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Thermophilc mold Sporotrichum thermophile secretes an acidstable and thermostable phytase, which finds application as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. Low extracellular phytase production by the mold is a major bottleneck for its application on a commercial scale. We have successfully overcome this problem by constitutive secretary expression of codon optimized rStPhy under glyceraldehyde phosphate dehydrogenase (GAP) promoter in Pichia pastoris. A ∼41-fold improvement in rStPhy production has been achieved. Circular Dichroism (CD) spectra revealed that rStPhy is composed of 26.65% α-helices, 5.26% β-sheets and 68.09% random coils at pH 5.0 and 60°C, the optima for the enzyme activity. The melting temperature (T m ) of the enzyme is ∼73°C. The 3D structure of rStPhy displayed characteristic signature sequences (RHGXRXP and HD) of HAP phytase. The catalytically important amino acids (Arg74, His75, Arg78, His368 and Asp369) were identified by docking and site directed mutagenesis. Fluorescence quenching by N-bromosuccinimide (NBS) and CsCl exposed tryptophan residues surrounded by negative charges, which play a key role in maintaining structural integrity of rStPhy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The codon-optimized phytase gene of the thermophilic mold Sporotrichum thermophile (St-Phy) was expressed in Pichia pastoris. The recombinant P. pastoris harboring the phytase gene (rSt-Phy) yielded a high titer of extracellular phytase (480 ± 23 U/mL) on induction with methanol. The recombinant phytase production was ~40-fold higher than that of the native fungal strain. The purified recombinant phytase (rSt-Phy) has the molecular mass of 70 kDa on SDS-PAGE, with K m and V max (calcium phytate), k cat and k cat/K m values of 0.147 mM and 183 nmol/mg s, 1.3 × 10(3)/s and 8.84 × 10(6)/M s, respectively. Mg(2+) and Ba(2+) display a slight stimulatory effect, while other cations tested exert inhibitory action on phytase. The enzyme is inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward's reagent K and 2,3-bunatedione, but resistant to both pepsin and trypsin. The rSt-Phy is useful in the dephytinization of broiler feeds efficiently in simulated gut conditions of chick leading to the liberation of soluble inorganic phosphate with concomitant mitigation in antinutrient effects of phytates. The addition of vanadate makes it a potential candidate for generating haloperoxidase, which has several applications.

Sporotrichum thermophile produces very low titres of phytase (St-Phy) extracellularly, which is acidstable, thermostable, and protease insensitive with broad substrate specificity, and therefore, the gene encoding phytase (St-Phy) has been cloned and expressed in E. coli. The purified recombinant phytase (rSt-Phy) has the molecular mass of 55 kDa with Km and Vmax (calcium phytate), kcat and kcat/Km of 0.143 mM, 185.05 nmoles mg(-1)  s(-1), 5.1 × 10(3) s(-1), and 3.5 × 10(7) M(-1) s(-1), respectively. Mg(2+) and Ba(2+) display slight stimulatory effect on the enzyme, while it is inhibited by other ions to a varied extent. The enzyme is also inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward's reagent K, and 2,3-butanedione but resistant to both pepsin and trypsin. The rSt-Phy is useful in dephytinization of tandoori and naan (unleavened flat Indian breads), and bread, liberating soluble inorganic phosphate that mitigates anti-nutrient effects of phytic acid.

The phytase of the yeast Pichia anomala (PPHY) is a suitable biocatalyst as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. The cell-bound nature and low phytase titres are the main bottlenecks for its utility in food and feed industries. In this investigation, we have overcome the problems by constitutive secretory expression of PPHY under glyceraldehyde phosphate dehydrogenase (GAP) promoter. A ~44-fold increase in rPPHY titre has been achieved after optimization of cultural variables by one-variable-at-a-time approach and two factorial statistical design. The use of GAP promoter makes the cultivation of the recombinant P. pastoris straight forward and eliminates the requirement of methanol for induction and hazards associated with its storage. Among metal-phytate complexes, Ca(2+) phytate is hydrolyzed more efficiently by rPPHY than Co(2+), Mn(2+), Mg(2+), Fe(3+) and Zn(2+) phytates. The enzyme is effective in dephytinizing whole wheat unleavened flat Indian breads (naan and tandoori) and different broiler feeds, thus mitigating anti-nutritional effects of phytates.

