Synthesis of beta-glucanases during sporulation in Saccharomyces cerevisiae: formation of a new, sporulation-specific 1,3-beta-glucanase.

A biphasic synthesis of 1,3-beta-glucanase occurred when cells of Saccharomyces cerevisiae AP-1 (a/alpha) were incubated in sporulation medium. The capacity to degrade laminarin increased very slowly during the first 7 h but at a much faster rate thereafter. Changes occurring during the first period were not sporulation specific since the moderate increase in activity against laminarin was insensitive to glutamine and hydroxyurea and also took place in the nonsporulating strain S. cerevisiae AP-1 (alpha/alpha). However, the changes taking place after 7 h must be included in the group of sporulation-specific events since they were inhibited by glucose, glutamine, and hydroxyurea and did not occur in the nonsporulating diploid. Consequently, only when the cells had been incubated for at least 7 h in sporulation medium did full induction of activity against laminarin take place upon shift to a medium which favored vegetative growth. Changes in the relative proportions of the vegetative glucanases, namely, endo- and exo-1,3-beta-glucanase, and the formation of a new sporulation-specific 1,3-beta-glucanase account for the observed events and are the consequence of the expression of the sporulation program.     

Purification and partial characterization of an exo-beta-glucanase from the yeast Kluyveromyces aestaurii.

The intracellular-periplasmic exo-1,3-beta-glucanase (EC 3.2.1.58) has been extracted from the yeast Kluyveromyces aestuarii and purified to immunoelectrophoretic homogeneity by ion-exchange and gel-exclusion chromatography. The kinetic constants and activation energies for laminarin, p-nitrophenyl-beta-D-glucoside, and pustulan have been determined, along with the effect of pH. Evidence is presented indicating that the enzyme is composed of a single polypeptide chain, about 24% carbohydrates, and its molecular weight was estimated to be 43 000.     

The Saccharomyces cerevisiae SPR1 gene encodes a sporulation-specific exo-1,3-beta-glucanase which contributes to ascospore thermoresistance.

A number of genes have been shown to be transcribed specifically during sporulation in Saccharomyces cerevisiae, yet their developmental function is unknown. The SPR1 gene is transcribed during only the late stages of sporulation. We have sequenced the SPR1 gene and found that it has extensive DNA and protein sequence homology to the S. cerevisiae EXG1 gene which encodes an exo-1,3-beta-glucanase expressed during vegetative growth (C. R. Vasquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebrada, E. Mendez, and F. del Ray, Gene 97:173-182, 1991). We show that spr1 mutant cells do not hydrolyze p-nitrophenyl-beta-D-glucoside or laminarin in a whole-cell assay for exo-1,3-beta-glucanases. In addition to the absence of this enzymatic activity, spr1 mutant spores exhibit reduced thermoresistance relative to isogenic wild-type spores. These observations are consistent with the notion that SPR1 encodes a sporulation-specific exo-1,3-beta-glucanase.     

Structure of beta-glucan oligomer from laminarin and its effect on human monocytes to inhibit the proliferation of U937 cells

We analyzed the human monocyte-stimulating ability of laminarin from Eisenia bicyclis, lichenan from Cetraria islandica, and their oligomers depolymerized with endo-1,3-beta-glucanase from Arthrobacter sp. The respective beta-glucan oligomers with different degrees of polymerization (DP) were fractionated from hydrolytic products of laminarin and lichenan using gel-filtration chromatography. The monocyte-conditioned medium pre-cultured in the presence of a fraction of beta-glucan oligomer (DP>/=8) from laminarin exhibited inhibitory activity against the proliferation of human myeloid leukemia U937 cells, while those pre-cultured with other beta-glucan oligomers and the original laminarin and lichenan showed little or no activity. NMR analysis indicated that the beta-glucan oligomer (DP>/=8) has an average DP value of 13, and its ratio of beta-1,3- to beta-1,6-linkages in glucopyranose units was estimated to be 1.3:1. These results indicate that the beta-1,3-glucan oligomer with a higher content of beta-1,6-linkage stimulates monocytes to inhibit the proliferation of U937 cells.     

