Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.     

Cellulose derivatives: An enzymatic approach to their modification

Carboxymethyl cellulose (CMC) with different degrees of substitution (DS) and molecular weights (MW) have been successfully hydrolyzed by cellulases sourced from different microorganisms. The extent of enzymatic hydrolysis of CMC was shown to decrease with increasing DS. According to chromatographic analyses, the best enzymatic degradation by the crude enzymic preparations employed was 47% when cellulase T from Trichoderma species acted on a CMC of DS = 0·7. However, the complete hydrolysis, required for a quantitative analysis, was reached when CMCs with DS up to 0·7 were degraded by cellulase P, a purified form of celluclast from Trichoderma reesei.     

Synergistic hydrolysis of carboxymethyl cellulose and acid-swollen cellulose by two endoglucanases (CelZ and CelY) from Erwinia chrysanthemi

Erwinia chrysanthemi produces a battery of hydrolases and lyases which are very effective in the maceration of plant cell walls. Although two endoglucanases (CelZ and CelY; formerly EGZ and EGY) are produced, CelZ represents approximately 95% of the total carboxymethyl cellulase activity. In this study, we have examined the effectiveness of CelY and CelZ alone and of combinations of both enzymes using carboxymethyl cellulose (CMC) and amorphous cellulose (acid-swollen cellulose) as substrates. Synergy was observed with both substrates. Maximal synergy (1.8-fold) was observed for combinations containing primarily CelZ; the ratio of enzyme activities produced was similar to those produced by cultures of E. chrysanthemi. CelY and CelZ were quite different in substrate preference. CelY was unable to hydrolyze soluble cellooligosaccharides (cellotetraose and cellopentaose) but hydrolyzed CMC to fragments averaging 10.7 glucosyl units. In contrast, CelZ readily hydrolyzed cellotetraose, cellopentaose, and amorphous cellulose to produce cellobiose and cellotriose as dominant products. CelZ hydrolyzed CMC to fragments averaging 3.6 glucosyl units. In combination, CelZ and CelY hydrolyzed CMC to products averaging 2.3 glucosyl units. Synergy did not require the simultaneous presence of both enzymes. Enzymatic modification of the substrate by CelY increased the rate and extent of hydrolysis by CelZ. Full synergy was retained by the sequential hydrolysis of CMC, provided CelY was used as the first enzyme. A general mechanism is proposed to explain the synergy between these two enzymes based primarily on differences in substrate preference.     

Purification and properties of an endo-1,4-beta-glucanase from Clostridium josui.

An enzyme active against carboxymethyl cellulose (CMC) was purified from the stationary-phase-culture supernatant of Clostridium josui grown in a medium containing ball-milled cellulose. The purification in the presence of 6 M urea yielded homogeneous enzyme after an approximately 50-fold increase in specific activity and a 13% yield. The enzyme had a molecular mass of 45 kilodaltons. The optimal temperature and pH of the enzyme against CMC were 60 degrees C and 6.8, respectively. The enzyme hydrolyzed cellotetraose, cellopentaose, and cellohexaose to cellobiose and cellotriose but did not hydrolyze cellobiose or cellotriose. A microcrystalline cellulose, Avicel, was also hydrolyzed significantly, but the extent of hydrolysis was remarkably less than that of CMC. On the basis of these results, the enzyme purified here is one of the endo-1,4-beta-glucanases. The N-terminal amino acid sequence of the enzyme is Tyr-Asp-Ala-Ser-Leu-Lys-Pro-Asn-Leu-Gln-Ile-Pro-Gln-Lys-Asn-Ile-Pro-Asn- Asn-Asp-Ala-Val-Asn-Ile-Lys.     

A Thermostable Xylanase from a Thermophilic Acidophilic Bacillus sp.

