C-terminal Cellulose-binding Domain Research Articles

Small-angle X-ray scattering and structural modeling of full-length: cellobiohydrolase I from Trichoderma harzianum

Cellobiohydrolases hydrolyze cellulose releasing cellobiose units. They are very important for a number of biotechnological applications, such as, for example, production of cellulosic ethanol and cotton fiber processing. The Trichoderma cellobiohydrolase I (CBH1 or Cel7A) is an industrially important exocellulase. It exhibits a typical two domain architecture, with a small C-terminal cellulose-binding domain and a large N-terminal catalytic core domain, connected by an O-glycosylated linker peptide. The mechanism by which the linker mediates the concerted action of the two domains remains a conundrum. Here, we probe the protein shape and domain organization of the CBH1 of Trichoderma harzianum (ThCel7A) by small angle X-ray scattering (SAXS) and structural modeling. Our SAXS data shows that ThCel7A linker is partially-extended in solution. Structural modeling suggests that this linker conformation is stabilized by inter- and intra-molecular interactions involving the linker peptide and its O-glycosylations.

Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

The modular xylanase Cex (or CfXyn10A) from Cellulomonas fimi consists of an N-terminal catalytic domain and a C-terminal cellulose-binding domain, joined by a glycosylated proline-threonine (PT) linker. To characterize the conformation and dynamics of the Cex linker and the consequences of its modification, we have used NMR spectroscopy to study full-length Cex in its nonglycosylated ( approximately 47 kDa) and glycosylated ( approximately 51 kDa) forms. The PT linker lacks any predominant structure in either form as indicated by random coil amide chemical shifts. Furthermore, heteronuclear (1)H-(15)N nuclear Overhauser effect relaxation measurements demonstrate that the linker is flexible on the ns-to-ps time scale and that glycosylation partially dampens this flexibility. The catalytic and cellulose-binding domains also exhibit identical amide chemical shifts whether in isolation or in the context of either unmodified or glycosylated full-length Cex. Therefore, there are no noncovalent interactions between the two domains of Cex or between either domain and the linker. This conclusion is supported by the distinct (15)N relaxation properties of the two domains, as well as their differential alignment within a magnetic field by Pf1 phage particles. These data demonstrate that the PT linker is a flexible tether, joining the structurally independent catalytic and cellulose-binding domains of Cex in an ensemble of conformations; however, more extended forms may predominate because of restrictions imparted by the alternating proline residues. This supports the postulate that the binding-domain anchors Cex to the surface of cellulose, whereas the linker provides flexibility for the catalytic domain to hydrolyze nearby hemicellulose (xylan) chains.

Characterization and gene cloning of a cold-active cellulase from a deep-sea psychrotrophic bacterium Pseudoalteromonas sp. DY3

The celX gene encoding an extracellular cold-active cellulase was isolated from a psychrotrophic bacterium, which was isolated from deep-sea sediment and identified as a Pseudoalteromonas species. It encoded a protein consisting of 492 amino acids with a calculated molecular mass of 52.7 kDa. The CelX consisted of an N-terminal catalytic domain belonging to glycoside hydrolase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The long linker sequence connecting both domains was composed of 105 residues. The optimal temperature for cellulase activity of CelX was 40 degrees C. The enzyme was most active at pH 6-7 and showed better resistance to alkaline condition. The zymogram activity analysis indicated that the CelX consisted of single enzyme component. The cellobiose was main hydrolysate of CelX.

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.

Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans.

Clostridium cellulovorans produces a cellulase complex (cellulosome) as well as noncellulosomal cellulases. In this study, we determined a factor that affected the solubility of the cellulosomal cellulase EngB and the noncellulosomal EngD when they were expressed in Escherichia coli. The catalytic domains of EngB and EngD formed inclusion bodies when expressed in E. coli. On the other hand, both catalytic domains containing the C-terminal cellulose-binding domain (CBD) of EngD were expressed in soluble form. Fusion with the CBD of EngD also helped increased the solubility of cellulosomal cellulase EngL upon expression in E. coli. These results indicate that the CBD of EngD plays an important role in the soluble expression of the catalytic domains of EngB, EngL, and EngD. The possible mechanisms of solubilization by fusion of the catalytic domain with the CBD from EngD are discussed.

EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan.

A novel gene, eglC, encoding an endoglucanase, was cloned from Aspergillus niger. Transcription of eglC is regulated by XlnR, a transcriptional activator that controls the degradation of polysaccharides in plant cell walls. EglC is an 858-amino-acid protein and contains a conserved C-terminal cellulose-binding domain. EglC can be classified in glycoside hydrolase family 74. No homology to any of the endoglucanases from Trichoderma reesei was found. In the plant cell wall xyloglucan is closely linked to cellulose fibrils. We hypothesize that the EglC cellulose-binding domain anchors the enzyme to the cellulose chains while it is cleaving the xyloglucan backbone. By this action it may contribute to the degradation of the plant cell wall structure together with other enzymes, including hemicellulases and cellulases. EglC is most active towards xyloglucan and therefore is functionally different from the other two endoglucanases from A. niger, EglA and EglB, which exhibit the greatest activity towards beta-glucan. Although the mode of action of EglC is not known, this enzyme represents a new enzyme function involved in plant cell wall polysaccharide degradation by A. niger.

Cloning and transcription analysis of the Aspergillus aculeatus No. F-50 endoglucanase 2 ( cmc2) gene

The cmc2 gene, coding for an endoglucanase 2 (CMC2) of Aspergillus aculeatus, was cloned using both genomic and cDNA libraries, and sequenced. The gene consists of 1230 bp encoding a protein of 410 amino acid residues with a molecular mass of 43,697 Da. The CMC2, composed of an N-terminal catalytic domain belonging to the family 5 of glycosyl hydrolases and a C-terminal cellulose-binding domain (CBD) belonging to the family I of CBDs, showed identity with other fungal endoglucanases, particularly with that of A. niger, A. nidulans, A. kawachii and A. aculeatus. The transcription of the cmc2 gene in A. aculeatus cells that were grown on different carbon sources was measured. Analysis by the ribonuclease protection assay revealed that expression of the cmc2 gene is induced by cellulose and some disaccharides and repressed by glucose.

Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase cel5 (formerly EGZ) from Erwinia chrysanthemi

Erwinia chrysanthemi, a Gram-negative plant pathogen, secretes the cellulase Cel5 (formerly EGZ) via the type II secretion pathway (referred to as Out). Cel5 is composed of two domains, a large N-terminal catalytic domain (390 amino acid residues) and a small C-terminal cellulose-binding domain (62 amino acid residues) separated by a linker region. A combination of mutagenesis and structural analysis permitted us to investigate the structure/secretion relationships with respect to the catalytic domain of Cel5. The 3D structure of the catalytic domain was solved by molecular replacement at 2.3 Å resolution. Cel5 exhibits the (β/α)8 structural fold and two extra-barrel features. Our previous genetic study based upon tRNA-mediated suppression allowed us to predict positions of importance in the molecule in relation to structure and catalysis. Remarkably, all of the predictions proved to be correct when compared with the present structural information. Mutations of Arg57, which is located at the heart of the catalytic domain, allowed us to test the consequences of structural modifications on the secretion efficiency. The results revealed that secretability imposes remarkably strong constraints upon folding. In particular, an Arg-to-His mutation yielded a species that folded to a stable conformation close to, but distinct from the wild-type, which however was not secretable. We discuss the relationships between folding of a protein in the periplasm, en route to the cell exterior, and presentation of secretion information. We propose that different solutions have been selected for type II secreted exoproteins in order to meet the constraints imposed by their interaction with their respective secretion machineries. We propose that evolutionary pressure has led to the adaptation of different secretion motifs for different type II exoproteins.

Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide–propylene oxide copolymers

Endoglucanases (endo-1,4-β- d-glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide–propylene oxide random copolymer (EO 50PO 50) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro) 4 tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro) 2 tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro) 4 improved the partition properties the most but had lower TEF in comparison to (Trp-Pro) 2.

A gene encoding a novel multidomain beta-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp.

