Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

Summary The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellobiose dehydrogenase remain to be elucidated.     

Degradation of cellulosic materials by Sporotrichum thermophile culture filtrate for sugar production

Sporotrichum thermophile Apinis, was the most active carboxymethyl-cellulose (CMC)-ase producer among seven thermophilic and four thermotolerant fungal species isolated from Egyptian soil and screened for their ability to produce extracellular cellulase in culture media containing CMC as a sole carbon source. The fungus also efficiently hydrolysed filter paper cellulose. Comparison of various untreated and alkali-treated cellulosic and lignocellulosic materials as substrates for cellulase production by S. thermophile revealed the most easily degraded substrate was sugarcane bagasse at 2% concentration. This substrate when alkali treated was the most susceptible to enzymic hydrolysis by culture filtrates of S. thermophile grown on untreated bagasse. Optimum hydrolysis was obtained after 18 h incubation with the filtrate at pH 3·5–4 and 45°C. Alkali treatment of bagasse reduced its lignin content significantly and the culture filtrate of S. thermophile grown on untreated bagasse was found to contain xylanase and polygalacturonase in addition to cellulase and cellobiase.     

Isolation, purification and regeneration of protoplasts from Sporotrichum thermophile conidiospores

Protoplasts of uniform size were prepared from mononucleated conidiospores of Sporotrichum thermophile. Conidia were preincubated in glucose yeast extract medium at 45 C for 4 h. The conidia were collected resuspended in buffer containing 0.6 M KCl (as stabilizer), and incubated with Novozyme SP249 and Cellulase CP at 37 C for 6 h. The protoplasts were separated from cell wall fragments and intact conidia by centrifugation over 50% sucrose. The purified protoplasts were regenerated in glucose yeast extract broth after 7 h of incubation at 45 C.     

Xylanase,CM-cellulase and Avicelase production by the thermophilic fungus Sporotrichum thermophile

Biotechnology Letters - When wheat straw was used as carbon source, Sporotrichum thermophile produced large amounts of xylanase extracellularly in addition to CM-cellulase and Avicelase. These...     

Cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile.

Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE-Trisacryl chromatography and named cellobiose dehydrogenase I and II have been purified to homogeneity by different chromatographic techniques. Both enzymes are slightly glycosylated flavocytochrome-b proteins with similar catalytic properties but with distinct molecular masses (91 kDa and 192 kDa for enzymes I and II, respectively) and isoelectric point (4.1 versus 3.45). Examination by SDS/PAGE clearly showed that the larger enzyme II is a homodimer, whose subunit is close to, but different from dehydrogenase I which is homogeneous by this technique. After limited digestion of both enzymes with papain, two main fractions with residual activity are formed, one carrying the heme, the other being the flavin component; each fraction is characterized by its particular chromatographic behaviour. The flavin carrying component shows an atypical (for flavoprotein) three-banded spectrum indicative of the presence of a flavin derivative. Both enzymes react very slowly with oxygen clearly forming some superoxide radicals and possibly hydrogen peroxide. Cellobiose and other cellodextrins are oxidized at their reducing glycosyl moiety to the corresponding aldonic acid. With the use of the autooxidable phenazinemethosulphate, cellulose (either in a hydrated form or crystalline) is also oxidized at free reducing ends so that appreciable amounts of cellobionic acid are released upon enzymatic hydrolysis.     

Production of cellulolytic enzymes by immobilized Sporotrichum thermophile.

Spores of Sporotrichum thermophile were immobilized in agar, polyacrylamide, and sodium alginate to generate in situ mycelium for production of cellulolytic enzymes. Immobilized mycelium was considerably less effective than free cells for cellulase productivity. Of the three gel types, agar beads proved to be the best carrier for the immobilized spores and subsequently generated mycelium. Results of repeated batch experiments suggested that the immobilized mycelia could be reused but at much reduced efficiency.     

Thermal stability characteristics of cellulase enzymes produced by Sporotrichum thermophile

Summary The thermal stability characteristics of the cellulase enzymes present in culture filtrates of the thermophilic fungus Sporotrichum thermophile were investigated at different temperatures and at different times of exposure. Maximum enzymic activities under assay conditions were found at 68°C for the filter paper activity (FPA) and the Cx activity (carboxymethylcellulose), while the maxima for the C₁ activity (cotton) and -glucosidase activity (cellobiose) were found to be at 55°C and 72°C respectively. Culture filtrates were exposed to a given constant temperature for varying lengths of time to a maximum of 48 hrs. and then analyzed for residual enzymic activities under assay conditions. The exposure temperatures studied were 50°C, 60°C and 65°C. After 48 hrs. exposure time at 50°C the residual activities for the FPA, Cx and -glucosidase were found to be 88%, 98% and 93% of the original activities respectively.     

Factors influencing the production of cellulases by Sporotrichum thermophile.

