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Yang, F., Jin, E. S., Zhu, Y., Wu, S., Zhu, W., Jin, Y., and Song, J. (2015). "A Mini-review on the applications of cellulose-binding domains in lignocellulosic material utilizations," BioRes. 10(3), 6081-6094


This manuscript provides a mini review on the fundamentals of cellulose binding domains (CBDs) or cellulose binding modules (CBMs) and their applications using lignocellulosic materials. CBDs, the non-productive part of cellulases, have miscellaneous biological functions and have been widely applied in lignocellulose hydrolysis, protein engineering, structural support, metabolism, energy storage, antibiosis, immunological recognition, targeting, attachment, etc. due to their specific affinity to various substrates of lignocelluloses. Understanding of the properties and mechanisms of CBDs is of vital significance because it provides the basis for fine manipulation of cellulose-CBM interactions and eventually improves the bioconversion performance of lignocelluloses into fuels and desired chemicals. In this short review, the fundamentals of CBD, the definition of CBM family, and the structures of different CBM families are introduced. Then recent findings in the applications of CBDs are discussed relative to the lignocelllulosic utilizations.

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A Mini-review on the Applications of Cellulose-Binding Domains in Lignocellulosic Material Utilizations

Fang Yang, Er-suo Jin, Yangyang Zhu, Shufang Wu, Wenyuan Zhu, Yongcan Jin, and Junlong Song*

This manuscript provides a mini review on the fundamentals of cellulose binding domains (CBDs) or cellulose binding modules (CBMs) and their applications using lignocellulosic materials. CBDs, the non-productive part of cellulases, have miscellaneous biological functions and have been widely applied in lignocellulose hydrolysis, protein engineering, structural support, metabolism, energy storage, antibiosis, immunological recognition, targeting, attachment, etc. due to their specific affinity to various substrates of lignocelluloses. Understanding of the properties and mechanisms of CBDs is of vital significance because it provides the basis for fine manipulation of cellulose-CBM interactions and eventually improves the bioconversion performance of lignocelluloses into fuels and desired chemicals. In this short review, the fundamentals of CBD, the definition of CBM family, and the structures of different CBM families are introduced. Then recent findings in the applications of CBDs are discussed relative to the lignocelllulosic utilizations.

Keywords: Cellulose-binding domain; Cellulase; Cellulose; Lignocellulose; Utilization

Contact information: Jiangsu Provincial Key Lab of Pulp and Paper Science and Technology, Nanjing Forestry University, Nanjing, Jiangsu, 210037 P.R. China;

* Corresponding author:


Sustainable and renewable fuels and materials have received increased attention recently due to concerns about the environmental and unsustainable issues associated with fossil fuels and materials. Lignocellulosic biomass is a major alternative and renewable resource for the production of fuels and chemicals through bioconversion (Nimlos et al. 2007). Cellulose, the major component of lignocellulosic biomass and the most abundant biopolymer in this biosphere, can be degraded by enzymes, i.e. cellulases, into oligosaccharides and finally monosaccharides, which are then subjected to fermentation into fuels and other chemicals (Pauly and Keegstra 2008). The enzymatic hydrolysis of cellulose is a very complex process, which involves the interaction of enzymes, substrates, and enzymatic environment (Yoshida et al. 2008; Zhu et al. 2008; Hall et al. 2010). In order to complete hydrolysis of cellulose, at least three different enzymes act in a synergistic manner (Lynd et al. 2002). These cellulases include exo-β-(1,4)-D-glucanases or cellobiohydrolases (CBHs), endo-β-(1,4)-glucanases (EGs), and β-D-glucosidases. CBHs hydrolyze cellulose chains from the ends to produce mainly soluble cellobiose. EGs hydrolyze internal nicks of the cellulosic chains thereby adding new chain ends for CBHs to act on. To complete the conversion, β-D-glucosidases hydrolyze the soluble disaccharide cellobiose to monomeric glucose (Rabinovich et al. 2002b).

A typical cellulolytic enzyme consists of a distinct cellulose binding module or cellulose binding domain (CBM and CBD are interchangeable thereafter) and at least one of several distinct catalytic domains (CDs), which are structurally and functionally discrete units of proteins and are connected by an inter-domain linker peptide (Mattinen et al. 1997). CBMs play a very important role in the degradation of cellulose crystalline regions since they can concentrate the enzymes on the insoluble cellulosic surface (Boraston et al. 2004; Guillen et al. 2010; Herve et al. 2010). The specific interactions between CBM and cellulose have been taken advantage of extensively in recent years relative to their applications in biology, material, enzyme purification, and other fields. In regard to the advances of fundamental research in CBMs, there are some good review papers available (Linder and Teeri 1997; Levy and Shoseyov 2002; Boraston et al. 2004). Therefore, this mini-review will concentrate on the applications of CBDs in the lignocellulosic utilization, which have been achieved in recent years. A brief description of fundamentals of CBDs will be given in the beginning as well.

