Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans

Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.     

Role of endo-1,4-beta-glucanases from neisseria sicca SB in synergistic degradation of cellulose acetate

An enzyme hydrolyzing beta-1,4 bonds in cellulose acetate was purified 10.5-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which assimilate cellulose acetate as the sole carbon and energy source. The enzyme was an endo-1,4-beta-glucanase, to judge from the substrate specificity and hydrolysis products of cellooligosaccharides, we named it endo-1,4-beta-glucanase I (EG I). Its molecular mass was 50 kDa, 9 kDa larger than EG II from this strain, and its isoelectric point was 5.0. Results of N-terminal and inner-peptide sequences of both enzymes, and a similarity search, suggested that EG I contained a carbohydrate-binding module at the N-terminus and that EG II lacked this module. The pH and temperature optima of EG I were 5.0-6.0 and 45 degrees C. It hydrolyzed water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for these compounds were 0.296% and 1.29 micromol min(-1) mg(-1), and 0.448% and 13.6 micromol min(-1) mg(-1), respectively. Both glucanases and cellulose acetate esterase from this strain degraded water-insoluble cellulose acetate synergistically.     

Addition of cloned beta-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulose complex

A thermostable beta-glucosidase from Clostridium thermocellum which is expressed in Escherichia coli was used to determine the substrate specificity of the enzyme. A restriction map of the beta-glucosidase gene cloned in plasmid pALD7 was determined. Addition of the E. coli cell extract (containing the beta-glucosidase) to the cellulase complex from C. thermocellum increased the conversion of crystalline cellulose (Avicel) to glucose. The increase was specifically due to hydrolysis of the accumulated cellobiose. A cellulose degradation process using beta-glucosidase to assist the potent cellulase complex of C. thermocellum, as shown here can open the way for industrial saccharification of cellulose to glucose.     

Temporal changes in wood crystalline cellulose during degradation by brown rot fungi

The degradation of wood by brown rot fungi has been studied intensely for many years in order to facilitate the preservation of in-service wood. In this work we used X-ray diffraction to examine changes in wood cellulose crystallinity caused by the brown rot fungi Gloeophyllum trabeum, Coniophora puteana, and two isolates of Serpula lacrymans. All fungi increased apparent percent crystallinity early in the decay process while decreasing total amounts of both crystalline and amorphous material. Data also showed an apparent decrease of approximately 0.05 Å in the average spacing of the crystal planes in all degraded samples after roughly 20% weight loss, as well as a decrease in the average observed relative peak width at 2θ = 22.2°. These results may indicate a disruption of the outer most semi-crystalline cellulose chains comprising the wood microfibril. X-ray diffraction analysis of wood subjected to biological attack by fungi may provide insight into degradative processes and wood cellulose structure.     

Studies on the anaerobic degradation of crystalline cellulose by Clostridium thermocellum using a new assay

Abstract The anaerobic degradation of microcrystalline cellulose by thermostable cellulolytic enzyme complexes from Clostridium thermocellum JW20 (ATCC 31449) was monitored. For quantitative investigations as enzyme-coupled spectrophotometric assay has been developed. The assay allows for the evaluation of the release of cellubiose-/glucose-units from native cellulose. Kinetic studies revealed that the anaerobic breakdown of crystalline cellulose (CC) at 60°C follows Michaelis-Menten kinetics K _m CC values have been determined for different aggregation states of the cellulolytic complex. The presented assay seems well suited to screen for CC-degrading enzymes of various sources, and to further explore the mechanism of CC-breakdown.     

Differences in crystalline cellulose modification due to degradation by brown and white rot fungi