Cellulase production and growth of a strain of Sporotrichum thermophile were studied by using a mineral salts medium supplemented with yeast extract and insoluble cellulose. The effects of cultural conditions, such as pH, nitrogen source, substrate concentration, and temperature, were examined. Maximum production of C1 and CX cellulases occurred at 45 C in 2 to 4 days, in the presence of 1% Solka/Floc as substrate, when NaNO3 or urea used as sources of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appeared to be similar to that reported for Trichoderma viride grown in a favorable environment. However, comparable yields of cellulase were produced by S. thermophile in less than one-quarter the time required by mesophilic fungi.     

Factors influencing the production of cellulases by Sporotrichum thermophile.

Cellulase production and growth of a strain of Sporotrichum thermophile were studied by using a mineral salts medium supplemented with yeast extract and insoluble cellulose. The effects of cultural conditions, such as pH, nitrogen source, substrate concentration, and temperature, were examined. Maximum production of C1 and CX cellulases occurred at 45 C in 2 to 4 days, in the presence of 1% Solka/Floc as substrate, when NaNO3 or urea used as sources of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appeared to be similar to that reported for Trichoderma viride grown in a favorable environment. However, comparable yields of cellulase were produced by S. thermophile in less than one-quarter the time required by mesophilic fungi.     

Purification,characterization and mass spectrometric identification of two thermophilic xylanases from Sporotrichum thermophile

Two xylanases were purified to electrophoretic homogeneity from the thermophilic fungus Sporotrichum thermophile grown in a submerged liquid culture using wheat straw as carbon source. The enzymes, StXyn1 and StXyn2, have molecular masses of 24 kDa and 48 kDa, respectively, and are optimally active at pH 5 and at 60 °C. Both enzymes displayed remarkable stability up to 50 °C for 1 h, exhibiting a half-life of 60 min (StXyn1) and 115 min (StXyn2) at 60 °C. Biochemical characterization of the two xylanases against poly- and oligosaccharides indicated that StXyn1 and StXyn2 hydrolytic profiles match those of xylanase family 11 and family 10, respectively. LC–MS/MS analysis provided peptide mass and sequence information that assisted the identification of the corresponding xylanase genes from the S. thermophile genome and the classification of the two purified StXyn1 and StXyn2 as a family GH11 and GH10 endo-1,4-β-xylanases, respectively.     

Production and partial characterization of xylanase by Sporotrichum thermophile under solid-state fermentation

A number of factors affecting production of xylanase, by the thermophilic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimized. Solid state fermentation in a laboratory horizontal bioreactor using the optimized medium allowed the production of 320 U g^–1 of carbon source which compared favourably with those reported for other microorganisms. Optimal xylanase activity was observed at pH 5 and 70 °C. Chromogenic (fluorogenic) 4-methylumbelliferyl -glycoside of xylobiose (MUX₂) was used to characterize the xylanase multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major xylanase fraction exhibiting pI and molecular mass values 4 and 90–120 kDa, respectively.     

Cloning and characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. CDH contains one heme b and one FAD per molecule and oxidizes cellobiose to cellobionolactone in the presence of cytochrome c. In this report, a thermostable CDH from the thermophilic ascomycete Sporotrichum thermophile has been purified, cloned, and characterized. The temperature optimum for this CDH reaction was 60 degrees C, and the activation energy for the reaction was 26.3 kJ/mol. The Km and kcat were temperature-dependent and increased as reaction temperature increased. These kinetic properties prove that this CDH is truly thermophilic. A 2.8-kb cDNA was isolated by screening an expression library of S. thermophile with a polyclonal antisera raised against Phanerochaete chrysosporium CDH. The cDNA encoded an 807-amino-acid protein with a predicted mass of 86,332 Da. S. thermophile CDH is organized into three domains, an N-terminal flavin domain, a middle heme domain, and a C-terminal cellulose-binding domain, which shows sequence similarity with the cellulose-binding domains of endoglucanases and cellobiohydrolases from Trichoderma reesei. Comparison with the CDH sequences of P. chrysosporium and Trametes versicolor identified Met 95 and His 143 as potential heme coordinations. EFIG, LGGPM, and VNSTH motifs in the heme domain and the XRXPXTDXPSXDGXRY motif in the flavin domain were identified as CDH-specific motifs. With regard to the amino acid composition, S. thermophile CDH has more disulfide linkages and acidic and basic amino acids compared to CDHs from P. chrysosporium and T. versicolor.     

Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile

The cellulolytic system of the thermophilic fungus Sporotrichum thermophile contains a recently discovered esterase that may hydrolyze the ester linkage between the 4- O -methyl- d -glucuronic acid of glucuronoxylan and lignin alcohols. The glucuronoyl esterase named St GE1 was purified to homogeneity with a molecular mass of M _r 58 kDa and pI 6.7. The enzyme activity was optimal at pH 6.0 and 60 °C. The esterase displayed a narrow pH range stability at pH 8.0 and retained 50% of its activity after 430 and 286 min at 50 and 55 °C, respectively. The enzyme was active on substrates containing glucuronic acid methyl ester, showing a lower catalytic efficiency on 4-nitrophenyl 2- O -(methyl-4- O -methyl-α- d -glucopyranosyluronate)-β- d -xylopyranoside than its mesophilic counterparts reported in the literature, which is typical of thermophilic enzymes. St GE1 was proved to be a modular enzyme containing a noncatalytic carbohydrate-binding module. LC-MS/MS analysis provided peptide mass and sequence information that facilitated the identification and classification of St GE1 as a family 15 glucuronoyl esterase that showed the highest homology with the hypothetical glucuronoyl esterase CHGG_10774 of Chaetomium globosum CBS 148.51. This work represents the first example of the purification and identification of a thermophilic glucuronoyl esterase from S. thermophile .     

