Production and localization of cellulases and β-glucosidase from the thermophilic fungusThielavia terrestris

Summary The enzyme production and localization ofThielavia terrestris strains C464 and NRRL 8126 were compared to determine their optimum temperature and pH for cellulase activity. High levels of intracellular -glucosidase activity were detected in the former strain. The intracellular -glucosidase of both strains were more thermostable than the extracellular enzyme; the half life ofT.terrestris (C464) endoglucanase activity at 60°C was greater than 96 hrs.     

Production of cellulase and β-glucosidase activities following growth of Streptomyces hygroscopicus on cellulose containing media

The actinomycete, Streptomyces hygroscopicus was shown to be capable of producing extracellular cellulase and cell associated -glucosidase activity during growth on cellulose containing media. Cell homogenates were shown to contain a -glucosidase fraction which was stable for up to 24h. at 30°C and had half-lives of 480min. and 220min. at 40 and 50°C, respectively. The enzyme fraction was also shown to be optimally active at pH 4.0 suggesting that it might represent a suitable supplement for fungal cellulase systems, deficient in -glucosidase activity.     

Continuous glucose production using encapsulated mycelial-associated β-glucosidase from Trichoderma E58

Summary Cellobiose and salicin were continuously hydrolysed in a packed bed reactor containing Trichoderma sp. E-58, encapsulated in calcium alginate beads. Continuous -glucosidase activities were measured using inlet substrate concentrations of 10 mM and 35 mM for cellobiose and salicin respectively. Maximal activities achieved using the described immobilization procedure were between 70 and 90 moles substrate reacted per minute per liter of bead volume. Immobilized mycelial-associated -glucosidase activity was shown to have a half-life of greater than 1000 hours when operating continuously at 50°C.     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

Production and characterization of cellulolytic and xylanolytic enzymes from the ligninolytic white-rot fungus Phanerochaete chrysosporium grown on sugarcane bagasse

The ligninolytic white-rot fungus Phanerochaete chrysosporium BKM-F-1767 produced extracellular cellulolytic enzymes (carboxymethylcellulase, CMCase and -glucosidase) and xylanolytic enzymes (xylanase and -xylosidase) in liquid medium containing 1.0% sugarcane bagasse with or without 1.0% glucose. The changes in pH and soluble protein content were monitored in the culture filtrates. The results obtained showed that the pH decreased after 3 days and then increased. The soluble protein content increased and reached the maximum value after 12 days. The results showed that the activities of enzymes were higher in the case of sugarcane bagasse without glucose. The characterization study indicated that the optimum pH values were 4.6, 4.2, 5.0 and 5.0 for CMCase, -glucosidase, xylanase and -xylosidase, respectively and the optimum temperatures were 60, 70, 65 and 60 °C for the investigated enzymes, respectively. The results showed also that after prolonged heating (5 h) at 60 °C, CMCase, -glucosidase, xylanase and -xylosidase retained 81.2, 86.8, 51.5 and 27.4% activity, respectively.     

Production,purification, and properties of β-glucosidase from Candida wickerhamii

Summary Candida wickerhamii growing on cellobiose produced -glucosidase with high activity against -nitrophenyl glucoside (PNPG) but low activity against cellobiose. -glucosidase production was constitutive, and was repressed by -glucosides and glucose. -glucosides containing an aromatic moiety in the aglycon were the best substrates for -glucosidase indicating that the enzyme is an aryl--glucosidase. A -glucosidase from C. wickerhamii cells was purified by (NH₄)₂SO₄ precipitation, dialysis, ion-exchange chromatography and gel filtration. The purified enzyme was homogeneous as shown by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme hydrolysed PNPG but not cellobiose. The K_m of the enzyme was 0.185 mM. Glucose inhibited the enzyme competitively and the K_i was 7.5 mM. The apparent molecular mass was 97,000. The optimum pH and temperature for enzyme activity were between pH 7 and 7.4 and 40°C respectively. At temperatures of 45°C and greater the enzyme was inactivated. The activation energy of the enzyme was 29.4 kJ · mol^-1.     

Production and properties ofβ-glucosidase byNeurospora sitophila

Growth at 25°C and pH 5.50 favour the production of-glucosidase. De-fatted oilseed flour and Tween 80 enhanced the production of-glucosidase, Lactose, gentibiose, gentibiose-acetate, laminarabiose and xylobiose induced-glucosidase activity. Precipitation of the culture filtrate with (NH₄)₂SO₄ resulted in 26-fold purification with 67% recovery. The optimum pH and temperature for activity were 5.0 to 5.4 and 55°C respectively. The enzyme was stable at 40°C with half-life at 12 h at 50°C. TheK _m andV _max for the hydrolysis ofp-nitrophenyl--d-glucoside at 40°C H 5.0 are 0.28mm and 0.60 U/mg protein, respectively.     

Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Two intracellular -glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for -glucosidase A and -glucosidase B, respectively. -Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (-glucosidase A, 29.2%; -glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for -glucosidases A and B, respectively. Both purified -glucosidases. especially -glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both -glucosidases were highly activated by CuCl₂, and inhibited by SnCl₂ and KMnO₄. Hg²⁺ and Ag⁺ also inhibited severely -glucosidase B. The K _m and V _max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2mol min^–1 mg^–1 for -glucosidase A and 2.76 mM and 143.5 mol min^–1 mg^–1 for -glucosidase B.     

The stromacentre inAvena plastids: an aggregation ofβ-glucosidase responsible for the activation of oat-leaf saponins

The stromacentre, a particular structure in the plastids of mostAvena species, was isolated from etioplasts ofAvena sativa and then characterized to determine its biological function. When comparing differentAvena species with or without stromacentre, it was shown that the stromacentre, a 63-kDa protein, and saponins (characteristic compounds ofAvena sativa) either occur together or not at all. This linkage was confirmed by demonstrating a transformation of saponins by the isolated stromacentre protein: avenacosides were hydrolyzed to 26-desgluco-avenacosides. Therefore, the stromacentre protein had to be regarded as a-glucosidase. Enzyme assays usingp-nitrophenyl--d-glucopyranoside as substrate showed that this-glucosidase has a pH optimum at pH 6.0. The calculatedK _m value for this substrate was 2.2·10^-3 M. Antibodies against the stromacentre protein inhibited-glucosidase activity. The determination of the molecular weight of the-glucosidase by sodium dodecyl sulfate-gel electrophoresis showed that it consists of subunits of 63 kDa. After gel electrophoresis under non-denaturing conditions, enzymatically active molecules were shown to consist of at least two of these subunits. Molecules aggregated up to about 10⁶ Da also had enzyme activity. Enzyme assays using avenacosides as substrate showed a pH optimum at pH 6.0. The calculatedK _m value for this substrate was 1.2·10^-5 M. The high affinity to the avenacosides and the high specificity for the C-26 bound glucose indicate that avenacosides are the natural substrates for this-glucosidase. Assuming that the avenacosides in oat leaves play a role as preformed chemical inhibitory substances against phytopathogenic microorganisms, a model is presented showing the stromacentre with a central role in activating the fungitoxicity of avenacosides.     

Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

Studies of Antarctic yeast for β-glucosidase production

Moss and lichen samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeasts. Six pure cultures were obtained. They were screened for -glucosidase production and two of them were selected. These were identified as Cryptococcus albidus AL₂ and C. albidus AL₃, according to their morphology, reproductive behaviour, and growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. These strains were examined for biosynthesis of -glucosidase on different carbon sources under aerobic conditions. High exocellular and endocellular activities were obtained when they were grown on cellobiose, methyl--D-glucopyranoside and salicin. The time course of growth and -glucosidase production of the yeast was examined by cultivation in a medium with cellobiose under aerobic conditions at temperatures 18 and 24 °C for 96 h. Cryptococcus albidus AL₂ and C. albidus AL₃ synthesized exocellular enzyme, respectively 58.33 and 55.83 U/ml and endocellular enzyme 137.75 and 205.34 U/ml at 24 °C for 72 h of the cultivation.     

Properties of theβ-glucosidase fromCellulomonas flavigena and fromEscherichia coli harboring the recombinant plasmid pJS3

Summary Plasmid-coded -glucosidase produced byEscherichia coli was characterized and compared to the enzyme produced byCellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl--d-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The -glucosidase was assayed for cellobiose andp-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50°C for the enzyme fromC. flavigena and at 37°C for that fromE. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37°C for both cell extracts. Most of the -glucosidase activity was intracellular. When cultures ofC. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of -glucosidase was increased considerably.E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. TheK _m values for cellobiose and PNPG indicated that the -glucosidase activity ofC. flavigena had a higher affinity for cellobiose as substrate, whereas the -glucosidase fromE. coli pJS3 showed higher affinity for PNPG.     

β-Glucosidase biosynthesis inThermomonospora curvata

Summary -Glucosidase biosynthesis was induced inThermomonospora curvata during early exponential growth on cellobiose, cellulose and protein-extracted lucerne fibre in mineral salts minimal medium at 53°C. Only about 3% of the total culture -glucosidase was found in cell-free fluid when the actinomycete was grown on cellobiose or purified cellulose. A variety of non-lytic agents including detergents, salts, proteinase and electroporation failed to release the cellbound enzyme. However, cells grown on the fibre released more than 50% of their -glucosidase. The maximal amount of extracellular accumulation was dependent on the initial concentration of fibre in the medium. Thermal instability at 53°C was the major cause for low exracellular -glucosidase activity in cellobiose- and cellulose-grown cultures. In cultures grown on the fibre, the extracellular enzyme was stabilized against thermal denaturation by the composition of the cell-free fluid, but was degraded by transient proteinase activity. Proteolysis decreased the average beta-glucosidase specific activity from about 460mU/mg extracellular protein to about 80mU/mg within 1 day after the appearance of the proteinase.

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Biosynthèse de -glucosidase chez Thermomonospora curvata

Résumé La biosynthèse de -glucosidase a été induite chezThermomonospora curvata pendant la phase exponentielle précoce de croissance sur cellobiose, sur cellulose et sur fibre de lucerne dont on avait extrait les protéines, en milieu minéral minimum à 53°C. A peine 3% de la -glucosidase totale dans la culture a été retrouvée dans le milieu acellulaire lorsque l'actinomyète a crû sur cellobiose ou sur cellulose purifiée. Une foule d'agents non-lytiques, comprenant des détergents, des sels, la protéinase et l'électroporation n'ont pas réussi à libérer l'enzyme liée aux cellules. Toutefois, les cellules développées sur la fibre ont libéré plus de 50% de leur -glucosidase. La quantité maximum d'accumulation extra-cellulaire a dépendu de la concentration initiale de fibre dans le milieu. L'instabilité thermique à 53°C a été la casue majeure des seuils faibles de -glucosidase extra-cellulaire dans les cultures sur cellobiose ou cellulose. Dans les cultures développées sur fibre, l'enzyme extra-cellulaire était stabilisée contre la dénaturation thermique par la composition du milieu acellulaire, mais elle était dégradée par l'activité transitoire de la protéinase. La protéolyse a décru l'activité spécifique moyenne de la -glucosidase d'environ 460 mU/mg de protéine extra-cellulaire à environ 80 mU/mg endéans la journée après l'apparition de la protéinase.

     

Evidence for the direct involvement ofβ-hemolysin inAeromonas hydrophila enteropathogenicity

Culture filtrates of 19 of 21 (90%) -hemolytic isolates ofAeromonas hydrophila caused fluid accumulation in permanently ligated rabbit ileal loops, whereas no fluid was accumulated with filtrates of eight non--hemolytic isolates. Antiserum to purified -hemolysin neutralized the ileal loop activity of culture filtrates from four of four -hemolytic isolates, and treatment at 56°C for 10 min eliminated the loop activity of six additional isolates. These results support the conclusion that -hemolysin alone causes significant changes in intestinal permeability and that it is a more common pathogenic mechanism than the heat-stable cytotonic enterotoxin. Electrophoretic and serological assays showed evidence for production of only one species of -hemolysin byA. hydrophila.     

Controlling simultaneous production of endoglucanase and beta-glucosidase by Fusarium oxysporum in submerged culture

Summary The simultaneous production of endoglucanase and -glucosidase by Fusarium oxysporum was investigated in submerged culture. Consecutive optimization of growth conditions resulted in the correction of large activity differences, observed during production of enzymes, and substantially enhanced low enzyme yields. At optimum growth conditions yields as high as 1650 and 232 U per g of carbon source of endoglucanase and -glucosidase were obtained respectively competing favourably with those reported for microorganisms grown on the same carbon source. The most important kinetic characteristics of the enzymes were the high temperature optima of endoglucanase (60°C) and -glucosidase (65°C) and the exceptionally high thermostability of endoglucanase. The latter enzyme retained 50% of the activity at pH 5.0 after approximately 6.5 h at 70°C     

Changes in carp myosin ATPase induced by temperature acclimation

Summary Myosins were isolated from dorsal ordinary muscles of carp acclimated to 10°C and 30°C for a minimum of 5 weeks and examined for their ATPase activities. Ca²⁺-ATPase activity was different between myosins from cold-and warm-acclimated carp, especially at KCl concentrations ranging from 0.1 to 0.2 M, when measured at pH 7.0. The highest activity was 0.32 mol P_i·min^-1·mg^-1 at 0.2 M KCl for cold-acclimated carp and 0.47 mol P_i·min^-1·mg^-1 at 0.1 M KCl for warm-acclimated fish. The pH-dependency of Ca²⁺-ATPase activity at 0.5 M KCl for both carp was, however, similar exhibiting two maxima around 0.3 mol P_i·min^-1·mg^-1 at pH 6 and 0.4 mol P_i·min^-1·mg^-1 at pH 9. K⁺(EDTA)-ATPase activity at pH 7.0 neither exhibited differences between both myosins. It increased with increasing KCl concentration showing the highest value of about 0.4 mol P_i·min^-1·mg^-1 at 0.6–0.7 M KCl. Actin-activated myosin Mg²⁺-ATPase activity was markedly different between cold-and warm-acclimated carp. The maximum initial velocity was 0.53 mol P_i·min^-1·mg^-1 myosin at pH 7.0 and 0.05 M KCl for cold-acclimated carp, which was 1.6 times as high as that for warm-acclimated carp. These differences were in good agreement with those obtained with myofibrillar Mg²⁺-ATPase activity between both carp. No differences were, however, observed in myosin affinity to actin. Differences in myosin properties between cold- and warm-acclimated carp were further evidenced by its thermal stability. The inactivation rate constant of myosin Ca²⁺-ATPase was 25·10^-4·s^-1 at 30°C and pH 7.0 for cold-acclimated carp, which was about 4 times as high as that for warm-acclimated carp. Light chain composition did not differ between both carp myosins. The differences in a primary structure of the heavy chain subunit was, however, clearly demonstrated between both myosins by peptide mapping.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5 dithio-bis-2-nitrobenzoic acid - DTT dithiothreitol - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Cellulase production byPenicillium janthinellum

The maximal carboxymethyl cellulase, filter paper (FP) cellulase and -glucosidase activities achieved byPenicillium janthinellum grown in a fermenter were 60, 5 and 9 U/ml, respectively. Enzymic hydrolysis of 5m NaOH-pre-treated straw, cotton and FP was 57 to 58% in 48 h at 50°C, with glucose as the major product.     

A comparison between the cellulase systems of Trichoderma harzianum E58 and Trichoderma reesei C30

Summary Nearly all of the filter paper, endoglucanase and -glucosidase activities of T. harzianum E58 were located extracellularly, with low amounts of these activities detected in the cell extracts and relatively little associated with the cell wall. Most of the filter paper and endoglucanase activities of T. reesei C30 were detected extracellularly. The half lives of the different cellulase activities were assayed at various temperatures over a period of time. When the pH of the filtrate was adjusted to 4.8, the cellulase activities were considerably enhanced, with the average half-life at 50°C extended to 25 hrs. When various lignocellulosic substrates were hydrolyzed by T. harzianum E58 cellulases approximately 90% of the reducing sugars were present as glucose while 50–60% of the reducing sugars were detected as glucose when T. reesei C30 cellulases were used.     

Cellobiose hydrolysis using glutaraldehyde-treated mycelia of Aspergillus terreus Thom

Summary The use of glutaraldehyde-treated mycelial pellets of Aspergillus terreus Thorn as a reusable form of -glucosidase was studied. The -glucosidase activity of these pellets has a pH optimum of 4.8 and as compared to untreated mycelia show decreased leakage of enzyme, higher Vmax, greater half-life at 65°C and increased reusability.     

Purification and properties of an extracellular β-glucosidase from the cellulolytic thermophile Clostridium stercorarium

Summary Clostridium stercorarium cultures grown on cellobiose contain both an extracellular and a cell-bound -glucosidase activity. A substantial portion of the cell-bound enzyme could be extracted by osmotic shock, suggesting a periplasmic localization. The -glucosidase present in culture supernatants was purified to homogeneity. It was found to be identical in all aspects tested with the cell-bound -glucosidase. The enzyme exists as a monomer with an apparent molecular weight of 85.000 (SDS-PAGE) and a pI of 4.8. It shows optimal activity as pH 5.5 and 65° C. Thiol groups are essential for enzyme activity. In the presence of reducing agents and divalent cations the half-life of the purified enzyme was more than 5 h at 60°C. The enzyme hydrolyses at different rates a wide range of substrates including aryl--glucosides, cellobiose, and disordered cellulose. K _m values were determined as 0.8 mM for p-nitrophenyl--glucoside (PNPG) and 33 mM for cellobiose. The cellular localization and the substrate specificity pattern are consistent with a dual role of the C. stercorarium -glucosidase in cellulose saccharification: (1) Cleavage of cellobiose formed by exoglucanase and (2) degradation of cellodextrins produced by endoglucanase action.