Enzymatic properties,crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans

A high-isoelectric-point (pI), alkaline endo-1,4-beta-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 degrees C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-beta-glucanase. However, this enzyme was not active on p-nitrophenyl beta-D-cellotrioside and p-nitrophenyl beta-D-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl(2) as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 A resolution. It belongs to the space group P2(1)2(1)2(1) with unit cell parameters of a=62.5 A, b=71.7 A, and c=88.6 A.     

Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-β-galactosidase-catalyzed hydrolytic and transglycosylation reactions

Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine β-glycosides GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)_nGalβ1,4GlcNAcβ-pNP (n=1–4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-β-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl β-glycosides GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)₁Galβ1,4GlcNAcβ-pNP (2), GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)₂Galβ1,4GlcNAcβ-pNP (3), GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)₃Galβ1,4GlcNAcβ-pNP (4) and GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)₄Galβ1,4GlcNAcβ-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide β-glycoside 4 to heptasaccharide β-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-β-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-β-galactosidase-catalyzed hydrolytic and transglycosylation reactions.     

An apparatus for safe and convenient handling of anhydrous, liquid hydrogen fluoride at controlled temperatures and reaction times. Application to the generation of oligosaccharides from polysaccharides

β-d-Gal-(1 → 4)-β-d-GlcNAc-OC₆H₄NO₂-p (p-nitrophenyl N-acetyl-β-lactosaminide) and β-d-Gal-(1 → 6)-β-d-GlcNAc-OC₆H₄NO₂-p (p-nitrophenyl N-acetyl-β-isolactosaminide) were regioselectively synthesized from lactose and p-nitrophenyl 2-acetamido-2-deoxy-glucopyranoside, employing transglycosylation by the β-d-galactosidase from Bacillus circulans and by controlling the concentration of organic solvent in the reaction system. The (1 → 4)-linked disaccharide was formed exclusively when the concentration of organic solvent was high, whereas the (1 → 6)-linked isomer was produced with a low concentration. Further utilization of the transglycosylation by the enzyme led to the regioselective formation of β-d-Gal-(1 → 4)-d-GalNAc and β-d-Gal-(1 → 4)-β-d-GalNAc-OC₆H₄NO₂-p. With the enzyme, β-d-galactosyl transfer occurred preferentially at the O-4 position of GlcNAc and GalNAc, regardless of the configuration of the hydroxyl group.     

Purification,Characterization, and Gene Analysis of Cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both β-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the β-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to β-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Purification and characterization of a novel alkaline β-1,3-1,4-glucanase (lichenase) from thermophilic fungus Malbranchea cinnamomea

A novel alkaline β-1,3-1,4-glucanase (McLic1) from a thermophilic fungus, Malbranchea cinnamomea, was purified and biochemically characterized. McLic1 was purified to homogeneity with a purification fold of 3.1 and a recovery yield of 3.7 %. The purified enzyme was most active at pH 10.0 and 55 °C, and exhibited a wide range of pH stability (pH 4.0–10.0). McLic1 displayed strict substrate specificity for barley β-glucan, oat β-glucan and lichenan, but did not show activity towards other tested polysaccharides and synthetic p-nitrophenyl derivates, suggesting that it is a specific β-1,3-1,4-glucanase. The K _m values for barley β-glucan, oat β-glucan and lichenan were determined to be 0.69, 1.11 and 0.63 mg mL^?1, respectively. Moreover, the enzyme was stable in various non ionic surfactants, oxidizing agents and several commercial detergents. Thus, the alkaline β-1,3-1,4-glucanase may have potential in industrial applications, such as detergent, paper and pulp industries.     

Purification and characterization of a protease-resistant cellulase from Aspergillus niger

