Isolation and properties of a (1,3)-beta-D-glucanase from Ruminococcus flavefaciens

A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and Characterization of an Endo-(1,3)-beta-d-Glucanase from Trichoderma longibrachiatum

A laminarinase [endo-(1,3)-beta-d-glucanase] has been purified from Trichoderma longibrachiatum cultivated with d-glucose as the growth substrate. The enzyme was found to hydrolyze laminarin to oligosaccharides varying in size from glucose to pentaose and to lesser amounts of larger oligosaccharides. The enzyme was unable to cleave laminaribiose but hydrolyzed triose to laminaribiose and glucose. The enzyme cleaved laminaritetraose, yielding laminaritriose, laminaribiose, and glucose, and similarly cleaved laminaripentaose, yielding laminaritetraose, laminaritriose, laminaribiose, and glucose. The enzyme cleaved only glucans containing beta-1,3 linkages. The pH and temperature optima were 4.8 and 55 degrees C, respectively. Stability in the absence of a substrate was observed at temperatures up to 50 degrees C and at pH values between 4.9 and 9.3. The molecular mass was determined to be 70 kilodaltons by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, and the pI was 7.2. Enzyme activity was significantly inhibited in the presence of HgCl(2), MnCl(2), KMnO(4), and N-bromosuccinimide. The K(m) of the enzyme on laminarin was 0.0016%, and the V(max) on laminarin was 3,170 mumol of glucose equivalents per mg of the pure enzyme per min.     

Isolation and characterization of two types of β-1,3-glucanases from the common sea hare Aplysia kurodai

Two types of β-1,3-glucanases, AkLam36 and AkLam33 with the molecular masses of 36 kDa and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare Aplysia kurodai. AkLam36 was regarded as an endolytic enzyme (EC 3.2.1.6) degrading laminarin and laminarioligosaccharides to laminaritriose, laminaribiose, and glucose, while AkLam33 was regarded as an exolytic enzyme (EC 3.2.1.58) directly producing glucose from polymer laminarin. AkLam36 showed higher activity toward β-1,3-glucans with a few β-1,6-linked glucose branches such as Laminaria digitata laminarin (LLam) than highly branched β-1,3-glucans such as Eisenia bicyclis laminarin (ELam). AkLam33 showed moderate activity toward both ELam and LLam and high activity toward smaller substrates such as laminaritetraose and laminaritriose. Although both enzymes did not degrade laminaribiose as a sole substrate, they were capable of degrading it via transglycosylation reaction with laminaritriose. The N-terminal amino-acid sequences of AkLam36 and AkLam33 indicated that both enzymes belong to the glycosyl hydrolase family 16 like other molluscan β-1,3-glucanases.     

Production of 1, 3-beta-glucanase by Bacillus No. 221. An alkalophilic microorganism.

A 1, 3-beta-glucanase of Bacillus No. 221 has been extensively purified by a DEAE-cellulose column followed by a Sephadex G-75 gel filtration, and crystallized in ammonium sulfate solution. The crystalline enzyme is homogenous on the basis of polyacrylamide gel electrophoresis, sedimentation in ultracentrifuge (3.2 S), Ampholine electrofocusing (pI=4.1) and dodecylsulfate-polyacrylamide gel electrophoresis (Mr=36 000). The enzyme has an optimum pH for enzyme action at 8.5 which is higher than those of other 1, 3-beta-glucanases so far reported. The enzyme is very thermostable; about 90% of activity remains after being heated at 70 degrees C for 10 min, and no effect of Ca-2's obversed. The enzyme does not hydrolyse laminaritriose, but hydrolyses laminaritetraose, and yields glucose and laminaritriose. The enzyme splits laminaran at random and yields glucose, laminaribiose, laminaritriose and higher oligosaccharides. From these results, this enzyme is a type of endo-1, 3-beta-glucanase.     

Identification,cloning, and characterization of β-glucosidase from <Emphasis Type="Italic">Ustilago esculenta</Emphasis>

Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.     

A thermostable alkaline β-1,3-glucanase produced by alkalophilic Bacillus sp. AG-430

Summary Alkalophilic Bacillus sp. AG-430 was isolated from soil and found to produce an extracellular -1,3-glucanase. This enzyme was purified by chromatography on DEAE-sepharose CL-6B, Sephadex G-75 and hydroxyapatite. The enzyme was extremely thermostable and lost only 10% of the original activity after incubation at 100°C for 10 min. The optimum temperature and pH for activity were 60°–65°C and 9–10, respectively. The molecular weight was estimated at about 35 000 on sodium dodecyl sulphate-polyacrylamide gel eletrophoresis and the pI was about 3.8. The enzyme hydrolysed laminaritetraose, but not laminaritriose, and the end-products detected in the hydrolysate were identified as glucose, laminaribiose and laminaritriose. The enzyme split laminarin at random and yielded glucose, laminaribiose, laminaritriose and higher oligosaccharides. The enzyme is a type of endo--1,3-glucanase.Offprint requests to: Y. Nogi     

Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12.

Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5.     

The laminarinase system of Streptomyces sp., ATCC 11238

Summary Extracellular proteins from Streptomyces sp. ATCC 11238 grown on fungal mycelia and chitin as C- and N-sources were concentrated by ultrafiltration and acetone precipitation. The crude preparation containing chitin and laminarin degrading enzymes was fractionated by repeated gel filtrations. Three different types of -1,3-glucanases were found. Besides oligomeric breakdown products laminaritriose is the main product of laminarin hydrolysis by one endo--1,3-glucanase. A second laminarin degrading (exo-splitting) enzyme yields predominantly laminaribiose. Another exo--1,3-glucanase liberates glucose but no, oligosaccharides from the nonreducing end of laminarin.     

Hydrolase and transferase activities of the beta-1,3-exoglucanase of Candida albicans.

The exo-beta-1,3-glucanase of Candida albicans (Exg) has a marked specificity for beta-1,3-glucosidic linkages as judged by the kinetic constants for p-nitophenyl beta-glucoside, beta-linked disaccharides of glucose (laminaribiose, gentiobiose, and cellobiose), oligosaccharides of the laminari series, laminarin and pustulan. The kcat/Km ratios for a series of laminari oligosaccharides from -biose to -heptaose showed that Exg has an extended substrate-binding site which contains at least five binding sites for sugar residues. Binding at position +2 (the third sugar residue) increases the kcat twofold while positions +3 and +4 lower the Km value further and thereby increase the catalytic efficiency. Exg catalyses an efficient transglucosylation reaction with high concentrations of laminari-oligosaccharides which specifically form beta-1,3 linkages and with yields up to 50%. The rate of the transglucosylation is concentration-dependent and can be more than 10 times faster than the hydrolytic reaction with excess donor substrates such as laminaritriose and laminarihexaose. The kinetics of Exg and the predicted substrate-binding site for up to five sugar residues are consistent with a recent structural analysis of the enzyme-binding site.     

Purification and properties of a beta-1,6-clucosidase from Flavobacterium.

An intracellular beta-1,6-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced semiconstitutively by Flavobacterium M64. This enzyme was purified 180-fold by fractionation with ammonium sulfate followed by chromatographies on carboxymethylcellulose, hydroxyapatite and Sephadex G-100. The final preparation appeared homogeneous on disc electrophoresis on polyacrylamide gel. The molecular weight of the enzyme was determined to be ca. 59 000 by Sephadex G-100 gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8 and the optimum temperature was 40 degrees C. The enzyme readily hydrolyzed oligomers with beta-a,6-glucosidic linkages, converting them to glucose. The Km values for gentio-biose, -triose, -tetraose and -pentaose were 2.8, 3.0, 4.2 and 4.6 times 10- minus 4 M, respectively. The rates of their hydrolyses decreased with increase in their chain lengths. The enzyme was concluded to be a beta-1,6-glucosidase from its substrate specificity, production of glucose, transferring ability and inhibition by glucono-delta-lactone. The enzyme activity was inhibited by Hg-2+, Cu-2+, Ag-+, Fe-3+, p-chloromercuribenzoate, N-ethylmaleimide, glucose and trishydroxyaminomethane (Tris) but not by ethylenediaminetetraacetic acid.     

Isolation and properties of an extracellular beta-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2.

An extracellular beta-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a M(r) of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pI of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40 degrees C are 0.25 mM and 27.1 mumol.min-1 x mg-1, respectively; with PNPG as the substrate, the corresponding values are of 0.35 mM and 27.7 mumol.min-1 x mg-1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 1.68 x 10(-2) and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50 degrees C. The enzyme has high activity against sophorose (beta-1,2-glucobiose) and laminaribiose (beta-1,3-glucobiose) but has no activity against gentiobiose (beta-1,6-glucobiose). The activity of the beta-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.     

Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum

Trichoderma asperellum produces at least two extracellular beta-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A beta-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-beta-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS-electrophoresis slab gel. The enzyme was only active toward glucans containing beta-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The K(m) and V(max) values for exo-beta-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml(-1) and 0.246 U min(-1), respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55 degrees C. Hg(2+) strongly inhibited the purified enzyme.     

