Cellulase production and its localization inTrichoderma harzianum

The ability ofT. harzianum to produce the three components of cellulase,viz. filter paper activity, carboxymethylcellulase and β-glucosidase was investigated. The optimum pH and temperature for all the enzymes in the culture nitrate was 5–7 and 27°C, respectively. The shaken culture conditions gave low yields of the enzyme as compared to static cultures. Most of the FP-activity and CM-cellulase were located extracellularly but reasonable amount of β-glucosidase was retained in the cell debris fraction.     

Saprotrophic basidiomycete mycelia and their interspecific interactions affect the spatial distribution of extracellular enzymes in soil

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of α-glucosidase, β-glucosidase, cellobiohydrolase, β-xylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, α-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of β-xylosidase, β-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal:bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content.     

The Relationship of Some Glycosidases to the Endogenous and IAA-induced Growth of Light-grown Cucumber Hypocotyls

Some glycosidases in light-grown cucumber (Cucumis sativus L. cv. Aonaga-jibae) hypocotyl sections were examined with respect to their localization and relation to endogenous and IAA-induced growth. Frozen-thawed sections were used directly for measurement of enzyme activities, and β-glucosidase, α- and β-galactosidases and β-xylosidase were assayed by using p- or o-nitro-phenylglycopyranosides as substrates. The order of the activity of these enzymes were β -glucosidase > β -galactosidase =α-galactosidase > β-xylosidase. No activity of α-glucosidase was detected. High glycosidase activities were found in the youngest region of the hypocotyl, where the endogenous growth rate was highest. However, there was no significant difference in the activities of this region between seedlings at different growth stages. Among the enzymes tested, β -glucosidase showed a high correlation with the endogenous growth rate. β-glucosidase was found to be mostly associated with the cell wall fraction, while β-galactosidase was rather found in the soluble fraction of the cell. Separation of the epidermis from the section showed that a very high activity of β-glucosidase was associated with the epidermis. In both whole sections and isolated cell wall fractions, IAA was shown to have no effect on the activities of β-glucosidase and β-galactosidase.     

Mechanism of Formation of Gentiobiose from Curdlan by Enzymes of Streptomyces sp.

The enzyme system (culture filtrate) from Streptomyces sp. W19-1 formed gentiobiose from curdlan (β-1,3-glucan). The mechanism of the formation of gentiobiose was investigated in this study.

Two kinds of enzymes, β-1,3-glucanase and β-glucosidase (transglucosidase), were isolated from the culture filtrate of the strain by hydroxylapatite column chromatography. The β-1,3-glucanase hydrolyzed curdlan to glucose and laminari-oligosaccharides, and the β-glucosidase formed gentiobiose by transglucosylation from the resultant laminari-oligosaccharides, especially laminaribiose. The two enzymes took part in the formation of gentiobiose from curdlan.     

The bgl1 gene of Trichoderma reesei QM 9414 encodes an extracellular, cellulose-inducible β-glucosidase involved in cellulase induction by sophorose

We have investigated the effect of disruption of the bgl1-(β-glucosidase l-encoding) gene of Trichoderma reesei on the formation of other β-glucosidase activities and on the induction of cellulases. To this end the bgl1 locus was disrupted by insertion of the Aspergillus nidulans amdS (acetamidase-encoding) gene. The bgl1-disrupted strain did not produce the 75kDa extracellular β-glucosidase on cellulose or lactose, but still formed β-glucosidase activity on glucose, cellobiose, xylan or β-1,3-glucan, suggesting that the enzyme(s) exhibiting this β-glucosidase activity is (are) not encoded by bgl1. The cellulose-inducer sophorose induced the bgl1-encoded β-glucosidase, whereas the remaining β-glucosidase activity was induced by methyl-β-D-glucoside. The bgl1-gene product was mainly secreted into the medium, whereas the other β-glucosidase activity was mainly associated with the cells. A bgl1-multicopy strain formed higher amounts of cellulases than the parent strain. Nonsaturating concentrations of sophorose efficiently induced cellobiohydrolase I formation in the bgl1-multicopy strain, but less efficiently in the bgl1-disrupted strain. The multicopy strain and the parent strain were comparably efficient at saturating sophorose concentrations. The β-glucosidase inhibitor nojirimycin strongly inhibited induction in all strains. These data suggest that the bgl1-encoded β-glucosidase is not identical to the plasma-membrane-bound, constitutive, methyl-β-glucoside inducible β-glucosidase, but represents an extracellular cellulose-induced enzyme. Both enzymes contribute to rapid induction of cellulases by modifying the inducer sophorose.     