Fundamentals of cellulose binding domains

Typical cellulolytic enzymes contain a CD and one or more CBMs; these are connected by an inter-domain linker peptide (Mattinen et al. 1997). Only cellulases originating from a few microorganisms and higher plants have no such domains (Herve et al. 2010). According to the amino acid sequence similarity and 3D structure of adsorption module, the different types of cellulases from different sources have been classified into families (Rabinovich et al. 2002a). In the latest update of the CAZY database, CBMs are grouped into 71 families, which show notable variations in substrate specificity and show miscellaneous biological functions ( CBM1_all.html). That database includes all the adsorption modules of carbohydrate binding modules. That is to say, the CBMs listed in the CAZy data not only contain cellulose binding domains, but also include some starch or other polysaccharides binding domains. The relative molecular weight of CBM is between 0.4 × 104 and 2.0× 104 Daltons (Levy and Shoseyov 2002). Most CBDs have β-sandwich structure and have different number of residues, while some families have β-trefoil, hevein, unique, cysteine knot, β-barrel, and lectin-like folds (Boraston et al. 2004; Cantarel et al. 2009). Three-dimensional structures of CBM and the protein data bank (PDB) code of main proteins have been summarized in the article by Boraston et al. (2004) For example, CBM1, the CBM from family 1, which is found almost exclusively in fungi, consists of 36 amino acid residues, and it is found either at the N-terminal or at the C-terminal extremity of the enzymes (Linder and Teeri 1997). CBMs 2 ~ 6 are mainly found in bacteria.

The structure of CBM1, especially the CBM from CBH I of Trichoderma reesei, has been investigated thoroughly. Its NMR structure, as shown in Fig. 1, shows a contiguous independently wedge-shaped structure. One face of the wedge contains three highly conserved aromatic amino acids Y5, Y31, and Y32, which are involved in cellulose recognition and binding. There are two more polar residues (Q7 and N29) that can potentially form hydrogen bonds to cellulose (Beckham et al. 2010). This wedge-shaped structure is likely to be shared by all fungal CBDs (Mattinen et al. 1997). The specificity of CBMs can be changed through simple mutations (von Schantz et al. 2014). It was suggested that tryptophan residues contribute to higher binding affinity than tyrosine residues. Trp→Tyr mutation would decrease the affinity and increase the off-rate of Cel6A CBM1 on crystalline cellulose (Lehtio et al. 2003). However, this effect took place only in the case of one Trp residue mutation. Two Trp residues’ mutation was found to yield no further improvement in binding.

Functions of CBMs relative to cellulosic materials embody mainly three aspects (Boraston et al.2004): (i) proximity effects: to increase accessibility of enzyme and insoluble substrates. Proximity effects have been confirmed by multiple mutation studies (Linder et al. 1995; Reinikainen et al. 1992). (ii) substrate targeting: to target specific carbohydrates in complex structures of plant cell walls, such as CBMs from xylanases or mannanases, which have been shown on large crystals of cellulose with immuno-gold-labeled CBMs (Lehtio et al. 2003). (iii) nonhydrolytic cellulose disruption: to disrupt cellulose regular rigid supramolecular structures. Some bacterial CBMs have indirectly confirmed this function (Kataeva et al. 2002), such as family 1 CBD and family 2 (Tomme et al. 1995).

Fig. 1. Carbohydrate-binding module from T. reesei Cel7A with Y5, Q7, N29, Y31, and Y32 shown. (a) Side view. (b) Top view. Reprinted from (Beckham et al. 2010) with permission from American Chemical Society.

CBM-cellulose binding involves both hydrophobic interactions and polar interaction. But there is not enough evidence to prove that CBM can modify cellulose substrates. The VDW interactions (McGuffee and Elcock 2006; McGuffee and Elcock 2010; Mereghetti and Wade 2011) play a more important role in stabilizing the CBM-fiber binding, whereas CBM and fiber can form a number of hydrogen bonds because of electrostatic interactions (Elcock 2004; Alekozai et al. 2013), which may explain why the diffusion of the CBM is hindered on the fiber surfaces.

CBMs have a high specificity and functional diversity. They come from different sources, and their functions are diverse from each other. It was established that the binding sites of families 1, 2, and 3 CBDs are adapted to bind to a surface, while family 4 CBD binds to a single molecule (Shoseyov et al.2006). For instance, CBM2, similar to CBM1 from fungi, can bind with the bacterial microcrystalline cellulose and partially crystalline phosphate expanding fibers (PASC) (Rabinovich et al. 2002b); whereas CBM4, with an obvious coupling groove structure as shown in Fig. 2, cannot bind bacterial microcrystalline cellulose, but it can combine with the amorphous region of PASC (Jervis et al. 1997; McLean et al. 2002). In addition, CBM adsorption and desorption involving cotton cellulose allomorphs (I, II, and III) has been investigated (Ciolacu et al. 2014). The results indicate that CBM1 has the highest adsorption capacity on cellulose I. There are some CBDs that have two or more multi-specific CBMs with similar sequences. For example, Xylanase 10A from Rhodothermus marinus has two CBMs, i.e. CBM4-1 and CBM4-2 (Abou-Hachem et al. 2003), though the cited article does not specify whether they have the same functions. Another enzyme, Cel9B from Paenibacillus barcinonensis, is composed of the two cellulose-binding domains CBM3b and CBM3c. Chiriac et al.(2010) demonstrated that CBM3b and CBM3c have different functions; CBM3b shows the ability to bind to cellulose while CBM3c does not.

Fig. 2. A cleft structure of the hydrophobic residues (yellow) of carbohydrate-binding module family 4. Reproduced with permission from (Prates et al. 2013).

Applications of CBDs in Lignocellulosic utilizations

CBDs’ natural function is involved in some biological processes such as structural support, metabolism, energy storage, antibiosis, immunological recognition, targeting, and attachment, etc.(Guillen et al. 2010).

