Molecular cloning of a gene encoding endo-beta-D-1,4-glucanase PCE1 from Phycomyces nitens

We previously cloned three endoglucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota. In this study, an endoglucanase gene, designated a pce1 gene, was cloned by plaque hybridization with the codon usage-optimized rce1 gene as a probe from Phycomyces nitens, a member of the subdivision Zygomycota. The pec1 gene had an open reading frame of 1,038 nucleotides encoding an endoglucanase (PCE1) of 346 amino acid residues. The amino acid sequence deduced from the pce1 gene consisted of a cellulose-binding domain (CBD) at the N terminus and of a catalytic domain belonging to family 45 glycoside hydrolase at the C terminus. PCE1 was purified to apparent homogeneity from the culture supernatant of P. nitens and the molecular mass was found to be 45 kDa. The optimum pH for the CMCase activity of PCE1 was 6.0, and the optimum temperature was 50 degrees C, the lowest among the family 45 endoglucanases.     

Molecular cloning of endo-beta-D-1,4-glucanase genes,rce1, rce2, and rce3, from Rhizopus oryzae

Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision ZYGOMYCOTA: All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one. rce1, rce2, and rce3 had various lengths of linker sequences.     

Specific characteristics of family 45 endoglucanases from Mucorales in the use of textiles and laundry

We examined the characteristics of family 45 endoglucanases (glycoside hydrolases family 45; GH45) from Mucorales belonging to Zygomycota in the use of textiles and laundry. The defibrillation activities on lyocell fabric of family 45 endoglucanases from Mucorales, such as RCE1 and RCE2 from Rhizopus oryzae, MCE1 and MCE2 from Mucor circinelloides, and PCE1 from Phycomyces nitens, were much higher than those of the other family 45 endoglucanases. By contrast, family 45 endoglucanases from Mucorales were less resistant to anionic surfactant and oxidizing agent, main components in detergents, than the other family 45 endoglucanases. RCE1 consists of two distinct modules, a catalytic module and a carbohydrate-binding module family 1 (CBM1), and these common specific characteristics were considered to due to the catalytic module, but not to the CBM1.     

The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities

The microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the beta1,4-beta1,3-mixed linked glucans lichenan and barley beta-glucan, cello-oligosaccharides, insoluble forms of cellulose, the beta1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the beta1,4,-beta1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site.     

A novel carbohydrate-binding protein is a component of the plant cell wall-degrading complex of Piromyces equi

The recycling of photosynthetically fixed carbon by the action of microbial plant cell wall hydrolases is a fundamental biological process that is integral to one of the major geochemical cycles and, in addition, has considerable industrial potential. Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Anaerobic fungi that colonize herbivores are the most efficient plant cell wall degraders known, and this activity is vested in a high molecular weight complex that binds tightly to the plant cell wall. To investigate whether plant cell wall attachment is mediated by noncatalytic proteins, a cDNA library of the anaerobic fungus Piromyces equi was screened for sequences that encode noncatalytic proteins that are components of the cellulase-hemicellulase complex. A 1.6-kilobase cDNA was isolated encoding a protein of 479 amino acids with a M(r) of 52548 designated NCP1. The mature protein had a modular architecture comprising three copies of the noncatalytic dockerin module that targets anaerobic fungal proteins to the cellulase-hemicellulase complex. The two C-terminal modules of NCP1, CBM29-1 and CBM29-2, respectively, exhibit 33% sequence identity with each other but have no homologues in protein data bases. A truncated form of NCP1 comprising CBM29-1 and CBM29-2 (CBM29-1-2) and each of the two individual copies of CBM29 bind primarily to mannan, cellulose, and glucomannan, displaying the highest affinity for the latter polysaccharide. CBM29-1-2 exhibits 4-45-fold higher affinity than either CBM29-1 or CBM29-2 for the various ligands, indicating that the two modules, when covalently linked, act in synergy to bind to an array of different polysaccharides. This paper provides the first report of a CBM-containing protein from an anaerobic fungal cellulase-hemicellulase complex. The two CBMs constitute a novel CBM family designated CBM29 whose members exhibit unusually wide ligand specificity. We propose, therefore, that NCP1 plays a role in sequestering the fungal enzyme complex onto the plant cell wall.     

Binding preferences, surface attachment, diffusivity, and orientation of a family 1 carbohydrate-binding module on cellulose

Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 μs of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.     