An endo-β-1,4-glucanase (EC 3.2.1.4) was purified from a culture filtrate of Aspergillus niger IFO31125 by column chromatography through TSK-gel DEAE-3SW and TSK-gel DEAE-5PW, and by gel filtration through TSK-gel G2000SW by high performance liquid chromatography. The enzyme was estimated to have a molecular weight of about 40 kDa by both gel filtration and SDS-polyacrylamide gel electrophoresis, and appeared to consist of a monomeric protein. It contained 8.9% carbohydrate. The optimal pH for activity was 6.0–7.0, and the stable pH range was 5.0–10.0. The optimum temperature at pH 6.0 was around 70°C. The enzyme was very thermally stable and no loss of original activity was found on incubation at 60°C for 2 h. The enzyme efficiently hydrolyzed carboxymethylcellulose and lichenan, but crystalline forms of cellulose, curdlan, laminarin, cellobiose, p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside were barely hydrolyzed. The activity of the enzyme was inhibited by Hg²⁺ and Cu²⁺ but was not affected by other inhibitors of thiol enzymes such as p-chloromercuribenzoate and N-ethylmaleimide. N-Bromosuccinimide showed a strong inhibitory effect, suggesting that a tryptophan residue is essential for the activity of the enzyme. The N-terminal amino acid sequence of the enzyme showed considerable homology to those of endo-β-1,4-glucanases from some other microorganisms, including Sclerotinia sclerotiorum and Schizophyllum commune. The enzyme had very strong protease-resistance, and showed no loss of activity when incubated with proteases such as Savinase at 40°C, even for 2 weeks.     

Expression ofBacillus subtilis JA18 endo-β-1,4-glucanase gene inEscherichia coli and characterization of the recombinant enzyme

Endo-β-1,4-glucanase encoded byBacillus subtilis JA18 was expressed inEscherichia coli. The recombinant enzyme was purified and characterized. The purified enzyme showed a single band of 50 kDa by SDS-PAGE. The optimum pH and temperature for this endo-β-1,4-glucanase was pH 5.8 and 60 °C. The endo-β-1,4-glucanase was highly stable in a wide pH range, from 4.0 to 12.0. Furthermore, it remained stable up to 60 °C. The endo-β-1,4-glucanase was completely inhibited by 2 mM Zn²⁺, Cu²⁺, Fe³⁺, Ag⁺, whereas it is activated in the presence of Co²⁺. In addition, the enzyme activity was inhibited by 1 mM Mn²⁺ but stimulated by 10 mM Mn²⁺. At 1% concentration, SDS completely inhibited the enzyme. The enzyme hydrolysed carboxymethylcellulose, lichenan but no activity was detected with regard to avicel, xylan, chitosan and laminarin. For carboxymethylcellulose, the enzyme had a Km of 14.7 mg/ml.     

Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237

Thermostable alkaline cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val-Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6–9.0 and displayed maximum activity at 45°C. The alkaline enzyme was stable up to 50°C and more than 30% of the original activity was detectable after heating at 100°C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (β-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H₃PO₄-swollen cellulose, NaOH-swollen cellulose, curdlan (β-1,3-linkage), laminarin (β-1,3;1,6-linkage), and xylan were barely hydrolyzed at all. Received: April 28, 1997 / Accepted: May 24, 1997     

Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island,Indian Ocean

The digestive ability of four sympatric land crabs species (the gecarcinids, Gecarcoidea natalis and Discoplax celeste and the anomurans, Birgus latro and Coenobita perlatus) was examined by determining the activity of their digestive enzymes. The gecarcinids are detritivores that consume mainly leaf litter; the robber crab, B. latro, is an omnivore that preferentially consumes items high in lipid, carbohydrate and/or protein; C. perlatus is also an omnivore/detritivore. All species possess protease, lipase and amylase activity for hydrolysing ubiquitous protein, lipid and storage polysaccharides (glycogen and starch). Similarly all species possess enzymes such as N-acetyl-β-d-glucosaminidase, the cellulases, endo-β-1,4-glucanase and β-glucohydrolase and hemicellulases, lichenase and laminarinase for the respective hydrolysis of structural substrates chitin, cellulose and hemicelluloses, lichenan and laminarin. Except for the enzyme activities of C. perlatus, enzyme activity could not be correlated to dietary preference. Perhaps others factors such as olfactory and locomotor ability and metabolic status may determine the observed dietary preferences. The digestive fluid of C. perlatus possessed higher endo-β-1,4-glucanase, lichenase and laminarinase activities compared to that of the other species. Thus, C. perlatus may be efficient at digestion of cellulose and hemicellulose within plant material. Zymography indicated that the majority of protease, lipase, phosphatase, amylase, endo-β-1,4-glucanase, β-glucohydrolase and N-acetyl-β-d-glucosaminidase isozymes were common to all species, and hence were inherited from a common aquatic ancestor. Differences were observed for the phosphatase, lipase and endo-β-1,4-glucanase isozymes. These differences are discussed in relation to phylogeny and possible evolution to cope with the adoption of a terrestrial diet.     

