Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium.

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.

The thermostable alpha-L-rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial alpha-L-rhamnoside hydrolase, a new type of inverting glycoside hydrolase.

An alpha-L-rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized. It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis. At pH 7.5 it is optimally active at 60 degrees C and insensitive to moderate concentrations of Triton X100, ethanol and EDTA. It hydrolysed p-nitrophenyl-alpha-L-rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg-1 respectively. Hydrolysis occurs by inversion of the anomeric configuration as detected using 1H-NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by incubation with a thermostable beta-glucosidase. The secondary structure of RamA consists of 27% alpha-helices and 50% beta-sheets, as detected by circular dichroism. The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family.

Active-site mutations which change the substrate specificity of the Clostridium stercorarium cellulase CelZ implications for synergism.

CelZ from the cellulolytic thermophile Clostridium stercorarium has been described as a 'monomeric' cellulase able to effect both the endoglucanolytic hydrolysis of internal glycosidic linkages and the exoglucanolytic degradation from the chain ends in a processive mode of action. The putative catalytic residues of this family 9 cellulase, Asp84 and Glu447 located within the N-terminal domain of the modular protein, were replaced by site-directed mutagenesis. A minimized CelZ derivative (CelZC') comprising the catalytic domain and the adjacent cellulose-binding domain (CBD) family IIIc domain C' was used as target for mutagenesis. Six mutant enzymes and the unmodified CelZC' protein were purified to homogeneity and compared with respect to thermoactivity, substrate specificity, product profile and synergism. CD studies revealed that no major changes to the overall structure of the proteins had occurred. Replacement of either one or both catalytic residues completely eliminated the ability of CelZ to attack insoluble Avicel preparations indicative of the exo-activity, whereas the endo-activity measured via hydrolysis of CM-cellulose was retained upon substitution of the catalytic base Asp84. Thus, endo-active CelZ mutants defective in the exo-activity were available for co-operativity studies with the C. stercorarium exoglucanase CelY. Synergism was found to be dependent on the endo-activity of CelZ. Mutants Asp84Gly and Asp84Glu were able to enhance the degradation of crystalline cellulose significantly, although no products could be released from this substrate by individual action of the mutants.

The modular cellulase CelZ of the thermophilic bacterium Clostridium stercorarium contains a thermostabilizing domain.

The non-catalytic region of the Clostridium stercorarium cellulase CelZ (Avicelase I) comprises two protein segments (C and C') grouped into different subfamilies of cellulose-binding domain (CBD) family III. The C-terminally located family IIIb domain C was identified as a true cellulose-binding domain responsible for anchoring the CelZ enzyme to cellulose. The family IIIc domain C' immediately adjacent to the catalytic domain was unable to mediate binding to cellulose. A deletion study revealed a lack of independence of this pair of domains: almost the entire C' domain was required to maintain the catalytic activity and the thermostability of the enzyme.

Intramolecular synergism in an engineered exo-endo-1,4-beta-glucanase fusion protein.

Exoglucanase CelY and endoglucanase CelZ from the cellulolytic thermophile Clostridium stercorarium act in synergism to hydrolyse cellulosic substrates. To increase the efficiency of the hydrolytic degradation process, an artificial multienzyme carrying both enzymatic activities on one polypeptide chain was constructed by gene fusion. A segment of CelZ, CelZdeltaBB'C (designated CelZC'), comprising the catalytic domain and the adjacent domain C' homologous to the cellulose-binding domain family IIIc, was fused to the C-terminus of CelY, yielding the fusion protein CelY-CelZC', designated CelYZ. The large fusion protein (170 kDa) could be isolated from a recombinant Escherichia coli strain in its intact form retaining the pronounced thermostability of the fusion partners. As a true multienzmye, CelYZ exhibited both exoglucanase and endoglucanase activities. The cellulolytic activity of the fusion protein was three- to fourfold higher than the sum of the individual activities. Dilution experiments showed that the enhanced cellulolytic activity of the multienzyme resulted from intramolecular synergism of the fusion partners. The product profiles and the kinetic constants of cellulose hydrolysis support a new mechanistic model proposed for explaining the co-operativity of the two catalytic domains within the multienzmye.

Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA.

The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like beta-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.

Purification and properties of an amylopullulanase, a glucoamylase, and an alpha-glucosidase in the amylolytic enzyme system of Thermoanaerobacterium thermosaccharolyticum.

Thermoanaerobic bacteria are of considerable interest as producers of thermostable amylolytic enzymes. The soluble amylolytic enzyme system of Thermoanaerobacterium thermosaccharolyticum DSM 571 was fractionated into a pullulanase, a glucoamylase, and an alpha-glucosidase. The enzymes were purified to homogeneity and their physical and catalytic properties were studied. The pullulanase, which cleaved both alpha-1,4- and alpha-1,6-glucosidic bonds, was an amylopullulanase closely related to similar enzymes from other thermoanaerobic bacteria. Partial amino acid sequences of the glucoamylase were identical with the corresponding sequences deduced from the cga gene encoding the glucoamylase from Clostridium sp. strain G0005. The alpha-glucosidase was identified as an isomaltase belonging to a group of structurally related exo-alpha-1,4-glucosidases and oligo-1,6-glucosidases from bacilli. Comparison of enzyme activities indicated that the glucoamylase had the major amylolytic activity of T. thermosaccharolyticum, with amylopullulanase and alpha-glucosidase assisting in the cleavage of alpha-1,6-glucosidic bonds.

Genes encoding two different beta-glucosidases of Thermoanaerobacter brockii are clustered in a common operon.

A 5.9-kb fragment of chromosomal DNA coding for beta-glucosidase activity of the thermophilic anaerobe Thermoanaerobacter brockii was sequenced. Two genes, cglT and xglS, encoding a cellodextrin-cleaving beta-glucosidase and a xylodextrin-degrading xylo-beta-glucosidase, respectively, were located directly adjacent to each other. The 5' region contained two additional genes, cglF and cglG, whose products exhibited similarity to integral membrane proteins of metabolite transport systems. The two beta-glucosidases, CglT and XglS, with deduced molecular masses of 52 and 81 kDa, belong to different families of glycosyl hydrolases. Both enzymes were overexpressed in Escherichia coli and could be detected after protein gel electrophoresis and activity staining. The enzyme CglT was purified by fast protein liquid chromatography and identified by N-terminal sequencing. The enzyme was thermostable at 60 degrees C for at least 24 h, and the temperature optimum was 75 degrees C. The ki for glucose inhibition was calculated to 200 mM. The enzyme released glucose from the nonreducing end of beta-1,4-cello oligomers as well as from various disaccharides. CglT was active on glucosides, galactosides and on fucosides, while XglS cleaved beta-glucosides and beta-xylosides as well. The cglT gene was also expressed in Bacillus subtilis, and the enzyme was mainly intracellular during exponential growth but was efficiently released into the supernatant after cultures entered the stationary phase.

Purification and properties of a cellobiose phosphorylase (CepA) and a cellodextrin phosphorylase (CepB) from the cellulolytic thermophile Clostridium stercorarium.

Two phosphorolytic enzymes displaying activity towards the soluble cellulose degradation products cellobiose and cellodextrins were purified from the crude extract of the cellulolytic thermophile Clostridium stercorarium. Both phosphorylases have monomeric structures with molecular masses of 93 and 91 kDa, respectively. Although the N-terminal amino acid sequences are highly similar, a clear distinction of the two enzymes could be made on the basis of their substrate specificities: the enzyme designated cellobiose phosphorylase cleaved exclusively the disaccharide substrate, whereas the enzyme designated cellodextrin phosphorylase accepted only oligosaccharides as substrates. Kinetic constants were determined for the cleavage of cellobiose and cellodextrins. Maximal activity was observed at 65 degrees C in the pH range 6.0-7.0 for both enzymes. The sequences of the genes cepA and cepB encoding the cellobiose phosphorylase and the cellodextrin phosphorylase, respectively, have been submitted to the GenBank database.

A novel type of thermostable alpha-D-glucosidase from Thermoanaerobacter thermohydrosulfuricus exhibiting maltodextrinohydrolase activity.