Recognition and hydrolysis of noncrystalline cellulose

Cellulase Cel5A from alkalophilic Bacillus sp. 1139 contains a family 17 carbohydrate-binding module (BspCBM17) and a family 28 CBM (BspCBM28) in tandem. The two modules have significantly similar amino acid sequences, but amino acid residues essential for binding are not conserved. BspCBM28 was obtained as a discrete polypeptide by engineering the cel5A gene. BspCBM17 could not be obtained as a discrete polypeptide, so a family 17 CBM from endoglucanase Cel5A of Clostridium cellulovorans, CcCBM17, was used to compare the binding characteristics of the two families of CBM. Both CcCBM17 and BspCBM28 recognized two classes of binding sites on amorphous cellulose: a high affinity site (K(a) approximately 1 x 10(6) M(-1)) and a low affinity site (K(a) approximately 2 x 10(4) M(-1)). They did not compete for binding to the high affinity sites, suggesting that they bound at different sites on the cellulose. A polypeptide, BspCBM17/CBM28, comprising the tandem CBMs from Cel5A, bound to amorphous cellulose with a significantly higher affinity than the sum of the affinities of CcCBM17 and BspCBM28, indicating cooperativity between the linked CBMs. Cel5A mutants were constructed that were defective in one or both of the CBMs. The mutants differed from the wild-type enzyme in the amounts and sizes of the soluble products produced from amorphous cellulose. This suggests that either the CBMs can modify the action of the catalytic module of Cel5A or that they target the enzyme to areas of the cellulose that differ in susceptibility to hydrolysis.     

Cloning, sequencing, and expression of a Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) with novel carbohydrate-binding modules, and properties of Cel5A

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.     

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln¹⁰⁶ is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.     

Cellulose-binding modules from extracellular matrix proteins of Dictyostelium discoideum stalk and sheath.

Cellulose-binding modules (CBMs) of two extracellular matrix proteins, St15 and ShD, from the slime mold Dictyostelium discoideum were expressed in Escherichia coli. The expressed proteins were purified to > 98% purity by extracting inclusion bodies at pH 11.5 and refolding proteins at pH 7.5. The two refolded CBMs bound tightly to amorphous phosphoric acid swollen cellulose (PASC), but had a low affinity toward xylan. Neither protein exhibited cellulase activity. St15, the stalk-specific protein, had fourfold higher binding affinity toward microcrystalline cellulose (Avicel) than the sheath-specific ShD CBM. St15 is unusual in that it consists of a solitary CBM homologous to family IIa CBMs. Sequence analysis of ShD reveals three putative domains containing: (a) a C-terminal CBM homologous to family IIb CBMs; (b) a Pro/Thr-rich linker domain; and (c) a N-terminal Cys-rich domain. The biological functions and potential role of St15 and ShD in building extracellular matrices during D. discoideum development are discussed.     

Sequence of celQ and properties of CelQ, a component of the Clostridium thermocellum cellulosome

The nucleotide sequence of the Clostridium thermocellum F1 celQ gene, which codes for the endoglucanase CelQ, consists of 2,130 bp encoding 710 amino acids. The precursor form of CelQ has a molecular weight of 79,809 and is composed of a signal peptide, a family 9 cellulase domain, a family IIIc carbohydrate-binding module (CBM), and a dockerin domain. Truncated derivatives of CelQ were constructed: CelQdeltadoc consisted of the catalytic domain and the CBM; CelQcat consisted of the catalytic domain only. CelQdeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan and low activity toward Avicel, acid-swollen cellulose, lichenan, and xylan. The Vmax and Km values were 235 micromol/min/mg and 3.3 mg/ml, respectively, for CMC. By contrast, CelQcat, which was devoid of the CBM, showed negligible activity toward CMC, i.e., about 1/1,000 of the activity of CelQdeltadoc, supporting the previously proposed idea that family IIIc CBMs participate in the catalytic function of the enzyme. Immunological analysis using an antiserum raised against CelQdeltadoc confirmed that CelQ is a component of the C. thermocellum cellulosome.     

Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.     

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum

Most cellulases contain carbohydrate-binding modules (CBMs) that largely contribute to their activity for insoluble substrates. Clostridium thermocellum Cel5E is an endoglucanase having xylanolytic activity. The Cel5E originally has a family 11 CBM preferentially binding to β-1,4- and β-1,3-1,4-mixed linkage glucans. In this study, we replaced the CBM with a different type of CBM, either a family 3 microcrystalline cellulose-directed CBM from Clostridium josui scaffoldin, or a family 6 xylan-directed CBM from Clostridium stercorarium xylanase 11A. Chimeric endoglucanases showed enhanced activity that was affected by CBM binding specificity. These chimeric enzymes could efficiently degrade milled lignocellulosic materials, such as corn hulls, because of heterologous components in the plant cell wall, indicating that diverse CBMs play roles in degradation of lignocellulosic materials.     

The location of the ligand-binding site of carbohydrate-binding modules that have evolved from a common sequence is not conserved.

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 A. The protein is a beta-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.     