Fungal phytases are histidine acid phosphatases, a subclass of acid phosphatases, which catalyse the hydrolysis of phytic acid resulting in the release of phosphate moieties and thus mitigate its antinutritional properties. The supplementation of feed with phytases increases the bioavailability of phosphorus and minerals in non-ruminant animals and reduces the phosphorus pollution due to phosphorus excretion in the areas of intensive livestock production. Although phytases are reported in plants, animals and micro-organisms, fungal sources are used extensively for the production of phytases on a commercial scale. Phytases have been produced by fungi in both solid-state fermentation (SSF) and submerged fermentation (SmF). The fungal phytases are high molecular weight proteins ranging from 35 to 500 kDa. They are optimally active within pH and temperature ranges between 4.5 and 6.0, and 45 and 70 °C respectively. Phytate degradation leads to amelioration in the nutritional status of foods and feeds by improving the availability of minerals, phosphorus and proteins in non-ruminant animals and human beings and thus mitigates the environmental phosphorus pollution. Our article focuses on the role of fungal phytases in improving nutritional value of foods and feeds with concomitant increase in growth of non-ruminant animals and mitigating environmental phosphorus pollution., (Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.)

The phytase of the yeast Pichia anomala is a histidine acid phosphatase based on signature sequences and catalytic amino acids identified by site-directed mutagenesis. Among modulators, N-bromosuccinimide and butanedione inhibit phytase, while Ca(2+) and Ni(2+) stimulate slightly. Vanadate exhibits competitive inhibition of phytase, making it bifunctional to act as haloperoxidase. Molecular docking supports vanadate to share its binding site with phytate. The T 1/2, activation energy (E a ), temperature quotient (Q 10), activation energy of thermal inactivation (Ed), and enthalpy (ΔH d (0) ) of the enzyme are 4.0 min (80 °C), 27.72 kJ mol(-1), 2.1, 410.62 kJ mol(-1), and ∼407.8 kJ mol(-1) (65-80 °C), respectively. The free energy of the process (ΔG d (o) ) increases from 49.56 to 71.58 kJ mol(-1) with rise in temperature, while entropy of inactivation (ΔS d (0) ) remains constant at ∼1.36 kJ mol(-1) K(-1). The supplementation of whole wheat dough with rPPHY resulted in 72.5 % reduction in phytic acid content of bread. These characteristics confirm that the phytase has adequate thermostability for its applicability as a food and feed additive.

The phytase (PPHY) of Pichia anomala has the requisite properties of thermostability and acidstability, broad substrate spectrum, and protease insensitivity, which make it a suitable candidate as a feed and food additive. The 1,389-bp PPHY gene was amplified from P. anomala genomic DNA, cloned in pPICZαA, and expressed extracellularly in P. pastoris X33. Three copies of PPHY have been detected integrated into the chromosomal DNA of the recombinant P. pastoris. The size exclusion chromatography followed by electrophoresis of the pure rPPHY confirmed that this is a homohexameric glycoprotein of ~420 kDa with a 24.3 % portion as N-linked glycans. The temperature and pH optima of rPPHY are 60 °C and 4.0, similar to the endogenous enzyme. The kinetic characteristics K(m), V(max), K(cat), and K(cat)/K(m) of rPPHY are 0.2 ± 0.03 mM, 78.2 ± 1.43 nmol mg(-1) s(-1), 65,655 ± 10.92 s(-1), and 328.3 ± 3.12 μM(-1) s(-1), respectively. The optimization of medium components led to a 21.8-fold improvement in rPPHY production over the endogenous yeast. The rPPHY titer attained in shake flasks could also be sustained in the laboratory fermenter. The rPPHY accounts for 57.1 % of the total secreted protein into the medium. The enzyme has been found useful in fractionating allergenic protein glycinin from soya protein besides dephytinization.