Bacillus sp. 11-IS, a strain of thermophilic acidophilic bacteria, produced an extracellular xylanase during growth on xylan. The enzyme purified from the culture supernatant solution was homogeneous on disc-gel electrophoresis. The molecular weight was calculated to be 56,000 by SDS-gel electrophoresis. The enzyme had a pH optimum for activity at 4.0, and its stability range was pH 2.0 ~ 6.0. The temperature optimum was 80°C (10-min assay); however, the enzyme retained full activity after incubation at 70°C for 15 min. The enzyme acted on carboxymethyl cellulose (CMC) and cellulose, as well as on xylan. The Michaelis constants for larchwood xylan and CMC were calculated to be 1.68 mg xylose eq/ml and 0.465 mg glucose eq/ml, respectively. The predominant hydrolysis products from larchwood xylan were xylobiose, xylotriose, and xylose; the release of arabinose from rice-straw arabinoxylan was not detected. CMC was cleaved to cellobiose and larger oligosaccharides. Thus, the enzyme is considered to be an endoenzyme which degrades the β-1,4-glycosyl linkages in xylan and cellulose.     

Rheology and Hydrodynamic Properties of Tolypocladium inflatum Fermentation Broth and Its Simulation

A physico-chemical, two phase simulated pseudoplastic fermentation (SPF) broth was investigated in which Solka Floc cellulose fibre was used to simulate the filamentous biomass, and a mixture of 0.1% (w/v) carboxymethyl cellulose (CMC) and 0.15 M aqueous sodium chloride was used to simulate the liquid fraction of the fermentation broth. An investigation of the rheological behaviour and hydrodynamic properties of the SPF broth was carried out, and compared to both a fungal Tolypocladium inflatum fermentation broth and a CMC solution in a 50 L stirred tank bioreactor equipped with conventional Rushton turbines. The experimental data confirmed the ability of the two phase SPF broth to mimic both the T. inflatum broth bulk rheology as well as the mixing and mass transfer behaviour. In contrast, using a homogeneous CMC solution with a similar bulk rheology to simulate the fermentation resulted in a significant underestimation of the mass transfer and mixing times. The presence of the solid phase and its microstructure in the SPF broth appear to play a significant role in gas holdup and bubble size, thus leading to the different behaviours. The SPF broth seems to be a more accurate simulation fluid that can be used to predict the bioreactor mixing and mass transfer performance in filamentous fermentations, in comparison with CMC solutions used in some previous studies.     

Determination of the degree of substitution and its distribution of carboxymethylcelluloses by capillary zone electrophoresis

A method based on capillary zone electrophoresis (CZE) has been developed to determine the degree of substitution (DS) of carboxymethylcellulose (CMC). Separations were performed with borate buffer (pH 9, ionic strength 20 mM) as background electrolyte in capillaries of 75 microm ID, with an applied voltage of 10 kV, and for detection UV absorption at 196 nm was measured. The use of an internal standard (phthalic acid) to correct for mobility variations resulted in a strong improvement of the precision of the DS determination. Experiments with indirect UV detection indicated that the peak widths obtained actually reflect the variation in mobility, and with that of the DS value, of CMC samples. With the proposed method not only the average DS value but also its dispersity could be established for technical CMC samples. A small but definite effect of the polymeric size on the mobilities was observed. Therefore, DS calibration curves will have to be determined for a specific MM range. Since the size effect is small, a classification of CMCs as low-, middle-, or high MM will be sufficient to obtain accurate data on the DS distribution.     

New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 2. Comparison of various enzyme preparations