Occurrence of an endo-1,3-beta-glucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity.

The endo-1,3-beta-glucanase (EC 3.2.1.6) secreted into the culture medium by cells of Candida utilis was isolated and purified to homogeneity on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (s20,w = 1.97S). The purified enzyme represented only 0.001% of the total 1,3-beta-glucanase activity, the remainder being due to an exo-1,3-beta-glucanase enzyme, and behaved as an acidic glycoprotein (pI 3.3) in isoelectric-focusing experiments. The mol.wt. was estimated to be 21 000 by gel filtration and polyacrylamide-gel electrophoresis. Studies on the hydrolysis of different substrates showed that the enzyme was only able to break down (1 leads to 3)-beta-linkages, by an endo-splitting mechanism. Glucono-delta-lactone, D-glucoronolactone and heavy metal ions such as Hg2+ were inhibitors of the enzyme activity. The function of this endo-beta-glucanase in C. utilis is discussed.     

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     

Substrate binding to a GH131 β-glucanase catalytic domain from Podospora anserina

β-Glucanases have been utilized widely in industry to treat various carbohydrate-containing materials. Recently, the Podospora anserina β-glucanase 131A (PaGluc131A) was identified and classified to a new glycoside hydrolases GH131 family. It shows exo-β-1,3/exo-β-1,6 and endo-β-1,4 glucanase activities with a broad substrate specificity for laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Here we report the crystal structures of the PaGluc131A catalytic domain with or without ligand (cellotriose) at 1.8 Å resolution. The cellotriose was clearly observed to occupy the +1 to +3 subsites in substrate binding cleft. The broadened substrate binding groove may explain the diverse substrate specificity. Based on our crystal structures, the GH131 family enzyme is likely to carry out the hydrolysis through an inverting catalytic mechanism, in which E99 and E139 are supposed to serve as the general base and general acid.     

Purification of an exo-1,3-beta-glucanase from Candida utilis.

An exo-1,3-beta-glucanase (EC 3.2.1.-) has been purified from the culture fluid of the yeast Candida utilis, and its biochemical properties have been studied. The amino acid analysis revealed a high content of acidic amino acids. The purified enzyme had 20% carbohydrate and a net negative charge showing higher affinity for laminarin than for p-nitrophenyl-beta-D-glucopyranoside and yeast cell-wall 1,3-beta-glucans. In addition, the enzyme hydrolyzed the substrates starting from the nonreducing ends, releasing glucose as the exclusive hydrolysis product. The enzyme activity was strongly inhibited by lactones and also by some heavy-metal ions.     

Purification and properties of a 1,3-β-glucanase from Penicillium oxalicum autolysates

High 1,3-beta-glucanase activity was detected during autolysis in a culture medium containing Penicillium oxalicum. It was due to the combined action of four enzymes. The purification process for the major enzyme produced a homogeneous band in the SDS polyacrylamide gel that corresponded to a molecular weight of 79,400 daltons. The enzyme pI was 6.3 and it was only active against 1,3-beta-glucans, with a S0.5 of 0.23 mg ml-1 against laminarin. The enzymatic optima were found at pH 4 and 55 degrees C, and instability was evident when pH and temperature were altered. The enzyme was not active against oxidated laminarin and was barely inhibited by glucono-D-lactone. Hg2+, Ag+ and Fe2+ were effective inhibitors. The enzyme was adsorbed by concanavalin-A-sepharose.     

Isolation, enzymatic properties, and mode of action of an exo-1,3-beta-glucanase from Trichoderma viride.