An alpha-glucosidase with the ability to attack polymeric substrates was purified to homogeneity from culture supernatants of Thermoanaerobacter thermohydrosulfuricus DSM 567. The enzyme is apparently a glycoprotein with a molecular mass of 160 kDa. Maximal activity is observed between pH5 and 7 at 75 degrees C. The alpha-glucosidase is active towards p-nitrophenyl-alpha-D-glucoside, maltose, malto-oligosaccharides, starch and pullulan. Highest activity is displayed towards the disaccharide maltose. In addition to glucose, maltohexaose and maltoheptaose can be detected as the initial products of starch hydrolysis. After short incubations of pullulan, glucose is found as the only product. At high substrate concentrations, maltose and malto-oligosaccharide, but not glucose, are used as acceptors for glucosyl-transfer. These findings indicate that the T. thermohydrosulfuricus enzyme represents a novel type of alpha-glucosidase exhibiting maltase, glucohydrolase and 'maltodextrinohydrolase' activity.

Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes.

Recombinant Escherichia coli clones displaying thermostable beta-glucanase activity were isolated from two different gene libraries of the hyperthermophilic bacterium Thermotoga maritima MSB8 (DSM 3109), and the nucleotide sequence of a 1,4-beta-glucanase gene designated celA was determined. Amino-terminal sequencing of cellulase I previously detected in T. maritima cells indicated that the celA gene encodes this beta-glucanase, which is now designated CelA. CelA, which has a calculated molecular mass of 29,732 Da, was purified from a recombinant E. coli strain to apparent homogeneity as judged by SDS-PAGE with a 44% yield. The enzyme was most active against soluble substrates such as mixed-linkage beta-glucan and CM-cellulose. CelA displayed remarkable thermostability, which was enhanced in the presence of high concentrations of salt. Downstream of the celA gene we found a second open reading frame, celB, whose nucleotide sequence was 58% identical to celA. Experimental proof that celB also encodes a beta-glucanase was obtained by separation from celA and expression in E. coli under the control of an efficient host promoter. According to the deduced amino acid sequences, CelB, in contrast to CelA, contains a signal peptide at the amino terminus. CelB and CelA had similar substrate specificities and temperature optima, but differed in their pH optima. Also, the addition of salt had a less stabilizing effect on CelB than on CelA. Nine 30 bp direct repeats, each itself representing a sequence with imperfect dyad symmetry, were detected upstream of the celA-celB cellulase gene cluster.

Affinity purification of cellulose-binding enzymes of Clostridium stercorarium.

Cellulose-affinity chromatography proved to be a fast and efficient purification procedure for exoenzymes of the thermophilic anaerobe Clostridium stercorarium, suitable for the preparation of large amounts of enzymes for technical applications. The cellulose-binding enzymes could be identified as the C. stercorarium; cellulolytic enzymes Avicelase I and Avicelase II characterized previously as endo-1,4-beta-glucanase and exo-1,4-beta-glucanase. A third protein was identified as xylanase A, the major endo-1,4-beta-xylanase of this organism.

Debranching of arabinoxylan: properties of the thermoactive recombinant alpha-L-arabinofuranosidase from Clostridium stercorarium (ArfB).

The gene arfB encoding alpha-L-arabinofuranosidase B of the cellulolytic thermophile Clostridium stercorarium was expressed in Escherichia coli from a 2.2-kb EcoRI DNA fragment. The recombinant gene product ArfB was purified by fast-performance liquid chromatography. It has a tetrameric structure with a monomeric relative molecular mass of 5200. The optima for temperature and pH are 70 degrees C and 5.0 respectively. The enzyme appears to have no metal cofactor requirement and is sensitive to sulfhydryl reagents. It hydrolyzes aryl and alkyl alpha-L-arabinofuranosides and cleaves arabinosyl side-chains from arabinoxylan (oat-spelt xylan) and from xylooligosaccharides produced by recombinant endoxylanase XynA from the same organism. The identify of the N-terminal amino acid sequences indicates that ArfB corresponds to the major alpha-arabinosidase activity present in the culture supernatant of C. stercorarium.

alpha-D-glucuronidases from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum.

