Phosphoric Acid-swollen Avicel Research Articles

Direct fermentation of cellulose to ethanol by Saccharomyces cerevisiae displaying a bifunctional cellobiohydrolase gene from Orpinomyces sp. Y102

Abstract A cellobiohydrolase gene, cbhC16, isolated from the cDNA library of a rumen fungus. The recombinant CbhC16 demonstrated a specific activity of 7.0 U mg−1 protein against phosphoric acid-swollen avicel, and cellobiose was the main product in the reaction mixture. Glucose was further released from cellobiose by the action of CbhC16 and fermented to ethanol by Saccharomyces cerevisiae. This phenomenon, possessing activities of both cellobiohydrolase and β-glucosidase, is rarely observed in the hydrolysis by typical cellobiohydrolases. Yeast cells displaying CbhC16 were inoculated into YNB–CAA broth containing carboxymethyl cellulose, β-glucan, ammonia fibre explosion-treated rice straw, or wheat bran, and incubated at 30 °C for 3 d. Under the same conditions, 0.20, 0.61, 0.15, and 0.61 g/L of ethanol were detected in the culture supernatant of the strain with the recombinant plasmid containing cbhC16, whereas wild-type yeasts without the recombinant plasmid containing cbhC16 did not produce detectable ethanol levels. The results indicated that yeast strains with cbhC16 could directly saccharify and ferment cellulose to produce ethanol in singular step. This gene encoding both cellobiohydrolase and β-glucosidase activities will enable developing a simple approach for producing a consolidated microorganism that will directly convert cellulosic feedstocks to ethanol.

Complete Saccharification of Cellulose at High Temperature Using Endocellulase and β-Glucosidase from Pyrococcus sp.

We investigated a potential for glucose production from cellulose material using two kinds of hyperthermophilic enzymes, endo-cellulase (EG) and beta-glucosidase (BGL). Two BGLs from hyperthermophile Pyrococcus furiosus and mesophile Aspergillus aculeatus were compared for complete hydrolysis of cellulose with P. horikoshii endo-cellulase (EGPh). The combination reactions by each BGL enzyme and EGPh could produce only glucose without the other oligosaccharides from phosphoric acid swollen Avicel (PSA). The combination of the both hyperthermophilic cellulases, BGLPf and EGPh, will be adaptable to high efficient system to produce glucose at high temperature.

Specificity and affinity of substrate binding by a family 17 carbohydrate-binding module from Clostridium cellulovorans cellulase 5A.

The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulovorans cellulase 5A is a beta-1,4-glucan binding module with a preference for soluble chains. CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (+/-0.2) x 10(5) and 1.6 (+/-0.2) x 10(5) M(-1), respectively. The capacity values for PASA and Avicel were 11.9 and 0.4 micromol/g of cellulose, respectively. CBM17 did not bind to crystalline cellulose. CBM17 bound tightly to soluble barley beta-glucan and the derivatized celluloses HEC, EHEC, and CMC. The association constants for binding to barley beta-glucan, HEC, and EHEC were approximately 2.0 x 10(5) M(-1). Significant binding affinities were found for cello-oligosaccharides greater than three glucose units in length. The affinities for cellotriose, cellotetraose, cellopentaose, and cellohexaose were 1.2 (+/-0.3) x 10(3), 4.3 (+/-0.4) x 10(3), 3.8 (+/-0.1) x 10(4), and 1.5 (+/-0.0) x 10(5) M(-1), respectively. Fluorescence quenching studies and N-bromosuccinimide modification indicate the participation of tryptophan residues in ligand binding. The possible architecture of the ligand-binding site is discussed in terms of its binding specificity, affinity, and the participation of tryptophan residues.

Binding site analysis of cellulose binding domain CBD(N1) from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis.

Endoglucanase C (CenC), a beta1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBD(N1) and CBD(N2). In this work, the contributions of 10 amino acids within the binding cleft of CBD(N1) were evaluated by single site-directed mutations to alanine residues. Each isolated domain containing a single mutation was analyzed for binding to an insoluble amorphous preparation of cellulose, phosphoric acid swollen Avicel (PASA), and to a soluble glucopyranoside polymer, barley beta-glucan. The effect of any given mutation on CBD binding was similar for both substrates, suggesting that the mechanism of binding to soluble and insoluble substrates is the same. Tyrosines 19 and 85 were essential for tight binding by CBD(N1) as their replacement by alanine results in affinity decrements of approximately 100-fold on PASA, barley beta-glucan, and soluble cellooligosaccharides. The tertiary structures of unbound Y19A and Y85A were assessed by heteronuclear single quantum coherence (HSQC) spectroscopy. These studies indicated that the structures of both mutants were perturbed but that all perturbations are very near to the site of mutation.

Cloning and expression of the Clostridium thermocellum celS gene in Escherichia coli.

