Strains Of Cellulomonas Research Articles

Harnessing Lignocellulolytic and Electrogenic Potential: Insights from Shewanella oneidensis MR-1 and Cellulomonas Strains on Lignocellulosic Biomass

The biodegradable and renewable nature of lignocellulosic biomass (LCB) has gained significant interest in recent years. This study explores the lignocellulolytic and electrogenic potential of Shewanella oneidensis MR-1, Cellulomonas fimi ATCC 484, and Cellulomonas biazotea NBRC 12680 on LCB. Two strategies were tested: assessing strains LCB degradation ability under non-electrochemical and electrochemical conditions. Strain selection was based on literature, and bioinformatical analyses were conducted to predict CAZymes and carbohydrate degradation pathways. Cellulomonas strains have a potential to degrade LCB due to high CAZyme count and specific metabolic pathways. Strains growth capacity on LCB was evaluated by culturing without electrodes on LCB for 12 days, showing superior growth on wheat bran compared to wheat straw. Enzymatic assays indicate laccase activity in all strains, highest in C. biazotea NBRC 12680 (11.66 IU). The strains ability to form electrogenic biofilms on carbon cloth anodes polarized at +0.2 V (vs Ag/AgCl) was evaluated. The results indicate that bioanodes can function with wheat bran (max current density: 14.92 mA m−2), with voltammograms showing redox activities. Attenuated total reflection Fourier transform infrared spectroscopy shows lignin and protein degradation in both electrochemical and non-electrochemical experiments. These findings suggest potential use of these strains in electro-microbial systems with LCB.

Genomic Insights Into Cadmium Resistance of a Newly Isolated, Plasmid-Free Cellulomonas sp. Strain Y8.

Our current knowledge on bacterial cadmium (Cd) resistance is mainly based on the functional exploration of specific Cd-resistance genes. In this study, we carried out a genomic study on Cd resistance of a newly isolated Cellulomonas strain with a MIC of 5 mM Cd. Full genome of the strain, with a genome size of 4.47 M bp and GC-content of 75.35%, was obtained through high-quality sequencing. Genome-wide annotations identified 54 heavy metal-related genes. Four potential Cd-resistance genes, namely zntAY8, copAY8, HMTY8, and czcDY8, were subjected to functional exploration. Quantitative PCR determination of in vivo expression showed that zntAY8, copAY8, and HMTY8 were strongly Cd-inducible. Expression of the three inducible genes against time and Cd concentrations were further quantified. It is found that zntAY8 responded more strongly to higher Cd concentrations, while expression of copAY8 and HMTY8 increased over time at lower Cd concentrations. Heterologous expression of the four genes in Cd-sensitive Escherichia coli led to different impacts on hosts’ Cd sorption, with an 87% reduction by zntAY8 and a 3.7-fold increase by HMTY8. In conclusion, a Cd-resistant Cellulomonas sp. strain was isolated, whose genome harbors a diverse panel of metal-resistance genes. Cd resistance in the strain is not controlled by a dedicated gene alone, but by several gene systems collectively whose roles are probably time- and dose-dependent. The plasmid-free, high-GC strain Y8 may provide a platform for exploring heavy metal genomics of the Cellulomonas genus.

Optimisation of xylanases production by two Cellulomonas strains and their use for biomass deconstruction

One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides.Key points• Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised.• Biomass-induced extracellular enzymes were identified by proteomic profiling.• Combinations of extra and intracellular extracts were used for barley straw hydrolysis.

Cellulomonas endophytica sp. nov., isolated from Gastrodia elata Blume.

