Studies on a wild strain of Schizophyllum commune: Cellulase and xylanase production and formation of the extracellular polysaccharide Schizophyllan

A wild strain of schizophyllum commune (Fr:Fr:) isolated in Bangladesh produced cellulase and xyianase in high yields as well as the exobiopolymer schizophyllan. It was found experimentally that concentrations of 4% Avicel, 3.5% peptone, and 0.5% Ca(NO(3))(2).4H(2)O were optimal for growth and product formation. Bacto-peptone was found to be the most suitable substrate of a number of casein, mycological, and meat peptone preparations for enzyme production. Young plate-culture inocula (4 days) were found to be better than comparatively aged fungal cultures (14 days). With the optimized medium, 5 units filter paper (FP) cellulase, 1244 units xylanase, 108 units beta-glucosidase, and 65 units of carboxymethyl (CM) cellulase per mL culture filtrate were obtained in shake flasks. In a laboratory fermentor the respective enzyme activities were 4.5 units FP-cellulase, 1200 units xylanase, 100 units beta-glucosidase, and 60 units CM-cellulase per mL culture filtrate. A biopolymer, reported to be active against can cerous cells, was an additional product in addition to the enzymes.     

Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilaris.

A cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 micro mol.min-1.(mg protein)-1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 micro mol.min-1.(mg protein)-1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods.     

Induction of cellulolytic enzymes in Trichoderma reesei by sophorose.

Sophorose (2-O-beta-glucopyranosyl-D-glucose) induces carboxymethyl cellulase in Trichoderma reesei QM6a mycelium with 1.5 to 2 h. The induction response to sophorose concentration, although complicated by the metabolism of sophorose, shows saturation kinetics. Most of the cellulase appears after most of the sophorose has been taken up, but the presence of an inducer is required to maintain cellulase synthesis because enzyme production ceases after separation of the mycelium from the induction medium. Cellulase appears simultaneously in the medium and in the mycelium, and no appreciable levels accumulate in the mycelium. Response to pH suggest either that synthesis and secretion of the enzyme are closely associated or concurrent events affected by surface interactions with the medium. Effects of temperature and pH on cellulase induction by sophorose are similar to those reported for induction by cellulose. The kinetics of absorption by mycelium differs from that of other beta-linked saccharides and glucose, the uptake of sophorose being much slower. Under our cultural conditions, sophorose appears to induce an incomplete array of cellulase enzymes, as indicated by enzymatic and electrophoretic studies.     

Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3-O-methylglucoside. Cellodextrins (C₂ to C₆) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [³H]cellobiose, [³H]sophorose, and [¹⁴C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3-O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [³H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.     

Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.

Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.     

Induction and regulation of cellulase synthesis in Trichoderma pseudokoningii mutants EA3-867 and N2-78

Two mutants, EA₃-867 and N₂-78, with high cellulase yields were obtained from wild strains of Trichoderma pseudokoningii Rifai, 1096 and Mo₃, respectively, by mutagenic treatments with a linear accelerator, ⁶⁰Co, u.v., nitrosoguanidine (NTG) and diethylsulphate (DTS). The mutants grew slowly to produce small colonies on agar plates with synthetic medium. On agar plates of peptone-yeast extract, the small colonies were as large as those of wild strains. The cellulase activities of these mutants in Koji extracts, shake flask culture filtrates, and enzyme preparations were markedly higher than those of their parents. The mutant N₂-78 reached quite high cellulase activity level when cultured for 60 h in shake flasks in a simple medium containing milled straw, wheat bran, mineral salts plus waste glucose molasses. The cellulase saccharifying activities on CMC, filter paper and cotton, were 255, 8.2 and 13.4 mg glucose/ml enzyme, respectively, or 11, 4.3 and 6 times more than those of its parent Mo₃.The cellulase synthesis of EA₃-867 and N₂-78 was strongly induced by sophorose, isolated from pods of Sophora japonica L., and was inhibited by glucose, sugar phosphates, glycerol and organic acids. We conclude that cellulase synthesis of the mutants is regulated by catabolite repression as well as by induction. The increase in cellulase production by both mutants results from changes in the regulatory systems for cellulase synthesis, i.e. the mutants showed higher sensitivity to inducer and lower susceptibility to catabolite repression than did the wild types.A cellulase preparation of Trichoderma pseudokoningii Rifai N₂-78 induced by sophorose was fractionated by DEAE-Sephadex A-50 and Sephadex G-100 column chromatography, selective inactivation and polyacrylamide gel electrophoresis. The components C₁(exo-β1,4-glucanase), C_x(endo-β1,4-glucanase) and β-glucosidase were separated, and their molecular weights were estimated to be 67 000, 62 000 and 42 000 respectively. The homogeneity of C₁ was verified by polyacrylamide gel electrophoresis, immunoelectrophoresis and ultracentrifugal analysis. It is a glycoprotein and is rich in glycine, aspartic acid, threonine, serine and glutamic acid. The C₁ showed a strong synergistic action with C_x in the degradation of cotton, Avicel and Walseth cellulose.A poly(A)-RNA, induced by sophorose in N₂-78 mycelium, was isolated by oligo(dT)-cellulose affinity chromatography.     

