Phytase production by a thermophilic mould Sporotrichum thermophile in solid state fermentation and its potential applications

Phytase production by a thermophilic mould Sporotrichum thermophile Apinis was investigated in solid state fermentation (SSF) using sesame oil cake as the substrate. Scanning electron microscopy of the fermented sesame oil cake revealed a dense growth of the mould with abundant conidia. Glucose, ammonium sulphate and incubation period were identified as the most significant factors by Plackett-Burman design. The optimum values of the critical components determined by central composite design of response surface methodology for the maximum phytase production were glucose 3%, ammonium sulphate 0.5% and incubation period 120 h. An overall 2.6-fold improvement in phytase production was achieved due to optimization. Highest enzyme production (348.76 U/g DMR) was attained at a substrate bed depth of 1.5 cm in enamel coated metallic trays. The enzyme liberated inorganic phosphate from wheat flour and soymilk with concomitant dephytinization and liberation of soluble inorganic phosphate.     

Improved phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation due to statistical optimization

Culture variables affecting phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation were optimized. Soluble starch, peptone, Tween-80 and sodium phytate were identified by Plackett-Burman design as the most significant factors to affect phytase production. The 2(4) full factorial central composite design of response surface methodology was applied for optimizing the concentrations of the significant variables and to delineate their interactions. Starch, Tween-80, peptone and sodium phytate at 0.4%, 1.0%, 0.3% and 0.3% supported maximum enzyme titres, respectively. An overall 3.73-fold improvement in phytase production was achieved due to optimization. When sodium phytate was substituted with wheat bran (3%), the phytase titre in the former was comparable with that in the latter.     

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.     

Optimization of Xylanase Production by Sporotrichum thermophile Using Corn Cobs and Response Surface Methodology

A 3² central composite experimental design was performed with the aim of optimizing the production of xylanase by Sporotrichum thermophile grown on corn cobs in submerged cultures. Various carbon and nitrogen sources were consecutively optimized, and corn cobs and ammonium phosphate concentrations were selected as substrates to test the effect of two variables, i.e., both substrate concentrations, on xylanase production. A second‐order quadratic model and a response surface method showed that the optimum conditions for xylanase production were 2.7 % [w/v] corn cobs and 0.7 % [w/v] ammonium phosphate. These optimum conditions were transferred to 7 L bioreactors, where activities as high as 56 U/mL were obtained.     

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.     

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 .     

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.     

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.     

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.     

A marked enhancement in phytase production by a thermophilic mould Sporotrichum thermophile using statistical designs in a cost-effective cane molasses medium

AIMS: Statistical optimization of phytase production by a thermophilic mould Sporotrichum thermophile in a cost-effective cane molasses medium. METHODS AND RESULTS: Sporotrichum thermophile secreted phytase in cane molasses medium at 45 degrees C and 250 rev min(-1) after 5 days. The important factors identified by Plackett-Burman design (magnesium sulfate, Tween 80, ammonium sulfate and incubation period) were further optimized by response surface methodology (RSM). An overall 107% improvement in phytase production was achieved due to optimization. Supplementation of the medium with inorganic phosphate repressed the enzyme synthesis. When inorganic phosphate was reduced from the cane molasses medium by treatment with calcium chloride, the enzyme production increased. The phytase activity was not affected by the enzyme treatment with trypsin and pepsin. CONCLUSIONS: A twofold increase in phytase production was achieved due to optimization using statistical designs in a cost-effective cane molasses medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase production was doubled due to optimization. The enzyme, being resistant to trypsin and pepsin, thermostable and acid stable, can find application in animal feed industry for improving nutritional status of the feed and combating environmental phosphorus pollution.     

Phytase production by the thermophilic fungus Rhizomucor pusillus

A thermophilic fungus, Rhizomucor pusillus, isolated from composting soil, was studied for phytase production using solid-state fermentation. The optimization of phytase production was carried out by Box–Behnken design of experiments, using three independent variables (pH of medium, culture age and incubation period), resulting in a maximal level of phytase (9.18 units/g substrate). The partially purified phytase was optimally active at 70 °C and pH 5.4, though the enzyme showed 80% activity over a wide pH range, 3.0–8.0. The phytase was found to have broad substrate specificity.     

Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysosporium) thermophile Apinis

Summary Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture. Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%–100%. The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium. The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads. Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium. A minimum of 8 h regeneration period was necessary for lipase synthesis. Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts. Tween 80 enhanced lipase activity of the immobilized protoplasts. Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion. Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity.     

Sporotrichum thermophile Growth, Cellulose Degradation, and Cellulase Activity

The activity of components of the extracellular cellulase system of the thermophilic fungus Sporotrichum thermophile showed appreciable differences between strains; β-glucosidase (EC 3.2.1.21) was the most variable component. Although its endoglucanase (EC 3.2.1.4) and exoglucanase (EC 3.2.1.91) activities were markedly lower, S. thermophile degraded cellulose faster than Trichoderma reesei. The production of β-glucosidase lagged behind that of endoglucanase and exoglucanase. The latter activities were produced during active growth. When growth was inhibited by cycloheximide treatment, the hydrolysis of cellulose was lower than in the control in spite of the presence of both endoglucanase and exoglucanase activities in the culture medium. Degradation of cellulose was a growth-associated process, with cellulase preparations hydrolyzing cellulose only to a limited extent. The growth rate and cell density of S. thermophile were similar in media containing cellulose or glucose. A distinctive feature of fungal development in media incorporating cellulose or lactose (inducers of cellulase activity) was the rapid differentiation of reproductive units and autolysis of hyphal cells to liberate propagules which were capable of renewing growth immediately.     

Characterization of a cellobiose dehydrogenase in the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile.

An extracellular enzyme from culture filtrates of Sporotrichum (Chrysosporium) thermophile (A.T.C.C. 42 464) after growth on cellulose or cellobiose was shown to oxidize cellobiose to cellobionic acid in vitro. Lactose and cellodextrins were also efficiently oxidized, but the enzyme was not active against most mono- and di-saccharides. Several redox substances could act as electron acceptors, but molecular oxygen, tetrazolium salts and NAD(P) were not reduced. Activity was stimulated up to 2-fold in the presence of 0.05 M-Mg2+. The pH optimum of the enzymic reaction was acidic when the activity was tested with dichlorophenol-indophenol or Methylene Blue, but was neutral to alkaline for 3,5-di-t-butyl-1,2-benzoquinone or phenazine methosulphate as electron acceptors. As the enzyme was formed inductively in parallel with the endocellulase, its possible function in relation to cellulolysis is discussed.     

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.     

Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus

The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h^–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH₄Cl 600 mg l^–1) to Regime III (NH₄CI 1200 mg l^–1), whereas production of protease was highest in Regime II (600 mg l^–1 < NH₄Cl <1200 mg l^–1).     

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.