Cloning and expression of a β -1,3-glucanase gene from Bacillus circulans KCTC3004 in Escherichia coli

Summary A new gene encoding the -1,3-glucanase(laminarinase) of Bacillus circulans KCTC3004 was cloned into Escherichia coli using pUC19 as a vector. The gene localized in the 5.3 kb PstI DNA fragment was expressed independently of its orientation in the cloning vector showing enzyme activity about 33 times greater than that produced by the original B. circulans. The optimum pH and temperature of the cloned enzyme were pH 5.4 and 50°C, respectively. The molecular weight of the enzyme was about 38,000 and the processing of the enzyme molecule within the E. coli cell was not observed. The enzyme hydrolyzed laminarin to produce laminaritriose, laminaribiose, and glucose as main products, but it was inactive for lichenan, CMC, or xylan.     

A wall-bound exo-1,3-β-d-glucanase from Acacia cultured cells

Several wall-bound exo-1,3-β-d-glucanases have been solubilized by 4 M LiCl from suspension-cultured Acacia cells. One exhibits both exo-laminarinase (EC 3.2.1.39) and β-d-glucosidase (EC 3.2.1.21) activities and has been purified up to 30-fold by anion-exchange chromatography, gel filtration and flat-bed electrofocusing. This enzyme hydrolyses laminarin, laminaribiose and p-nitrophenyl-β-d-glucopyranoside. The enzyme, with a pI of 4.6, is apparently homogenous, since it behaves as a single protein with an apparent molecular weight of 62000 on SDS-polyacrylamide gel electrophoresis. Its K_m value in 0.1 M acetate buffer (pH 5.0) with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.27 mM; with laminarin as substrate the K_m expressed in glucosyl residue concentration was 0.64 mM. Other kinetic experiments showed that exo-laminarinase and β-d-glucosidase activities correspond to two distinct catalytic sites in the same protein.     

Purification and characterization of laminaran hydrolases from Trichoderma viride

At least three extracellular laminaran hydrolases which hydrolyzed laminaran (beta-1,3:1,6-glucan) from Eisenia bicyclis were secreted in wheat bran solid medium by Trichoderma viride U-1. These three enzymes, lam AI, AII, and B, were purified to electrophoretic homogeneity. Their molecular masses were estimated to be 70.1, 70.4, and 45.0 kDa for lam AI, AII, and B, respectively, by SDS-PAGE. Whereas both lam AI and AII could hydrolyze laminarin from Laminaria digitata, lam AII showed higher activity against Laminaria laminarin rather than Eisenia laminaran. On the other hand, lam B preferentially hydrolyzed pustulan, a beta-1,6-glucan. Laminarioligosaccharide was hydrolyzed by lam AI and AII but not B, whereas gentiooligosaccharide was hydrolyzed by only lam B. It showed that lam AI and AII were specific for beta-1,3-linkages, but lam B was specific for beta-1,6-linkages. These results indicated that T. viride U-1 has a multiple glucanolytic enzyme system.     

Purification and Characterization of Wall-bound Exo-l,3-{beta}-D-Glucanase from Barley (Hordeum vulgare L.) Seedlings

A ß-D-glucanase activity hydrolyzing 1,3:1,4-ß-D-glucanwas released from the cell walls of barley by 3M LiCl treatment.It was purified by sequential cation-exchange, gel-filtrationand hydrophobic chromatography. The molecular mass of the glucanasewas 66 kDa as determined by SDS-polyacrylamide gel electrophoresis.Sequence determination of the first thirty amino acids of theN-terminus revealed a high homology of this enzyme to the Pseudomonasl,4-ß-D-glucosidase (56.5%). The purified ß-D-glucanasehas a pH optimum at 5.0, and hydrolyzes oligosaccharides containingß-D-1,3 or ß-D-1,4 linkage. The glucanaseshowed maximum hydrolytic activity toward laminaritetraose,the rate being about two times that of cellotetraose and aboutfour times that of gentiobiose. Polysaccharides such as lichenan,l,3:l,4-ß-D-glucan (from barley), laminarin and pustulanare also hydrolyzed, but not carboxylmethyl-curdlan, carboxymethyl-cellulose,xyloglucan and maltose. The purified ß-D-glucanaseyielded monomeric glucose from laminarihexaose, and exhibitedcharacteristics of an exo-l,3-ß-D-glucanase (EC 3.2.1.58 [EC] ).The activity and biochemical characteristics of this enzymesuggest that it is an exo-l,3-ß-D-glucanase involvedin the rapid turnover of l,3:l,4-ß-D-glucan in barleycell walls during seedling growth. (Received September 24, 1996; Accepted December 9, 1996)     

Purification to homogeneity and characterization of a 1,3-beta-glucan (callose) synthase from germinating Arachis hypogaea cotyledons.

