Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1.

The extracellular endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 were analyzed by high-performance liquid chromatography (HPLC) by using DEAE ion-exchange, hydroxylapatite, and gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). Two endo-1,4-beta-glucanase peaks were resolved by DEAE-HPLC and termed endoglucanases A and B. Carboxymethyl cellulose (CMC) zymograms were achieved by enzyme separation using nondenaturing PAGE followed by incubation of the gel on top of a CMC-agarose gel. This revealed no less than 13 and 5 endo-1,4-beta-glucanase components present in endoglucanases A and B, respectively. Hydroxylapatite chromatography of endoglucanases A and B revealed one activity peak for each preparation, which contained 4 and 5 endo-1,4-beta-glucanase components, respectively. Gel filtration chromatography of endoglucanase A following hydroxylapatite chromatography resolved the most active carboxymethylcellulase (CMCase) component from other endo-1,4-beta-glucanase activities. Gel filtration of endoglucanase B following hydroxylapatite chromatography showed one CMCase activity peak. Protein stains of sodium dodecyl sulfate-PAGE and nondenaturing PAGE gels of endoglucanases A and B from hydroxylapatite and gel filtration chromatography revealed multiple protein components. When xylan was substituted for CMC in zymograms, identical separation patterns for CMCase and xylanase activities were observed for both endoglucanases A and B. These data suggest that both 1,4-beta linkage-hydrolyzing activities reside on the same polypeptide or protein complex. The highest endo-1,4-beta-glucanase-specific activities were observed following DEAE-HPLC chromatography, with 16.2 and 7.5 mumol of glucose equivalents per min per mg of protein for endoglucanases A and B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple forms of endo-1,4-beta-glucanases in the endosperm of Euphorbia heterophylla L

Germinating seeds of Euphorbia heterophylla L. contain endo-1,4-beta-glucanases which degrade carboxymethylcellulose (CMC). The activity decreased approximately 66% in extracts of endosperm containing isopropanol or ethanol. The endoglucanases were isolated from endosperm extracts using ammonium sulphate fractionation followed by Sephacryl S-100-HR chromatography resulting in two main peaks: I and II. Peak I endoglucanase was further purified about 15-fold on DEAE-Sephadex A50 and then by affinity chromatography (CF11-cellulose). Peak II endoglucanases were further purified 10-fold on CM-cellulose chromatography. The results indicated the occurrence of a 66 kDa endoglucanase (fractionated by SDS-PAGE and visualized by activity staining using Congo Red). Several acidic (pI 3.0 to 5.7) and basic (pI 8.5 to 10.0) forms from both peaks which differed in their capacities for degrading CMC or xyloglucans from Copaifera langsdorffii or Hymenaea courbaril were detected.     

Formation, Location, and Regulation of Endo-1,4-beta-Glucanases and beta-Glucosidases from Cellulomonas uda

The formation and location of endo-1,4-beta-glucanases and beta-glucosidases were studied in cultures of Cellulomonas uda grown on microcrystalline cellulose, carboxymethyl cellulose, printed newspaper, and some mono- or disaccharides. Endo-1,4-Glucanases were found to be extracellular, but a very small amount of cell-bound endo-1,4-beta-glucanase was considered to be the basal endoglucanase level of the cells. The formation of extracellular endo-1,4-beta-glucanases was induced by cellobiose and repressed by glucose. Extracellular endoglucanase activity was inhibited by cellobiose but not by glucose. beta-Glucosidases, on the other hand, were formed constitutively and found to be cell bound. beta-Glucosidase activity was inhibited noncompetitively by glucose. Some characteristics such as the optimal pH for and the thermostability of the endoglucanases and beta-glucosidases and the end products of cellulose degradation were determined.     