CBDs also can play an important role in other applications, such us the fusion of CBM and other proteins to produce complexes that offer the possibility of developing paper-based diagnostic tools (Kong and Hu 2012) for infectious diseases (Rosa et al. 2014). Such structures can target immobilization of antibodies (Cao et al. 2007; Hussack et al. 2009), proteins (Ofir et al. 2005), bacteriophages (Tolba et al. 2008), and bacteria (Wang et al. 2002) onto celluloses. It is possible even to detect multiple targets.

CBD can also be employed in the utilization of lignocellulosic biomass. During the application of lignocelluloses, enzymes are usually used to break down or modify a cellulose surface for improved properties. In order to lower the cost of enzymes, it is necessary to modify the enzymes for improved performance. Due to the specific binding between CBD and cellulose, it is rational to modify the CBDs of enzymes. By this means one can fine-tune the interactions between CBD and substrate cellulose for better performance. In this review, these applications are grouped according to the methodology by which they are used and the type of CBMs (CBM family) they employ.

Applications of CBDs Solely

Even though there are a number of applications of CBDs for bioconversion in the literature, there are only a few reports on the applications of CBDs solely, i.e. using only the CBD portion of enzyme and without the CD segment involved. Hall et al. (2011a) utilized the CBDs of cellulases from T. reesei to pretreat biomass prior to enzymatic hydrolysis. CBDs of cellulases from T. reesei had a noticeable impact on cellulose structure upon incubation. The crystallinity of different types of cellulose (microcrystalline and fibrous) was reduced after pretreatment with CBDs, rendering the substrates less recalcitrant to enzymatic hydrolysis, and an increase up to 25% in glucose concentration during hydrolysis was obtained. In addition, the bleached chemical pulp properties, viscosity and strength after PFI refining, can be equally affected by  the presence of CBD in intact cellulases (Suurnäkki et al.2000).

Mello and Polikarpov (2014) cloned, expressed, and purified the CBM from CBH I from Trichoderma harzianum. Then it was used, in combination with either a commercial cellulase preparation, T. reeseiCBH I, or its separate catalytic domain, to hydrolyze filter paper. In all cases the amount of glucose released was increased, reaching up to 30% gain when the carbohydrate-binding module was added to the reaction.

In another report (Kitaoka and Tanaka 2001), CBDs was added into the pulp with anionic polyacrylamides (A-PAM). Wet strength and dry tensile strengths of the paper sheets were significantly improved. Yokota et al. (2009) went further and grafted CBMs derived from T. viride and T. reesei, and Thermomyces lanuginosus, respectively, into anionic polyacrylamide via a peptide condensation reaction. The prepared CBM-conjugated A-PAMs displayed different retention behaviors, depending on the kind of pulp substrates, i.e. hardwood and softwood fibers.

Applications of Proteins with Fused CBDs

Fusion of one or more CBDs to a neutral enzyme

A neutral enzyme, which has low activity in its original state, can be enhanced significantly in its performance by fusion one or more CBDs into the neutral enzyme. For instance, Cel5A, an endoglucanase (EG), contains a single catalytic domain but no distinct cellulose-binding domain. It has been reconstructed by attaching one or two CBDs for enhanced performance by Tang et al. (2014). They reconstructed it by fusion of a CBM containing a linker from Corticium rolfsii. Results showed that the degradation of cellulose by redesigned EG was about double that by original enzyme; while the optimal pH and temperature, pH stability, and heat stability of redesigned EG were similar to the original one. Telke et al. (2013) fused a family 6 CBM from Saccharophagus degradans to the C-terminus of a mutant of Cel5A, which resulted in a 7-fold increase in thermostability than the native Cel5A. The final fusion protein showed much higher affinity and 7-fold higher hydrolytic activity than the native Cel5A on Avicel and filter paper.

Due to the specific adsorption of CBD to cellulose substrates, an efficient purification process has been developed for some proteins by tagging a CBD. This scalable method can purify the CBM-tag protein by using a simple unit operation, involving centrifugation or filtration. For example, the cathelicidin-derived human peptide LL37, which has a broad spectrum of antimicrobial and immunomodulatory activities, was cloned to the N- and C-termini of a CBM3 fused to the linker sequence (LK-CBM3) from Clostridium thermocellum (Ramos et al. 2010). In the course of processing, the purification was achieved using cellulose CF11 fibers, taking advantage of the CBM3’s specific affinity for cellulose. To improve the performance of cellulose substrate in the CBM-tagged protein purification process, Hong et al. (2008) developed an ultra-high capacity cellulosic adsorbent regenerated amorphous cellulose (RAC), with a binding capacity of up to 365 mg protein per gram of RAC, enabling it to efficiently capture (dilute) proteins.

Fusion of one more CBD to an enzyme for improved activity

Due to the specific affinity between CBD and cellulose, one or more CBDs can be fused to an enzyme that has its own CBDs for improved affinity and other properties.

The family 1 cellulose-binding module (CBM1) from fungi is the most extensively investigated CBM. It has been fused with glucose-tolerant β-glucosidase (BGL) (Zhao et al. 2013), CSCMCase (Thongekkaew et al. 2013), endo-1,4-mannanases (Tuan Anh et al. 2010), and laccase (Ravalason et al. 2009), for improved properties. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose, and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied; the yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188. Compared with CSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL® and Avicel) was higher than that of the native enzyme. The fused mannanase–CBM enzyme was more thermostable, and showed that mannanase–CBM improved the glucose yield compared to wild-type and recombinant mannanases for softwood hydrolysis when it was used in association with a T. reeseienzymatic cocktail. Laccase-CBM was able to bind to a cellulosic substrate and, to a greater extent, to softwood kraft pulp. Laccase-CBM was further investigated for its softwood kraft pulp bio-bleaching potential and compared with the P. cinnabarinus laccase. Addition of a CBM was shown to greatly improve the delignification capabilities of the laccase in the presence of 1-hydroxybenzotriazole (HBT). In addition, ClO2 reduction using 5U of chimeric enzyme per gram of pulp was almost double than that observed using 20U of P. cinnabarinus laccase per gram of pulp.