Cellulase formation by Aspergillus nidulans

The optimum pH, temperature and concentration of the substrate, carboxymethyl-cellulose (CMC), for the production of cellulases by Aspergillus nidulans were found to be 3.05, 37°C and 1%, respectively. When grown on CMC under optimum conditions, it produced the three components of the cellulase complex, exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase, both in cell free as well as cell-associated states. The enzyme yields in shake cultures were lower than those obtained during stationary cultivation. Among the defined substrates, lactose emerged as the best inducer for exo-glucanase and endo-glucanase, while β-glucosidase was best induced by pectin. Endo-glucanase production increased significantly when A. nidulans was grown on insoluble delignified lognocellulosic substrates, with the maximum being on paddy straw.It appears that the synthesis of individual components of the cellulase system of A. nidulans may not be regulated in a strictly coordinated manner.     

The purification and properties of an aryl beta-hexosidase from bovine liver.

An aryl β-hexosidase was purified 800-fold from bovine liver. The purified enzyme hydrolyzed p-nitrophenyl glycosylpyranoside derivatives of β-d-galactose, β-d-glucose, β-d-xylose, β-d-mannose, and α-l-arabinose, but did not hydrolyze several other p-nitrophenyl glycosides. The enzyme also catalyzed hydrolysis of a variety of plant arylglucosides. Disaccharides, polysaccharides, glycolipids, glycoproteins, and glycosaminoglycans containing terminal nonreducing β-d-galactopyranosyl or β-d-glucopyranosyl residues were not hydrolyzed. The pH optima for the several substrates tested ranged from 7.0 to 9.5. The purified enzyme was homogeneous by disc gel electrophoresis and had a molecular weight of 41,000 by Sephadex gel filtration and 46,000 by disc gel electrophoresis performed in the presence of sodium dodecyl sulfate. The enzyme readily transferred glycosyl residues from susceptible β-galactosides or β-glucosides to other sugars; the resulting products were not hydrolyzed by the enzyme. Methyl α-d-glucopyranoside was the most efficient carbohydrate acceptor compound tested. The enzyme exhibited a K_m for p-nitrophenyl β-d-galactopyranoside of 1.78 × 10^?3m and for p-nitrophenyl β-d-glucopyranoside, 2.50 × 10^?3m when incubations were conducted in the presence of 0.15 m methyl α-d-glucopyranoside. Aryl β-hexosidase was found in the cytosol of all mammalian livers tested, but could not be detected in liver of birds, reptiles, or fish; low levels were detected in frog liver. Analysis of bovine extracts indicated that the enzyme occurred in liver, kidney, and intestinal mucosa; it was not detected in testis, spleen, serum, or muscle.     

Family 28 carbohydrate-binding module of the thermostable endo-1,4-β-glucanase CelD from <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> maximizes enzyme activity and irreversibly binds to amorphous cellulose

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second encoding a 749 aa multimodular endo-1,4-β-glucanase CelD (85019 Da). The N-terminal region of the protein includes a signal peptide and a catalytic module of glycoside hydrolase family 5 (GH5), followed by a carbohydrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic module and CBM28, were produced in E. coli cells and purified to homogeneity. An analysis of the catalytic properties showed CelD to be an endo-1,4-β-glucanase with maximum activity on barley β-glucan at pH 6.2 and 70°C. The enzyme was stable at 50°C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 was decreased on cellulose substrates, and its thermostability has dropped. Binding of CBM28 to amorphous cellulose has been almost irreversible as it could not be removed from this substrate in a range of pH of 4–11, temperatures of 0–75°C, and NaCl concentrations of 0–5 M. Only 100% formamide or 1% SDS have been able to remove the protein.     

Influence of environmental factors on endo-β-1,4-glucanase production by Bacillus HR68, isolated from a Zimbabwean hot spring

The production of endo-β-1,4-glucanase by a Bacillus strain isolated from a hot spring in Zimbabwe was studied in batch culture, chemostat culture, and carbon dioxide-regulated auxostat (CO₂-auxostat). The bacteria produced the enzyme in the presence of excess glucose or sucroso, but not under carbon-limited conditions in a chemostat using mineral medium. There was a specific growth rate dependent linear increase in enzyme production in glucose excess, nitrogen-limited chemostat cultures. A high specific growth rate of 2.2 h^-1 and a high rate of enzyme production of 362 nkat (mg dry mass h)^-1 were attained under nutrient rich conditions in the CO₂-auxostat. The bacteria had the highest specific growth rate and endo-β-1,4-glucanase enzyme production at 50° C. The maximum specific growth rate and the rate of enzyme production increased when yeast extract and tryptone were added in increasing amounts to the mineral medium used for cultivation in separate experiments. Increasing the glucose concentration in the CO₂-auxostat cultures increased the rate of enzyme production but did not affect the specific growth rate.     

Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.     

High-level production and characterization of a novel β-1,3-1,4-glucanase from Aspergillus awamori and its potential application in the brewing industry

A novel β-1,3-1,4-glucanase gene (AaBglu12A) from Aspergillus awamori was extracellularly expressed in Pichia pastoris. AaBglu12A showed amino acid identity of 96 % with a glycoside hydrolase family 12 cellulase from A. kawachii and 48 % with a β-1,3-1,4-glucanase from Magnaporthe oryzae. The highest β-1,3-1,4-glucanase activity of 159,500 ± 500 U/mL with protein concentration of 31.7 ± 0.3 g/L was achieved in a 5-L fermentor. AaBglu12A was purified until homogeneous with recovery yield of 92 %. Its maximal activity was found at 55 °C and pH 5.0. The enzyme was stable up to 60 °C and within the pH range of 2.0-9.0. It also demonstrated strict substrate specificity towards oat- and barley-glucans as well as lichenan. The K_m values for oat-, barley-glucans, and lichenan were 2.82, 3.51, and 2.53 mg/mL, respectively. The V_max values for oat-, barley-glucans, and lichenan were 12,068, 10,790, and 7236 μmol/min·mg, respectively. AaBglu12A hydrolyzed oat- and barley-β-glucans to produce tetra- and tri-saccharides. However, lichenan was hydrolyzed to yield trisaccharides as the main end product. The addition of AaBglu12A to the mashing process substantially decreased filtration time by 34.5 % and viscosity by 9.6 %. Therefore, the high-level production of AaBglu12A might be a promising strategy for the brewing industry owing to its favorable properties.     

Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

A Bacillus subtilis gene coding for an endo-β-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage λ and plasmid vectors. The gene is contained within a 1.6-kb EcoRI-PvuI DNA fragment and directs the synthesis in E. coli of a β-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates.     

Distribution and Properties of Endo-β-1,4-glucanase from a Lower Termite,Coptotermes formosanus (Shiraki)

A multi-enzyme distribution of endo-β-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract.

EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and K _m were 48 kDa, 6.0, 50°C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-β-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-β-1,4-glucanases of family 9.     

Hydrolysis of β-Galactosyl Ester Linkage by β-Galactosidases

p-Hydroxybenzoyl β-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of β-galactosyl ester linkage by β-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl β-galactoside (pNP-Gal), a usual substrate with a β-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that β-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H₂ ¹⁸O to liberate galactose containing ¹⁸O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.     

Characterization of a Thermostable Endo-β-1,4-D-galactanase from the Hyperthermophile Thermotoga maritima

A putative endo-β-1,4-D-galactanase gene of Thermotoga maritima was cloned and overexpressed in Escherichia coli. The recombinant enzyme hydrolyzed pectic galactans and produced D-galactose, β-1,4-D-galactobiose, β-1,4-D-galactotriose, and β-1,4-D-galactotetraose. The enzyme displayed optimum activity at 90 °C and pH 7.0. It was slowly inactivated above pH 8.0 and below pH 5.0 and stable at temperatures up to 80 °C.     

Endo-beta-N-acetylglucosaminidases acting on carbohydrate moieties of glycoproteins: purification and properties of the two enzymes with different specificities from Clostridium perfringens.

Two endo-β-N-acetylglucosaminidases (C_I and C_I) acting on carbohydrate moieties of glycoproteins were highly purified from the culture fluid of Clostridium perfringens. C_I had the substrate specificity indistinguishable from that of endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae. C_II showed the specificity similar to that of endo-β-N-acetylglucosaminidase H from Streptomyces griseus but is distinct from the streptomyces enzyme with respect to the relative activity toward ovalbumin glycopeptides and Unit A glycopeptides of thyroglobulin. Both enzymes from C. perfringens were most active at neutral pH and were inhibited by p-chloromercuriphenylsulfonate.