Purification and Some Properties of an Endo-β,1,6-glucanase from Neurospora crassa

An endo-β-1,6-glucanase (E.C. 3.2.1.75) was purified from the culture filtrate of Neurospora crassa IFO-6O68 by chromatographies on CM-cellulofine, Con-A Sepharose 4B, and Sepharose Cl-6B followed by preparative affinity gel electrophoresis. The purified enzyme had an apparent molecular weight of 47,000. The pH and temperature optima for the activity were 5.0 and 50°C. The enzyme acted on β-1,6-glucan (Pustulan) and yielded a series of gentio-oligosaccharides with endo- type action, and finally, glucose and gentiobiose were produced. The enzyme was also able to act on N. crassa cell wall β-glucan, and a small amount of hydrolysis fragments were liberated without apparent change of the cell wall glucan molecules.     

Purification and properties of Acinetobacter sp. endo-β-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins

A novel endo-β-N-acetylglucosaminidase capable of acting on complex type sugar chains of glycoproteins was found in the culture broth of a bacterium which was isolated from soil and identified as Acinetobacter sp. The enzyme was purified to homogeneity on polyacrylamide gel electrophoresis by successive purification procedures involving ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxylapatite and Sephadex G-150. Its molecular weight was about 35,000 on gel filtration. The optimum pH was 3.0–3.5, and the enzyme was stable in the pH range from 6–8. The enzyme had high activity on dansyl ovalbumin glycopeptide, and also could hydrolyze dansyl asialotransferrin glycopeptide and dansyl transferrin glycopeptide containing complex type sugar chains. The K_m value for dansyl asialotransferrin glycopeptide as the substrate of enzyme assay was 0.68 mM. The enzyme could release complex type sugar chains from intact asialotransferrin without the addition of any detergent.     

First Thermostable Endo-β-1,4-Glucanase from Newly Isolated Xanthomonas sp. EC102

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 μmol min^?1 mg^?1, V _max for CMC was 1.44 μmol mg^?1 min^?1, and K _m values was 25.6 mg mL^?1. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²⁺ and Co²⁺. This is the first report about thermostable endoglucanase from Xanthomonas sp.     

Purification and some properties of cellulose 1,4-β-cellobiosidase from a strain of Penicillium sp.

A cellulase was purified from the culture supernatant of a strain of Penicillium sp. The purified enzyme was homogenous on polyacrylamide disc gel electrophoresis. It was a glycoprotein with a molecular weight of 52,000 estimated by gel filtration. The optimum pH was about 4.0 and the optimum temperature was 60°C. The enzyme was stable in the pH range of 3.0–10.0 at 6°C for 48 h and on heating at 60°C for 10 min. The activity of the enzyme toward Avicel was about 3 times higher than toward carboxymethyl cellulose. The enzyme showed a low activity for cotton, newspaper, filter paper and cellulose powder. The main product from Avicel was cellobiose, with a trace of glucose.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Purification and properties ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524

Summary Purification and properties of two -fructofuranosidases, which produce 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, fromAureobasidium sp. ATCC 20524 are reported. The enzymes were purified to homogeneity by fractionations involving ethanol, calcium acetate and ammonium sulfate and DEAE-Cellulofine and Sephadex G-200 chromatography. Molecular weights of the enzymes were estimated to be about 318000 (P-1) and 346000 (P-2) daltons by gel filtration. The enzymes were glycoproteins that contained about 30% (w/v) (P-1) and 53% (w/v) (P-2) carbohydrate. The optimum pH for the enzymatic reactions were 4.5–5.5 (P-1) and 4.5–6 (P-2). The enzymes were stable over a wide pH range (4–9). The optimum reaction temperatures for both enzymes were 50–55°C and they retained more than 94% (P-1) and 98% (P-2) activities at 50°C after 15 min. TheK _m values for sucrose were 0.47 M (P-1) and 0.65 M (P-2). The enzymes were inhibited by mercury, copper and lead ions as well asp-chloromercuribenzoate.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

Purification and Properties of an Endo β-1,6-Glucanase from Rhizopus chinensis R-69

An endo β-1,6-glucanase (β-1,6-glucan glucanohydrolase, E, C. 3. 2. 1.) has been purified from the culture filtrate of a strain resembling Rhizopus chinensis in homogeneous form. The procedures involved ammonium sulfate fractionation followed by column chromatography of DEAE-cellulose, CM-Sephadex C–50 and BioGel P–60.