A novel gene encoding a swollenin-like protein, POSWOI, was isolated from the filamentous fungus Penicillium oxalicum by Thermal Asymmetric Interlaced PCR (TAIL-PCR) (Kang et al. 2013). It consisted of a CBM1 followed by a linker connected to a family 45 endoglucanase-like domain. Using the CBH I promoter, recombinant POSWOI was efficiently produced in T. reesei with a yield of 105 mg/L, and it showed significant disruptive activity on crystalline cellulose. Simultaneous reaction with both POSWOI and cellulases enhanced the hydrolysis of crystalline cellulose Avicel by approximately 50%. Using a POSWOI-pretreatment procedure, cellulases can produce nearly twice as many reducing sugars as without pretreatment.

A two-CBM1 strategy was also used to construct recombinants for improved performance. Linder et al. (1996) constructed a recombinant by fusing the CBDs of two T. reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers. The double CBDs exhibited much higher affinity on cellulose than either of the single CBD, indicating an interplay between the two components. In another report, Gundllapalli et al. (2007) claimed that they linked the β-glucosidase enzyme (BGL) from Saccharomycopsis fibuligera to the CBM1 of CBH II in T. reesei with single and double copies of CBD fused at the N-terminus of BGL. The results indicated that the recombinant enzymes of BGL-CBD displayed a 2- to 4-fold increase in their hydrolytic activity toward cellulosic substrates such as Avicel, amorphous cellulose, bacterial microcrystalline cellulose, and carboxymethyl cellulose in comparison with the native enzyme.

CBMs from other families are also employed to construct new proteins. The family 2 cellulose-binding module (CBM2) of xylanase CflXyn11A from Cellulomonas flavigena was fused to cellulase Cel7B from T. reesei, which has a CBM1 (Pavon-Orozco et al. 2012). The results showed that there is a high degree of synergy (6.3) between CflXyn11A and TrCel7B in hydrolysis of sugar cane bagasse after 12 h in the equimolar mixture.

The two CBDs of family 3 (CBM3) from Clostridium cellulovorans were fused together by Levy et al.(2002) to produce a cellulose cross-linked protein (CCP), which was found to improve the filter performance of paper. A very interesting aspect is that the single CBD protein can also improve the mechanical properties of paper, but the protein with double CBM3 yielded very poor results. This may be explained by the effect of linker length. Maybe in this CCP, the distance between two CBDs is too short and the two CBDs cannot fully develop their binding ability. Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with a novel molecular architecture among family 9 enzymes that comprises a catalytic domain (GH9), a family 3c cellulose-binding domain (CBM3c), a fibronectin III-like domain repeat (Fn31,2), and a C-terminal family 3b cellulose-binding domain (CBM3b). Chiriac et al. (2010) demonstrated that CBM3b and CMB3c have different functions. Deletion of CBM3c produced a notable reduction in hydrolytic activity, while it did not affect the cellulose-binding properties, as CBM3c did not show the ability to bind to cellulose. On the contrary, CBM3b exhibited binding to cellulose. Cadena et al. (2010) used this Cel9B as a highly efficient biocatalyst in pulp refining to reduce the associated energy. In their work, they found that the truncated form containing the catalytic domain (GH9-CBD3c) has a strong effect on fiber morphology. Comparing its effect with that of the whole cellulase (Cel9B), the truncated enzyme contributed to increasing paper strength through improved tensile strength, burst strength, and tear resistance. Therefore, the catalytic domain of Cel9B has biorefining action on pulp. Although cellulose binding domains (CBDs) are less efficient toward pulp refining, evidence obtained in their work suggested that CBD3b alters fiber surfaces and influences paper properties as a result.

The family 6 carbohydrate-binding module (CBM6) was used to fuse with xylanase (Qiao et al. 2014) to increase the ability to digest hemicelluloses. Bae et al. (2003) went further, fusing endoglucanase Cel5 of Ruminococcus albus at the C-terminus with the single-cellulose binding domain (CBM6) of Clostridium stercorarium xylanase A. The recombinant Cel5-CBM6 improved the affinity of enzyme to the insoluble substrate cellulose.

The family 22 of carbohydrate binding module (CBM22) binds xylan but not to cellulose. Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two CBMs that belong to family 22. Mamo et al. (2007) took advantage of this type CBM and fused to an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but it decreased the stability of the enzyme. Meanwhile, the optimum temperature and pH for the activity of the xylanase did not change.

Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibers during amorphogenesis (Gourlay et al. 2012). The extents of phosphoric acid-induced and swollenin-induced changes to cellulose accessibility were successfully quantified using this technique. The adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick and quantitative measure of the accessibility of cellulosic substrates.