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85 degrees C and pH 6.0 and was extremely thermostable at 70 degrees C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.

Characterization of a Neocallimastix patriciarum xylanase gene and its product.

A xylanase gene (xynC) isolated from the anaerobic ruminal fungus Neocallimastix patriciarum was characterized. The gene consists of an N-terminal catalytic domain that exhibited homology to family 11 of glycosyl hydrolases, a C-terminal cellulose binding domain (CBD) and a putative dockerin domain in between. Each domain was linked by a short linker domain rich in proline and alanine. Deletion analysis demonstrated that the CBD was essential for optimal xylanase activity of the enzyme, while the putative dockerin domain may not be required for enzyme function.

Cloning and Characterization of a Thermostable Cellobiose Dehydrogenase fromSporotrichum thermophile

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. CDH contains one hemeband one FAD per molecule and oxidizes cellobiose to cellobionolactone in the presence of cytochromec.In this report, a thermostable CDH from the thermophilic ascomyceteSporotrichum thermophilehas been purified, cloned, and characterized. The temperature optimum for this CDH reaction was 60°C, and the activation energy for the reaction was 26.3 kJ/mol. TheKmandkcatwere temperature-dependent and increased as reaction temperature increased. These kinetic properties prove that this CDH is truly thermophilic. A 2.8-kb cDNA was isolated by screening an expression library ofS. thermophilewith a polyclonal antisera raised againstPhanerochaete chrysosporiumCDH. The cDNA encoded an 807-amino-acid protein with a predicted mass of 86,332 Da.S. thermophileCDH is organized into three domains, an N-terminal flavin domain, a middle heme domain, and a C-terminal cellulose-binding domain, which shows sequence similarity with the cellulose-binding domains of endoglucanases and cellobiohydrolases fromTrichoderma reesei.Comparison with the CDH sequences ofP. chrysosporiumandTrametes versicoloridentified Met 95 and His 143 as potential heme coordinations. EFIG, LGGPM, and VNSTH motifs in the heme domain and the XRXPXTDXPSXDGXRY motif in the flavin domain were identified as CDH-specific motifs. With regard to the amino acid composition,S. thermophileCDH has more disulfide linkages and acidic and basic amino acids compared to CDHs fromP. chrysosporiumandT. versicolor.

Isolation and transcriptional analysis of a cellulase gene ( cell) from the basidiomycete Irpex lacteus

A gene (named cell) homologous to the cellobiohydrolase I gene ( cbhl) of Trichoderma reesei was isolated and sequenced from the white rot basidiomycete Irpex lacteus MC-2. The cell open reading frame consists of 1551 bp, which is interrupted by two introns, encoding a polypeptide of 517 amino acid residues with a calculated molecular mass of 54,522 Da. The deduced amino acid sequence showed that CEL1 (the protein encoded by cell) has a modular structure consisting of a catalytic domain of 449 amino acids and a C-terminal cellulose-binding domain (CBD) of 36 amino acids separated by a proline-, serine-, threonine-rich linker region of 32 amino acids. The CEL1 catalytic domain is homologous with fungal cellobiohydrolases (CBHs) belonging to family 7 of the glycosyl hydrolases. The transcription of cell was induced in the presence of various cellulosic substrates and repressed by glucose. It was therefore concluded that the reported sequence represents the first cellulase gene isolated from the basidiomycete Irpex.

Cloning, nucleotide sequence, and transcriptional analysis of Aspergillus aculeatus no. F-50 cellobiohydrolase I ( cbhI) gene

The cbhI gene, coding for a major cellobiohydrolase (CBHI) of Aspergillus aculeatus, was cloned and sequenced. The gene consists of 1620-bp and encodes a protein containing 540 amino acids with a calculated molecular mass of 56,723 Da. CBHI, composed of an N-terminal catalytic domain belonging to family 7 of the glycosyl hydrolases, and a C-terminal cellulose-binding domain (CBD) belonging to family I of the CBDs, showed high similarity with other fungal CBHIs, especially with that of Penicillium janthinellum. The cbhI gene transcription start points in A. aculeatus were defined by primer extension, and the putative promoter sequence was analyzed. This sequence was found to be closely related to the consensus sequences of various fungal genes. Transcription analysis by ribonuclease protection assay revealed that the cbhI gene is induced by low-molecular-weight cellooligosaccharide and repressed by glucose. The results emphasize the possibility that in the A. aculeatus cellulase system, cellobiose is the true inducer and the role of the cbhI gene lies within the cascade regulating cellulase induction.