An analysis of the extracellular xylanases and cellulases of Butyrivibrio fibrisolvens H17c

The extracellular xylanase and cellulase components of Butyrivibrio fibrisolvens H17c were investigated. Two major peaks of enzyme activity were eluted by hydroxylapatite chromatography and designated complex A (CA), having cellulase activity, and complex B (CB) having predominantly xylanase activity but with some activity on carboxymethyl cellulose (CMC). CB was further purified on a DE-52 column and subjected to gel filtration. The xylanase and CMCase activities eluted in a single peak with an apparent molecular mass greater than thyroglobulin (Mr 669,000). CMC xymograms of polyacrylamide gels electrophoresed under non-denaturing conditions indicated the presence of five bands with CMCase activity from CA and eight from CB. Xylan xymograms under the same conditions indicated the presence of four bands of activity in CB. Under mild denaturing conditions the xylanase activity in CB was found in 11 bands with molecular mass ranging from 45 to 180 and the CMCase activity in three bands with molecular mass ranging from 45 kDa to 60 kDa. This indicates that CB exists as a multi-subunit protein aggregate of xylanases, some of which also have cellulase activity.     

Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulose from Cellulomonas sp. ATCC 21399

Two endoglucanases (endoglucanase B and endoglucanase C) without affinity for cellulose were purified from the culture broth of Cellulomonas sp. ATCC 21399 using gelfiltration and ion exchange chromatography. Fused rocket immunoelectrophoresis was used to select the fractions with the highest content of endoglucanase and lowest content of contaminating proteins. The endoglucanases were purified to immunological homogeneity. In addition both endoglucanases were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights of endoglucanase B and endoglucanase C were 67000 and 25000, respectively). Endoglucanase B was homogeneous when studied by isoelectric focusing showing one protein band at pl 4.3. Both endoglucanases lacked activity against microcrystalline cellulose (Avicel) and showed similar endo action on carboxymethylcellulose (CMC). Endoglucanase B had a high specific activity against CMC, H(3)PO(4)-swollen Avicel and xylan, but showed no activity against galactomannan. In contrast, endoglucanase C showed activity against both CMC, xylan, and galactomannan all being polysaccharide substrates linked with beta-1-4-D-glucoside bonds. The specific activity of endoglucanase C against H(3)PO(4)-swollen Avicel was low.     

Cloning in escherichia coli of a bi‐functional cellulase/xylanase enzyme from ruminococcus flavefaciens FD‐1

Abstract

A genomic library of Ruminococcus fl avef aciens FD‐1 DNA was constructed using the Escherichia coli bacteriophage λ vector λDASH. A recombinant phage exhibiting activity against both Ostazin brilliant red‐hydroxyethyl cellulose (OBR‐HEC) and carboxymethyl cellulose (CMC) was isolated. This clone (designated FD1‐1) was further analyzed by restriction endonuclease mapping and Southern blot analysis. Substrate specificity data shows that the cloned gene(s) encodes both endoglucanase activity and endoxylanase activity. CMC and xylan zymograms of protein(s) produced by this clone and then separated by non‐denaturing PAGE suggest that the endoglucanase/endoxylanase activities reside on the same polypeptide or protein complex. An additional xylanase product lacking CMCase activity was also detected.     

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.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

Involvement of a conidial endoglucanase and a plasma-membrane-bound beta-glucosidase in the induction of endoglucanase synthesis by cellulose in Trichoderma reesei

The induction of endo-1,4-beta-glucanase synthesis by Trichoderma reesei QM 9414 was investigated in conidia, mycelia and protoplasts. Cellulose induced endoglucanase synthesis only in conidia, but not in glucose-grown mycelia or protoplasts. Cellooligosaccharides and sophorose induced endoglucanase synthesis in mycelia, conidia and protoplasts. Only conidia exhibited detectable basal endoglucanase levels, whereas beta-glucosidase activity was found in conidia, mycelia and protoplasts. The beta-glucosidase was inhibited in vitro by nojirimycin and glucono-delta-lactone. Addition of either of these inhibitors to the induction medium blocked de noro synthesis of endo-1,4-beta-glucanase with cellulose (conidia) or cellooligosaccharides (protoplasts and mycelia) as inducer, whereas induction by sophorose remained unaffected. The results are consistent with the assumption that basal constitutive levels of endoglucanase and beta-glucosidase are involved in the induction of cellulase synthesis by cellulose in T. reesei.     

Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4-beta-glucanase from Bacillus sp. KSM-330.

