Culture variables affecting phytase production by a thermophilic mould Sporotrichum thermophile i... more 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.
The phytase production by Sporotrichum thermophile TLR50 was recorded on all the commonly used an... more The phytase production by Sporotrichum thermophile TLR50 was recorded on all the commonly used animal feed ingredients tested to varying degrees in solid-state fermentation. Enzyme production increased to 180 U/g of dry moldy residue (DMR) in sesame oil cake at 120 h and 45°C at the initial substrate-to-moisture ratio of 1∶2.5 and a w of 0.95. Supplemenetation of sesame oil cake with glucose and ammonium sulfate further enhanced phytase titer (282 U/g of DMR). An overall 76% enhancement in phytase production was achieved owing to optimization. The mold secreted acid phosphatase, amylase, xylanase, and lipase along with phytase. By the action of phytase, inorganic phosphate was liberated efficiently, leading to dephytinization of sesame oil cake.
Aims: Statistical optimization of phytase production by a thermophilic mould Sporotrichum thermo... more 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°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 of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorgan... more Phytase of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorganic phosphate from Ca+2, Mg+2, and Co+2 phytates more efficiently than those of Al3+, Fe2+, Fe3+, and Zn2+. The hydrolysis rate was higher at 60 °C as compared to 26 °C. Among all the organic acids tested, citrate was more effective in enhancing solubilization of insoluble phytate salts by phytase than others. The dry weight and inorganic phosphate contents of the wheat plants were high when supplemented with phytase or fungal spores. The plants provided with 5 mg phytate per plant exhibited enhanced growth and inorganic phosphate. With increase in the dosage of phytase, there was increase in growth and inorganic phosphate of plants, the highest being at 20 U per plant. The compost made employing the combined native microflora of the wheat straw and S. thermophile promoted growth of the plants. The plant-growth-promoting effect was also higher with the compost made using S. thermophile than that from only the native microflora.
Phosphorus (P) is one of the major constituents in energy metabolism and biosynthesis of nucleic ... more Phosphorus (P) is one of the major constituents in energy metabolism and biosynthesis of nucleic acids and cell membranes with an important role in regulation of a number of enzymes. Soil phosphorous is an important macronutrient for plant growth. Phosphorus deficiency in soil is a major problem for agricultural production. Total soil P occurs in either organic or in organic form. Phytic acid as phytate (salts of phytic acid) is the major form of organic phosphorus in soil and it is not readily available to plants as a source of phosphorus because it either forms a complex with cations or adsorbs to various soil components. Phosphate solubilizing microorganisms are ubiquitous in soils and could play an important role in supplying P to plants. Microorganisms utilizing phytate are found in cultivated soils as well as in wetland, grassland and forest soils. Various fungi and bacteria (including plant growth promoting rhizobacteria) hydrolyze this organic form of phosphorus secreting phosphatases such as phytases and acidic/alkaline phosphatases. A large number of transgenic plants have been developed which were able to utilize sodium phytate as sole source of phosphorus. However, the recombinant phytases were similar to their wild type counterparts in terms of their properties. Increased phytase/phosphatase activity in transgenic plants may be an effective approach to promote their phytate-phosphorus utilization. The extracellular phytase activity of transgenic plant roots is a significant factor in the utilization of phosphorus from phytate. Furthermore, this indicated that an opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus. This review is focused on the role of phytases and phytase producing microbes in promoting the growth of different plants.
The Pichia anomala gene PPHY, which codes for a cell-bound phytase, was isolated from genomic DNA... more The Pichia anomala gene PPHY, which codes for a cell-bound phytase, was isolated from genomic DNA by PCR, using oligonucleotide sequences derived from the N-terminal region of the purified phytase protein (Pphyp) and a degenerate primer derived from conserved sequences of yeast and fungal phytases as primers. The gene harbours an ORF of 1389bp, encoding a 462-amino-acid protein. The deduced amino acid sequence has similarity, to a varied extent, with those of phosphatases from Pichia stipitis (62%), Candida dubliniensis (51%), Candida albicans (51%), Arxula adeninivorans (35%) and phytases from Debaryomyces castellii (50%) and Pichia fabianii (39%). The sequence contains the phytase consensus heptapeptide motif (-Arg-His-Gly-X-Arg-X-Pro-) as well as two phosphohistidine signature motifs found in histidine acid phosphatases. After transformation of PPHY into the yeasts Saccharomyces cerevisiae, A. adeninivorans and Hansenula polymorpha, the last species was selected as the most suitable for synthesis of recombinant Pphyp. The cell-bound enzyme activities produced by wild-type P. anomala and transgenic H. polymorpha strains bearing the PPHY gene placed under the control of the inducible H. polymorpha-derived FMD promoter were characterized. In both cases, a molecular mass of approximately 380kDa was determined for the native enzyme (corresponding to a hexamer); the pH and temperature optima for the activity were 4.0 and 60 degrees C, respectively. The enzyme was active on phytic acid, p-nitrophenylphosphate, glucose-6-phosphate, ADP, sodium pyrophosphate, AMP, 1-naphthylphosphate and ATP. Based on the K(m)/K(cat) and further biochemical parameters, the enzyme was classified as a cell-bound phytase with acid phosphatase activity and not as acid phosphatase, despite its strong similarity to the latter class of enzymes. The yeast biomass containing phytase has been demonstrated to be useful as a feed additive in poultry and aquaculture, and dephytinization of foods and feeds.