Various physicochemical and chemical characteristics of the enzyme have been made clear, including complete amino acid composition. Optimum pH, optimum temperature, apparent activation energy for activity, Km and V_max are 5.5~6.0, 60°C, 4.39 Cal per mole, 9.39×10^?3m glucose equivalents (0.169%) and 43.13 International Units, respectively. The enzyme required no metal ions for its activity, and it hydrolyzed β-1,6-glucan larger than gentiotetraose, forming gentiobiose and gentiotriose as main products.     

Purification and Some Properties of β-Glucosidase from Streptomyces sp

β-Glucosidases I, II, and III were isolated from the culture filtrate of a Streptomyces sp. by ammonium sulfate fractionation, hydroxylapatite column chromatography, filtration on Bio-Gel P-100, and DE-52 column chromatography. β-Glucosidase III had a single active band on disc-gel electrophoresis. Its optimum pH and temperature for activity were 6.0 and 60°C, respectively. The isoelectric point and molecular weight of the enzyme were pH 4.5 and 45,000, respectively. From an experiment using ¹⁴C-labeled glucose, gentiobiose seemed to be formed from laminaribiose as isomaltose is formed from maltose by fungal α-glucosidase. The enzyme showed transglucosylation and produced gentiobiose from β-gluco-disaccharides and 4-O-β-d-glucopyranosyl-d-manno-pyranose (epicellobiose). The enzyme acted on phenolic β-d-glucosides to produce unknown transfer products.     

Purification and properties of extracellularβ-fructofuranosidases fromAureobasidium sp. ATCC 20524

Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK _m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK _m values ofE-2 andP-2.     

An α-1, 3-Glucanase from Streptomyces sp. KI-8 Production and Purification

An α-l,3-glucanase was detected in the culture supernatant of a micro-organism, which was isolated from soil on agar medium containing α-l,3-glucan as sole carbon source. The isolated strain was characterized as a strain of Streptomyces, tentatively named KI-8. This enzyme required α-l,3-glucosidic linkage as an inducer. The optimum conditions for enzyme production were studied.

The enzyme was purified by (NH₄)₂SO₄ precipitation, column chromatography on DEAE-cellulose and P(phospho)-cellulose. To eliminate the concomitant β-l,3-glucanase activity, partially purified enzyme preparation was passed through a column packed with pachyman. Final purification was accomplished by the adsorption chromatography using Sephadex G-150 from which the α-l,3-glucanase was eluted with a solution of α-1,3-linked gluco-oligo-saccharides. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 78,000 by SDS-polyacrylamide gel electrophoresis.     

Purification and properties of a β-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp.

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Purification and properties of the Endo-1,4-β-glucanase from Ruminococcus albus and its gene product in Escherichia coli

Endoglucanases, EGI and EgI, were produced from the same Ruminococcus albus gene in R. albus and recombinant Escherichia coli, respectively. EGI was purified from R. albus culture supernatant and EgI was extracted from the transformant E. coli (JM101/pURA1) and purified. The purified enzymes EGI and EgI revealed maximum endoglucanase activity at a same pH of 6.8 and a temperature of 37°C. Both enzymes were stable at temperatures below 30°C. In addition, about 10% of their original activities were conserved even after boiling for 10 min. Amino acid sequences of both enzymes at the N-terminal (Ala-Ala-Asp-Glu-Ser-Glu-Thr-Glu-Asn-Val-Pro-Val-Ser-Gln-Thr-His--) were consistent with each other. The antiserum against EgI reacted with both EgI and EGI, indicating that both their protein moieties were the same immunologically. However, the molecular size of EGI (43,000) was larger than that of EgI (39,000) due to the presence of sugar moiety. The specific activity (54 units/mg) of EGI was almost double that (27 units/mg) of EgI. EGI was immunologically different from the endoglucanase purified in the previous paper [Ohmiya et al.: Carbohydrate Res., 166, 145–155 (1987)].     