In this part of our studies, dealing with new approaches to the analysis of enzymatically hydrolyzed methyl cellulose, five different enzymes or enzyme preparations containing endoglucanases (from Bacillus agaradhaerens Cel 5A, Trichoderma reesei, Trichoderma viride, and two obtained from Trichoderma longibrachiatum) were used to hydrolyze six different methyl celluloses (MCs). The main goal was to investigate whether enzymes could be used for determination of the heterogeneity of the substituent distribution along the cellulose chain. To obtain information about the heterogeneity, it was necessary to gather information on how the enzymes affect hydrolysis. Size exclusion chromatography with multi-angle light scattering and refractive index detection (SEC-MALS/RI) was used to estimate the molar mass distribution of the MCs before and after hydrolysis. A novel internal standard addition method in combination with electrospray ionization ion trap mass spectrometry (ESI-ITMS) was used to determine the amount of formed oligomers. Two MCs, one with a degree of substitution (DS) of 1.8 and one with DS 1.3, were hydrolyzed with all of the five enzymes. The yield of summarized di- and trisaccharides was approximately 2% of the hydrolysis products for the MC with DS 1.8, whereas the product mixture, obtained from a MC with a DS of 1.3, contained 7-16% di- and trisaccharides. By a novel sample preparation method in combination with ESI-IT tandem MS, outlined in part 1 of this work, it was shown that the enzymes produced oligomers with the reducing end bearing no or only one substituent. Comparison of the methyl pattern at the nonreducing ends of the dimers and trimers indicated that the -2 subsite of the active complex is less tolerant than subsites -3 and +1. All enzymes had similar general selectivity toward the methyl substituents but also showed some differences. From both SEC-MALS/RI and ESI-ITMS, differences with respect to substituent distribution of MCs could be recognized but not for each enzyme used. Basic considerations for enzymatic hydrolysis and analysis of methyl cellulose were listed as a consequence of the results from the work.     

Characterizing the influence of electron irradiation on scleroglucan

The electron irradiation effect on scleroglucans was investigated using different energy doses. Electron spin resonance spectra revealed radicals that were stable for several days. ¹H NMR, ¹³C NMR and Raman spectra indicated no differences in chemical backbone structure due to irradiation. In contrast, lower viscosities of aqueous solutions were received at higher energy doses. This was caused by polymer degradation. The irradiation also decreased the weight average molar masses observed by gel permeation chromatography and multi-angle light scattering. Beginning from raw materials exceeding 4 · 10⁶ Da, a number of main chain scissions of approximately 0.3 · 10⁻⁷ mol J⁻¹ was found. But for one scleroglucan quality the scission number decreased with higher doses. In addition, the characterization via asymmetrical flow field-flow fractionation proved the presence of low and high molar mass fractions. The electron irradiation led to a preferred scission of the high molar mass chains and increased the lower molar mass fraction. Due to this effect, the broadness of the molar mass distribution decreased.     

Enzyme biotechnology development for treating polymers in hydraulic fracturing operations

Carboxymethyl cellulose (CMC) is a polymer used in many different industrial sectors. In the oil and gas industry, CMC is often used during hydraulic fracturing (fracking) operations as a thickening agent for effective proppant delivery. Accumulations of CMC at fracture faces (known as filter cakes) can impede oil and gas recovery. Although chemical oxidizers are added to disrupt these accumulations, there is industrial interest in developing alternative, enzyme-based treatments. Little is known about CMC biodegradation under fracking conditions. Here, we enriched a methanogenic CMC-degrading culture and demonstrated its ability to enzymatically utilize CMC under the conditions that typify oil fields. Using the extracellular enzyme fraction from the culture, significant CMC viscosity reduction was observed between 50 and 80˚C, at salinities up to 20% (w/v) and at pH 5–8 compared to controls. Similar levels of viscosity reduction by extracellular enzymes were observed under oxic and anoxic conditions. This proof-of-concept study demonstrates that enzyme biotechnology holds great promise as a viable approach to treating CMC filter cakes under oilfield conditions.     

Degradation of cellulose by the major endoglucanase produced from the brown-rot fungus Fomitopsis pinicola

An endoglucanase that is able to degrade both crystalline and amorphous cellulose was purified from the culture filtrates of the brown-rot fungus Fomitopsis pinicola grown on cellulose. An apparent molecular weight of the purified enzyme was approximately 32 kDa by SDS-PAGE analysis. The enzyme was purified 11-fold with a specific activity of 944 U/mg protein against CMC. The partial amino acid sequences of the purified endoglucanase had high homology with endo-beta-1,4-glucanase of glycosyl hydrolase family 5 from other fungi. The K(m) and K(cat)values for CMC were 12 mg CMC/ml and 670/s, respectively. The purified EG hydrolyzed both cellotetraose (G4) and cellopentaose (G5), but did not degrade either cellobiose (G2) or cellotriose (G3).     