Aims: Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in... more Aims: Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in submerged fermentation and its application in bread.Methods and Results: The production of phytase by a thermophilic mould S. thermophile was investigated using free and immobilized conidiospores in cane molasses medium in shake flasks, and stirred tank and air-lift fermenters. Among surfactants tested, Tweens (Tween-20, 40 and 80) and sodium oleate increased phytase accumulation, whereas SDS and Triton X-100 inhibited the enzyme production. The mould produced phytase optimally at aw 0·95, and it declined sharply below this aw value. The enzyme production was comparable in air-lift and stirred tank reactors with a marked reduction in fermentation time. Among the matrices tried, Ca-alginate was the best for conidiospore immobilization, and fungus secreted sustained levels of enzyme titres over five cycles. The phytic acid in the dough was efficiently hydrolysed by the enzyme accompanied by the liberation of soluble phosphate in the bread.Conclusions: The phytase production by S. thermophile was enhanced in the presence of Tween-80 in cane molasses medium. A peak in enzyme production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks. Ca-alginate immobilized conidiospores germinated to produce fungal growth that secreted sustained levels of phytase over five cycles. The bread made with phytase contained reduced level of phytic acid and a high-soluble phosphate.Significance and Impact of the Study: The phytase accumulation by S. thermophile was increased by the surfactants. The sustainability of enzyme production in stirred tank and air-lift fermenters suggested the possibility for scaling up of phytase. The bread made with phytase contained low level of antinutrient, i.e. phytic acid.
Culture variables affecting phytase production by a thermophilic mould Sporotrichum thermophile i... more 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.
The phytase production by Sporotrichum thermophile TLR50 was recorded on all the commonly used an... more The phytase production by Sporotrichum thermophile TLR50 was recorded on all the commonly used animal feed ingredients tested to varying degrees in solid-state fermentation. Enzyme production increased to 180 U/g of dry moldy residue (DMR) in sesame oil cake at 120 h and 45°C at the initial substrate-to-moisture ratio of 1∶2.5 and a w of 0.95. Supplemenetation of sesame oil cake with glucose and ammonium sulfate further enhanced phytase titer (282 U/g of DMR). An overall 76% enhancement in phytase production was achieved owing to optimization. The mold secreted acid phosphatase, amylase, xylanase, and lipase along with phytase. By the action of phytase, inorganic phosphate was liberated efficiently, leading to dephytinization of sesame oil cake.
Aims: Statistical optimization of phytase production by a thermophilic mould Sporotrichum thermo... more 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°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 of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorgan... more Phytase of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorganic phosphate from Ca+2, Mg+2, and Co+2 phytates more efficiently than those of Al3+, Fe2+, Fe3+, and Zn2+. The hydrolysis rate was higher at 60 °C as compared to 26 °C. Among all the organic acids tested, citrate was more effective in enhancing solubilization of insoluble phytate salts by phytase than others. The dry weight and inorganic phosphate contents of the wheat plants were high when supplemented with phytase or fungal spores. The plants provided with 5 mg phytate per plant exhibited enhanced growth and inorganic phosphate. With increase in the dosage of phytase, there was increase in growth and inorganic phosphate of plants, the highest being at 20 U per plant. The compost made employing the combined native microflora of the wheat straw and S. thermophile promoted growth of the plants. The plant-growth-promoting effect was also higher with the compost made using S. thermophile than that from only the native microflora.