An exo-1,3-beta-glucanase has been isolated from cultural filtrate of T. viride AZ36. The N-terminal sequence of the purified enzyme (m = 61 +/- 1 kDa) showed no significant homology to other known glucanases. The 1,3-beta-glucanase displayed high activity against laminarins, curdlan, and 1,3-beta-oligoglucosides, but acted slowly on 1,3-1,4-beta-oligoglucosides. No significant activity was detected against high molecular mass 1,3-1,4-beta-glucans. The enzyme carried out hydrolysis with inversion of the anomeric configuration. Whereas only glucose was released from the nonreducing terminus during hydrolysis of 1,3-beta-oligoglucosides, transient accumulation of gentiobiose was observed during hydrolysis of laminarins. The gentiobiose was subsequently degraded to glucose. The Michaelis constants Km and Vmax have been determined for the hydrolysis of 1,3-beta-oligoglucosides with degrees of polymerization ranging from 2 to 6. Based on these data, binding affinities for subsites were calculated. Substrate binding site contained at least five binding sites for sugar residues.     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cullulose. Functional characterization of five endo-1,4-beta-glucanases and one exo-1,4-beta-glucanase.

A strong synergistic response was observed between the five endo-1,4-beta-glucanases and the exo-1,4-beta-glucanase obtained from culture solutions of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum), when these enzymes were allowed to degrade de-waxed cotton and Avicel. No synergism was observed if Walseth cellulose, an acid-swollen cullulose, was used. If de-waxed cotton was pretreated with endo-1,4-beta-glucanases, the exo-1,4-beta-glucanase enzyme released much more degradation products than from an untreated cotton...     

Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium

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

Cellulolytic activity of white, brown and gray wood rot fungi

Culture fluids obtained from submerged cultures of white, brown and gray wood rot fungi were assayed for the presence of cellulolytic activity complexes against the model substrated carboxymethylcellulose-Na and Standard Whatman cellulose and natural substrates, i.e. celluloses isolated from pine bark and sawdust. The cellulolytic activity of the examined fungal species was highly differentiated. The use of model and natural substrates allowed determination of the high substrate specificity of the cellulase complexes produced by the fungi. Not all the fungi were found to produce EC 3.2.1.4. endo-1, 4-beta-glucanase under the culture conditions employed. All the fungi were, however, able to produce a complex of EC 3.2.1.4. exo-1, 4-beta-glucanases. All the examined fungi were also able to degrade, although to a varied extent, such higher forms of cellulose as Standard Whatman cellulose or natural celluloses isolated from pine bark and sawdust. Determination of the cellulolytic activity of fungi against the above-mentioned specific natural substrates affords the possibility of their practical use.     

Beta-glucanases of the yeast Pichia polymorpha.

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl-beta-D-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A50 has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different beta-glucanases located in the periplasmic space is presented.     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cellulose. 3. Purification and physico-chemical characterization of an exo-1,4-beta-glucanase.

An exo-1,4-beta-glucanase from culture solution of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source has been extensively purified and characterized with respect to some physico-chemical properties. The purification has been carried out in a five-step procedure comprising chromatography on DEAE-Sephadex, gel filtration on polyacrylamide P-150, activation on a Dowex 2-X8 anion exchanger, chromatography on Concanavalin A-Sepharose and chromatography on SP-Sephadex. The purified enzyme was found to be pure and homogeneous by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical isoelectric focusing. A single symmetrical peak was obtained with the free zone electrophoresis method. The purification factor is about 15 and the yield of exo-1,4-beta-glucanase activity 7%. After purification, the enzyme showed no viscosity-decreasing activity towards carboxymethyl-cellulose solutions. The exo-1,4-beta-glucanase was isoelectric at pH 4.3 (4 degrees C). A molecular weight of 48600 was calculated on the basis of a knowledge of the partial specific volume, ultracentrifugation data and the amino acid composition. The enzyme contained no carbohydrate.     

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.     

The copurification of beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58

Two enzyme complexes, each with beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21), beta-xylosidase (beta-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-beta-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75,400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the beta-xylosidase, beta-glucosidase, and 1,3-beta-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl beta-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the beta-glucosidase and 1,3-beta-glucanase of the major complex had inhibition constants which differed by more than 80,000 times. In addition, the inhibition on the 1,3-beta-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.     

Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis

β-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0–5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on β-1,3-linked oligosaccharides than on β-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-β-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-β-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-β-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.