Solution structure of the cellulose-binding domain of the endoglucanase Z secreted by Erwinia chrysanthemi.

Two-dimensional proton nuclear magnetic resonance spectroscopy has been used to determine the three-dimensional structure of the 62 amino acid C-terminal cellulose-binding domain (CBD) of the endoglucanase Z (CBDEGZ), secreted by Erwinia chrysanthemi. An experimental data set comprising 958 interproton nOe-derived restraints was used to calculate 23 structures. The calculated structures have an average root-mean-square deviation between Cys4 and Cys61 of 0.91 +/- 0.11 A for backbone atoms and 1.18 +/- 0.12 A for the heavy atoms. The CBDEGZ exhibits a skiboot shape based mainly on a triple antiparallel beta-sheet perpendicular to a less-ordered summital loop. Three aromatic rings (Trp18, Trp43, and Tyr44) are localized on one face of the protein and are exposed to the solvent in a conformation compatible with a cellulose-binding site. Based on its original folding, we have been able to relate the CBD sequence to those of several domains of unknown function occurring in several bacterial chitinases as well as other proteins. This study also provides a structural basis for analyzing the secretion-related information specific to the CBDEGZ.

CDNA Cloning of a Trichoderma reesei Cellulase and Demonstration of Endoglucanase Activity by Expression in Yeast

A Trichoderma reesei cDNA encoding a previously unknown protein with a C-terminal cellulose-binding domain was obtained by complementation screening of a T. reesei cDNA library in a sec1 yeast mutant impaired in protein secretion. The T. reesei protein shows amino acid similarity over its entire length to the Agaricus bisporus cellulose-induced protein CEL1 whose function is not known. These two proteins form a new glycosyl hydrolase family, number 61. Expression of the T. reesei cDNA in yeast showed that it encoded a protein with endoglucanase activity and thus the protein was named EGIV and the corresponding gene egl4. Polyclonal antibodies were prepared against EGIV produced in Escherichia coli and detected a 56-kDa protein in the T. reesei culture supernatant. Northern hybridisation revealed that T. reesei egl4 is regulated in the same manner as other cellulase genes of this fungus.

Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: structure and stability of the recombinant enzyme and its isolated cellulose-binding domain.

The hyperthermophilic bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120-kDa endo-1,4-D-xylanase exhibiting high intrinsic stability and a temperature optimum approximately 90 degrees C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C-terminal part of XynA was previously shown to be responsible for cellulose binding (Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose-binding domain within the multi-domain 120 kDa Xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431-444). In order to characterize the domain organization and the stability of XynA and its C-terminal cellulose-binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand-free form, was expressed as a glutathione S-transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield > 80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far-UV circular dichroism in order to characterize protein stability, guanidine-induced unfolding of XynA leads to biphasic transitions, with half-concentrations c1/2 (GdmCl) approximately 4 M and > 5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine-transition from 5 to 7 M GdmCl. This can be tentatively attributed to the cellulose-binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi-state behavior of XynA. In the case of CBD, a temperature-induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far-UV CD in the native state.

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp.

Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi

The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a polypeptide of 1090 amino acids. Cellobiohydrolase B (CbhB) is 1037 amino acids long, with a calculated molecular mass of 109765 Da. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose-binding domain of 104 amino acids. The catalytic domain belongs to family 48 of glycosyl hydrolases. CbhB has a very low activity on CM-cellulose. Viscometric analysis of CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase. Cellobiose is the major product of hydrolysis of cellulose. In common with two other exoglycanases from C. fimi, CbhB has low but detectable endoglucanase activity. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases.

Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end.

Linker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter.