beta-Glucosidases from cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum as typical glycosidases

By ethanol precipitation (v/v) and chromatography on Sephadex SP, DEAE (or DEAE-cellulose), and G-200 beta-glucosidases (EC 3.2.1.21) from the culture filtrates of cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum grown on the medium with cellulose containing materials were isolated. The enzymes were homogenous as shown by different techniques. The substrate specificities of the obtained enzymes were studied. beta-Glucosidases had higher affinity for p-nitrophenyl-beta-D-glucopyranoside than for cellobiose (Km 1.25, 0.34, 0.20 and 5.4, 2.0, 1.2 mM, respectively) and were able to hydrolyze both laminaribiose and gentiobiose; but they were unable to cleave cotton fiber, carboxymethylcellulose, and other glycans to reducing sugars. They showed transglycosylase activity. Ki values for arylglucosidase activity of beta-glucosidases from A. terreus, G. candidum, and T. longibrachiatum in the presence of either glucose or glucono-1,5-lactone were 12.2, 6.0, 2.1 and 0.20, 0.19, 0.07 mM, respectively. The Mr's were estimated by gel filtration and by sedimentation equilibrium centrifugation to 200,000, 200,000, 350,000, respectively. The isoelectric points of beta-glucosidases were 4.8, 5.9, and 4.2, respectively. The optimum temperatures and pH's were 60, 50, and 50 degrees C and at pH 4.5, 4.5, and 4.8-5.7, respectively. These properties appear to relate beta-glucosidases obtained in the present study to typical glycosidases.     

Beta-glucosidase from Penicillium purpurogenum: purification and properties.

beta-Glucosidase was purified from the culture supernatant of Penicillium purpurogenum. The purified enzyme was homogeneous on both nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme is a monomeric glycoprotein with M(r) of 90,000 as determined by gel filtration on Bio-Gel P-300 and SDS-polyacrylamide gels. Two enzyme forms were resolved by chromatofocusing and isoelectric focusing, and the pI values obtained with both methods were 4.2 (major form) and 6.0. The major form was characterised further. Enzyme activity was optimal at pH 3.5 and at 60 degrees C. The enzyme was stable in the pH range 2.5-9.5 for 24 h at 4 degrees C. Kinetic analysis gave Kms of 0.8 mM for cellobiose and 85 microM for p-nitrophenyl-beta-D-glucopyranoside. The enzyme hydrolyses a wide range of substrates including aryl-beta-glucosides, cellobiose, and amygdalin. Glucose inhibits competitively and glucono-delta-lactone is a mixed inhibitor of the enzyme.     

Purification and partial characterization of a cellodextrin glucohydrolase (beta-glucosidase) from Trichoderma reesei strain QM 9414

A beta-glucosidase (E.C. 3.2.1.21) was isolated from the culture filtrate of fungus Trichoderma reesei QM 9414 grown in continuous culture with biomass retention. The crude extracellular enzyme preparation was fractionated by a three-step purification procedure [chromatography on Fractogel HW-55 (S) and Bio-Gel A 0.5 plus final preparative isoelectric focusing] to yield three beta-glucosidases with isoelectric points at pH 8.4, 8.0, and 7.4. Only one enzyme (pi 8.4) met the stringent criterion of being homogeneous according to titration curve analysis. This enzyme was then characterized not to be a glycoprotein, although the native protein contained 35% carbohydrate (as glucose). It was found to have an apparent molar mass of 7 x 10(4) g/mol (SDS-PAGE), exhibited its optimum activity towards cellobiose at pH 4.5 and 70 degrees C (30 min test), and lost less than 3% activity at 50 degrees C over a period of 7 h. The K(M) values towards cellobiose and p-nitrophenyl-beta-D-glucopyranoside were determined to be 0.5mM and 0.3mM, respectively. The enzyme hydrolyzed cellodextrins (cellotriose to cellooctaose) by sequentially splitting off glucose units from the nonreducing end of the oligomers. The extent of the observed transfer reactions varied with the initial substrate concentration. No enzyme activity towards microcrystalline cellulose or carboxymethylcellulose could be detected. The classification of the enzyme as beta-glucosidase or exo-beta-1,4-glucan glucohydrolase is discussed with respect to the exhibited hydrolytic activities.     

Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C.

1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid.     

Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.     