Synthesis and localization of α-amylase and α-glucosidase in Bacillus licheniformis grown in batch and continuous culture

In batch and continuous cultures of Bacillus licheniformis NC1B 6346 α-amylase was invariably extracellular and could not be detected in the cytoplasm or cell surface. α-Glucosidase however, was largely intracellular but at the end of exponential growth and during slow growth under Mg²⁺ limitation it was detected in the culture fluid. Both enzymes were susceptible to catabolite repression and glucose totally inhibited their synthesis in batch culture. In maltose-limited chemostat culture, synthesis of both enzymes was maximal at D = 0.2/h and declined at higher growth rates. α-Amylase synthesis was constitutive but α-glucosidase synthesis was induced by maltose and maltotriose but not by methyl-α-D-glucoside or phenyl-α-D-glucoside. α-Amylase was synthesized at pH 6.5 and above in maltose-limited chemostat culture but not below this pH. Intracellular α-glucosidase synthesis varied little with pH. Increasing temperature decreased the synthesis of both enzymes in chemostat culture to the extent that α-glucosidase was undetectable at 50° C. Polar lipid composition varied with pH and temperature but there was no correlation between this and enzyme secretion. Moreover cerulenin, an antibiotic that inhibits protein secretion in some bacteria by interacting with the membrane had no effect on α-amylase secretion but decreased the release of α-glucosidase upon protoplast formation.     

Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3-O-methylglucoside. Cellodextrins (C₂ to C₆) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [³H]cellobiose, [³H]sophorose, and [¹⁴C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3-O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [³H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.     

Cellulase secretion by immobilized protoplasts of Trichoderma reesei

Protoplasts from Trichoderma reesei were immobilized in alginate and induced to produce cellulase (endoglucanase and β-glucosidase) enzymes. The specific activities of the synthesized enzymes were higher in immobilized protoplasts than in both free and immobilized mycelia. Immobilized protoplasts show an enhanced rate of exocellular β-glucosidase production compared to intact mycelia due to the lack of cell wall. The ratio of the exocellular/intracellular β-glucosidase was 5.9 for immobilized protoplasts and 0.32 for free mycelia.     

Production of a xylose-stimulated β-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of β-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 ± 411.2 U g(-1), while β-glucosidase production was increased about 2.6-fold, reaching 20.7 ± 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis β-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for β-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The β-glucosidase maintained about 95 % of its activity after 26 h in water at 55 °C, with half-lives of 15.7 h at 60 °C and 5.1 h at 65 °C. The presence of xylose during heat treatment at 65 °C protected β-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 °C. Xylose stimulated β-glucosidase activity up to 1.7-fold, at 200 mmol L(-1). The notable features of both xylanase and β-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.     

Induction of β-Glucosidase in Botrytis cinerea by Cell Wall Fractions of the Host Plant

The pathogenicity of Botrytis cinerea has been found to correlate positively with the β-glucosidase activity. In this report, the relationship between the induction of β-glucosidase and the components of host plant tissues was studied by the use of tissue fractions and cellulose-related compounds.

The most active enzyme induced by the crude fiber fraction and Avicel was β-glucosidase, among the cell wall degrading enzymes tested. The β-glucosidase was very inducible in the strains with strong pathogenicity, and intensively degraded the fiber fraction made from apple fruit tissues. The same degradation of the cell wall fraction was demonstrated with the purified enzyme.     