CBD not only exhibits specific adsorption to cellulose substrate, but also shows evidence of acting as a thermostabilizing domain for enzymes. The CBD of cellobiohydrolase Cel7A from T. reesei melted at 65 to 66 oC, the catalytic domain obtained via papain-catalyzed proteolysis was shown to denature at 51 oC, while the whole enzyme displayed a melting point of 59 oC (Hall et al. 2011b). Its thermostability has also been demonstrated in xylanase 2 from T. reesei (He et al. 2009) .


Lignocellulose, which has been the main raw material for many commodities closely related to people’s life for centuries, is one of the richest sources for renewable fuels and chemicals for future. How to utilize it efficiently and effectively is an issue of great importance. Enzymes can catalyze the reactions associated with lignocellulose and its components of cellulose, hemicellulose, and lignin efficiently and effectively. Due to the specific affinity between CBD and cellulose, as well as the matrix structure of lignocellulose, CBDs adsorbed on cellulose surface concentrate the enzymes on the insoluble materials and therefore can improve the performance of not only cellulases, but also mananases, xylanase, laccases, and other enzymes. CBD can be fused to a neutral enzyme without a CBD or to a typical enzyme, which has its own CBD for improved affinity and activity. It has been fused to cellulases, mananases, xylanase, laccases, and other enzymes to promote the degradation of the components of lignocellulose for biofuels and biochemicals. Therefore, understanding of CBMs properties and the mechanisms in ligand binding is vital to the comprehensive utilization of lignocellulose.

Due to the versatile functions of CBMs, there is a large pool of CBMs available for potential utilization. This offers numerous choices to improve the performance of enzymes by fusion of one or more CBMs to an existing enzyme to fine-tune its affinity to the substrate and therefore to manipulate its performance as desired. As a consequence of the activity improvement, the consumption and the cost of enzymes can be reduced dramatically. So far, the most extensively studied CBM is the CBM from family 1, and some scientific reports and commercial applications are related to family 3 CBM, but only a few applications have involved the CBMs from other families. The interactions between CBMs and cellulose is not fully understood yet, especially the CBMs from other families.

Better understanding of CBMs properties and the mechanisms in ligand binding will promote development of new cellulose-recognition technologies and provide the basis for fine manipulation of the CBM-cellulose interactions. Such efforts will benefit the utilization of lignocellulose materials eventually.


The authors are grateful for the support of Special Fund for Forestry Scientific Research in the Public Interest (201404510), National Natural Science Foundation of China (31270613, 31200454), Qing-Lan Project, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.


Abou-Hachem, M., Olsson, F., and Nordberg Karlsson, E. (2003). “Probing the stability of the modular family 10 xylanase from Rhodothermus marinus,” Extremophiles: Life under Extreme Conditions 7(6), 483-491. DOI: 10.1007/s00792-003-0348-1

Alekozai, E. M., GhattyVenkataKrishna, P. K., Uberbacher, E. C., Crowley, M. F., Smith, J. C., and Cheng, X. (2013). “Simulation analysis of the cellulase Cel7A carbohydrate binding module on the surface of the cellulose Iβ,” Cellulose 21(2), 951-971. DOI: 10.1007/s10570-013-0026-0

Bae, H. J., Turcotte, G., Chamberland, H., Karita, S., and Vezina, L. P. (2003). “A comparative study between an endoglucanase IV and its fused protein complex Cel5-CBM6,” FEMS Microbiol. Lett.227(2), 175-181. DOI: 10.1016/S0378-1097(03)00593-7

Beckham, G. T., Matthews, J. F., Bomble, Y. J., Bu, L., Adney, W. S., Himmel, M. E., Nimlos, M. R., and Crowley, M. F. (2010). “Identification of Amino acids responsible for processivity in a family 1 carbohydrate-binding module from a fungal cellulase,” J. Phys. Chem. B 114(3), 1447-1453. DOI: 10.1021/jp908810a

Boraston, A. B., Bolam, D. N., Gilbert, H. J., and Davies, G. J. (2004). “Carbohydrate-binding modules: Fine-tuning polysaccharide recognition,” Biochem. J. 382, 769-781. DOI: 10.1042/BJ20040892

Cadena, E. M., Chriac, A. I., Pastor, F. I. J., Diaz, P., Vidal, T., and Torres, A. L. (2010). “Use of cellulases and recombinant cellulose binding domains for refining TCF kraft pulp,” Biotechnol. Prog.26(4), 960-967. DOI: 10.1002/btpr.411

Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V., and Henrissat, B. (2009). “The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics,” Nucleic Acids Res. 37, D233-D238. DOI: 10.1093/nar/gkn663

Cao, Y., Zhang, Q., Wang, C., Zhu, Y. Y., and Bai, G. (2007). “Preparation of novel immunomagnetic cellulose microspheres via cellulose binding domain-protein A linkage and its use for the isolation of interferon alpha-2b,” J. Chromatogr. A 1149(2), 228-235. DOI: 10.1016/j.chroma.2007.03.032

Chiriac, A. I., Cadena, E. M., Vidal, T., Torres, A. L., Diaz, P., and Pastor, F. I. J. (2010). “Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture,” Appl. Microbiol. Biotechnol. 86(4), 1125-1134. DOI: 10.1007/s00253-009-2350-8

Ciolacu, D., Chiriac, A. I., Pastor, F. I., and Kokol, V. (2014). “The influence of supramolecular structure of cellulose allomorphs on the interactions with cellulose-binding domain, CBD3b from Paenibacillus barcinonensis,” Bioresour. Technol. 157, 14-21. DOI: 10.1016/j.biortech.2014.01.027