alpha-D-Glucuronidases were purified from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum. This enzyme activity was found to be intracellular in each organism, with T. saccharolyticum producing much greater total activity. The specific activities of the purified enzymes (10 U mg-1 T. saccharolyticum; 1.7 U mg-1 C. stercorarium) differed by a factor of approximately 5. For the determination of enzyme activities, 4-O-methyl-alpha-D-glucuronosyl-xylotriose was used as a substrate and the glucuronic acid released by alpha-D-glucuronidase action was quantified by a colorimetric procedure. 4-O-Methyl-alpha-D-glucuronosyl-xylotriose was the hydrolysis product that accumulated after exhaustive degradation of 4-O-methyl-alpha-D-glucuronoxylan with xylanases of C. stercorarium. Hydrolysis of side chains in high-molecular-mass glucuronoxylan could not be detected. Neither of the enzymes was able to hydrolyse the chromogenic aryl-substrate p-nitrophenyl-alpha-D-glucuronoside. Both alpha-D-glucuronidases have a dimeric structure, with monomeric molecular masses of 72 and 76 kDa for C. stercorarium and of 71 kDa for T. saccharolyticum. The pI was estimated to be 4.3 for each enzyme. While both enzymes exhibited a similar pH optimum (pH 5.5-6.5) they differed in their thermostabilities. At 60 degrees C, half-lives of 14 and 2.5 h, respectively, were determined for the alpha-D-glucuronidases of C. stercorarium and T. saccharolyticum. This description of alpha-D-glucuronidase activity in thermophilic anaerobic bacteria extends our knowledge of these enzymes, previously purified and characterized only in fungi.

Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials.

A separation procedure for the analysis of the enzyme components of the hyperthermophilic bacterium Thermotoga maritima involved in cellulose and xylan degradation was developed. Resolution of the enzymes was achieved by a combination of fast protein liquid chromatography anion exchange and hydrophobic interaction chromatography. Enzyme fractions were assayed for hydrolysis of Avicel, carboxymethylcellulose (CMC), beta-glucan, laminarin, xylan, p-nitrophenyl-beta-D-glucoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-xyloside, p-nitrophenyl-alpha-L-arabinofuranoside, and 4-O-methyl-glucuronosyl-xylotriose. The activities of two cellulases, one laminarinase, one xylanase, two putative beta-D-xylosidases, alpha-D-glucuronidase, and alpha-L-arabinosidase were identified. Because of their selective retardation on a Superdex gel filtration column, the two cellulases could be purified to homogeneity. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular masses of 27 and 29 kDa, respectively, were determined for cellulase I and cellulase II. Maximal activities of both enzymes were observed at 95 degree C between pH 6.0 and 7.5. In the presence of 2.5 M NaC1 the purified enzymes retained about 90% of their initial activities after a 6-h incubation at 80 degree C. On the basis of its activity towards CMC, cellulase I was classified as endo-beta-1,4-glucanase. Cellulase II was able to attack Avicel in addition to CMC, beta-glucan, and p-nitrophenyl-beta-D-cellobioside. It releases cellobiose and cellotriose from Avicel. The latter product is further cleaved into glucose and cellobiose. Cellulase II may therefore be classified as exo-beta-1,4-glucanase.

Purification and properties of a novel type of exo-1,4-beta-glucanase (avicelase II) from the cellulolytic thermophile Clostridium stercorarium.

Avicelase II was purified to homogeneity from culture supernatants of Clostridium stercorarium. A complete separation from the major cellulolytic enzyme activity (avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective retardation of avicelase II. The enzyme has an apparent molecular mass of 87 kDa and a pI of 3.9. Determination of the N-terminal amino acid indicates that avicelase II is not a proteolytically processed product of avicelase I. Maximal activity of avicelase II is observed between pH 5 and 6. In the presence of Ca2+, the enzyme is highly thermostable, exhibiting a temperature optimum around 75 degrees C. Hydrolysis of avicel occurs at a linear rate for three days at 70 degrees C. Avicelase II is active towards unsubstituted celluloses, cellotetraose and larger cellodextrins. It lacks activity towards carboxymethylcellulose and barley beta-glucan. Unlike other bacterial exoglucanases, avicelase II does not hydrolyze aryl-beta-D-cellobiosides. Avicel is degraded to cellobiose and cellotriose at a molar ratio of approximately 4:1. With acid-swollen avicel as substrate, cellotetraose is also formed as an intermediary product, which is further cleaved to cellobiose. The degradation patterns of reduced cellodextrins differ from that expected for a cellobiohydrolase attacking the non-reducing ends of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while cellohexaitol is initially cleaved into cellobiitol and cellotetraose. These findings, taken together, indicate that avicelase II represents a novel type of exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of the substrate, releases cellotetraose, cellotriose, or cellobiose from the non-reducing end of the cellulose chains.

