cellobiose competitive product inhibition of the reaction is Ki = (1.13 +/- 0.37) X 10(-3) M, (ii) the adsorption of CBH on fibrous alpha-cellulose and its subsequent reaction conform to kinetic equations developed in conjunction with the Langmuir adsorption isotherm, (iii) the rate-pH curve has a maximum at pH 5.2 and decreases at higher and lower pH values, exhibiting enzyme pK values of 3.8 and 6.5, and (iv) the energy of activation of the overall reaction between 5 and 60 degrees C is 5.3 +/- 0.3 kcal mol-1 at pH 5.2. Studies of the time course of the reaction over extended periods of time up to 40% hydrolysis of the cellulose show that (v) the data fit better to a competitive product inhibition model than to models of anticompetitive product inhibition or noncompetitive product inhibition.     

Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: adsorption, sugar production pattern, and synergism of the enzymes

Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40 degrees C) and enzyme loading (0.16 micromol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 micromol/g at 4 degrees C and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60-90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40 degrees C when the enzymes were added together they produced 27-45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4 degrees C, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11-15%, 82-89%, and <6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain.     

Intrinsic Fluorescence in Endoglucanase and Cellobiohydrolase from Trichoderma pseudokiningii S-38: Effects of pH, Quenching Agents, and Ligand Binding

To gain further insight into the difference in substrate specificity between endoglucanase and cellobiohydrolase, the intrinsic fluorescence properties of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from Trichoderma pseudokiningii S-38 were investigated. The results for the spectral characteristics, ligand binding and fluorescence quenching suggest that the fluorescence of two enzymes comes from tryptophan residues, and that tryptophan residue(s) may be involved in the function of the two enzymes. The results also suggest that the binding tryptophan in EG I may be more exposed to solvent than that in CBH I. This interpretation is supported by the observations that the effects of pH upon the fluorescence of EG I are greater than that of CBH I; spectral shifts are different in EG I and CBH I under various conditions, and fluorescence lifetime changes caused by cellobiose binding are larger for EG I than for CBH I.     

Intrinsic Fluorescence in Endoglucanase and Cellobiohydrolase from Trichoderma pseudokiningii S-38: Effects of pH, Quenching Agents, and Ligand Binding

To gain further insight into the difference in substrate specificity between endoglucanase and cellobiohydrolase, the intrinsic fluorescence properties of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from Trichoderma pseudokiningii S-38 were investigated. The results for the spectral characteristics, ligand binding and fluorescence quenching suggest that the fluorescence of two enzymes comes from tryptophan residues, and that tryptophan residue(s) may be involved in the function of the two enzymes. The results also suggest that the binding tryptophan in EG I may be more exposed to solvent than that in CBH I. This interpretation is supported by the observations that the effects of pH upon the fluorescence of EG I are greater than that of CBH I; spectral shifts are different in EG I and CBH I under various conditions, and fluorescence lifetime changes caused by cellobiose binding are larger for EG I than for CBH I.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Reaction specificity and thermodynamics of interactions of small substrates and ligands with the 1,4-beta-glucan cellobiohydrolase II

The 1,4-beta-glucan cellobiohydrolase II (CBH II) from Trichoderma reesei QM 9414 catalyses the hydrolysis of the 4-methylumbelliferyl beta-D-glycosides derived from cellotriose, cellotetraose and cellopentaose [MeUmb(Glc)n; n = 3 - 5]. The reaction has been followed by quantitative high-performance liquid chromatography. Specific activity for cellobiose removal at apparent substrate saturation were determined as (0.8 +/- 0.2) min-1 for MeUmb(Glc)3 and (9 +/- 2) min-1 for MeUmb(Glc)4. The enzyme showed a deviant specificity with MeUmb(Glc)5 as substrate. Two chromophoric products were formed simultaneously [MeUmb(Glc)3 and MeUmb(Glc)2] with turn-over numbers (17 +/- 4) min-1 and (21 +/- 6) min-1, respectively. Methylumbelliferyl beta-glucoside (MeUmbGlc) and the corresponding cellobioside [MeUmb(Glc)2] were used in equilibrium binding experiments. Both ligands yielded one binding site per molecule of Mr = 54000 upon forced flow dialysis (diafiltration). The association constants found were in fair agreement with those determined from MeUmb fluorescence quenching titrations. Quenching was total at all temperatures investigated for MeUmb(Glc)2, whereas for MeUmbGlc it increased from 80% to 100% between 2 degrees C and 20 degrees C. The association constants fitted linear van't Hoff plots in both cases. MeUmb(Glc)2 and MeUmbGlc were also used as indicator ligands to determine the association constants and thermodynamic parameters of several non-chromophoric ligands of CBH II. The binding of glucose increased the affinity for MeUmb(Glc)2 whereas it displaced MeUmbGlc from its complex. A putative binding site of the CBH II containing four subsites can be proposed. The thermodynamic data for methyl beta-D-glucopyranoside and cellobiose as ligands also point at an extended binding site.     

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.     

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.     

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.     