Yersinia kristensenii: A new species of enterobacteriaceae composed of sucrose-negative strains (formerly called atypicalYersinia enterocolitica orYersinia enterocolitica-Like)

Induction of cellulase was observed inFusarium sp. with reduction in lag period by lactose-pregrown cells as compared with glucose-pregrown cells. Insoluble cellulose (Sigmacell) induced maximum cellulase production in the induction medium. Supplementation of the culture growing on cellulose by cellobiose or glucose resulted in increased cellular growth and decreased cellulase production. Stepfeeding of cellobiose to the culture growting on carboxymethyl cellulose resulted in decreased cellulase production. Significant cellulase activity was detected in the culture filtrate of cells growing on Sigmacell supplemented with glucose, only when the glucose disappeared from the medium. This suggests that cellulase production may in part be regulated by catabolite repression.     

Retinol and retinal metabolism. Relationship to the state of differentiation of cultured human keratinocytes.

The modes of action of the five major endo-(1----4)-beta-D-glucanases (I, II, III, IV and V) purified from Penicillium pinophilum cellulase were compared by h.p.l.c. analysis, with normal, 1-3H-labelled and reduced cello-oligosaccharides and 4-methylumbelliferyl glycosides as substrates. Significant differences were observed in the preferred site of cleavage even when substrates with the same number of glycosidic bonds were compared. Thus, although endoglucanase I was unable to attack normal cello-oligosaccharides shorter than degree of polymerization 6, it hydrolysed reduced cellopentaose to yield cellotriose and cellobi-itol, and it produced cellotriose and 4-methylumbelliferyl glucoside from 4-methylumbelliferyl cellotetraoside. Endoglucanase IV hydrolysed [1-3H]cellotriose but did not attack either cellotri-itol or 4-methylumbelliferyl cellobioside. These and other anomalous results indicated clearly that modification of the reducing glycosyl residue on the cello-oligosaccharides induces in an apparent change in the mode of action of the endoglucanases. It is suggested that, although cello-oligosaccharide derivatives are useful for differentiating and classifying endoglucanases, conclusions on the mechanism of cellulase action resulting from these measurements should be treated cautiously. Unequivocal information on the mode of endoglucanase action on cello-oligosaccharides was obtained with radiolabelled cello-oligosaccharides of degree of polymerization 3 to 5. Indications that transglycosylation was a property of the endoglucanases were particularly evident with the 4-methylumbelliferyl cello-oligosaccharides. Turnover numbers for hydrolysis of the umbelliferyl cello-oligosaccharides were calculated, and these, along with the other analytical data collected on the products of hydrolysis of the normal, reduced and radiolabelled cello-oligosaccharides, suggested that the various endoglucanases had different roles to play in the overall hydrolysis of cellulose to sugars small enough to be transported through the cell membrane.     

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.     

Enzymatic hydrolysis of cellulosic materials by Sclerotium rolfsii culture filtrate for sugar production.