Purification and properties of two β-glucosidases isolated from Aspergillus niger

The cellulase complex of the fungus Aspergillus niger (strain CBS 554.65 = ATCC 16 888) was fractionated by gel filtration yielding six pronounced peaks. Only proteins from the fraction corresponding to the first peak (96 kDa) showed β-glucosidase activity vs. the substrate 4-nitrophenyl-β-D-glucopyranoside (pNPG). These proteins have been fractionated by chromatofocusing, yielding two β-glucosidases (I and II) which are shown to be homogeneous in isoelectric focusing experiments (pI = 4.6 and 3.8, respectively). Kinetic experiments with pNPG, MU-glucopyranoside and cellobiose revealed that both types of β-glucosidases behave like aryl-β-glucosidases. β-Glucosidase-I acting on pNPG exhibits a split kinetics characterized by high and low substrateconcentration kinetics which are differentiated by different values of V and of K_m. In addition, β-glucosidase-II is shown to be an exo-glucohydrolase as deduced from experiments with MU-cellobiopyranoside. Experimental features should be emphasized; usual soft-gel ion-exchange materials did not work in the chromatofocusing separation of the two β-glucosidases, in contrast to the 10μ-Si 500 = DEAE exchange material (Serva) typically used in HPLC-experiments. Furthermore, protein content determinations based on different procedures yielded widely differing values.     

Purification and properties of cellulase enzymes from Robillarda sp. Y-20

One endo-β-1,4-glucanase (EC 3.2.1.4) and two unique β-glucosidases (EC 3.2.1.21) have been isolated from culture filtrates Robillarda sp. Y-20 by combinations of DEAE A-50 column chromatography and isoelectric focusing. These enzymes were homogeneous on gel filtration, isoelectric focusing and polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate (SDS). The molecular weights of endoglucanase, and the two β-glucosidases, I and II by SDS-polyacrylamide gel electrophoresis were 59000, 76000 and 54000, respectively. The pI values were 3.5, 7.5, and 3.8 for endoglucanase, β-glucosidase I and II, respectively. The major β-glucosidase I was a glycoprotein, but the endoglucanase and β-glucosidase II were not. The endoglucanase rapidly reduced the viscosity of carboxymethyl (CM) cellulose with concomitant production of reducing sugar. The enzyme had very low activity with crystalline cellulose such as insoluble acid treated cellulose, Avicel and filter paper. The endoglucanase attacked celloheptaose to cellotetraose more readily than cellotriose, but did not hydrolyze cellobiose. Both β-glucosidases attacked celloheptaose to cellotetraose more readily than cellotriose and cellobiose, but did not hydrolyze CM-cellulose and insoluble acid treated cellulose. Strong synergism was observed for hydrolysis of CM-cellulose by the endoglucanase and β-glucosidases.     

beta-Glucanases of Geotrichum candidum

The formation of (1-4)-, (1-3)- and (1-6)-beta-glucanases and beta-glucosidases was studied during the growth of the fungus Geotrichum candidum under the conditions of submerged cultivation in a medium optimal for the production of cellulolytic enzymes. Endo-(1-4)-beta-glucanases and C1 enzyme, as well as (1-3)- and (1-6)-beta-glucanases appeared in the medium as soon as by the 45th hour of growth. However, the maximal concentration of the enzymes in the medium was observed at different periods of the fermentation: between 75th and 105th, 70th and 95th, 55th and 100th, 80th and 105th hours, respectively. The content of the enzymes abruptly decreased by the 160th hour of the growth. The activity of beta-glucosidases, which was low at the beginning of the growth, sharply increased by the 70th hour and remained at the same level by the 160th hour of the growth. The accumulation of beta-glucanases was an uneven process, consistent with irregular changes in the content of DNA and protein in the biomass. The isoelectric points of beta-glucanases and beta-glucosidases were studied in the filtrate of the cultural broth after 96 h of the cultivation. The high activity of endo-(1-4)-beta-glucanase was found at the pH 4.6, 4.1 and 3.8; its low activity was detected at the pH 6.4, 3.2, 1.6 and 1.3. Other glucanases behaved also as acid proteins. During isoelectric focusing, (1-3)-beta-glucanase showed the peaks of activity at the pH 4.4, 4.0, 3.8 and 2.9; (1-6)-beta-glucanase, at the pH 5.0, 3.7, 3.5, 3.1 and 2.0; beta-glucosidases were distributed over a broad pH range from 6.7 to 2.0, with the maximal activity at the pH 6.2, 4.8 and 3.7.     

Glucosidases specific for the cyanogenic glucoside triglochinin. Purification and characterization of beta-glucosidases from Alocasia macrorrhiza Schott]

beta-Glucosidases have been isolated from Alocasia macrorrhiza plants. The enzymes are highly specific for the hydrolysis of the cyanogenic glucoside triglochinin endogenous to this plant. Upon chromatography of protein extracts on cation exchange resins and Sephadex G-200, separation into various enzymatically active bands was observed. The main fractions possess molecular weights of approximately 310000 and 105 000, as shown by preparative ultracentrifugation in a linear saccharose gradient. The beta-glucosidases are composed of subunits (molecular weight 55 000 to 60 000), as revealed by sodium dodecylsulfate gel electrophoresis. The result of alkaline disc electrophoresis and isoelectric focusing in polyacrylamide gel suggest that the beta-glucosidase fraction with molecular weight 105 000 is a dissociation product of the 310 000 molecular-weight species. The isoelectric points of the various beta-glocusidase bands, obtained by isoelectric focusing, vary between pH 4.5 and 5.0. The beta-glucosidases show a pronounced specificity for triglochinin. The Km for this substrate (3 times 10(-5) M) is 50 to 100-fold lower than for all other substrates hydrolyzed. Of the other cyanogenic glycosides, only those with an aromatic aglycone, (S)-configuration at the asymmetric carbon atom of the aglycone and glucose as sugar moiety were hydrolyzed to a measurable extent. The pH optimum of the enzyme reaction is 5.5, the temperature optimum around 50 degrees C. Cu2 ions and glucono-1,5-lactone inhibit beta-glucosidase activity approximately 50% at a concentration of 5 times 10(-4) M, while Hg2,Ag and p-chloromercuribenzoate show the same percent inhibition at 5 times 10(-7) M. Lipophilic solvents (ethanol, ethylene glycol monomethylether) activate the beta-glucosidase activity, preferentially by influencing the V values of the enzymes.     

Purification of cat submaxillary kallikrein.

The cat submaxillary gland contains 1,000--2,400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6--7 forms with pI's between 4.2 and 5.1. One mg of the purified kallikrein contained 930--1,260 KU in the dog vasodilator assay, and hydrolyzed 15--25 and 9--12 mumol of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-alpha-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in 1 min at 25 degrees C and pH 8.0. The Km's of the purified kallikrein with BAEE and TAME were 0.67 and 0.34 mM, respectively. Hydrolysis of N-alpha-benzoyl-L-tyrosine ethyl ester (BTEE), N-alpha-benzoylarginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5 X 10(4) by Sephadex G-100 gel filtration and 4.7 X 10(4) by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18.5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.     

Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second beta-glucosidase.

A second extracellular beta-glucosidase (betalarge) of Aspergillus fumigatus was purified to homogeneity and shown to be a glycoprotein, as determined by polyacrylamide gel electrophoresis followed by staining for protein and for carbohydrate. Its molecular weight was approximately 340,000 by gel filtration, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave an apparent molecular weight of 170,000, suggesting that the enzyme has two subunits. The glucosidase contained covalently bound sugars consisting of about 2 mol of glucosamine and 16 mol of mannose per mol of protein. The carbohydrate was found to be attached to the peptide via glucosaminyl leads to peptide linkage, possibly to asparagine residues. At pH 4.5 this enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.88 mM) and cleaved two glucose disaccharides: gentiobiose (beta,1 leads to 6; Km = 0.75 mM) and cellobiose (beta,1 leads to 4; Km = 0.84 mM). Although its activity is similar to that of a previously purified beta-glucosidase (betasmall), the two enzymes differ with respect to their pH activity profiles, substrate specificities, and molecular weights. Also double diffusion tests with anti-betasmall antiserum and both purified beta-glucosidases revealed a nonidentical cross-reaction. Microcomplement fixation of native and periodate-oxidized betasmall suggested that the oligosaccharide chain(s) was not a major antigenic site.     

Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D.

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.     

Neuraminidase in Bacteroides fragilis.

A neuraminidase from Bacteroides fragilis was purified 542-fold by isoelectric focusing, adsorption chromatography on Affi-Gel 202, and gel filtration chromatography on Sephadex G-200. On isoelectric focusing the neuraminidase was resolved into three differently charged fractions with pI values of 6.8, 7.1, and 7.4. The major component of pI 7.1 was used for further purification. The purified enzyme had optimal activity at pH 6.4 with N-acetylneuraminlactose as the substrate. Its molecular weight, determined by Sephadex G-200 gel filtration chromatography, was 92,000. The neuraminidase hydrolyzed terminal neuraminic acid residues from N-acetylneuraminlactose, fetuin, bovine submaxillary mucin, and porcine stomach lining mucin. A new method for the detection of neuraminidase activity is described which is based on rocket affinoelectrophoresis. It utilizes the differences in the interaction of sialylated and desialylated mucin with Helix pomatia lectin, enzymatic activity being detected by formation of affinorockets after incubation of the neuraminidase with bovine submaxillary mucin.     