In vitro expression of Penicillium janthinellum cellobiohydrolase I gene in a coupled transcription-translation system

An enzymatically-active fungal cellobiohydrolase I (CBH I) was first synthesized in a coupled reticulocyte lysate system lacking of glycosylation modification by the template DNA(Cbh1) in the presence of T7 RNA polymerase. The synthesized CBH I had the expected size (57 kDa) and catalyzed the substrate of p-nitrophenyl-beta-D-cellobioside (pNPC), and had no activity against carboxymethyl cellulose (CMC-Na). The K(m) and V(max) values of the CBH I for pNPC were 0.82 mmol and 0.067 micromol min(-1) per microg enzyme, respectively. The results indicated that glycosylation may not be necessary for enzymatic activity of fungal cellulases.     

纤维二糖脱氢酶的纤维素降解中的作用研究

裂褶菌纤维二糖脱氢酶（ｃｅｌｌｏｂｉｏｓｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ,ＣＤＨ）可以提高纤维素酶对纤维素的降解。以纤维二糖为电子供体,ＣＤＨ作用于羧甲基纤维可降低其溶液的粘度,作用纤维素ＣＦ１１和磷酸膨胀纤维素,分别使其悬浊液的浊度提高７％和１４．４％。ＣＤＨ与纤维二糖水解酶或切纤维素酶在降解棉花纤维素时没有表现出协同作用。但若棉花事先在纤维二糖存在下用ＣＤＨ预处理,则变得易于被水解。     

Strategy for Identification of Novel Fungal and Bacterial Glycosyl Hydrolase Hybrid Mixtures that can Efficiently Saccharify Pretreated Lignocellulosic Biomass

A rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, was applied in order to develop tailored enzyme cocktails to efficiently hydrolyze pretreated lignocellulosic biomass. The fungal cellulases include cellobiohydrolase I (CBH I; GH family 7A), cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B), and β-glucosidase (βG; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), β-glucosidase (LβG; GH family 1), endoxylanases (LX1 and LX2; GH family 10), and β-xylosidase (LβX; GH family 52) from multiple sources were cloned, expressed, and purified. Enzymatic hydrolysis for varying enzyme combinations was carried out on ammonia fiber expansion (AFEX)-treated corn stover at three total protein loadings (i.e., 33, 16.5, and 11 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary to maximize both glucan and xylan yields was determined using a suitable design of experiments. The optimal hybrid enzyme mixtures contained fungal cellulases (78% of total protein loading), which included CBH I (loading ranging between 9-51% of total enzyme), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LX1 (13%) and LβX (9%). The hybrid mixture was effective at 50°C, pH 4.5 to maximize saccharification of AFEX-treated corn stover resulting in 95% glucan and 65% xylan conversion. This strategy of screening novel enzyme mixtures on pretreated lignocellulose would ultimately lead to the development of tailored enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.     

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.     

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.     

Recombinant Cellulase Accumulation in the Leaves of Mature,Vegetatively Propagated Transgenic Sugarcane

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.     

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.     

Kinetic analysis of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase using an amperometric biosensor

An amperometric biosensor for the detection of cellobiose has been introduced to study the kinetics of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase. By use of a sensor in which pyrroloquinoline quinone-dependent glucose dehydrogenase was immobilized on the surface of electrode, direct and continuous observation of the hydrolysis can be achieved even in a thick cellulose suspension. The steady-state rate of the hydrolysis increased with increasing concentrations of the enzyme to approach a saturation value and was proportional to the amount of the substrate. The experimental results can be explained well by the rate equations derived from a three-step mechanism consisting of the adsorption of the free enzyme onto the surface of the substrate, the reaction of the adsorbed enzyme with the substrate, and the liberation of the product. The catalytic constant of the adsorbed enzyme was determined to be 0.044+/-0.011s(-1).     

Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass

Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput fast protein liquid chromatography (HT-FPLC)-based method has been developed to quantify cellobiohydrolase I (CBH I or Cel7A), cellobiohydrolase II (CBH II or Cel6A), and endoglucanase I (EG I or Cel7B) present in hydrolyzates of untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated corn stover (CS). This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall-derived components. The binding isotherms for CBH I, CBH II, and EG I were obtained after incubation for 2 h at 4 °C. Both AFEX and dilute acid pretreatment resulted in increased cellulase binding compared with untreated CS. Cooperative binding of CBH I and/or CBH II in the presence of EG I was observed only for AFEX treated CS. Competitive binding between enzymes was found for certain other enzyme-substrate combinations over the protein loading range tested (i.e., 25-450 mg/g glucan). Langmuir single-site adsorption model was fitted to the binding isotherm data to estimate total available binding sites E(bm) (mg/g glucan) and association constant K(a) (L/mg). Our results clearly demonstrate that the characteristics of cellulase binding depend not only on the enzyme GH family but also on the type of pretreatment method employed.     

Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production

An acidic Cel6A, cellobiohydrolase (CBH) II, was purified from Penicillium decumbens and designated as PdCel6A. The deduced internal amino acid sequence of the novel CBH has a high degree of sequence identity with the CBH II from Aspergillus fumigatus. Surprisingly, PdCel6A exhibits characteristics comparable to that of CBH I, as well as CBH II. Similar to CBH I, the novel CBH has a specific activity of 1.9 IU/mg against p-nitrophenyl-β-d-cellobioside. The enzyme retains about 80% of its maximum activity after 4h of incubation at pH 2.0. Using delignified corncob residue as the substrate, ethanol concentration increased by 20% during simultaneous saccharification and fermentation when supplemented with low doses of PdCel6A (0.2mg/g substrate). To our knowledge, this is the first report involving a CBH I-like CBH II. The present paper provides new insight into the role of CBH II in cellulose degradation.