A single gene codes for two forms of rat nucleolar protein B23 mRNA

Protein B23 (38 kDa, pI = 5.1) is an abundant RNA-associated nucleolar phosphoprotein and putative ribosome assembly factor. A full length cDNA clone (lambda JH1) encoding a major expressed form of rat protein B23, now designated B23.1, was reported recently (Chang, J. H., Dumbar, T. S., and Olson, M. O. J. (1988) J. Biol. Chem. 263, 12824-12837). In this paper the isolation from a rat brain library and sequence of a cDNA clone (lambda JH2) coding for a second form (B23.2) of protein B23 is reported. Isoforms B23.1 and B23.2 are polypeptides of 292 and 257 amino acids, respectively. The 5'-untranslated regions of the two cDNAs and the amino-terminal 255 amino acids of the proteins are identical in the two isoforms. However, the 3'-untranslated regions of the mRNAs are completely different, and the dipeptide Gly-Gly in B23.1 (residues 256 and 257) is replaced by Ala-His in B23.2 indicating that the former is not a precursor of the latter. The finding of AGGT sequences in the 3' regions of lambda JH1 suggest the presence of intron-exon boundaries at the point where the two cDNAs begin to differ. To investigate the origin of the two isoforms, two rat genomic libraries were screened with oligonucleotide probes based on sequences from the unique regions of the two cDNAs. One of the genomic clones isolated (lambda JH125) contained a 6.5-kilobase fragment encoding the 3' end of both cDNAs. lambda JH125 contains four exons designated W, X, Y, and Z in the order indicated. Exons W and X encode 36 amino acids at the carboxyl terminus of B23.2, whereas exons W, Y, and Z encode the carboxyl-terminal 71 amino acid residues of B23.1. Exons X and Z each contain distinct 3'-untranslated sequences in which are found polyadenylation signals. These data suggest that two different mRNAs are formed by alternative splicing of separate 3' segments onto a common 5' region.     

The Role of Pseudo-Endoglucanases in the Evolution of Nematode Cell Wall-Modifying Proteins

In this article, the characterization and evolution of pseudo-endoglucanases and a putative expansin-like gene in the migratory nematode Ditylenchus africanus are described. Four genes were cloned with a very high similarity to the endoglucanase Da-eng1, which, however, lack a part of the catalytic domain most probably due to homologous recombination. Owing to this deletion, at least one of the catalytic residues of the corresponding protein is missing, and hence these genes are possibly pseudogenes. In two of the pseudo-endoglucanase genes, the deletions cause a frameshift (Da-engdel2, Da-engdel4), while two others (Da-engdel1, Da-engdel3) code for protein sequences with an intact carbohydrate-binding module (CBM). Recombinant proteins for Da-ENG1, Da-ENGDEL1, and Da-ENGDEL3 were demonstrated to bind to cellulose, while only Da-ENG1 showed cellulose-degrading activity. This indicates that Da-ENGDEL1 and Da-ENGDEL3 which lack cellulase activity, could still exert a function similar to cellulose-binding proteins (CBPs). Next to the pseudo-endoglucanases, a putative expansin-like gene (Da-exp1) was identified, consisting of a signal peptide, an expansin-like domain, and a CBM. This domain structure was never found before in nematode expansin-like proteins. Interestingly, the CBM of the expansin-like gene is very similar to the endoglucanase CBMs, and a conserved intron position in the CBM of nematode endoglucanases, expansin-like genes, and CBPs indicates a common origin for these domains. This suggests that domain shuffling is an important mechanism in the evolution of cell wall-modifying enzymes in nematodes.     

Cloning of the Ruminococcus albus cel5D and cel9A genes encoding dockerin module-containing endoglucanases and expression of cel5D in Escherichia coli

An EcoRI chromosomal DNA fragment of Ruminococcus albus F-40 that conferred endoglucanase activity on Escherichia coli was cloned. An open reading frame (ORF1) and another incomplete reading frame (ORF2) were found in the EcoRI fragment. The ORF2 was completed using inverse PCR genome walking technique. ORF1 and ORF2, which confront each other, encoded cellulases belonging to families 5 and 9 of the glycoside hydrolases and were designated cel5D and cel9A respectively. The cel5D gene encodes 753 amino acids with a deduced molecular weight of 83,409. Cel5D consists of a signal peptide of 24 amino acids, a family-5 catalytic module, a dockerin module, and two family-4 carbohydrate-binding modules (CBMs). The cel9A gene encodes 936 amino acids with a deduced molecular weight of 104,174, consisting of a signal peptide, a family-9 catalytic module, a family-3 CBM, and a dockerin module. The catalytic module polypeptide (rCel5DCat) derived from Cel5D was constructed, expressed, and purified from a recombinant E. coli. The truncated enzyme hydrolyzed cellohexaose, cellopentaose, and cellotetraose to yield mainly cellotriose and cellobiose with glucose as a minor product, but the enzyme was less active toward cellotriose and not active toward cellobiose, suggesting that this enzyme is a typical endoglucanase. rCel5DCat had a Km of 3.9 mg/ml and a Vmax of 37.2 micromol/min/mg for carboxymethycellulose.     

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.