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

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, 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 native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.     

Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

The C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. The Listeria monocytogenes Ply118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure of Listeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding using L. monocytogenes serovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundant tagO homologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of either tagO1 (EGDe lmo0959) or tagO2 (EGDe lmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficient Listeria cells. Substitution of tagO from an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from other Listeria phage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.     

Characterization and affinity applications of cellulose-binding domains

Cellulose-binding domains (CBDs) are discrete protein modules found in a large number of carbohydrolases and a few nonhydrolytic proteins. To date, almost 200 sequences can be classified in 13 different families with distinctly different properties. CBDs vary in size from 4 to 20 kDa and occur at different positions within the polypeptides; N-terminal, C-terminal and internal. They have a moderately high and specific affinity for insoluble or soluble cellulosics with dissociation constants in the low micromolar range. Some CBDs bind irreversibly to cellulose and can be used for applications involving immobilization, others bind reversibly and are more useful for separations and purifications. Dependent on the CBD used, desorption from the matrix can be promoted under various different conditions including denaturants (urea, high pH), water, or specific competitive ligands (e.g. cellobiose). Family I and IV CBDs bind reversibly to cellulose in contrast to family II and III CBDs which are in general, irreversibly bound. The binding of family II CBDs (CBD_Cex) to crystalline cellulose is characterized by a large favourable increase in entropy indicating that dehydration of the sorbent and the protein are the major driving forces for binding. In contrast, binding of family IV CBDs (CBD_N1) to amorphous or soluble cellulosics is driven by a favourable change in enthalpy which is partially offset by an unfavourable entropy change. Hydrogen bond formation and van der Waals interactions are the main driving forces for binding. CBDs with affinity for crystalline cellulose are useful tags for classical column affinity chromatography. The affinity of CBD_N1 for soluble cellulosics makes it suitable for use in large-scale aqueous two-phase affinity partitioning systems.     

Use of High-Affinity Cell Wall-Binding Domains of Bacteriophage Endolysins for Immobilization and Separation of Bacterial Cells

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.     

Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects

In insects, chitin is a major structural component of the cuticle and the peritrophic membrane (PM). In nature, chitin is always associated with proteins among which chitin-binding proteins (CBPs) are the most important for forming, maintaining and regulating the functions of these extracellular structures. In this study, a genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin-binding domains (CBDs) was conducted. A total of 53 genes encoding 56 CBPs were identified, including 15 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 11 CPAP3s (CPAPs with 3 CBDs) and 17 PMPs (PM proteins) with a variable number of CBDs, which are structural components of cuticle or of the PM. CBDs were also identified in enzymes of chitin metabolism including 6 chitinases and 7 chitin deacetylases encoded by 6 and 5 genes, respectively. RNA-seq analysis confirmed that PMP and CPAP genes have differential spatial expression patterns. The expression of PMP genes is midgut-specific, while CPAP genes are widely expressed in different cuticle forming tissues. Phylogenetic analysis of CBDs of proteins in insects belonging to different orders revealed that CPAP1s from different species constitute a separate family with 16 different groups, including 6 new groups identified in this study. The CPAP3s are clustered into a separate family of 7 groups present in all insect orders. Altogether, they reveal that duplication events of CBDs in CPAP1s and CPAP3s occurred prior to the evolutionary radiation of insect species. In contrast to the CPAPs, all CBDs from individual PMPs are generally clustered and distinct from other PMPs in the same species in phylogenetic analyses, indicating that the duplication of CBDs in each of these PMPs occurred after divergence of insect species. Phylogenetic analysis of these three CBP families showed that the CBDs in CPAP1s form a clearly separate family, while those found in PMPs and CPAP3s were clustered together in the phylogenetic tree. For chitinases and chitin deacetylases, most of phylogenetic analysis performed with the CBD sequences resulted in similar clustering to the one obtained by using catalytic domain sequences alone, suggesting that CBDs were incorporated into these enzymes and evolved in tandem with the catalytic domains before the diversification of different insect orders. Based on these results, the evolution of CBDs in insect CBPs is discussed to provide a new insight into the CBD sequence structure and diversity, and their evolution and expression in insects.     