Purification of a cellulase from kidney bean abscission zones

The purification of a cellulase isoenzyme with a pI of 9.5 from kidney bean abscission zones is described. An important step in the purification involved the adsorption of the cellulase isoenzyme onto an affinity column of CF-11 cellulose and the subsequent elution with cellobiose. Native and SDS polyacrylamide gel electrophoresis established that there was only one component in the purified cellulase samples. Antibodies raised against the purified pI 9.5 cellulase precipitated this isoenzyme from crude or purified solutions but did not cross react with pI 4.5 cellulase from 2,4-D-treated abscission zones. The antibody was shown to be monospecific by immunoelectrophoresis and by the fact that it precipitated only a single ¹⁴C-labeled protein from an abscission zone extract heavily labeled with ¹⁴C amino acids.     

粗毛栓菌胞外锰过氧化物酶的纯化及性质

培养于麦草粉上的白腐担子菌粗毛栓菌分泌胞外木质纤维素降解酶(纤维素酶、木聚糖酶、漆酶、锰过氧化物酶和木质素过氧化物酶)。经过超滤、盐析、离子交换层析、凝胶过滤和活性聚丙烯酰胺凝胶电泳等步骤,获得了初步纯化的锰过氧化物酶组分。利用变性聚丙烯酰胺凝胶电泳和等电点聚焦技术所测定的锰过氧化物酶的相对分子质量和等电点分别为35.7 ku和pI 2.8。研究结果表明,所纯化的锰过氧化物酶在407nm处具有最大光吸收峰,该酶最适作用pH值和温度分别为pH 5.3和35℃。     

Chromato-focussing of plasma low density proteins

A few alternatives of the binding of healthy patients plasma low density lipoproteins (LDL) with anion exchanger PBE-94 were revealed. In the first case the main part of LDL did not bind to the gel and the isoelectric points of the minor subfractions were 5.8 and 5.3, and pI 4.1. In the second case about half of lipoproteins did not bind to the gel, and the isoelectric points of subfractions were 5.7 and 5.0; and pI 4.1. In the third case when all lipoproteins bound to the PBE-94, there were much more subfractions and their isoelectric points were 6.2, 5.8, 5.2, 4.9, 4.5 and pI 4.1. All LDL of the patients with ischemic heart disease bound to anion exchanger, and the part of subfraction with pI 4.1 was three or five times as great as the one of the healthy person. Increasing of the LDL subfraction with pI 4.1 was observed at prolonged keeping of the LDL obtained from the healthy person plasma. LDL isoelectric point distribution of the persons with carcinoma uterine cervix did not differ from the one of the healthy persons. Acetylation and hexanol modification resulted in the isoelectric point shift from 5.7 to 4.6 and to 4.3 in the case of LDL subfraction to be obtained preparatively using the chromatofocusing.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two beta-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50 degrees C and 65 degrees C for exo-glucanases and endo-glucanases but 35 degrees C and 65 degrees C for beta-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40-50 degrees C. Exo-II and Exo-III (Km, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I (Km, 50.00 mg/ml). The Km values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities (Vmax, 12.0 and 10.0 IU/mg protein) were comparable. beta-Glucosidases exhibited Km values of 0.24 and 0.12 mmol and Vmax values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29,000; Exo-II, 72,500; Exo-III, 138,000; Endo-I, 25,000; Endo-II, 32,500; beta-Gluco-I, 14,000 and beta-Gluco-II, 26,000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas beta-glucosidases did not. Hg2+ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

Separation and Some Properties of Two Intracellular beta-Glucosidases of Sporotrichum (Chrysosporium) thermophile

Intracellular, inducible beta-glucosidase from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile (ATCC 42464) was fractionated by gel chromatography or isoelectric focusing into components A and B. Enzyme A (molecular weight 440,000) had only aryl-beta-glucosidase activity, whereas enzyme B (molecular weight 40,000) hydrolyzed several beta-glucosides but had only low activity against o-nitrophenyl-beta-d-glucopyranoside (ONPG). Both enzymes had temperature optima of about 50 degrees C. The pH optimum was 5.6 for enzyme A and 6.3 for enzyme B, respectively. The K(m) (ONPG) value for enzyme A was 0.5 mM, and the corresponding values for enzyme B were 0.18 mM (ONPG) and 0.28 mM (cellobiose). Enzyme B, when tested with ONPG, showed substrate inhibition at a substrate concentration above 0.4 mM which could be released by cellobiitol and other alditols. Enzyme A was isoelectric at pH 4.48, and enzyme B was isoelectric at pH 4.64. Several inhibitors were tested for their action on the activity of enzymes A and B. Both enzymes were found to be concomitantly induced in cultures with either cellobiose or cellulose as carbon source.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).     

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.