Wood-decaying basidiomycetes are some of the most effective bioconverters of lignocellulose in nature, however the way they alter wood crystalline cellulose on a molecular level is still not well understood. To address this, we examined and compared changes in wood undergoing decay by two species of brown rot fungi, Gloeophyllum trabeum and Meruliporia incrassata, and two species of white rot fungi, Irpex lacteus and Pycnoporus sanguineus, using X-ray diffraction (XRD) and ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy. The overall percent crystallinity in wood undergoing decay by M. incrassata, G. trabeum, and I. lacteus appeared to decrease according to the stage of decay, while in wood decayed by P. sanguineus the crystallinity was found to increase during some stages of degradation. This result is suggested to be potentially due to the different decay strategies employed by these fungi. The average spacing between the 200 cellulose crystal planes was significantly decreased in wood degraded by brown rot, whereas changes observed in wood degraded by the two white rot fungi examined varied according to the selectivity for lignin. The conclusions were supported by a quantitative analysis of the structural components in the wood before and during decay confirming the distinct differences observed for brown and white rot fungi. The results from this study were consistent with differences in degradation methods previously reported among fungal species, specifically more non-enzymatic degradation in brown rot versus more enzymatic degradation in white rot.     

Structure and characteristics of an endo-beta-1,4-glucanase, isolated from Trametes hirsuta, with high degradation to crystalline cellulose

Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-beta-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain.     

Cellobiohydrolase hydrolyzes crystalline cellulose on hydrophobic faces

Biodegradation of plant biomass is a slow process in nature, and hydrolysis of cellulose is also widely considered to be a rate-limiting step in the proposed industrial process of converting lignocellulosic materials to biofuels. It is generally known that a team of enzymes including endo- and exocellulases as well as cellobiases are required to act synergistically to hydrolyze cellulose to glucose. The detailed molecular mechanisms of these enzymes have yet to be convincingly elucidated. In this report, atomic force microscopy (AFM) is used to image in real-time the structural changes in Valonia cellulose crystals acted upon by the exocellulase cellobiohydrolase I (CBH I) from Trichoderma reesei. Under AFM, single enzyme molecules could be observed binding only to one face of the cellulose crystal, apparently the hydrophobic face. The surface roughness of cellulose began increasing after adding CBH I, and the overall size of cellulose crystals decreased during an 11-h period. Interestingly, this size reduction apparently occurred only in the width of the crystal, whereas the height remained relatively constant. In addition, the measured cross-section shape of cellulose crystal changed from asymmetric to nearly symmetric. These observed changes brought about by CBH I action may constitute the first direct visualization supporting the idea that the exocellulase selectively hydrolyzes the hydrophobic faces of cellulose. The limited accessibility of the hydrophobic faces in native cellulose may contribute significantly to the rate-limiting slowness of cellulose hydrolysis.     

Corn stover pretreatment by inorganic salts and its effects on hemicellulose and cellulose degradation

Inorganic salts, NaCl, KCl, CaCl₂, MgCl₂, FeCl₂, FeSO₄, FeCl₃, and Fe₂(SO₄)₃, were studied as catalysts for the degradation of hemicellulose in corn stover. FeCl₃ significantly increased the hemicellulose degradation in aqueous solutions heated between 140 and 200 °C with high xylose recovery and low cellulose removal, amounting to 90% and <10%, respectively. Hemicellulose removal increased 11-fold when the corn stover was pretreated with 0.1 M FeCl₃ compared to pretreatment with hot water under otherwise the same conditions, which was also 6-fold greater than pretreatment with dilute sulfuric acid at the same pH. Optimum pretreatment conditions were found where the corn stover was pretreated with 0.1 M FeCl₃ at 140 °C for 20 min. Under such conditions, 91% of hemicellulose was removed, and the recovery of monomeric and oligomeric xylose in liquid fraction achieved 89%, meanwhile, only 9% of cellulose was removed.     

Enzymology of cellulose degradation

In the last few years there has been a considerable improvement in the understanding of the mechanisms involved in the microbial degradation of cellulose, but there are still many uncertainties. As presently understood, it would appear that different mechanisms may operate in the various types of microorganism. Thus degradation of crystalline cellulose is effected by anaerobic bacteria by large Ca-dependent and thiol-dependent multicomponent endoglucanase-containing complexes (cellulosomes) located on concerted action of endo- and exo-glucanases which act some distance from the cell which renders cellulose soluble. All of the endo- and exo-glucanases possess a bifunctional domain structure: one contains the catalytic site, the other is involved in binding the enzyme to crystalline cellulose.     