The gene encoding an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The recombinant plasmid contained a 3.1 kb HindIII insert, 1.8 kb of which was sufficient for the expression of endoglucanase activity in E. coli HB101. Nucleotide sequencing of this region (1816 bp) revealed an open reading frame of 1389 bp. The protein deduced from this sequence was composed of 463 amino acids with an Mr of 51882. The deduced amino acid sequence from amino acids 56 through 75 coincided with the amino-terminal sequence of the endoglucanase, Endo-K, purified from culture of Bacillus sp. KSM-330. The deduced amino acid sequence of Endo-K had 30% homology with that of the celA enzyme from Clostridium thermocellum NCIB 10682 and 25% homology with that of the enzyme from Cellulomonas uda CB4. However, the Endo-K protein exhibited no homology with respect to either the nucleotide or the amino acid sequences of other endoglucanases from Bacillus that had been previously characterized. These results indicate that the gene for Endo-K in Bacillus sp. KSM-330 has evolved from an ancestral gene distinct from that of other Bacillus endoglucanases.     

Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus

Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56 kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1,4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012 bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194 bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.     

Purification and properties of an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330

A novel acid cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) was found in a culture of Bacillus sp. KSM-330 isolated from soil. One-step chromatography on a column of CM-Bio-Gel A yielded a homogeneous enzyme, as determined by silver staining of both sodium dodecyl sulphate (SDS) and nondenaturing gels. The enzyme had a molecular mass of 42 kDa, as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was higher than pH 10. The N-terminal amino acid sequence of the enzyme was Val-Ala-Lys-Glu-Met-Lys-Pro-Phe-Pro-Gln-Gln-Val-Asn-Tyr-Ser-Gly-Ile-Leu- Lys-Pro . This enzyme had an optimum pH for activity of 5.2, being active over an extremely narrow range of pH values, from 4.2 to 6.9; below and above these pH values no activity was detectable. The optimum temperature at pH 5.2 was around 45 degrees C. The enzyme efficiently hydrolysed carboxymethylcellulose (CMC) and lichenan, but more crystalline forms of cellulose, curdlan, laminarin, 4-nitrophenyl-beta-D-glucopyranoside and 4-nitrophenyl-beta-D-cellobioside were barely hydrolysed. The enzymic activity was inhibited by Hg2+ but was not affected by other inhibitors of thiol enzymes, such as 4-chloromercuribenzoate. N-ethylmaleimide and monoiodoacetate. N-Bromosuccinimide abolished the enzymic activity, and CMC protected the enzyme from inactivation by this tryptophan-specific oxidant. It is suggested that a tryptophan residue(s) is involved in the mechanism of action of the Bacillus cellulase and that the inhibition of enzymic activity by Hg2+ is ascribable to interactions with the tryptophan residue(s) rather than with thiol group(s).     

白蚁内切-β—1，4-葡聚糖酶基因的克隆、表达、纯化及酶学性质的研究

提取了台湾家白蚁总RNA并反转录获得eDNA,PCR扩增出白蚁内切葡聚糖酶的基因,并将目的基因分别克隆到大肠杆菌和酿酒酵母载体中,构建了产内切-β-1,4-葡聚糖酶的基因工程菌。由于大肠杆菌会有少量的泄漏表达,而所用的酿酒酵母表达载体是本实验室构建带有INU信号肽的表达载体,故都可采用刚果红平板染色法筛选具有羧甲基纤维素酶（CMCase）活性的重组转化子。利用金属镍亲和层析对大肠杆菌表达的内切-β-1,4-葡聚糖酶进行纯化,CMC酶活检测显示纯化酶的最适温度和最适pH值分别为42℃、6．5;内切-β-1,4-葡聚糖酶的Vmax为0．071mg／mL·min,Km值为80．2712mg／mL。     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cellulose. 1. Separation, purification and physico-chemical characterization of five endo-1,4-beta-glucanases.