Interaction between Basic Residues of Epstein-Barr Virus EBNA1 Protein and Cellular Chromatin Mediates Viral Plasmid Maintenance

The Epstein-Barr virus (EBV) genome is episomally maintained in latently infected cells. The viral protein EBNA1 is a bridging molecule that tethers EBV episomes to host mitotic chromosomes as well as to interphase chromatin. EBNA1 localizes to cellular chromosomes (chromatin) via its chromosome binding domains (CBDs), which are rich in glycine and arginine residues. However, the molecular mechanism by which the CBDs of EBNA1 attach to cellular chromatin is still under debate. Mutation analyses revealed that stepwise substitution of arginine residues within the CBD1 (amino acids 40–54) and CBD2 (amino acids 328–377) regions with alanines progressively impaired chromosome binding activity of EBNA1. The complete arginine-to-alanine substitutions within the CBD1 and -2 regions abolished the ability of EBNA1 to stably maintain EBV-derived oriP plasmids in dividing cells. Importantly, replacing the same arginines with lysines had minimal effect, if any, on chromosome binding of EBNA1 as well as on its ability to stably maintain oriP plasmids. Furthermore, a glycine-arginine-rich peptide derived from the CBD1 region bound to reconstituted nucleosome core particles in vitro, as did a glycine-lysine rich peptide, whereas a glycine-alanine rich peptide did not. These results support the idea that the chromosome binding of EBNA1 is mediated by electrostatic interactions between the basic amino acids within the CBDs and negatively charged cellular chromatin.     

Chloroplast protein import : quantitative analysis of precursor binding

The first step of chloroplast protein import is binding of a precursor protein to the surface of the organelle. Precursor binding for the small subunit of ribulose-1,5-bisphosphate carboxylase to isolated pea chloroplasts was investigated using a receptor-ligand binding assay. Translocation of precursors was blocked by conducting the binding assays at 0°C. Binding of precursor was judged to be receptor mediated by the following criteria: (a) precursor binding was saturable at between 1500 and 3500 molecules per chloroplast; (b) binding is a high affinity interaction with a dissociation constant of 6 to 10 nanomoles; (c) binding is physiologically productive since most of the bound precursors could be imported from the bound state; and (d) precursor binding was sensitive to both protease and the sulfhydryl modifying reagent N-ethylmaleimide. The effects of these two reagents differed in that protease reduced the total number of binding sites from the surface of chloroplasts but had little effect on binding affinity, whereas N-ethylmaleimide reduced the binding affinity but had little or no effect on receptor density.     

Cellulose-binding domains: biotechnological applications

Many researchers have acknowledged the fact that there exists an immense potential for the application of the cellulose-binding domains (CBDs) in the field of biotechnology. This becomes apparent when the phrase "cellulose-binding domain" is used as the key word for a computerized patent search; more then 150 hits are retrieved. Cellulose is an ideal matrix for large-scale affinity purification procedures. This chemically inert matrix has excellent physical properties as well as low affinity for nonspecific protein binding. It is available in a diverse range of forms and sizes, is pharmaceutically safe, and relatively inexpensive. Present studies into the application of CBDs in industry have established that they can be applied in the modification of physical and chemical properties of composite materials and the development of modified materials with improved properties. In agro-biotechnology, CBDs can be used to modify polysaccharide materials both in vivo and in vitro. The CBDs exert nonhydrolytic fiber disruption on cellulose-containing materials. The potential applications of "CBD technology" range from modulating the architecture of individual cells to the modification of an entire organism. Expressing these genes under specific promoters and using appropriate trafficking signals, can be used to alter the nutritional value and texture of agricultural crops and their final products.     

Development of binding assays to screen ligands for Plasmodium falciparum thioredoxin and glutathione reductases by ultrafiltration and liquid chromatography/mass spectrometry

To identify potential lead compounds for malaria drug discovery, ultrafiltration and liquid chromatography and mass spectrometry (UF and LC/MS) based binding assays were developed for the first time for Plasmodium falciparum thioredoxin (PfTrxR) and glutathione (PfGR) reductases. In the binding assays, curcuminoids (bis-demethoxycurcumin 1, demethoxycurcumin 2, and curcumin 3) were used to study the binding affinity for PfTrxR and PfGR enzymes. The optimum binding was observed when the curcumimoids mixture (1 μM) was incubated with 1 μM PfTrxR and 0.5 μM PfGR enzymes separately for 60 min at 25 °C. The peak areas of the ligands in the chromatogram corresponding to incubation with active PfTrxR and PfGR enzymes increased by 1.6- and 2.0-fold respectively compared to the chromatogram of test compounds incubated with denatured enzymes. Further, binding assay experiments were carried out for compound 2 under non-competitive and competitive incubation conditions with 1 μM PfTrxR and 0.5 μM PfGR enzymes, separately. The binding affinity of compound 2 was higher for both the enzymes under non-competitive incubation conditions. To validate the binding assay developed, we have tested bis-2,4-dinitrophenyl sulfide (4) which is reported as an inhibitor of PfTrxR and PfGR enzymes. Compound 4 showed greater binding affinity for both enzymes under competitive incubation conditions. The relative peak area of compound 4 increased by 3.2- and 6-fold when incubated with active PfTrxR (1 μM) and PfGR (0.5 μM) enzymes respectively compared to the peak areas of the compound in control experiments. The current method developed has a potential for automated high-throughput screening to rapidly determine the binding affinity of ligands for these enzymes.     