The effect of oxidative pretreatment on cellulose degradation by Poria placenta and Trichoderma reesei cellulases

The possible role of hydrogen peroxide in brown-rot decay was investigated by studying the effects of pretreatment of spruce wood and microcrystalline Avicel cellulose with H₂O₂ and Fe²⁺ (Fenton's reagent) on the subsequent enzymatic hydrolysis of the substrates. A crude endoglucanase preparation from the brown-rot fungus Poria placenta, a purified endoglucanase from Trichoderma reesei and a commercial Trichoderma cellulase were used as enzymes. Avicel cellulose and spruce dust were depolymerized in the H₂O₂/Fe²⁺ treatment. Mainly hemicelluloses were lost in the treatment of spruce dust. The effect of the pretreatment on subsequent enzymatic hydrolysis was found to depend on the nature of the substrate and the enzyme preparation used. Pretreatment with H₂O₂/Fe²⁺ clearly increased the amount of enzymatic hydrolysis of spruce dust with both the endoglucanases and the commercial cellulase. In all cases the amount of hydrolysis was increased about threefold. The hydrolysis of Avicel with the endoglucanases was also enhanced, whereas the hydrolysis with the commercial cellulase was decreased. Received: 23 December 1996 / Received revision: 17 April 1997 / Accepted: 19 April 1997     

Shift in microbial population in response to crystalline cellulose degradation during enrichment with a semi-desert soil

Addition of crystalline cellulose to semi-desert soil shifts the microbial population; this was assessed by following the 16S rRNA gene, glycosyl hydrolase, and measuring its functional diversity in the bacterial population. Quantification of the glycosyl hydrolase gene showed an increase from 1 × 10⁴ g⁻¹ of unamended soil to 3 × 10⁴ g⁻¹ of crystalline-cellulose-amended soil by the 15th day of crystalline cellulose utilization. The indigenous glycosyl hydrolase community in unamended soil was dominated by the clone families that were closely related to the glycosyl hydrolases from Betaproteobacteria and Firmicutes. The addition of crystalline cellulose induced a shift in the glycosyl hydrolase population toward an increase in the relative abundance of the glycosyl hydrolase that was consistent with those of Bacteroidetes and Flavobacteria. The population shift of glycosyl hydrolase was also supported by the comparison of the 16S rRNA gene families in unamended and crystalline-cellulose-amended soil libraries. The most abundant 16S rRNA gene sequences retrieved in the unamended soil were identical to Pseudomonas, Massilia, Paenibacillus, and Bacillus spp., while Cytophaga and Flavobacterium spp. dominated in crystalline-cellulose-amended soil.     

Biomass photochemistry: XI. Photochemical pretreatment of cellulose and its fungal degradation

In order to study the single-cell proteins and cellulase production, fungal degradation of alpha-cellulose was tested. Photochemical pretreatment of alpha-cellulose at lambda > 254 nm increases by over 100% the cellulase production by Chrysonilia sitophila (TFB strain). This is reflected in a larger production of soluble proteins in the filtrate with higher cellulase activity when compared with unirradiated cellulose. Single-cell protein production (insoluble proteins) was also improved after preirradiation of cellulose or even on rice hull as the only carbon source after inoculation with the spore of C. sitophila. This pretreatment is a valuable method for increasing the digestibility of lignocellulosic materials.     

The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose.

CenA is a bacterial cellulase (beta-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide. The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed. CenA and CBD.PTCenA' (the CBD plus linker) adsorbed rapidly to cellulose at 30 degrees C, and no net desorption of protein was observed during the following 16.7 h. There was no detectable adsorption of the catalytic domain. Scatchard plots of adsorption data for CenA and for CBD.PTCenA were nonlinear (concave upward). The adsorption of CenA and CBD.PTCenA exceeded 7 and 8 mumol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed. A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues). A relative equilibrium association constant (Kr) of 40.5 and 45.3 liter.g cellulose-1 was estimated for CenA and CBD.PTCenA, respectively, according to this model. A similar Kr value (33.3 liter.g-1) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both beta 1,4-xylosidic and beta-1,4-glucosidic bonds. It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface.     