Purification and properties of endo-(1→4)-β-d-glucanase from Ruminococcus albus

An enzyme active against O-(carboxymethyl)cellulose (CMC) was purified from a synthetic medium containing ball-milled cellulose wherein Ruminococcus albus had been cultivated for 70 h. After 570-fold purification, a homogeneous enzyme was obtained in a yield of 3%. The enzyme degraded CMC (molecular weight, 180,000; degree of substitution, 0.6) to a smaller polymer having a molecular weight of ∼20,000, and generated a small proportion of glucose, but negligible proportions of such cello-saccharides as cellobiose, cellotriose, cellotetraose, or cellopentaose. The fact that the enzyme could produce water-insoluble fragments was discovered by dissolving substrate and products in Cadoxen solution. No water-soluble cello-oligomers were detected by thin-layer chromatography after degradation of water-insoluble cellulose by the purified enzyme. Therefore, the enzyme was classified as an endo-(1→4)-β-d-glucanase.     

Purification and characterization of wall-localized cellulase from maize coleoptiles

Wall-localized cellulase was partially purified from freeze-dried maize coleoptiles by a combination of DEAE-Sepharose, Superdex-200 gel filtration and Hydroxyapatite column chromatography. Activity was measured by both reducing sugar assay and dot assay on agarose gel containing carboxymethylcellulose(CMC). In situ activity staining on a nondenaturing gel overlaid on agarose gel containing CMC turned out to be a quite reliable method to detect cellulase activity. The molecular mass of partially-purified cellulase was determined to be about 53 kD based on SDS-PAGE, and the N-terminal amino acid sequence of this cellulase was NH₂-AGAKGANXLGGLXRA. The enzyme hydrolyzed CMC with an optimal pH of 4.5 and optimal temperature of 40°C. It also catalyzed carboxymethylcellulose with aK _m of 2.02 mg/mL and aV _max of 160 ng/h/mL The β-1,4-glucosyl linkages of CMC, fibrous cellulose and lichenan were cleaved specifically by this enzyme. Reducing reagents such as cysteine-HCI, dithiothreitol and glutathione strongly enhanced the activity, suggesting that SH-groups of the enzyme were protected from oxidation. N-ethylmaleimide which is a sulfhydryl-reacting reagent did not seem to inhibit the activity, indicating that cysteine residues were not located near the active site of the enzyme. These results will be valuable in understanding the structure of wall-localized cellulase in maize coleoptiles and in predicting its possible function in the cell wall.     

Characterization of motor patterns in isolated human colon: are there differences in patients with slow-transit constipation?

The patterns of motor activity that exist in isolated full-length human colon have not been described. Our aim was to characterize the spontaneous motor patterns in isolated human colon and determine whether these patterns are different in whole colons obtained from patients with slow-transit constipation (STC). The entire colon (excluding the anus), was removed from patients with confirmed STC and mounted longitudinally in an organ bath ～120 cm in length, containing oxygenated Krebs' solution at 36°C. Changes in circular muscle tension were recorded from multiple sites simultaneously along the length of colon, by use of isometric force transducers. Recordings from isolated colons from non-STC patients revealed cyclical colonic motor complexes (CMCs) in 11 of 17 colons, with a mean interval and half-duration of contractions of 4.0 ± 0.6 min and 51.5 ± 15 s, respectively. In the remaining six colons, spontaneous irregular phasic contractions occurred without CMCs. Interestingly, in STC patients robust CMCs were still recorded, although their CMC pacemaker frequencies were slower. Intraluminal balloon distension of the ascending or descending colon evoked an ascending excitatory reflex contraction, or evoked CMC, in 8 of 30 trials from non-STC (control) colons, but not from colons obtained from STC patients. In many control segments of descending colon, spontaneous CMCs consisted of simultaneous ascending excitatory and descending inhibitory phases. In summary, CMCs can be recorded from isolated human colon, in vitro, but their intrinsic pacemaker frequency is considerably faster in vitro compared with previous human recordings of CMCs in vivo. The observation that CMCs occur in whole colons removed from STC patients suggests that the intrinsic pacemaker mechanisms underlying their generation and propagation are preserved in vitro, despite impaired transit along these same regions in vivo.     