Physical and chemical mutagens improved Sporotrichum thermophile,strain ST20 for enhanced Phytase activity

ObjectivePhosphorous is an essential micronutrient of plants and involved in critical biological functions. In nature, phosphorous is mostly present in immobilized inorganic mineral and in the fixed organic form including phytic acid and phosphoesteric compounds. However, the bioavailability of bound phosphorous could be enhanced by the use of phosphate solubilizing microorganisms such as bacteria and fungi. The phytases are widespread in an environment and have been isolated from different sources comprising bacteria and fungi.MethodologyIn current studies, we show the successful use of gamma rays and EMS (Ethyl Methane Sulphonate) mutagenesis for enhanced activity of phytases in a fungal strain Sporotrichum thermophile.ResultsWe report an improved strain ST2 that could produce a clear halo zone around the colony, up to 24 mm. The maximum enzymatic activity was found of 382 U/mL on pH 5.5. However, the phytase activity was improved to 387 U/ml at 45 °C. We also report that the mutants produced through EMS showed the greater potential for phytase production.ConclusionThe current study highlights the potential of EMS mutagenesis for strain improvement over physical mutagens.     

Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis

The production of polygalacturonase (PGase) by Sporotrichum thermophile Apinis in stirred submerged fermentation (SmF) was high in comparison with that in static conditions. Yeast extract (0.25%) and citrus pectin (2%) at pH 7.0 and 45 degrees C supported a high enzyme production in flasks agitated at 200 rpm. An overall 1.75-fold enhancement in PGase production was achieved as a result of optimization. The enzyme was optimally active at pH 7.0 and 55 degrees C, and exhibited t(1/2) of 4 h at 65 degrees C. The Km and Vmax values of the enzyme (for pectin) were 0.416 mg ml(-1) and 0.52 micromol mg(-1)min(-1), respectively. The PGase activity was stimulated by Mn(2+) and Fe(2+), but inhibited strongly by Mg(2+), and slightly by Tween 80 and Triton X-100. Among the additives tested, beta-mercaptoethanol exerted a strong inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. An increase in the yield of banana, grape and apple juices was recorded due to the treatment of fruit pulps with the mixture of enzymes (pectinase, xylanases and cellulase) of S. thermophile as compared to that with only pectinase. The yield of fruit juices did not increase with enhanced titre of cellulase in the enzyme mixture.     

Calorimetric versus Growth Microbial Analysis of Cellulase Enzymes Acting on Cellulose

Assay of cellulase enzymology on cellulose was investigated by two methods: (i) plate colony counting to determine microbial growth and (ii) microbial calorimetry. These methods were chosen because they accept raw samples and have the potential to be far more specific than spectrophotometric reducing sugar assays. Microbial calorimetry requires ca. 0.5 to 1 h and 10 to 100 μM concentrations of cellulolytic lower sugars (glucose and cellobiose). Growth assay (liquid culture, plating, colony counting) requires 15 to 20 h and ca. 0.5 mM sugars. Microbial calorimetry requires simply aerobic metabolism, whereas growth assay requires completion of the cell cycle. A stripping technique is described for use in conjunction with the calorimetric method to enable separate analysis of the two sugars. Mixtures of glucose and cellobiose are equilibrated with Escherichia coli and spun out to remove glucose. The supernatant is calorimetrically combusted with Klebsiella sp. to quantitate cellobiose, and the same organism combusting the nonstripped mixture gives heat proportional to the sum of the two sugars. Calorimetry of cellulolysis products from individual exo- and endocellulases, and from their reconstituted mixture, was carried out to develop a microbial calorimetric means for demonstrating enzyme synergism.     

Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency

Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.     

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.     

Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential

Myceliophthora thermophila syn. Sporotrichum thermophile is a ubiquitous thermophilic mould with a strong ability to degrade organic matter during optimal growth at 45?°C. Both genome analysis and experimental data have suggested that the mould is capable of hydrolyzing all major polysaccharides found in biomass. The mould is able to secrete a large number of hydrolytic enzymes (cellulases, laccases, xylanases, pectinases, lipases, phytases and some other miscellaneous enzymes) employed in various biotechnological applications. Characterization of the biomass-hydrolyzing activity of wild and recombinant enzymes suggests that this mould is highly efficient in biomass decomposition at both moderate and high temperatures. The native enzymes produced by the mould are more efficient in activity than their mesophilic counterparts beside their low enzyme titers. The mould is able to synthesize various biomolecules, which are used in multifarious applications. Genome sequence data of M. thermophila also supported the physiological data. This review describes the biotechnological potential of thermophilic mould, M. thermophila supported by genomic and experimental evidences.