Elcock, A. H. (2004). “Molecular simulations of diffusion and association in multimacromolecular systems,” Method. Enzymol. 383, 166-198. DOI: 10.1016/s0076-6879(04)83008-8

Gourlay, K., Arantes, V., and Saddler, J. N. (2012). “Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis,” Biotechnol. Biofuels 5, 51. DOI: 10.1186/1754-6834-5-51

Guillen, D., Sanchez, S., and Rodriguez-Sanoja, R. (2010). “Carbohydrate-binding domains: multiplicity of biological roles,” Appl. Microbiol. Biotechnol. 85(5), 1241-1249. DOI: 10.1007/s00253-009-2331-y

Gundllapalli, S. B., Pretorius, I. S., and Otero, R. R. C. (2007). “Effect of the cellulose-binding domain on the catalytic activity of a beta-glucosidase from Saccharomycopsis fibuligera,” J. Ind. Microbiol. Biotechnol. 34(6), 413-421. DOI: 10.1007/s10295-007-0213-9

Hall, M., Bansal, P., Lee, J. H., Realff, M. J., and Bommarius, A. S. (2010). “Cellulose crystallinity – A key predictor of the enzymatic hydrolysis rate,” Febs J. 277(6), 1571-1582. DOI: 10.1111/j.1742-4658.2010.07585.x

Hall, M., Bansal, P., Lee, J. H., Realff, M. J., and Bommarius, A. S. (2011a). “Biological pretreatment of cellulose: Enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases,” Bioresour. Technol. 102(3), 2910-2915. DOI: 10.1016/j.biortech.2010.11.010

Hall, M., Rubin, J., Behrens, S. H., and Bommarius, A. S. (2011b). “The cellulose-binding domain of cellobiohydrolase Cel7A from Trichoderma reesei is also a thermostabilizing domain,” J. Biotechnol.155(4), 370-376. DOI: 10.1016/j.jbiotec.2011.07.016

He, J., Yu, B., Zhang, K., Ding, X., and Chen, D. (2009). “Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2.” New Biotechnol. 26(1-2), 53-59. DOI: 10.1016/j.nbt.2009.04.002

Herve, C., Rogowski, A., Blake, A. W., Marcus, S. E., Gilbert, H. J., and Knox, J. P. (2010). “Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects,” Proc. Natl. Acad. Sci. USA, 107(34), 15293-15298. DOI: 10.1073/pnas.1005732107

Hong, J., Ye, X., Wang, Y., and Zhang, Y. H. P. (2008). “Bioseparation of recombinant cellulose-bindning module-proteins by affinity adsorption on an ultra-high-capacity cellulosic adsorbent,” Anal. Chim. Acta 621(2), 193-199. DOI: 10.1016/j.aca.2008.05.041

Hussack, G., Luo, Y., Veldhuis, L., Hall, J. C., Tanha, J., and MacKenzie, R. (2009). “Multivalent anchoring and oriented display of single-domain antibodies on cellulose,” Sensors-Basel 9(7), 5351-5367. DOI: 10.3390/s90705351

Jervis, E. J., Haynes, C. A., and Kilburn, D. G. (1997). “Surface diffusion of cellulases and their isolated binding domains on cellulose,” J. Biol. Chem. 272(38), 24016-24023. DOI: 10.1074/jbc.272.38.24016

Kang, K., Wang, S., Lai, G., Liu, G., and Xing, M. (2013). “Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose,” BMC Biotechnol. 13(1), 42. DOI: 10.1186/1472-6750-13-42

Kataeva, I. A., Seidel, R. D., Shah, A., West, L. T., Li, X. L., and Ljungdahl, L. G. (2002). “The fibronectin type 3-like repeat from the Clostridium thermocellum cellobiohydrolase CbhA promotes hydrolysis of cellulose by modifying its surface,” Appl. Environ. Microb. 68(9), 4292-4300. DOI: 10.1128/AEM.68.9.4292-4300.2002

Kitaoka, T., and Tanaka, H. (2001). “Novel paper strength additive containing cellulose-binding domain of cellulase,” J. Wood Sci. 47(4), 322-324. DOI: 10.1007/BF00766721

Kong, F. Z., and Hu, Y. F. (2012). “Biomolecule immobilization techniques for bioactive paper fabrication,” Anal. Bioanal. Chem. 403(1), 7-13. DOI: 10.1007/s00216-012-5821-1

Kraulis, J., Clore, G. M., Nilges, M., Jones, T. A., Pettersson, G., Knowles, J., and Gronenborn, A. M. (1989). “Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing,” Biochem. 28(18), 7241-7257. DOI: 10.1021/bi00444a016

Lehtio, J., Sugiyama, J., Gustavsson, M., Fransson, L., Linder, M., and Teeri, T. T. (2003). “The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules,” Proc. Natl.l Acad. Sci. USA 100(2), 484-489. DOI: 10.1073/pnas.212651999

Levy, I., Nussinovitch, A., Shpigel, E., and Shoseyov, O. (2002). “Recombinant cellulose crosslinking protein: A novel paper-biomaterial,” Cellulose 9(1), 91-98. DOI: 10.1023/A:1015848701029

Levy, I., and Shoseyov, O. (2002). “Cellulose-binding domains: Biotechnological applications,” Biotechnol. Adv. 20(3), 191-213. DOI: 10.1016/s0734-9750(02)00006-x