Detection and characterization of the specific and nonspecific endoglucanases of Trichoderma reesei: Evidence demonstrating endoglucanase activity by cellobiohydrolase II

The cellulase enzyme system of Trichoderma reesei RUT C-30 has been separated by DEAE ion exchange chromatography into four fractions. Their specificity towards substituted cellulose and cellooligosaccharides was revealed by analytical IEF and activity stains. Fraction EGI (26% of the total protein) exhibited mainly endoglucanase activity on carboxymethylcellulose (CMC) whereas endoglucanases EGII and EGIII (15% of the total protein) showed high activity towards CMC as well as xylan, 4-methylumbelliferyl cellobioside [MeUmb(Glc)₂] and p-nitrophenyl lactoside (pNPL). A subfraction of EGI (pI 5.9) which has been described in the literature as a cellobiohydrolase (CBHII) was isolated by preparative isoelectric focusing, and was shown to have only 3 U CMCase activity per milligram. Turbidimetric measurements and phase contrast microscopy demonstrated differences between endoglucanase and cellobiohydrolase behaviour during the hydrolysis of purified cellulose (Solka Floc BW-40). Treatment of the purified cellulose with endoglucanases resulted in fibre breakdown into small particles. This was contrasted with no morphological change to the fibres when contacted with the cellobiohydrolase. By this technique it was revealed that the EGI subfraction (pI 5.9) behaves as an endoglucanase and not as a cellobiohydrolase. Incubation of this enzyme with acid-swollen cellulose resulted in cellotriose production, as it did with other endoglucanases which exhibited CMCase activities >; 100 U mg⁻¹. Cellotriose was not present during the hydrolysis of acid-swollen cellulose with the CBHI fraction.     

Cellulase Enzyme from Aspergillus niger

Cellulase enzyme was produced by a selected strain of Aspergillus niger isolated from deteriorated wood and grown on different carbon sources. Filter paper gave the highest yield, followed by carboxymethyl cellulose (CMC). Cellobiose as well as glucose gave a low yield, while the yield from lactose was negligible. The concentration of filter paper cellulose that induced the maximum yield of the enzyme was 1%. Both soluble cellulose (CMC) and cotton cellulose treated with phosphoric acid (swollen) were easily hydrolyzed by cellulase; an increase in cellulase concentration lead to more hydrolysis of CMC and gave linearity in the reaction velocity. At certain concentrations of the enzyme, increase in CMC concentration, (up to 1%) resulted in more reducing sugar. Beyond this point no more hydrolysis occur.     

Three Native Cellulose-Depolymerizing Endoglucanases from Solid-Substrate Cultures of the Brown Rot Fungus Meruliporia (Serpula) incrassata

Three extracellular cellulose-depolymerizing enzymes from cotton undergoing decay by the brown rot fungus Meruliporia (Serpula) incrassata were isolated by anion-exchange and hydrophobic interaction chromatographies. Depolymerization was detected by analyzing the changes in the molecular size distribution of cotton cellulose by high-performance size-exclusion chromatography. The average degree of polymerization (DP; number of glucosyl residues per cellulose chain) was calculated from the size-exclusion chromatography data. The very acidic purified endoglucanases, Cel 25, Cel 49, and Cel 57, were glycosylated and had molecular weights of 25,200, 48,500, and 57,100, respectively. Two, Cel 25 and Cel 49, depolymerized cotton cellulose and were also very active on carboxymethyl cellulose (CMC). Cel 57, by contrast, significantly depolymerized cotton cellulose but did not release reducing sugars from CMC and only very slightly reduced the viscosity of CMC solutions. Molecular size distributions of cotton cellulose attacked by the three endoglucanases revealed single major peaks that shifted to lower DP positions. A second smaller peak (DP, 10 to 20) was also observed in the size-exclusion chromatograms of cotton attacked by Cel 49 and Cel 57. Under the reaction conditions used, Cel 25, the most active of the cellulases, reduced the weight average DP from 3,438 to 315, solubilizing approximately 20% of the cellulose. The weight average DP values of cotton attacked under the same conditions by Cel 49 and Cel 57 were 814 and 534; weight losses were 9 and 11% respectively.     