Sequence analysis of the Clostridium stercorarium celZ gene encoding a thermoactive cellulase (Avicelase I): identification of catalytic and cellulose-binding domains.

The nucleotide sequence of the celZ gene coding for a thermostable endo-beta-1,4-glucanase (Avicelase I) of Clostridium stercorarium was determined. The structural gene consists of an open reading frame of 2958 bp which encodes a preprotein of 986 amino acids with an Mr of 109,000. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase I purified from C. stercorarium culture supernatants. The recombinant protein expressed in Escherichia coli is proteolytically cleaved into catalytic and cellulose-binding fragments of about 50 kDa each. Sequence comparison revealed that the N-terminal half of Avicelase I is closely related to avocado (Persea americana) cellulase. Homology is also observed with Clostridium thermocellum endoglucanase D and Pseudomonas fluorescens cellulase. The cellulose-binding region was located in the C-terminal half of Avicelase I. It consists of a reiterated domain of 88 amino acids flanked by a repeated sequence about 140 amino acids in length. The C-terminal flanking sequence is highly homologous to the non-catalytic domain of Bacillus subtilis endoglucanase and Caldocellum saccharolyticum endoglucanase B. It is proposed that the enhanced cellulolytic activity of Avicelase I is due to the presence of multiple cellulose-binding sites.

Identification of Mycelium-Associated Cellulase from Streptomyces reticuli.

Among 180 Streptomyces strains tested, 25 were capable of hydrolyzing microcrystalline cellulose (Avicel) at 30 degrees C. Streptomyces reticuli was selected for further studies because of its ability to grow at between 30 and 50 degrees C on Avicel. Enzymatic activities degrading Avicel, carboxymethyl cellulose, and cellobiose were found both in the culture supernatant and in association with the mycelium and crystalline substrate. The bound enzymes were efficiently solubilized by repeated washes with buffer of low ionic strength (50 mM Tris hydrochloride [pH 7.5]) and further purified by fast protein liquid chromatography. A high-molecular-weight Avicelase of >300 kilodaltons could be separated from carboxymethyl cellulase (CMCase) and beta-glucosidase activities (molecular mass, 40 to 50 kilodaltons) by gel filtration on Superose 12. The CMCase fraction was resolved by Mono Q anion-exchange chromatography into two enzymes designated CMCase 1 and CMCase 2. The beta-glucosidase activity was found to copurify with CMCase 2. The purified cellulase components showed optimal activity at around pH 7.0 and temperatures of between 45 and 50 degrees C. Avicelase (but not CMCase) activity was stimulated significantly by the addition of CaCl(2).

Activity staining of cellulases in polyacrylamide gels containing mixed linkage beta-glucans.

Endoglucanase and cellobiohydrolase components of thermophilic cellulases can be detected in situ after gel electrophoresis in the presence of sodium dodecyl sulfate by incorporating a mixed linkage beta-glucan (barley beta-glucan, lichenan) in the separation gel. Zymograms are prepared after a renaturation treatment and incubation by staining the gel with Congo red. This method is suitable for the detection of beta-glucanases with different substrate specificities cleaving beta-1,4-, beta-1,4-1,3-, or beta-1,3-glucans. Cellobiohydrolase activities can be detected by adding 4-methylumbelliferyl-beta-D-cellobioside to the incubation buffer. The gels are subsequently stained with Coomassie blue to establish identical molecular weights of beta-glucanase and protein bands. Applications of this technique for the comparison of cellulases and for the identification of cellulase components expressed from recombinant clones are presented.