The processive endoglucanase EngZ is active in crystalline cellulose degradation as a cellulosomal subunit of Clostridium cellulovorans

Clostridium cellulovorans produces an efficient enzyme complex for the degradation of lignocellulosic biomass. In our previous study, we detected and identified protein spots that interacted with a fluorescently labeled cohesin biomarker via two-dimensional gel electrophoresis. One novel, putative cellulosomal protein (referred to as endoglucanase Z) contains a catalytic module from the glycosyl hydrolase family (GH9) and demonstrated higher levels of expression than other cellulosomal cellulases in Avicel-containing cultures. Purified EngZ had optimal activity at pH 7.0, 40°C, and the major hydrolysis product from the cellooligosaccharides was cellobiose. EngZ's specific activity toward crystalline cellulose (Avicel and acid-swollen cellulose) was 10-20-fold higher than other cellulosomal cellulase activities. A large percentage of the reducing ends that were produced by this enzyme from acid-swollen cellulose were released as soluble sugar. EngZ has the capability of reducing the viscosity of Avicel at an intermediate-level between exo- and endo-typing cellulases, suggesting that it is a processive endoglucanase. In conclusion, EngZ was highly expressed in cellulolytic systems and demonstrated processive endoglucanase activity, suggesting that it plays a major role in the hydrolysis of crystalline cellulose and acts as a cellulosomal enzyme in C. cellulovorans.     

Purification and characterization of an endo-1,4-beta-glucanase from Neisseria sicca SB that hydrolyzes beta-1,4 linkages in cellulose acetate

An enzyme catalyzing hydrolysis of beta-1,4 bonds in cellulose acetate was purified 18.3-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The molecular mass of the enzyme was 41 kDa and the isoelectric point was 4.8. The pH and temperature optima of the enzyme were 6.0-7.0 and 60 degrees C. The enzyme catalyzed hydrolysis of water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for water-soluble cellulose acetate and carboxymethyl cellulose were 0.242% and 2.24 micromol/min/mg, and 2.28% and 12.8 micromol/min/mg, respectively. It is estimated that the enzyme is a kind of endo-1,4-beta-glucanase (EC 3.2.1.4) from the substrate specificity and hydrolysis products of cellooligosaccharides. The enzyme and cellulose acetate esterase from Neisseria sicca SB degraded water-insoluble cellulose acetate by synergistic action.     

Mechanisms of wood degradation by brown-rot fungi: chelator-mediated cellulose degradation and binding of iron by cellulose

Iron, hydrogen peroxide, biochelators and oxalate are believed to play important roles in cellulose degradation by brown-rot fungi. The effect of these compounds in an 'enhanced' Fenton system on alpha-cellulose degradation was investigated specifically in regard to molecular weight distribution and cellulose-iron affinity. This study shows that the degradative ability of an ultrafiltered low molecular weight preparation of chelating compounds isolated from the brown-rot fungus Gloeophyllum trabeum (termed 'Gt chelator') increased with increasing Gt chelator concentration when the FeIII to Gt chelator ratio was greater than about 30:1. When this ratio was less than 30:1, increasing Gt chelator concentration did not accelerate cellulose degradation. In excess hydrogen peroxide, cellulose degradation increased and then decreased with increasing iron concentration when FeIII was present in excess of the Gt chelator. The critical ratio of FeIII to Gt chelator varied depending on the concentration of hydrogen peroxide in the system. Increasing iron concentration above a critical iron:chelator ratio inhibited cellulose degradation. The optimum pH for cellulose degradation mediated by Gt chelator was around 4.0. A comparison of the effects of 2,3-DHBA (a chelator that reduces iron similarly to Gt chelator) and Gt chelator with respect to cellulose degradation demonstrated the same pattern of cellulose degradation. Cellulose-iron affinity studies were conducted at three pH levels (3.6, 3.8, 4.1), and the binding constants for cellulose-FeIII, cellulose-FeII and cellulose-FeIII in the presence of Gt chelator were calculated. The binding constants for cellulose-FeIII at all three pH levels were much higher than those for cellulose-FeII, and the binding constants for cellulose-FeIII in the presence of Gt chelator were very close to those for cellulose-FeII. This is probably the result of FeIII reduction to FeII by Gt chelator and suggests that chelators from the fungus may be able to sequester iron from cellulose and reduce it in near proximity to the cellulose and thereby better promote depolymerization. The free radical generating system described has potential for use in a variety of industrial processing and pollution control applications.