Domain shuffling and module engineering of Listeria phage endolysins for enhanced lytic activity and binding affinity

Bacteriophage endolysins are peptidoglycan hydrolases employed by the virus to lyse the host at the end of its multiplication phase. They have found many uses in biotechnology; not only as antimicrobials, but also for the development of novel diagnostic tools for rapid detection of pathogenic bacteria. These enzymes generally show a modular organization, consisting of N‐terminal enzymatically active domains (EADs) and C‐terminal cell wall‐binding domains (CBDs) which specifically target the enzymes to their substrate in the bacterial cell envelope. In this work, we used individual functional modules of Listeria phage endolysins to create fusion proteins with novel and optimized properties for labelling and lysis of Listeria cells. Chimaeras consisting of individual EAD and CBD modules from PlyPSA and Ply118 endolysins with different binding specificity and catalytic activity showed swapped properties. EAD118–CBDPSA fusion proteins exhibited up to threefold higher lytic activity than the parental endolysins. Recombineering different CBD domains targeting various Listeria cell surfaces into novel heterologous tandem proteins provided them with extended recognition and binding properties, as demonstrated by fluorescent GFP‐tagged CBD fusions. It was also possible to combine the binding specificities of different single CBDs in heterologous tandem CBD constructs such as CBD500‐P35 and CBDP35‐500, which were then able to recognize the majority of Listeria strains. Duplication of CBD500 increased the equilibrium cell wall binding affinity by approximately 50‐fold, and the enzyme featuring tandem CBD modules showed increased activity at higher ionic strength. Our results demonstrate that modular engineering of endolysins is a powerful approach for the rational design and optimization of desired functional properties of these proteins.     

Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Ni²⁺-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni²⁺-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni²⁺-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.     

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 μM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.     

Improved immobilization of fusion proteins via cellulose-binding domains

Cellulose-binding domains (CBDs) are structurally and functionally independent, noncatalytic modules found in many cellulose or hemicellulose degrading enzymes. Recent biotechnological applications of the CBDs include facilitated protein immobilization on cellulose supports. In some occasions there have been concerns about the stability of the CBD driven immobilization. Here we have studied the chromatographic behavior of variants of the Trichoderma reesei cellobiohydrolase I CBD belonging to family I. Both CBDs fused to antibody fragments and isolated CBDs were studied and compared. Tritium labeling by reductive methylation was used as a sensitive detection method. The fusion protein as well as the isolated CBD was found to leak from the column at a rate of 0.3-0.5% of the immobilized protein per column volume. However, the leakage could be overcome by using two CBDs instead of a single CBD for the immobilization. In this way leakage was reduced to less than 0.01% per column volume. The improved immobilization could also be seen as a decreased migration of the protein down the column in extended washes.     

Assay of the acetylcholine receptor by affinity labeling

An accurate and sensitive assay for nicotinic acetylcholine receptor binding sites is described which is based on the specificities of receptor both for an affinity label, 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA), and for α-neurotoxins from Naja venoms. It has been demonstrated that MBTA reacts exclusively with one type of subunit of the acetylcholine receptors isolated from the electric tissue of Electrophorus electricus and Torpedo californica and that this reaction is blocked in the presence of Naja naja siamensis α-neurotoxin and of other ligands of the acetylcholine binding site. Thus, in this assay the difference in the extent of labeling by MBTA in the absence and presence of N. n. siamensis toxin is considered the specific labeling of receptor. Although this assay is more complicated than direct α-neurotoxin binding, it is justified by the wellestablished site specificity of the labeling. The specific activities of several different receptor preparations determined using this assay are one-half of those determined using toxin binding. It is possible to assay accurately as little as 0.25 μg of receptor in the presence of 100-fold as much other protein.     