The hydrolysis of purified celluloses (cotton, Avicel, Cellulose-123, Solka Floc SW40) and cellulosic wastes (rice straw, sugarcane bagasse, wood powders, paper factory effluents) by Sclerotium rolfsii CPC 142 culture filtrate was studied. Factors which effect saccharification such as pH, temperature, enzyme concentration, substrate concentration, produce inhibition, adsorption, and inactivation of enzyme and particle size were studied. Virtually no inhibition (less than 3%) of cellulose hydrolysis by the culture filtrate was observed by cellobiose and glucose up to 100 mg/mL. Filter paper degrading enzyme(s) (but neither carboxymethylcellulase nor beta-glucosidase) was adsorbed on cellulose. The n value in the S. rolfsii system was calculated to be 0.32 for Avicel P.H. 101 and 0.53 for alkali-treated (AT) rice straw indicating penetration of cellulase into AT rice straw. In batch experiments at 10% substrate level, solutions containing 6 to 7%, 3.8 to 4.7%, 4.0 to 5.1%, and 4.2 to 4.9% reducing sugars were produced in 24 to 48 from AT rice straw. AT bagasse, alkali - peracetic acid treated mesta wood and paper factory sedimented sludge effluent, respectively. The main constituent in the hydrolysate from cellulose was glucose with little or no cellobiose, probably due to the high cellobiase content in the culture filtrate.     

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.     

Enhanced Expression of Endochitinase in Trichoderma harzianum with the cbh1 Promoter of Trichoderma reesei

Production of extracellular endochitinase could be increased 5-fold in the mycoparasite fungus Trichoderma harzianum by using the cellulase promoter cbh1 of Trichoderma reesei, whereas the total endochitinase activity increased 10-fold. The cbh1 promoter was not expressed on glucose and sucrose in T. harzianum and was induced by sophorose and on cellulase-inducing medium. The endogenous endochitinase gene was expressed at a low basal level on glucose and sucrose. No specific induction by crab shell chitin or sophorose was observed.     

Cellulases: Biosynthesis and applications

Strains of Trichoderma, particularly T. reesei and its mutants, are good sources of extracellular cellulase suitable for practical saccharification. They secrete a complete cellulase complex containing endo- and exo-glucanases plus β-glucosidase (cellobiase) which act syngergistically to degrade totally even highly resistant crystalline cellulose to soluble sugars. All strains investigated to date are inducible by cellulose, lactose, or sophorose, and all are repressible by glucose. Induction, synthesis and secretion of the β-glucanase enzymes appear to be closely associated. The composition and properties of the enzyme complex are similar regardless of the strain or inducing substrate although quantities of the enzyme secreted by the mutants are higher. Enzyme yields are proportional to initial cellulose concentration. Up to 15 filter paper cellulase units (20 mg of cellulase protein) per ml and productivities up to 80 cellulase units (130 mg cellulase protein) per litre per hour have been attained on 6% cellulose. The economics of glucose production are not yet competitive due to the low specific activity of cellulase (0.6 filter paper cellulase units/mg protein) and poor efficiency (about 15% of predicted sugar levels in 24 h hydrolyses of 10–25% substrate). As hydrolysis proceeds, the enzyme reaction slows due to increasing resistance of the residue, product inhibition, and enzyme inactivation. These problems are being attacked by use of pretreatments to increase the quantity of amorphous cellulose, addition of β-glucosidase to reduce cellobiose inhibition, and studies of means to overcome instability and increase efficiency of the cellulases. Indications are that carbon compounds derived from enzymatic hydrolysis of cellulose will be used as fermentation and chemical feedstocks as soon as the process economics are favourable for such application.     

Functional cello-oligosaccharides production from the corncob residues of xylo-oligosaccharides manufacture

An integrated process has been developed, consisting of the “adsorption–separation” of cellulase enzymes to selectively remove β-glucosidase, and multi-stage enzymatic hydrolysis of corncob residues from xylo-oligosaccharides manufacture with the β-glucosidase deficient cellulase, aiming to obtain a high yield of cello-oligosaccharides production. After the “adsorption–separation” process, 79.50% of the endo-glucanase was retained in substrate, whereas 90.67% of β-glucosidase was removed with the separated liquid fraction, utilizing the different adsorbability of these enzymes to the substrate. A three-stage enzymatic hydrolysis of corncob residues with the β-glucosidase deficient cellulase was proposed in which the first, the second and the third stage were conducted for 6, 6 h and 12 h, respectively. Analysis indicated that the removal of hydrolysis products (glucose and cello-oligosaccharides) at each stage improved cello-oligosaccharides productivity and enzymatic hydrolysis yield. The cello-oligosaccharides yield and enzymatic hydrolysis yield in three-stage enzymatic hydrolysis were significantly improved to 51.78% and 75.56%, respectively, which were 36.00% and 25.10% higher than single-stage hydrolysis with original cellulase enzymes.     

Production of cellulases by Thermomonospora sp.

Glucose, cellulose, Avicel, and Solka Floc were utilized as substrates for growth of Thermomonospora sp in order to study the induction–repression characteristics of its associated cellulase system. While glucose proved to be an effective repressor of the cellulase enzymes, the other three substrates induced relatively high levels of enzyme activity as measured by the filter paper assay. On a unit cell mass basis the highest values of cellulase activity were obtained when Avicel was utilized as the carbon and energy source. The nature of the cellulosic material and its initial concentration were identified as two very important parameters of the induction process.     

Sorbose mediated enhancement of cellulase biosynthesis inTrichoderma reesei

Addition of L-sorbose, a non-metabolizable non-inducing ketohexose, toTrichoderma reesei cultures growing on cellobiose or Avicel-cellulose lead to increased cellulase activities. Addition of sorbose resulted in a 6-fold increase in cellodextrins (cellotriose, cellotetraose, cellopentaose) concentration on day 3 in cellobiose cultures and 1.3-fold increase in cellodextrins concentrations on day 4 in Avicel cellulose cultures. This increase in intracellular cellodextrins concentration matched closely with the increase in endoglucanase activity at these time points. Treatment of the cell-free extracts with cellulase preparation led to disappearance of the cellodextrins and increase of glucose. These observations suggested a more direct involvement of cellodextrins in cellulase induction process. The cellulases produced in sorbose-supplemented cellobiose medium hydrolyzed microcrystalline cellulose as effectively as the ones produced on Avicel cellulose medium.     

Fermentation of Cellulosic Substrates in Batch and Continuous Culture by Clostridium thermocellum

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was compared in batch and continuous cultures. Maximum specific growth rates per hour obtained on cellulosic substrates were 0.1 in batch culture and >0.13 in continuous culture. Cell yields (grams of cells per gram of substrate) in batch culture were 0.17 for pretreated wood and 0.15 for Avicel. Ethanol and acetate were the main products observed under all conditions. Ethanol:acetate ratios (in grams) were approximately 1.8:1 in batch culture and generally slightly less than 1:1 in continuous culture. Utilization of cellulosic substrates was essentially complete in batch culture. A prolonged lag phase was initially observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuous culture with ~5 g of glucose equivalent per liter in the feed, substrate conversion relative to theoretical ranged from 0.86 at a dilution rate (D) of 0.05/h to 0.48 at a D of 0.167/h for Avicel and from 0.75 at a D of 0.05/h to 0.43 at a D of 0.11/h for pretreated wood. At feed concentrations of <4.5 g of glucose equivalent per liter, conversion of pretreated wood was 80 to 90% at D = 0.083/h. Lower conversion was obtained at higher feed substrate concentrations, consistent with a limiting factor other than cellulose. Free Avicelase activities of 12 to 84 mU/ml were observed, with activity increasing in this order: batch cellobiose, batch pretreated wood < batch Avicel, continuous pretreated wood < continuous Avicel. Free cellulase activity was higher at increasing extents of substrate utilization for both pretreated wood and Avicel under all conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity, are essentially the same for pretreated mixed hardwood and Avicel under a variety of conditions. Hydrolysis yields obtained with C. thermocellum cellulase acting either in vitro or in vivo were comparable to those previously reported for Trichoderma reesei on the same substrates.     

Cellulase kinetics as a function of cellulose pretreatment

Microcrystalline cellulose (Avicel) was subjected to three different pretreatments (acid, alkaline, and organosolv) before exposure to a mixture of cellulases (Celluclast). Addition of beta-glucosidase, to avoid the well-known inhibition of cellulase by cellobiose, markedly accelerated cellulose hydrolysis up to a ratio of activity units (beta-glucosidase/cellulase) of 20. All pretreatment protocols of Avicel were found to slightly increase its degree of crystallinity in comparison with the untreated control. Adsorption of both cellulase and beta-glucosidase on cellulose is significant and also strongly depends on the wall material of the reactor. The conversion-time behavior of all four states of Avicel was found to be very similar. Jamming of adjacent cellulase enzymes when adsorbed on microcrystalline cellulose surface is evident at higher concentrations of enzyme, beyond 400 U/L cellulase/8 kU/L beta-glucosidase. Jamming explains the observed and well-known dramatically slowing rate of cellulose hydrolysis at high degrees of conversion. In contrast to the enzyme concentration, neither the method of pretreatment nor the presence or absence of presumed fractal kinetics has an effect on the calculated jamming parameter for cellulose hydrolysis.