Clostridium thermocellum ATCC 27405 produces an extremely complicated multi-component cellulase aggregate (cellulosome) highly active on crystalline cellulose. From the cellulosome, two subunits, CelS (or Ss; M(r) = 82,000) and CelL (or SL, CipA; M(r) = 250,000), have been identified as essential for crystalline cellulose degradation [Wu et al. (1988) Biochemistry 27:1703]. We have determined the DNA sequence of the celS gene from four cloned DNA fragments encompassing this gene [Wang et al. (1993) J Bacteriol 175:1293]. To express the entire celS gene in Escherichia coli, the celS structural gene was amplified by the polymerase chain reaction (PCR) employing the PCR primers corresponding to sequences flanking the desired gene. This PCR product (2.1 x 10(3) bases; 2.1 kb) was cloned into an E. coli expression vector pRSET B. Subsequent expression of the cloned gene resulted in a fusion protein (rCelS; M(r) = 86,000) as inclusion bodies. The rCelS protein was recognized specifically by an anti-CelS antiserum in a Western blot analysis. The inclusion bodies were purified and solubilized in 5 M urea. The refolded rCelS produced very little reducing sugar from carboxymethylcellulose. However, it showed a higher activity on the crystalline cellulose (Avicel) and an even higher activity on phosphoric-acid-swollen Avicel. These results indicate that the CelS is an exoglucanase.

Induction of cellulase by cello-oligosaccharides in Trichoderma koningii G-39

A fraction of cello-oligosaccharides with a high degree of polymerization (Gx, degree of polymerization greater than 10) was found to be a potent cellulase inducer of Trichoderma koningii G-39. It was prepared by partial sulfuric acid hydrolysis of cellulose. A comparison of the induction potential and efficiency of Gx and sophorose indicated that the latent period of induction was longer and the maximal enzyme production lower for Gx than for sophorose. The optimal concentrations are 1% with Gx and 0.1% (w/v) with sophorose with a corresponding latent period and a maximum carboxymethylcellulase (CMCase) activity, which accumulated in the culture filtrate, of about 8 h and 5.4 units for Gx and 4 h and 7.2 units (in mg glucose equivalent per ml culture filtrate) for sophorose. The apparent differences between the two compounds are attributable to the interplay of catabolite repression in the inducing effect of Gx which is subjected to cellulase degradation with glucose as the major end products. HPLC sugar analysis of the culture filtrate incubated with Gx suggests that the inducing potential and efficiency of cello-oligosaccharides depend on its degree of polymerization. The protein patterns as revealed by SDS-polyacrylamide gel analysis of the culture filtrates from incubation with Avicel, phosphoric acid-swollen Avicel, Gx or sophorose showed no inducerspecific protein bands except variation in intensity and induction time. A dot hybridization analysis of poly(A) RNA from washed mycelium incubated with Gx or sophorose using a 650 basepair cDNA fragment of cellobiohydrolase I gene as the probe indicates that the inductive formation of cellulase by both inducers in this strain is operative at the transcriptional level.

β- d-glucosidases of Sclerotium rolfsii. Substrate specificity and mode of action

The substrate specificity and mode of action of the four pure β- d-glucosidase enzymes (EC 3.2.1.21) from Sclerotium rolfsii were studied and their contribution to cellulolysis is discussed. The enzymes are specific for substrates having the β- d configuration. The specificity of the enzymes is not restricted to the β- d-(1→4) linkage, as all four β- d-glucosidases hydrolyzed substrates having β- d-(1→6)-, -(1→3) and -(1→2) linkages. The enzymes require strictly a d- gluco configuration for activity. The β- d-glucosidases had no action on highly ordered cellulose, such as Avicel, but slowly hydrolyzed disordered cellulose (phosphoric acid-swollen Avicel) and carboxymethylcellulose, and rapidly cellodextrins, removing d-glucose residues from the nonreducing end. The pure enzymes behaved rather as exo-β- d-glucan glucohydrolase. The K m values of all four β- d-glucosidases decreased with increase in the chain length of cellodextrins. Cellopentaose was the preferred substrate for all four enzymes. The major route of d-glucose formation from cellulose by hydrolysis with S. rolfsii β- d-glucosidases proceeds through higher-molecular-weight cellodextrins.

The Mechanism of Enzymatic Cellulose Degradation

A cellulolytic enzyme (“C1” enzyme) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. The purification method is a four‐step procedure including chromatography on Bio‐Gel P‐10, DEAE‐Sephadex chromatography, isoelectric focusing and chromatography on Bio‐Gel P‐60. A yield of 144 mg enzyme was obtained per 100 g commercial cellulase. The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 5.0 and at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in the ultracentrifuge. No enzyme activity towards carboxymethylcellulose could be detected in the purified material under the assay conditions used. Similarly, there was no β‐glucosidase activity. The purified enzyme was associated with 3.3% carbohydrate and is assumed to be a glycoprotein. The enzyme was isoelectric at pH 3.79 (10°C). A molecular weight of 46000 was determined by chromatography of the reduced and alkylated enzyme on a calibrated column of Sepharose 6B in 6 M guanidine‐HCl. Crystalline cellulose (Avicel), phosphoric acid‐swollen Avicel and cellotetraose were degraded by the enzyme and in each case the principle reaction product was cellobiose. Evidence indicates that the purified enzyme is a β‐1,4‐glucan cellobiohydrolase.