Linder, M., Mattinen, M. L., Kontteli, M., Lindeberg, G., Ståhlberg, J., Drakenberg, T., Reinikainen, T., Pettersson, G., and Annila, A. (1995). “Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I,” Protein Sci. 4(6), 1056-1064. DOI: 10.1002/pro.5560040604

Linder, M., Salovuori, I., Ruohonen, L., and Teeri, T. T. (1996). “Characterization of a double cellulose-binding domain. Synergistic high affinity binding to crystalline cellulose,” J. Biol. Chem.271(35), 21268-21272. DOI: 10.1074/jbc.271.35.21268

Linder, M., and Teeri, T. T. (1997). “The roles and function of cellulose-binding domains,” J. Biotechnol. 57(1-3), 15-28. DOI: 10.1016/s0168-1656(97)00087-4

Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S. (2002). “Microbial cellulose utilization: Fundamentals and biotechnology,” Microbiol. Mol. Biol. R. 66(4), 739-739. DOI: 10.1128/MMBR.66.4.739.2002

Mamo, G., Hatti-Kaul, R., and Mattiasson, B. (2007). “Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7,” Extremophiles11(1), 169-177. DOI: 10.1007/s00792-006-0023-4

Mattinen, M.-L., Linder, M., Teleman, A., and Annila, A. (1997). “Interaction between cellohexaose and cellulose binding domains from Trichoderma reesei cellulases,” FEBS Lett. 407(3), 291-296. DOI: 10.1016/S0014-5793(97)00356-6

McGuffee, S. R., and Elcock, A. H. (2006). “Atomically detailed simulations of concentrated protein solutions: The effects of salt, pH, point mutations, and protein concentration in simulations of 1000-molecule systems,” J. Am. Chem. Soc. 128(37), 12098-12110. DOI: 10.1021/ja0614058

McGuffee, S. R., and Elcock, A. H. (2010). “Diffusion, crowding & protein stability in a dynamic molecular model of the bacterial cytoplasm,” Plos Comput. Biol. 6(3), e1000694. DOI: 10.1371/journal.pcbi.1000694

McLean, B. W., Boraston, A. B., Brouwer, D., Sanaie, N., Fyfe, C. A., Warren, R. A. J., Kilburn, D. G., and Haynes, C. A. (2002). “Carbohydrate-binding modules recognize fine substructures of cellulose,” J. Biol. Chem. 277(52), 50245-50254. DOI: 10.1074/jbc.M204433200

Mello, B. L., and Polikarpov, I. (2014). “Family 1 carbohydrate binding-modules enhance saccharification rates,” AMB Express, 4, 36-36. DOI: 10.1186/s13568-014-0036-9

Mereghetti, P., and Wade, R. C. (2011). “Diffusion of hydrophobin proteins in solution and interactions with a graphite surface,” Bmc Biophys. 4, 9. DOI:10.1186/2046-1682-4-9. DOI: 10.1186/2046-1682-4-9

Nimlos, M. R., Matthews, J. F., Crowley, M. F., Walker, R. C., Chukkapalli, G., Brady, J. W., Adney, W. S., Cleary, J. M., Zhong, L., and Himmel, M. E. (2007). “Molecular modeling suggests induced fit of Family I carbohydrate-binding modules with a broken-chain cellulose surface,” Protein Eng. Des. Sel. 20(4), 179-187. DOI: 10.1093/protein/gzm010

Ofir, K., Berdichevsky, Y., Benhar, I., Azriel-Rosenfeld, R., Larned, R., Barak, Y., Bayer, E. A., and Morag, E. (2005). “Versatile protein microarray based on carbohydrate-binding modules,” Proteomics5(7), 1806-1814. DOI: 10.1002/pmic.200401078

Pauly, M., and Keegstra, K. (2008). “Cell-wall carbohydrates and their modification as a resource for biofuels,” Plant J. 54(4), 559-568. DOI: 10.1111/j.1365-313X.2008.03463.x

Pavon-Orozco, P., Santiago-Hernandez, A., Rosengren, A., Eugenia Hidalgo-Lara, M., and Stalbrand, H. (2012). “The family II carbohydrate-binding module of xylanase CflXyn11A from Cellulomonas flavigena increases the synergy with cellulase TrCel7B from Trichoderma reesei during the hydrolysis of sugar cane bagasse,” Bioresour. Technol. 104, 622-630. DOI: 10.1016/j.biortech.2011.11.068

Prates, E. T., Stankovic, I., Silveira, R. L., Liberato, M. V., Henrique-Silva, F., Pereira, N., Jr., Polikarpov, I., and Skaf, M. S. (2013). “X-ray structure and molecular dynamics simulations of Endoglucanase 3 from Trichoderma harzianum: Structural organization and substrate recognition by endoglucanases that lack cellulose binding module,” PLoS ONE 8(3), e59069. DOI: 10.1371/journal.pone.0059069

Qiao, W., Tang, S., Mi, S., Jia, X., Peng, X., and Han, Y. (2014). “Biochemical characterization of a novel thermostable GH11 xylanase with CBM6 domain from Caldicellulosiruptor kronotskyensis,” J. Mol. Catal. B-Enzym. 107, 8-16. DOI: 10.1016/j.molcatb.2014.05.009

Rabinovich, M. L., Melnick, M. S., and Bolobova, A. V. (2002a). “The structure and mechanism of action of cellulolytic enzymes,” Biochemistry-Moscow 67(8), 850-871. DOI: 10.1023/A:1019958419032

Rabinovich, M. L., Melnik, M. S., and Boloboba, A. V. (2002b). “Microbial cellulases (Review),” Appl. Biochem. Microbiol. 38(4), 305-321. DOI: 10.1023/A:1016264219885