A Gram-stain-positive, facultatively anaerobic and non-motile strain, designated SYSUP0004T, was isolated from the tubers of Gastrodia elata Blume collected from Yunnan Province, PR China. The 16S rRNA gene sequence result showed that the strain SYSUP0004T shared low similarity (97.7 %) with the type strain of Cellulomonas marina. SYSUP0004T grew at pH 6.0-9.0 (optimum, pH 8.0), temperature 4-30 °C (optimum, 28 °C) and could tolerate NaCl up to 4 % w/v (optimum in the absence of NaCl). The cell-wall peptidoglycan type was A4β with an interpeptide bridge l-ornithine-d-glutamic acid. Cell-wall sugars were mannose, ribose, glucose, galactose and fucose. The menaquinone was MK-9(H4). The major fatty acids were anteiso-C15:0, anteiso-C15 : 1 A, C16 : 0 and anteiso-C17 : 0. The polar lipids of SYSUP0004T were diphosphatidylglycerol, unidentified phosphoglycolipid, phosphatidylinositol mannosides and unidentified glycolipid. The genomic DNA G+C content was 76.5 %. The average nucleotide identity values between SYSUP0004T and members of the genus Cellulomonas were below the cut-off level (95-96 %) recommended as the ANI criterion for interspecies identity. Thus, based on the above results strain SYSUP0004T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas endophytica sp. nov. is proposed. The type strain, SYSUP0004T (=KCTC 49025T=CGMCC 1.16405T).

Deconstruction of plant biomass by a Cellulomonas strain isolated from an ultra-basic (lignin-stripping) spring.

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched forsuch bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded β-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

Cellulomonas pakistanensis sp. nov., a moderately halotolerant Actinobacteria.

A rod-shaped, motile, facultatively anaerobic and moderately halotolerant plant-growth-promoting actinobacterial strain, designated NCCP-11(T), was isolated from paddy grains. To delineate its taxonomic position, the strain was subjected to a polyphasic characterization. Cells of strain NCCP-11(T) grew at 10-37 °C (optimum 28-32 °C), at pH 6-9 (optimum pH 7) and in 0-12% (w/v) NaCl (optimum 1-2%) in broth medium. Based on 16S rRNA gene sequence analysis, strain NCCP-11(T) showed highest similarity to the type strains of Cellulomonas hominis (98.99%) and Cellulomonas denverensis (98.09 %) and less than 97 % with other closely related taxa. The chemotaxonomic data [major menaquinone: MK-9(H4); cell-wall peptidoglycan: type A4β; major fatty acids: anteiso-C15 : 0, C16 : 0, C14 : 0 and anteiso-C17 : 0; major polar lipids: diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositolmannosides and two unknown polar lipids] also supported the affiliation of strain NCCP-11(T) to the genus Cellulomonas. The level of DNA-DNA relatedness between strain NCCP-11(T) and the two type strains mentioned above was less than 42.7%. On the basis of DNA-DNA relatedness, physiological and biochemical characteristics and phylogenetic position, strain NCCP-11(T) can be differentiated from species of the genus Cellulomonas with validly published names and thus represents a novel species, for which the name Cellulomonas pakistanensis sp. nov. is proposed. The type strain is NCCP-11(T) ( = DSM 24792(T) = JCM 18755(T) = KCTC 19798(T)).

Purification and Characterization of an Endo-d-arabinase Produced by Cellulomonas

An extracellular endo-D: -arabinase enzyme produced by the bacterial strain of Cellulomonas was purified 77.1-fold with 0.20% recovery for protein by DEAE Sepharose anion exchange, Sephacryl S-300 gel filtration and blue Sepharose affinity chromatography, and designated as CEDAase. The apparent molecular mass of CEDAase was 45kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CEDAase is an endoenzyme for arabinogalactan with the main and specific product of hexa-arabinofuranoside. It reacts optimally with its substrate, arabinogalactan, at approximately pH 8.0 and at 40°C. CEDAase shows stability in the pH range of 6.0-9.0 and at the temperature below 50°C. The K(m) measured for the CEDAase was 55.6μM, with an apparent V(max) of 0.083μmol/min. To our knowledge, for the first time, the current work obtains an extracellular Cellulomonas endo-D: -arabinase enzyme that might be potentially served as a tool enzyme for hydrolyzing specific cell wall such as Mycobacterium cell. It is purified as an important potential initial material basis for mass spectrometric sequencing and chemical gene synthesis. It may make it possible to clone and express this valuable endo-D: -arabinase and make it available to the mycobacteria scientific community.