Use of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization and separation of bacterial cells

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.     

Binding of leukemia inhibitory factor (LIF) to mutants of its low affinity receptor, gp190, reveals a LIF binding site outside and interactions between the two cytokine binding domains.

The gp190 transmembrane protein, the low affinity receptor for the leukemia inhibitory factor (LIF), belongs to the hematopoietin family of receptors characterized by the cytokine binding domain (CBD). gp190 is one of the very few members of this family to contain two such domains. The membrane-proximal CBD (herein called D2) is separated from the membrane-distal one (called D1) by an immunoglobulin-like (Ig) domain and is followed by three fibronectin type III repeats. We used truncated gp190 mutants and a blocking anti-gp190 monoclonal antibody to study the role of these repeats in low affinity receptor function. Our results showed that the D1Ig region was involved in LIF binding, while D2 appeared to be crucial for the proper folding of D1, suggesting functionally important interactions between the two CBDs in the wild-type protein. In addition, a point mutation in the carboxyl terminus of the Ig region strongly impaired ligand binding. These findings suggest that at least two distinct sites, both located within the D1Ig region, are involved in LIF binding to gp190, and more generally, that ligand binding sites on these receptors may well be located outside the canonical CBDs.     

Expression of the carbohydrate recognition domain of FimH and development of a competitive binding assay

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). In the first step of this infective process, the virulence factor FimH located on type 1 pili allows UPEC to specifically adhere to oligosaccharides, which are part of glycoproteins on the urinary bladder mucosa. This initial step prevents the clearance of E. coli from the urinary tract and enables the invasion of the host cells. Because FimH antagonists can block this interaction, they exhibit a promising therapeutic potential as anti-infectives. For the evaluation of their binding properties, a reliable, target-based affinity assay is required. Here, we describe the expression and purification of the carbohydrate recognition domain of FimH (FimH-CRD) as well as the development of a competitive binding assay. FimH-CRD linked with a thrombin cleavage site to a 6His-tag is recombinantly expressed and purified by affinity chromatography. For the evaluation of FimH antagonists, a cell-free binding assay based on the interaction of a biotinylated polyacrylamide glycopolymer with the FimH-CRD was developed. Complexation of the biotinylated glycopolymer with streptavidin coupled to horseradish peroxidase allows the quantification of the binding properties of FimH antagonists. The assay format was optimized and validated by a comparison with affinity data from reported assays.     

In Vivo Tissue-dependent Abscisic Acid Specific-binding Proteins with High Affinity in Cytosol of Developing Apple Fruits (in English)

The in vivo highly tissue-dependent abscisic acid (ABA) specific-binding sites localized in cytosol were identified and characterized in the flesh of developing apple ( Malus pumila L. cv. Starkrimon) fruits. ABA binding activity was scarcely detectable in the microsomes and the cytosolic fraction isolated from the freshly harvested fruits via an in vitro ABA binding incubation of the subcellular fractions. If, however, instead that the subcellular fractions were in vitro incubated in H-ABA binding medium, the flesh tissue discs were directly in vivo incubated in H-ABA binding medium, a high ABA binding activity to the cytosolic fraction isolated from these tissue discs was detected. The in vivo ABA binding capacity of the cytosolic fraction was lost if the tissue discs had been pretreated with boiling water, indicating that the ABA binding needs a living state of tissue. The in vivo tissue-dependent binding sites were shown to possess protein nature with both active serine residua and thiol-group of cysteine residua in their functional binding center. The ABA binding of the in vivo tissue-dependent ABA binding sites to the cytosolic fraction was shown to be saturable, reversible, and of high affinity. The scatchard plotting gave evidence of two different classes of ABA binding proteins, one with a higher affinity ( Kd =2.9 nmol/L) and the other with lower affinity ( Kd =71.4 nmol/L). Phaseic acid, 2- trans -4- trans -ABA or cis-trans -(－)-ABA had substantially no affinity to the binding proteins, indicating their stereo-specificity to bind physiologically active ABA. The time course, pH- and temperature-dependence of the in vivo tissue-dependent binding proteins were determined. It is hypothesized that the detected ABA-binding proteins may be putative ABA-receptors that mediate ABA signals during fruit development.     

Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus

Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and β-glucosidase from the fungus Aspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and β-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for β-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and β-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and β-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.