Phosphorus (P) is one of the major constituents in energy metabolism and biosynthesis of nucleic ... more Phosphorus (P) is one of the major constituents in energy metabolism and biosynthesis of nucleic acids and cell membranes with an important role in regulation of a number of enzymes. Soil phosphorous is an important macronutrient for plant growth. Phosphorus deficiency in soil is a major problem for agricultural production. Total soil P occurs in either organic or in organic form. Phytic acid as phytate (salts of phytic acid) is the major form of organic phosphorus in soil and it is not readily available to plants as a source of phosphorus because it either forms a complex with cations or adsorbs to various soil components. Phosphate solubilizing microorganisms are ubiquitous in soils and could play an important role in supplying P to plants. Microorganisms utilizing phytate are found in cultivated soils as well as in wetland, grassland and forest soils. Various fungi and bacteria (including plant growth promoting rhizobacteria) hydrolyze this organic form of phosphorus secreting phosphatases such as phytases and acidic/alkaline phosphatases. A large number of transgenic plants have been developed which were able to utilize sodium phytate as sole source of phosphorus. However, the recombinant phytases were similar to their wild type counterparts in terms of their properties. Increased phytase/phosphatase activity in transgenic plants may be an effective approach to promote their phytate-phosphorus utilization. The extracellular phytase activity of transgenic plant roots is a significant factor in the utilization of phosphorus from phytate. Furthermore, this indicated that an opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus. This review is focused on the role of phytases and phytase producing microbes in promoting the growth of different plants.
The Pichia anomala gene PPHY, which codes for a cell-bound phytase, was isolated from genomic DNA... more The Pichia anomala gene PPHY, which codes for a cell-bound phytase, was isolated from genomic DNA by PCR, using oligonucleotide sequences derived from the N-terminal region of the purified phytase protein (Pphyp) and a degenerate primer derived from conserved sequences of yeast and fungal phytases as primers. The gene harbours an ORF of 1389bp, encoding a 462-amino-acid protein. The deduced amino acid sequence has similarity, to a varied extent, with those of phosphatases from Pichia stipitis (62%), Candida dubliniensis (51%), Candida albicans (51%), Arxula adeninivorans (35%) and phytases from Debaryomyces castellii (50%) and Pichia fabianii (39%). The sequence contains the phytase consensus heptapeptide motif (-Arg-His-Gly-X-Arg-X-Pro-) as well as two phosphohistidine signature motifs found in histidine acid phosphatases. After transformation of PPHY into the yeasts Saccharomyces cerevisiae, A. adeninivorans and Hansenula polymorpha, the last species was selected as the most suitable for synthesis of recombinant Pphyp. The cell-bound enzyme activities produced by wild-type P. anomala and transgenic H. polymorpha strains bearing the PPHY gene placed under the control of the inducible H. polymorpha-derived FMD promoter were characterized. In both cases, a molecular mass of approximately 380kDa was determined for the native enzyme (corresponding to a hexamer); the pH and temperature optima for the activity were 4.0 and 60 degrees C, respectively. The enzyme was active on phytic acid, p-nitrophenylphosphate, glucose-6-phosphate, ADP, sodium pyrophosphate, AMP, 1-naphthylphosphate and ATP. Based on the K(m)/K(cat) and further biochemical parameters, the enzyme was classified as a cell-bound phytase with acid phosphatase activity and not as acid phosphatase, despite its strong similarity to the latter class of enzymes. The yeast biomass containing phytase has been demonstrated to be useful as a feed additive in poultry and aquaculture, and dephytinization of foods and feeds.
Aims: Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in... more Aims: Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in submerged fermentation and its application in bread.Methods and Results: The production of phytase by a thermophilic mould S. thermophile was investigated using free and immobilized conidiospores in cane molasses medium in shake flasks, and stirred tank and air-lift fermenters. Among surfactants tested, Tweens (Tween-20, 40 and 80) and sodium oleate increased phytase accumulation, whereas SDS and Triton X-100 inhibited the enzyme production. The mould produced phytase optimally at aw 0·95, and it declined sharply below this aw value. The enzyme production was comparable in air-lift and stirred tank reactors with a marked reduction in fermentation time. Among the matrices tried, Ca-alginate was the best for conidiospore immobilization, and fungus secreted sustained levels of enzyme titres over five cycles. The phytic acid in the dough was efficiently hydrolysed by the enzyme accompanied by the liberation of soluble phosphate in the bread.Conclusions: The phytase production by S. thermophile was enhanced in the presence of Tween-80 in cane molasses medium. A peak in enzyme production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks. Ca-alginate immobilized conidiospores germinated to produce fungal growth that secreted sustained levels of phytase over five cycles. The bread made with phytase contained reduced level of phytic acid and a high-soluble phosphate.Significance and Impact of the Study: The phytase accumulation by S. thermophile was increased by the surfactants. The sustainability of enzyme production in stirred tank and air-lift fermenters suggested the possibility for scaling up of phytase. The bread made with phytase contained low level of antinutrient, i.e. phytic acid.
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