Five endo-1,4-beta-glucanases (EC 3.2.1.4) have been separated from culture solutions OF THE ROT FUNGUS Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source. They have been extensively purified and characterized with regard to some physicochemical properties. The purifications have been carried out on a quantitative basis, the purity of the enzymes being tested in several ways. After purification they all showed one single protein band in analytical polyacrylamide electrophoresis, on dodecyl-sulphate gels and in analytical isoelectric focusing on flat-bed polyacrylamide gels. One exo-1,4-beta-glucanase has also been identified in the culture solution and separated from the endo-1,4-beta-glucanases. From the data obtained during the quantitative purification it has been possible to calculate that the ratio of activity between the five endoglucanases T1, T2a, T2b, T3a, and T3b in the culture solutions is 4:1:1:1:1. It has also been calculated that the weight ratio endoglucanase protein to exoglucanase protein is approximately 1:1. Flat-bed isoelectric focusing has been used for the identification of the individual endoglucanases and a new zymogram technique, useful for studies of carbohydrases in general, has been developed. The molecular weights, determined by ultracentrifugation, and calculated on the basis of a knowledge of the amino acid composition and carbohydrate content vary between 28200 and 37500. Small but significant differences in the amino acid compositions of the different endoglucanases have been found. The carbohydrate content varies between 0 and 10.5%, all but one of the enzymes being glycoproteins. For two of these the exact carbohydrate composition has been determined. Enzyme T1 contains 2 glucose and 19 mannose units per enzyme molecule while enzyme T2b contains 5 mannose, 7 galactose, 1 glucose and 1 arabinose unit per molecule.     

Co-purification and characterization of ATP-sulfurylase and adenosine-5'-phosphosulfate kinase from rat chondrosarcoma

The two sulfate-activating enzymes, ATP-sulfurylase (EC 2.7.7.4) and adenosine-5'-phosphosulfate kinase (adenylylsulfate kinase, EC 2.7.1.25), were each purified about 2000-fold from crude rat chondrosarcoma homogenate. Throughout a purification protocol which included Sephacryl S-300 gel filtration, DEAE-Sephadex ion exchange, hydroxylapatite, and ATP-agarose affinity chromatography, these two activities consistently co-purified. ATP-sulfurylase and adenosine-5'-phosphosulfate kinase each showed a pH optima of 7.0-7.4 and a bimodal temperature optima of 46 and 52-54 degrees C. Both activities preferred Mg2+ as their divalent cation source over Mn2+, Co2+, or Zn2+. The apparent Km values determined for adenosine 5'-phosphosulfate in both assays was 1-5 microM; the Km for pyrophosphate in the sulfurylase reaction was 40 microM and for ATP in the kinase reaction was 5 mM. Gel electrophoresis indicated major bands at Mr = 160,000 in nondenaturing systems and 35,000-37,000 and 60,000 under dissociative conditions, whereas gel filtration of the most highly purified fractions yielded a coincident peak in the molecular weight range 260,000.     

Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium

Activities of cellulolytic and hemicellulolytic enzymes endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, 1,4-beta-glucosidase, endo-1,4-beta-xylanase, 1,4-beta-xylosidase and 1,4-beta-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase were detected during the growth of the white-rot fungus Pleurotus ostreatus on wheat straw in the presence and absence of cadmium. The loss of substrate dry weight and Mn-peroxidase activity decreased with increasing Cd concentration, whereas the activities of endo-1,4-beta-glucanase, 1,4-beta-glucosidase and laccase were highly increased in the presence of metal. The onset of hemicellulose-degrading enzyme activity was delayed in the presence of cadmium. The degradation of a model synthetic dye Poly B-411 did not correspond to the activities of ligninolytic enzymes. This is the first report about 1,4-beta-mannosidase in P. ostreatus.     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

A highly thermostable,alkaline CMCase produced by a newly isolated Bacillus sp. VG1

An extracellular, highly thermostable and alkaline CMCase was purified from Bacillus sp. VG1 using ion exchange and gel filtration chromatography. Enzyme was optimally produced in a medium containing 1.0% CMC and 0.5% tryptone. The purified CMCase had a pH optimum of 9–10 and a half life of 12 min even at 100 °C. The enzyme activity was reduced by Hg²⁺ and stimulated by Co²⁺, Na⁺ and K⁺. Various detergents and proteinases moderately inhibited the CMCase activity. The molecular weight studies showed a single band on SDS–PAGE.     

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.     

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.