Isolation and some properties of two extracellular β-glucosidases from Trichosporon adeninovorans

The yeast Trichosporon adeninovorans secretes two multiple forms of β-glucosidase at a high rate if grown in a medium containing cellobiose. Following mutagenesis a mutant strain resistant to 2-deoxy-D-glucose was selected. This strain produced more β-glucosidase activity and had acquired a strong resistance against repression by glucose. The β-glucosidases were separated one from each other by chromatography on hydroxylapatite and by gel filtration. Both enzymes have similar properties. The optimal temperature for their activity was 60 to 63°C and the enzymes displayed highest activity at pH of 4.5. The molecular weight of β-glucosidase I was found to be 570,000 and that for β-glucosidase II was 525,000. The K_m value for cellobiose was determined to be 4.1 mM for β-glucosidase I and 3.0 mM for β-glucosidase II.     

Utilization of cellobiose and production of β-clucosidase by nine yeast species

A comparative study of 9 yeasts namely, Candida blankii,C.humicola,C.ishiwadae,C.rhagii,C.tropicalis,Hensenula subpelliculosa,Saccharomyces cerevisiae,Trichosporon cutaneum and Tr.pullulans was carried out for the production of extracellular and cell bound β-glucosidase using cellobiose as the substrate. Trichosporon cutaneum was found to be the best extracellular as well as cell bound β-clucosidase producer and the former activity was more than the latter. In the rest of the yeasts most of them showed more cell bound β-glucosidase as compared to the extracellular.     

Effects of the addition of laminaran on β-glucosidase production by Trichoderma viride

β-Glucosidase production by Trichoderma viride WU-36B was studied in media containing laminaran. By the addition of laminaran to the medium containing glycerol as a carbon source, extracellular activities of β-glucosidase and β-1,3-glucanase increased but CMCase activity was not detected during whole culture period. Extracellular activities of β-glucosidase, CMCase, and β-1,3-gluconase were higher in the medium containing both avicel and laminaran than in the media containing avicel or laminaran as a sole carbon source.     

I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates

Cultured skin fibroblasts from a patient with I-Cell disease (mucolipidosis II) were assayed for a number of lysosomal enzymes using both natural and synthetic substrates. The cells from this patient were found to have very low activity for galactosylceramide β-galactosidase, lactosylceramide β-galactosidases (using two assay methods that measure different enzymes), G_M1 ganglioside β-galactosidase and sphingomyelinase. Glucosylceramide β-glucosidase activity was found to be normal. Acid hydrolase activities toward many synthetic substrate were measured and all except β-glucosidase and acid phosphatase were found to be extremely low (as has been reported by others). Acid phosphatase and β-glucosidase were in the low normal range. These studies expand on previously published reports on I-Cell disease that only present data from synthetic substrates, and also report the fibroblast culture deficiencies of galactosyl-ceramide β-galactosidase (the Krabbe disease enzyme) and sphingomyelinase (the Niemann-Pick disease enzyme) activities for the first time. Those two enzymes do not have a readily available synthetic analog to assay. Acid β-galactosidase activity measured with both the 4-methylumbelliferyl derivative and G_M1 ganglioside was partially deficient in leukocytes prepared from this patient. New methods for measuring 4-methylumbelliferyl-β-D-glucoside and glucosylceramide β-glucosidase activities are also presented.     

Regular enzyme recovery enhances cellulase production by <Emphasis Type="Italic">Trichoderma reesei</Emphasis> in fed-batch culture

Objective

To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals.

Results

Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24–62%) and β-glucosidase (22–76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and β-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5–4.5 g l^?1.

