Characteristics of the endoglucanase encoded by a cel gene from Bacteroides succinogenes expressed in Escherichia coli.

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.     

Cloning of a cellulase gene from the rumen anaerobe Fibrobacter succinogenes SD35 and partial characterization of the gene product

N. OZCAN, C. CUNNINGHAM AND W.J. HARRIS. 1996. A gene encoding an enzyme which degrades cellulose ( end-1 ) was isolated from a library of Fibrobacter succinogenes SD35 DNA fragments and expressed in pUC18. The product of end-1 showed significant activity against carboxymethylcellulose but relatively minor activity against lichenan, xylan and avicel. The nucleotide sequence indicated a product of 388 amino acids with a molecular mass of 50.2 kDa. This was in agreement with the molecular size estimated by gel electrophoresis. No significant DNA sequence similarity was identified with any published endoglucanase.     

Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei.

There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.     

Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum

The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.     

Characterization and synergistic interactions of Fibrobacter succinogenes glycoside hydrolases

The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. succinogenes, Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.     

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.     

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1.

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.     

Electrophoretic and enzymatic studies on the crude extracellular enzyme system of the cellulolytic bacterium Cellulomonas sp. ATCC 21399

Cellulomonas sp. ATCC 21399 produced extracellular enzyme activities against Avicel, H(3)PO(4)-swollen Avicel, carboxymethylcellulose, (1-3, 1-4)-beta-D-heteroglucan, xylan, galactomannan, and amylose drying growth on microcrystalline cellulose. No extracellular cellobiase activity was produced. Crossed immunoelectrophoresis of the crude extracellular enzyme system revealed 15 immunologically distinct immunoprecipitates. The immunoprecipitates of endoglucanase A, endoglucanase B and the xylanase appeared heterogeneous with several optima, whereas the immunoprecipitates of endoglucanase C and the amylase appeared homogeneous. The heterogeneity of endoglucanase A, endoglucanase B and xylanase was also visualized using electrofocusing-immunoelectrophoresis. Electro-focusing could resolve the activity against carboxymethylcellulose into six peaks, whereas only one peak of activity against Avicel was observed. The later peak coincided with the major peak of activity against carboxymethylcellulose with isoelectric point between pH 4.0-5.0.     

Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter succinogenes S85 grown on different substrates

The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (beta-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endA(FS) genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. succinogenes.     

Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts

β-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of β-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or β-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for β-1,3-1,4 glucans, showing maximum activity on barley β-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley β-glucan and lichean were 3-O-β-cellotriosyl-D-glucose and 3-O-β-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for β-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase.     

Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei

Trichoderma reesei produces five known endoglucanases. The most studied are Cel7B (EG I) and Cel5A (EG II) which are the most abundant of the endoglucanases. We have performed a characterisation of the enzymatic properties of the less well-studied endoglucanases Cel12A (EG III), Cel45A (EG V) and the catalytic core of Cel45A. For comparison, Cel5A and Cel7B were included in the study. Adsorption studies on microcrystalline cellulose (Avicel) and phosphoric acid swollen cellulose (PASC) showed that Cel5A, Cel7B, Cel45A and Cel45Acore adsorbed to these substrates. In contrast, Cel12A adsorbed weakly to both Avicel and PASC. The products formed on Avicel, PASC and carboxymethylcellulose (CMC) were analysed. Cel7B produced glucose and cellobiose from all substrates. Cel5A and Cel12A also produced cellotriose, in addition to glucose and cellobiose, on the substrates. Cel45A showed a clearly different product pattern by having cellotetraose as the main product, with practically no glucose and cellobiose formation. The kinetic constants were determined on cellotriose, cellotetraose and cellopentaose for the enzymes. Cel12A did not hydrolyse cellotriose. The k(Cat) values for Cel12A on cellotetraose and cellopentaose were significantly lower compared with Cel5A and Cel7B. Cel7B was the only endoglucanase which rapidly hydrolysed cellotriose. Cel45Acore did not show activity on any of the three studied cello-oligosaccharides. The four endoglucanases' capacity to hydrolyse beta-glucan and glucomannan were studied. Cel12A hydrolysed beta-glucan and glucomannan slightly less compared with Cel5A and Cel7B. Cel45A was able to hydrolyse glucomannan significantly more compared with beta-glucan. The capability of Cel45A to hydrolyse glucomannan was higher than that observed for Cel12A, Cel5A and Cel7B. The results indicate that Cel45A is a glucomannanase rather than a strict endoglucanase.     

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3.

The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.     

Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture

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 (Fn3_1,2), and a C-terminal family 3b cellulose-binding domain (CBM3b). A series of truncated derivatives of endoglucanase Cel9B have been constructed and characterized. 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. The truncated forms devoid of CBM3b lost cellulose-binding ability and showed a reduced activity on crystalline cellulose, although activity on amorphous celluloses was not affected. Endoglucanase Cel9B produced only a small ratio of insoluble products from filter paper, while most of the reducing ends produced by the enzyme were released as soluble sugars (91%), indicating that it is a processive enzyme. Processivity of Cel9B resides in traits contained in the tandem of domains GH9–CBM3c, although the slightly reduced processivity of truncated form GH9–CBM3c suggests a minor contribution of domains Fn3_1,2 or CBM3b, not contained in it, on processivity of endoglucanase Cel9B.     

Evidence that the xylanase activity from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> Oα is encoded by the endoglucanase precursor gene (<Emphasis Type="Italic">sso1354</Emphasis>) and characterization of the associated cellulase activity

Sulfolobus solfataricus strain Oalpha was previously isolated for its ability to grow on minimal medium supplemented with xylan as a carbon source. The strain exhibited thermostable xylanase activity but several attempts to identify the gene encoding for the activity failed. Further studies showed that the xylanase displayed activity on carboxymethylcellulose (CMC) and the new activity was characterized. It exhibited an optimal temperature and pH of 95 degrees C and 3.5, respectively, and a half-life of 53 min at 95 degrees C. The enzyme, which was demonstrated to be glycosylated, hydrolyzed CMC in an endo-manner releasing cellobiose and other cello-oligomers. Analysis of the tryptic fragments by tandem mass spectrometry led to identification of the endoglucanase precursor, encoded by the sso1354 gene, as the protein possessing dual activity. The efficiency of the SSO1354 protein in degrading cellulosic and hemicellulosic fractions contained in agronomic residues was tested at low pH and high temperature. Cellulose and xylan were degraded to glucose and xylose at 90 degrees C, pH 4 by an enzyme mix consisting of SSO1354 and additional glycosyl hydrolases from S. solfataricus Oalpha. Given its role in saccharification processes requiring high temperatures and acidic environments, SSO1354 represents an interesting candidate for the utilization of agro-industrial waste for fuel production.     

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.     

Cel9D, an atypical 1,4-beta-D-glucan glucohydrolase from Fibrobacter succinogenes: characteristics, catalytic residues, and synergistic interactions with other cellulases

The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to beta-1-->4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased kcat/Km values for cello-oligosaccharides with higher degrees of polymerization. The kcat/Km value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.     

A comparative analysis of two cDNA clones of the cellulase gene family from anaerobic fungus Piromyces rhizinflata

Cellulase family and some other glycosyl hydrolases of anaerobic fungi inhabiting the digestive tract of ruminants are believed to form an enzyme complex called cellulosome. Study of the individual component of cellulosome may shed light on understanding the organization of this complex and its functional mechanism. We have analysed the primary sequences of two cellulase clones, cel5B and cel6A, isolated from the cDNA library of ruminal fungus, Piromyces rhizinflata strain 2301. The deduced amino acid sequences of the catalytic domain of Cel5B, encoded by cel5B, showed homology with the subfamily 4 of the family 5 (subfamily 5(4)) of glycosyl hydrolases, while cel6A encoded Cel6A belonged to family 6 of glycosyl hydrolases. Phylogenetic tree analysis suggested that the genes of subfamily 5(4) glycosyl hydrolases of P. rhizinflata might have been acquired from rumen bacteria. Cel5B and Cel6A were modular enzymes consisting of a catalytic domain and dockerin domain(s), but not a cellulose binding domain. The occurrence of dockerin domains indicated that both enzymes were cellulosome components. The catalytic domain of the Cel5B (Cel5B') and Cel6A (Cel6A') recombinant proteins were purified. The optimal activity conditions with carboxymethyl cellulose (CMC) as the substrate were pH 6.0 and 50 degrees C for Cel5B', and pH 6.0 and 37-45 degrees C for Cel6A'. Both Cel5B' and Cel6A' exhibited activity against CMC, barley beta-glucan, Lichenan, and oat spelt xylan. Cel5B' could also hydrolyse p-nitrophenyl-beta-d-cellobioside, Avicel and filter paper while Cel6A' did not show any activity on these substrates. It is apparent that Cel6A' acted as an endoglucanase and Cel5B' possessed both endoglucanase and exoglucanase activities. No synergic effect was observed for these recombinant enzymes in vitro on Avicel and CMC.     

Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure-function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in 'designer cellulosomes'. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.     

Functional analysis of a gene encoding endoglucanase that belongs to glycosyl hydrolase family 12 from the brown-rot basidiomycete Fomitopsis palustris

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and beta- glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.