Purification and characterization of a cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli

A cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli was purified 88-fold by chromatography on High Q and hydroxyapatite. N-terminal amino acid sequence analyses confirmed that the cellulase represented the product of the cellulase gene Cel B2. The purified enzyme possessed high activity toward barley beta-glucan, lichenan, carboxymethyl cellulose (CMC), xylan, but not toward laminarin and pachyman. In addition, the cloned enzyme was able to hydrolyze p-nitrophenyl (PNP)-cellobioside, PNP-cellotrioside, PNP-cellotetraoside, PNP-cellopentaoside, but not PNP-glucopyranoside. The specific activity of the cloned enzyme on barley beta-glucan was 297 units/mg protein. The purified enzyme appeared as a single band in SDS-polyacrylamide gel electrophoresis and the molecular mass of this enzyme (58000) was consistent with the value (56463) calculated from the DNA sequence. The optimal pH of the enzyme was 5.5, and the enzyme was stable between pH 5.0 and pH 7.5. The enzyme had a temperature optimum at 40 degrees C. The K(m) values estimated for barley beta-glucan and CMC were 0.32 and 0.50 mg/ml, respectively.     

Liquid chromatography combined with mass spectrometry for the investigation of endoglucanase selectivity on carboxymethyl cellulose

Endoglucanases are useful tools in the chemical structure analysis of cellulose derivatives. However, knowledge on the endoglucanase selectivity, which is of central importance for data interpretation, is still limited. In this study, new reverse-phase liquid chromatography mass spectrometry (LC–MS) methods were developed to investigate the selectivity of the endoglucanases Cel5A, Cel7B, Cel45A, and Cel74A from the filamentous fungus Trichoderma reesei. The aim was to improve the identification of the regioisomers in the complex mixtures that are obtained after enzymatic hydrolysis. Reduction followed by per-O-methylation was performed in order to improve the separation in reverse-phase LC, increase MS sensitivity, and to facilitate structure analysis by MS/MS of O-carboxymethyl glucose and cellooligosaccharides. The cellulose selective enzymes that were investigated displayed interesting differences in enzyme selectivity on CMC substrates.     

The cellulase system of a Cytophaga species.

Cellulases (EC 3.2.1.4) of a Cytophaga species WTHC 2421 (ATCC 29474) were found in the soluble portion of the cell (the periplasm and the cytoplasm) and on the membrane. Cell-free cellulases were not found. Most of the carboxymethylcellulase activity associated with reduction of viscosity was membrane bound, whereas most of the carboxymethylcellulose (CMC) saccharifying activity was soluble. The CMC-saccharifying activity was increased 534 X by purification procedures which included ammonium sulfate precipitation and molecular exclusion chromatography with Sephadex G-75 and Biogel p-100. Periplasmic carboxymethycellulase had a molecular weight of 6250 and cytoplasmic carboxymethylcellulase had a molecular weight of 8650. Analytical ultracentrifugation of the periplasmic carboxymethylcellulase (CMCase) indicated that it had a low molecular density. The chromatographic fraction containing periplasmic CMCase also contained enzyme activity against crystalline cellulose. The activity against crystalline cellulose was 238 X higher than the activity shown by the whole cell. The reaction of the enzyme with either CMC or dewaxed cotton produced only glucose. The enzyme was slightly inhibited by the presence of 0.01% (w/v) glucose, lactose, or cellobiose, but it was not affected by sucrose, and exhibited increased activity in the presence of xylose and fructose.