Conclusion

Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

     

A novel high molecular weight thermo-acidoactive β-glucosidase from <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

An extracellular high molecular weight β-glucosidase was secreted by a local strain P1 of Beauveria bassiana. The enzyme was produced in the presence of various carbon sources, namely glucose, maltose, lactose, glycerol, starch, wheat bran and gruel. The highest level of β-glucosidase activity was produced with wheat bran at the concentration of 3%. Glucose caused a repressor effect on the β-glucosidase expression in a dose-dependent manner. The highest enzyme production level was obtained at initial pH of 6.0 and 7.0 in the culture medium. The zymography analysis revealed that B. bassiana secreted a β-glucosidase with high molecular weight between 400 and 600 kDa. The enzymatic preparation was characterized and showed temperature and pH optima of 55°C and 5.0, respectively. The enzyme was stable at 40 and 50°C but its stability declined at 60°C. Interestingly, this β-glucosidase had high stability at acid and basic pH saving its initial activity after 24 h incubation at pH from 3.0 to 11.0. It was stable also in presence of monovalent Na⁺ and K⁺ ions saving 60% of its initial activity at 2 M salts. Bivalent metal ions preserved totally or partially the enzymatic activity; in addition, Ba²⁺ was revealed as an activator. This is the first report that focuses on the production and the biochemical characterization of a β-glucosidase from the entomopathogenic fungus, B. bassiana.     

The uptake and degradation of sheep thyroglobulin by macrophages in vitro.

The activities of seven lysosomal and three mitochondrial enzymes from isolated lysosomes and mitochondria of cultivated lymphoid cell lines, obtained from 3 patients with leukemia and from 6 normal individuals, were investigated. The lysosomal enzymes included: α-glucosidase, β-glucosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, aryl sulfatase and acid phosphatase. These enzymes are involved in the degradation of glycoprotein, glycolipids, mucopolysaccharide-protein complexes, polysaccharides, mucopolysaccharides, organic sulfates and phosphoric esters. In the mitochondrial fraction, glutamic, succinic and malic dehydrogenases were studied. The range of lysosomal enzyme activities obtained from cell lines of leukemic origin was found to be consistently higher than in the normal controls [200 % (aryl sulfatase) to 732% (β-glucosidase)]. The mitochondrial enzyme activities showed only slight differences between the leukemic and control cell lines. This study demonstrates that the lysosomal functions of lymphoid cells derived from patients with acute lymphoblastic leukemia are fundamentally different from those from healthy donors.     

Enhancement of <Emphasis Type="Italic">Penicillium echinulatum</Emphasis> glycoside hydrolase enzyme complex

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.     

Activity of cell wall-associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β-glucosidase (EC 3.2.1.21) α-galactosidase (EC 3.2.1.22). β-galactosidase (EC 3.2.1.23). α-arabinosidase (EC 3.2.1.55), α-mannosidase (EC 3.2.1,24). β-xylosidase (EC 3.2.1.37) and β-N-acetylglucosamidase (EC 3.2.1.30). as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer-soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-hound glycosidase During ripening there was a marked change in the percentages of covalently- and tonically linked activities of β-glucosidase and β-galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black-ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.     

Purification, characterization, and properties of beta-glucosidase enzymes from Sclerotium rolfsii

Four β-glucosidase enzymes were extensively purified from the culture filtrates of Sclerotium rolfsii and some of their physicochemical properties studied. All the enzymes showed a single protein band in sodium dodecyl sulfate-gel electrophoresis and in disc gel electrophoresis at pH 8.9 and 4.3. The purified β-glucosidases were free of endoglucanase (carboxymethyl cellulose viscosity-lowering activity). All the enzymes are glycoproteins and are composed of one polypeptide chain. The molecular weight of the four β-glucosidases varies between 90,000 and 107,000. The pH and temperature optima of the four β-glucosidases are 4.2 and 68 °C with p-nitrophenyl-β-d-glucoside and 4.5 and 65 °C with cellobiose as substrate. The isoelectric points for the enzymes are 4.10, 4.55, 5.10, and 5.55, respectively. The specific activities of the enzymes with cellobiose as substrate are 55, 78, 175, and 51 μmol glucose released per minute per milligram protein, respectively. The enzymes are inhibited by the reaction product glucose, and by glucono-δ-lactone and nojirimycin. A carboxylate group is implicated in the catalysis of β-glucosidase.