Effective extracellular trehalose production by Cellulosimicrobium cellulans

A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (alpha- d-glucopyranosyl-[1,1]-alpha- d-glucopyranose) in culture medium containing glucose. 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp. Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus. ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose. Effective extracellular trehalose production by ST26 was achieved by supplying 0.5-1% ammonium sulfate and 0.5-1% CaCO(3). The addition of CaCO(3) adjusted the pH of the culture to around 5.0. The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61%. The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium. ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism.

Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates.

The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).

Isolation and Identification of Cellulolytic Bacteria Involved in the Degradation of Natural Cellulosic Fibres

In search for bacterial cultures that are able to rapidly degrade cellulosic plant fibres in vitro, 77 cellulolytic strains were isolated from Belgian and Czech soils after enrichment on flax or sisal fibres as sole sources of carbon. The strains were characterized using fatty acid analysis, and 74 strains were grouped into three major clusters by numerical analysis. The first major cluster contained Cellulomonas strains. Within this cluster three subclusters could be delineated by principal component analysis, that were recognized by their fatty acid compositions as Cellulomonas gelida, Cellulomonas biazotea and Cellulomonas cellulans, containing 9, 8 and 13 strains respectively. The second major cluster, with 9 strains, was assigned to Flavobacterium johnsoniae. The 34 strains of the third cluster could not be identified by commercial identification systems on the basis of their fatty acid profiles and API 20NE profiles. On the basis of their phenotypic characteristics they met the description of the genus Cellvibrio, their fatty acid profiles were similar to those of four authentic Cellvibrio mixtus strains, and the 16S rRNA genes from four representatives showed up to 97.8% sequence similarity to 16S rDNA from Cellvibrio mixtus ACM 2603. Three non-clustered strains were assigned to Curtobacterium flaccumfaciens, Achromobacter piechaudii and Pseudomonas mendocina. Two strains assigned to Cellvibrio were able to degrade several flax, broom and cotton fibres very rapidly in a standardized in vitro test, causing mass losses of 40 to 86% within 13 days of incubation, but not jute.

Detection of a whitening fluorescent agent as an indicator of white paper biodegradation: a new approach to study the kinetics of cellulose hydrolysis by mixed cultures

A simple and reliable method to estimate paper degradation by cellulolytic bacteria is described. This method is based on the detection in the culture medium of a fluorescent whitening agent (FWA) added to white paper during the manufacturing process. Preliminary results using a Cellulomonas strain cultivated in a liquid medium containing FWA, indicated that this component is non-toxic at a final concentration of 0.01‰ (v/v) and that the fluorescence decreased during the first 24 h of incubation, i.e. during exponential growth phase, suggesting an adsorption of FWA on bacterial cells. Consequently, all experiments have been performed with a liquid medium containing FWA (0.01‰ v/v) and white paper (8.0 g/l) as cellulose source. Mixed bacterial populations (MBPs) were prepared from refuse samples. These MBPs, which mainly consisted of bacterial rod cells, were used as inocula and fluorescence was measured after 30 h of incubation, i.e. after the stationary phase was reached. A high linear correlation ( R 2=0.979) was found between the percentages of degraded paper (% P) deduced from residual paper weight and the fluorescence values ( F) of the culture medium and the following equation between % P and F was determined: % P=8.71·10 −5× F. An additional experiment using a second MBP showed a strong correlation ( R 2=0.990) between the measured % P and the % P estimated from F values, confirming the reproducibility of the method. Moreover, the time course of paper degradation by five replicate flasks from a unique MBP was set up. Paper degradation was detected 3 to 5 days after the beginning of the stationary phase. The average degradation rate between the 7th and the 11th day of incubation was 11.4% per day. Rates of paper degradation ranged from 31 to 60% after 10 days and from 77 to 88% after 3 weeks of incubation, depending on the inoculum.