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 properties of endo-1,4-β-xylanase from Trichosporon cutaneum

Summary The endoxylanase (1,4-D-xylan xylanohydrolase, EC 3.2.1.8) was purified 3,7 fold from the culture filtrate of the yeast Trichosporon cutaneum grown on oathusk xylan. The final enzyme preparation gave a single protein band on disc gel electrophoresis and has a molecular weight of approx. 45000. The enzyme has a pH optimum of 5.0 and a temperatur optimum of 50°C. Patterns of hydrolysis demonstrate that this xylanase is an endo-splitting enzyme able to break down xylans at random giving xylobiose, xylotriose and xylose as the main end-products. Since the enzyme seems not to be capable of liberating L-arabinose from arabino-xylan branched arabinose-containing xylooligosaccharides are formed, too. This enzyme contains carbohydrates in a noncovalent manner, indicating that this extracellular xylanase, is not a glycoprotein.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

Cyclic (1→2)-β-d-Glucan-hydrolyzing Enzymes from Acremonium sp. 15 Purification and Some Properties of Endo-( 1 → 2)-α-d-glucanase and β-d-Glucosidase

Acremonium sp. 15 a fungus isolated from soil, produces an extracellular enzyme system degrading cyclic (1→2)-β-d-glucan. This enzyme was found to be a mixture of endo-(1→2)-β-d-glucanase and β-d-glucosidase. The (1→2)-β-d-glucanase was purified to homogeneity shown by disc-electrophoresis after SP-Sephadex column chromatography, Sephadex G-75 gel filtration, and rechromatography on SP-Sephadex. The molecular weight of the enzyme was 3.6 × 10⁴ by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was pH 9.6. The enzyme was most active at pH 4.0—4.5, and stable up to 40°C in 20 mm acetate buffer (pH 5.0) for 2 hr of incubation. This enzyme hydrolyzed only (l→2)-β-d-glucan and did not hydrolyze laminaran, curdlan, or CM-cellulose. The hydrolysis products from cyclic (1→2)-β-d-glucan were mainly sophorose.

The β-d-glucosidase was purified about 4000-fold. The rate of hydrolysis of the substrates by this β-d-glucosidase decreased in the following order: β-nitrophenyl-β-d-glucoside, sophorose, phenyl-β-d-glucoside, laminaribiose, and salicin. This enzyme has strong transfer action even at the low concentration of 0.75 mm substrate.     

Characterization of Cryptopygus antarcticus Endo-β-1,4-Glucanase from Bombyx mori Expression Systems

Endo-β-1,4-glucanase (CaCel) from Antarctic springtail, Cryptopygus antarcticus, a cellulase with high activity at low temperature, shows potential industrial use. To obtain sufficient active cellulase for characterization, CaCel gene was expressed in Bombyx mori-baculovirus expression systems. Recombinant CaCel (rCaCel) has been expressed in Escherichia coli (Ec-CaCel) at temperatures below 10 °C, but the expression yield was low. Here, rCaCel with a silkworm secretion signal (Bm-CaCel) was successfully expressed and secreted into pupal hemolymph and purified to near 90 % purity by Ni-affinity chromatography. The yield and specific activity of rCaCel purified from B. mori were estimated at 31 mg/l and 43.2 U/mg, respectively, which is significantly higher than the CaCel yield obtained from E. coli (0.46 mg/l and 35.8 U/mg). The optimal pH and temperature for the rCaCels purified from E. coli and B. mori were 3.5 and 50 °C. Both rCaCels were active at a broad range of pH values and temperatures, and retained more than 30 % of their maximal activity at 0 °C. Oligosaccharide structural analysis revealed that Bm-CaCel contains elaborated N- and O-linked glycans, whereas Ec-CaCel contains putative O-linked glycans. Thermostability of Bm-CaCel from B. mori at 60 °C was higher than that from E. coli, probably due to glycosylation.     

Purification and Some Properties of β-1, 3-Glucanase of Suspension-cultured Tobacco Cells

A laminaran-hydrolyzing enzyme was purified from the homogenate of suspension-cultured tobacco ceils by the treatment with ion-exchangers and gel filtration. The purified enzyme was homogemous in disc-electrophoresis and was a basic protein. The optimal pH of the enzyme was 5.0. The enzyme was stable at temperature below 40°C. The inhibitory effect of Hg²⁺ Cu²⁺ and Ag⁺ was observed. Investigation of the hydrolysis product revealed that the enzyme attacked laminaran endo-wise to form laminari-tetraose, -triose, -biose and glucose.