A 1,3-beta-D-glucan (callose) synthase (CS) from a plasma membrane fraction of germinating peanut (Arachis hypogaea L.) cotyledons has been purified to apparent homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino-terminal analysis, and the Western blots pattern. The purification protocol involved preparation of a high specific activity plasma membrane fraction, selective solubilization of the enzyme from the membrane with 0.5% digitonin at a protein-to-detergent ratio of 1:6, sucrose gradient centrifugation, and chromatography on hydroxylapatite and DEAE-Sephadex A-50. The purified CS shows a molecular mass of approximately 48,000 by SDS-PAGE, pH optimum of 7.4, leucine as the amino-terminal residue, Km for UDP-glucose of 0.67 mM, and Vmax of 6.25 mumol/min/mg protein. The enzyme is specific for UDP-glucose as the glucosyl donor and required Ca2+, at an optimum concentration of 2-5 mM, for activity. The enzyme activity was inhibited by nucleotides (ATP, GTP, CTP, UTP, UDP, and UMP). The enzyme activity was also inhibited by the addition of EDTA or EGTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The reaction product formed during incubation of UDP-[14C]glucose and cellobiose with purified enzymes was susceptible to digestion by exo-(1,3)-beta-glucanase, but was resistant to alpha- and beta-amylases and to periodate oxidation, indicating that the polymer formed was 1,3-beta-glucan, and beta-1,4 and beta-1,6 linkages were absent.     

News & Notes: Production, Purification, and Properties of an Endo-1,3-β-Glucanase from the Basidiomycete Agaricus bisporus

Agaricus bisporus H 25 produced extracellular endo-1,3-β-glucanase when grown in a static culture at 25°C in a minimal synthetic medium supplemented with A. bisporus cell walls plus fructose. Endo-1,3-β-glucanase was purified 17.85-fold from 20-day-old culture filtrates by precipitation at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and preparative PAGE followed by electroelution. The purified enzyme yielded a single band in both native and SDS-polyacrylamide gels with a molecular mass of 32 kDa (SDS-PAGE) and 33.7 kDa (MALDI-MS), showing an isoelectric point of 3.7. The enzyme was active against β-1,3- linkages and, to a lesser extent, against β-1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase against laminarin and pustulan were observed between pH 4 and 5.5, and between 40° and 50°C for laminarin, and between 30° and 50°C for pustulan. The optimum pH and temperature were 4.5 and 45°C for both substrates. Received: 17 June 1998 / Accepted: 24 September 1998     

Purification and some properties of a beta-glucosidase from Flavobacterium johnsonae

Flavobacterium johnsonae was isolated as a microorganism that produced a beta-glucosidase with hydrolytic activity of beta-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45 degrees C, and the enzyme was stable below 35 degrees C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl beta-glucosides such as p-nitrophenyl beta-glucoside, phenyl betaglucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.     

Purification and properties of endo-alpha-1,3-glucanase from a Streptomyces chartreusis strain.

An enzyme hydrolyzing the water-insoluble glucans produced from sucrose by Streptococcus mutans was purified from the culture concentrate of Streptomyces chartreusis strain F2 by ion-exchange chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose columns and gel filtration on Bio-Gel A-1.5m. The purification achieved was 6.4-fold, with an overall yield of 27.3%. Electrophoresis of the purified enzyme protein gave a single band on a sodium dodecyl sulfate-polyacrylamide gel slab. Its molecular weight was estimated to be approximately 68,000, but there is a possibility that the native enzyme exists in an aggregated form or is an oligomer of the peptide subunits, have a molecular weight larger than 300,000. The pH optimum of the enzyme was 5.5 to 6.0, and its temperature optimum was 55 degrees C. The enzyme lost activity on heating at 65 degrees C for 10 min. The enzyme activity was completely inhibited by the presence of 1 mM Mn2+, Hg2+, Cu2+, Ag2+, or Merthiolate. The Km value for the water-insoluble glucan of S. mutans OMZ176 was an amount of glucan equivalent to 1.54 mM glucose, i.e., 0.89 mM in terms of the alpha-1,3-linked glucose residue. The purified enzyme was specific for glucans containing an alpha-1,3-glucosidic linkage as the major bond. The enzyme hydrolyzed the S. mutans water-insoluble glucans endolytically, and the products were oligosaccharides. These results indicate that the enzyme elaborated by S. chartreusis strain F2 is an endo-alpha-1,3-glucanase (EC 3.2.1.59).