Enhanced production of cellulases byCellulomonas strains grown on different cellulosic residues

Cellulomonas strains consumed commercial cellulose, cellulosic residues, xylan, cellobiose and carboxymethyl cellulose (CMC) as carbon sources in liquid culture, the growth being the most on cellobiose medium. All three components of the cellulase complex ofCellulomonas were produced when the organisms utilized all substrates as sole carbon and energy sources. The filter-paper cellulase (FPase) and endo-glucanase (CMCase) activities were higher in media containing alpha-cellulose and cellulosic residues than in media containing CMC, cellobiose, and xylan. Cell-free supernatants of all organisms exhibited greater CMC hydrolyzing activity than filter paper and beta-glucoside hydrolyzing activities. All strains synthesized beta-glucosidase maximally on cellobiose followed by commercial cellulose and cellulosic residues.C. biazotea produced the highest FPase and CMCase activity during growth on alpha-cellulose. It was followed byC. flavigena, C. cellasea, andC. fimi. Endo-glucanase and FPase from all organisms were secreted into the medium; 10-13 % became adsorbed on the surface of the insoluble substrates and could be successfully eluted using Tween 80. beta-Glucosidase was located in cell extracts from all organisms.C. biazotea produced FPase and beta-glucosidase activities several-fold greater than those produced by many other strains ofCellulomonas and some other cellulolytic bacteria and fungi.

Cellulase production by Cellulomonas biazotea cultured in media containing different cellulosic substrates

Production of filter paper cellulase (FPase), endo-β-glucanase and β-glucosidase by Cellulomonas biazotea was investigated during growth on different substrates. The organism utilized four different cellulosics, NaOH-pretreated ground plant material of four lignocellulosic (LC) substrates grown on saline lands, three agricultural wastes, carboxymethyl cellulose (CMC), cellobiose and xylan as carbon sources in Dubos salts liquid medium and produced the enzymes. The highest level of volumetric productivity ( Q p) of FPase occurred in the cell-free supernatants of C. biazotea during growth on α-cellulose followed by Leptochloa fusca (kallar grass), while that of endo-β-glucanase occurred on kallar grass followed by α-cellulose. Maximum β-glucosidase was produced in culture media containing cellobiose and kallar grass as carbon sources. Thus the production of these enzymes is influenced by the carbon source used. β-Glucosidase was produced mainly periplasmic and was several fold greater in quantity than that reported in other strains of Cellulomonas, as well as other bacteria. Kallar grass culture medium, during growth of C. biazotea, supported maximum Q p levels of 37.5, 17.5 and 6.1 IU/l/h for CMCase, FPase and β-glucosidase, respectively, with cell mass productivity of 0.235 g/l/h and was selected as a preferred substrate for cellulase production.

TOXICITY EVALUATION OF STRAINS OF CELLULOMONAS

ABSTRACT Five strains of Cellulomonas (ATCC 482, ATCC 488, ATCC 491, ATCC 15724 and ATCC 21399) were evaluated for toxicity. Extracts of cells in both water and oil as well as the cell free spent broth injected into fertile chicken eggs were not lethal to the embryos. Viable organisms inoculated in rats, mice and rabbits by oral, subcutaneous and intraperitoneal routes respectively, did not cause generalized or localized infection nor abnormal changes in the vital organs. The sexual maturity, fertility and the vital organs of adult rats as well as the vital data of their progeny were not affected by feeding 10% protein from all strains.

Identification of some clinical strains of CDC coryneform group A-3 and A-4 bacteria as Cellulomonas species and proposal of Cellulomonas hominis sp. nov. for some group A-3 strains

CDC coryneform group A-3 and A-4 bacteria were defined by Hollis and Weaver in 1981, but their taxonomic position is still unclear. By using biochemical and chemotaxonomical methods, four clinical strains belonging to CDC coryneform groups A-3 (n = 2) and A-4 (n = 2) were studied and could be assigned to the genus Cellulomonas, resulting in the first description of Cellulomonas strains isolated from clinical specimens. CDC coryneform group A-3 and A-4 strains were compared with the type strains of the seven species constituting the genus Cellulomonas at present as well as with the closely related species Oerskovia turbata, Oerskovia xanthineolytica, and Jonesia denitrificans, but their biochemical patterns were not compatible with the patterns of any of those species. Almost the entire sequences of the 16S rRNA genes of one representative strain of both CDC taxa were determined, and comparative sequence analysis confirmed the placement of the CDC coryneform group A-3 and A-4 strains studied in the Cellulomonas-Oerskovia subbranch of the actinomycetes. Both CDC taxa exhibited > 99% base pair homology within their 16S rDNAs. On the basis of phenotypic and molecular data, we formally propose a new species, Cellulomonas hominis sp. nov., for the CDC coryneform group A-3 bacteria examined. The type strain is DSM 9581. The precise taxonomic status of the CDC coryneform group A-4 strains studied remains to be established by quantitative DNA-DNA hybridizations.