Ramos, R., Domingues, L., and Gama, M. (2010). “Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum,” Protein Expr. Purif. 71(1), 1-7. DOI: 10.1016/j.pep.2009.10.016

Ravalason, H., Herpoel-Gimbert, I., Record, E., Bertaud, F., Grisel, S., de Weert, S., van den Hondel, C. A. M. J. J., Asther, M., Petit-Conil, M., and Sigoillot, J.-C. (2009). “Fusion of a family 1 carbohydrate binding module of Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulp biobleaching,” J. Biotechnol. 142(3-4), 220-226. DOI: 10.1016/j.jbiotec.2009.04.013

Reinikainen, T., Ruohonen, L., Nevanen, T., Laaksonen, L., Kraulis, P., Jones, T. A., Knowles, J. K. C., and Teeri, T. T. (1992). “Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.” Proteins: Struct., Funct., Bioinf. 14(4), 475-482. DOI: 10.1002/prot.340140408

Rosa, A. M., Louro, A. F., Martins, S. A., Inacio, J., Azevedo, A. M., and Prazeres, D. M. (2014). “Capture and detection of DNA hybrids on paper via the anchoring of antibodies with fusions of carbohydrate binding modules and ZZ-domains,” Anal. Chem., 86(9), 4340-4347. DOI: 10.1021/ac5001288

Shoseyov, O., Shani, Z., and Levy, I. (2006). “Carbohydrate binding modules: Biochemical properties and novel applications,” Microbiol. Mol. Biol. Rev. 70(2), 283-295. DOI: 10.1128/MMBR.00028-05

Suurnäkki, A., Tenkanen, M., Siika-Aho, M., Niku-Paavola, M.-L., Viikari, L., and Buchert, J. (2000). “Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp,” Cellulose 7(2), 189-209. DOI: 10.1023/A:1009280109519

Tang, Z., Chen, H., Chen, L., Liu, S., Han, X., and Wu, Q. (2014). “Improving endoglucanase activity by adding the carbohydrate-binding module from Corticium rolfsii,” J. Microbiol. Biotechnol. 24(4), 440-446. DOI: 10.4014/jmb.1311.11007

Telke, A. A., Zhuang, N., Ghatge, S. S., Lee, S.-H., Shah, A. A., Khan, H., Um, Y., Shin, H.-D., Chung, Y. R., Lee, K. H., and Kim, S.-W. (2013). “Engineering of Family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates,” PLoS ONE8(6), e65727. DOI: 10.1371/journal.pone.0065727

Thongekkaew, J., Ikeda, H., Masaki, K., and Iefuji, H. (2013). “Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates,” Enzyme Microb. Technol. 52(4-5), 241-246. DOI: 10.1016/j.enzmictec.2013.02.002

Tolba, M., Brovko, L. Y., Minikh, O., and Griffiths, M. W. (2008). “Engineering of bacteriophages displaying affinity tags on its head for biosensor applications,” Nsti Nanotech. (2), 449-452.

Tomme, P., Driver, D. P., Amandoron, E. A., Miller, R. C., Jr., Antony, R., Warren, J., and Kilburn, D. G. (1995). “Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain,” J. Bacter. 177(15), 4356-4363.

Tuan Anh, P., Berrin, J. G., Record, E., Kim Anh, T., and Sigoillot, J.-C. (2010). “Hydrolysis of softwood by Aspergillus mannanase: Role of a carbohydrate-binding module,” J. Biotechnol. 148(4), 163-170. DOI: 10.1016/j.jbiotec.2010.05.012

von Schantz, L., Hakansson, M., Logan, D. T., Nordberg-Karlsson, E., and Ohlin, M. (2014). “Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Pi interactions,” Proteins 82(12), 3466-3475. DOI: 10.1002/prot.24700

Wang, A. J. A., Mulchandani, A., and Chen, W. (2002). “Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase,” Appl. Environ. Microb. 68(4), 1684-1689. DOI: 10.1128/AEM.68.4.1684-1689.2002

Yokota, S., Matsuo, K., Kitaoka, T., and Wariishi, H. (2009). “Retention and paper strength characteristics of anionic polyacrylamides conjugated with carbohydrate-binding modules,” BioResources 4(1), 234-244.

Yoshida, M., Liu, Y., Uchida, S., Kawarada, K., Ukagami, Y., Ichinose, H., Kaneko, S., and Fukuda, K. (2008). “Effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides,” Biosci. Biotechnol. Biochem. 72(3), 805-810. DOI: 10.1271/bbb.70689

Zhao, L., Pang, Q., Xie, J., Pei, J., Wang, F., and Fan, S. (2013). “Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum beta-glucosidase fused to Clostridium cellulovoranscellulose binding domain and its application in hydrolysis of microcrystalline cellulose,” BMC Biotechnol. 13, 101. DOI: 10.1186/1472-6750-13-101

Zhu, L., O’Dwyer, J. P., Chang, V. S., Granda, C. B., and Holtzapple, M. T. (2008). “Structural features affecting biomass enzymatic digestibility,” Bioresour. Technol., 99(9), 3817-3828. DOI: 10.1016/j.biortech.2007.07.033

Article submitted: May 7, 2015; Peer review completed: June 10, 2015; Revised version received: June 19, 2015; Accepted: June 20, 2015; Published: June 26, 2015.

DOI: 10.15376/biores.10.3.Yang