Isolation and characterization of a new strain ofCellulomonas flavigena

A cellulolytic bacterium was isolated from leaf litter. Its nutritional characteristics and most of its morphological features closely resemble those of ATCC # 482, which is considered to be the type species ofCellulomonas flavigena (Stackebrandt and Keddie in Bergey's Manual of Systematic Bacteriology). However, when stationary phase cells from cultures using a minimal medium containing an excess carbon and energy source are compared, a very prominent morphological difference is manifest. Phase contrast microscopy and transmission electron microscopy indicate the presence of large swollen polar structures in the type species. The absence of such structures from the new isolate and the production of large amounts of an extracellular polysaccharide indicate that it is a new strain ofCellulomonas flavigena.

Mixed cultures fermentation for bioconversion of whole bagasse into microbial protein

AbstractTwo basidiomycetes cultures, BH1 and BW1, isolated from the biomanure sample of a bio‐gas plant belonging to Polyporus sp., were used in combination with celluolytic cultures viz. Trichoderma sp. and Cellulomonas strain R2 for microbial protein production from whole bagasse. The biomass yields obtained were much higher in mixed cultures fermentation than in corresponding controls. Combination of BH1/BW1 with Cellulomonas proved to be the better in comparison with Trichoderma. Simultaneous inoculation of the bacteria along with BH1 culture resulted in higher protein production.

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, β-glucosidase and β-xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, β-glucosidase and β-xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C.biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.

Protein enrichment of rice straw by fermentation with Cellulomonas sp.

Protein enrichment of rice straw was attempted by subjecting it to fermentation with a strain of Cellulomonas (CBS.7) which was originally isolated from decomposing cotton debris. Maximum cellulose utilisation (73·2%) was recorded with ammonium sulphate as nitrogen source, supplemented with biotin, at pH 7·5 within 72h of fermentation. Pretreatment of milled rice straw with a 2:1 mixture of NaOH (5%) and H 2O 2 (1%) resulted in lignin reduction from 13·32 to 3·52%. Treated straw yielded 189–195 mg of protein per 100 ml of medium in a period of 120 h of fermentation under shaker-grown condition. Growth in a bench-top fermenter resulted in a protein yield of 1183 mg of protein per litre of medium excluding straw-bound particulate protein. The nutrient profile of bacterial SCP produced is also reported.

Cellulolytic Activities of Strains of the Genus Cellulomonas

The effects of pH, temperature, the presence or absence of glucose and cellobiose, and culture medium on cellulase synthesis or activity or both by 12 strains of Cellulomonas were compared. The liquefaction of carboxymethyl cellulose (CMC) gels by eight of the strains was examined. The optimum temperature for hydrolysis of CMC during growth was 40°C, and the optimum pH was 7 to 7.5. The eight strains broke into two distinct groups with a fourfold difference in rates of CMC or filter paper hydrolysis during growth. The presence of either glucose or cellobiose in the culture medium stimulated growth but repressed either the synthesis or the activity of CMC and filter paper cellulases. This effect is the opposite of that reported previously for Bacillus CMC cellulase. The greatest rate of filter paper hydrolysis occurred in a synthetic medium. The cell-free carboxymethylcellulase of Cellulomonas flavigena ATCC 482Twas partially purified by molecular exclusion chromatography on Bio-Gel P-100 and Sephadex G-75. The partially purified enzyme had a molecular weight of 40,000 and optimum activity at pH 7.0. Cellobiose was a competitive inhibitor of the enzyme activity. The results obtained with the partially purified enzyme indicated that the results obtained by using CMC gels were valid.