Among several yeasts isolated from dried flowers of Woodfordia fruticosa, Pichia anomala produced a high titre of cell-bound phytase. The optimization of fermentation variables led to formulation of media and selection of cultural variables that supported enhanced phytase production. The enzyme productivity was very high in fed batch fermentation in air-lift fermentor as compared to that in stirred tank fermentor. Amelioration in the cell-bound phytase activity was observed when yeast cells were permeabilized with Triton-X-100. The enzyme is thermostable and acid stable with broad substrate specificity, the characteristics that are desirable for enzymes to be used in the animal feed industry. The phytase-encoding gene was cloned and sequenced. The 3D structure of the enzyme was proposed by comparative modeling using phytase of Debaryomyces occidentalis (50% sequence identity) as template. When broiler chicks, and fresh water and marine fishes were fed with the feed supplemented with yeast biomass containing phytase, improvement in growth and phosphorus retention, and decrease in the excretion of phosphorus in the faeces were recorded. The cell-bound phytase of P. anomala could effectively dephytinize wheat flour and soymilk.

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., (Copyright 2010 Elsevier B.V. All rights reserved.)

Aims: Whole cell permeabilization of Pichia anomala to ameliorate the cell-bound phytase activity and usability of permeabilized cells in dephytinization of soymilk., Methods and Results: The cells of P. anomala were subjected to permeabilization using the surfactant Triton X-100 to overcome the permeability barrier and prepare whole cell biocatalysts with high phytase activity. The statistical approach, response surface methodology (RSM) was used to optimize the operating conditions for permeabilization. The treatment of cells with 5% Triton X-100 for 30 min resulted in c. 15% enhancement in cell-bound phytase activity. The shrinkage of protoplast was observed, although cell viability and phytase stability were not significantly altered. The free as well as immobilized permeabilized cells hydrolysed soymilk phytate, and the latter could be reused over four consecutive cycles., Conclusions: Whole cell permeabilization of P. anomala using Triton X-100 led to enhancement in cell-bound phytase activity. The viability and integrity of yeast cells were not significantly affected because of permeabilization. The permeabilized P. anomala cells effectively dephytinized soymilk, and the permeabilized cells immobilized in alginate could be reused because of sustained phytase activity., Significance and Impact of the Study: This is the first report on the use of permeabilized yeast cells for mitigating phytate content of soymilk. Alginate entrapment of permeabilized P. anomala allows reuse of cells for soymilk dephytinization, thus suggesting a potential application in food industry.

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 Al(3+), Fe(2+), Fe(3+), and Zn(2+). The hydrolysis rate was higher at 60 degrees C as compared to 26 degrees Celsius. 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.

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of approximately 456kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 degrees C with a T(1/2) of 16h at 60 degrees C and 1.5h at 80 degrees C. The activation energy of the enzyme reaction is 48.6KJmol(-1) with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg(2+) exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3mM. The maximum hydrolysis rate (V(max)) and apparent Michaelis-Menten constant (K(m)) for sodium phytate were 83nmolmg(-1)s(-1) and 0.156mM, respectively. The catalytic turnover number (K(cat)) and catalytic efficiency (K(cat)/K(m)) of phytase were 37.8s(-1) and 2.4x10(5)M(-1)s(-1), respectively. Based on the N-terminal and MALDI-LC-MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.

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 a(w) 0.95, and it declined sharply below this a(w) 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.

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.

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.

Phytases hydrolyze phytates to liberate soluble and thus readily utilizable inorganic phosphate. Although phytases are produced by various groups of microbes, yeasts being simple eukaryotes and mostly non-pathogenic with proven probiotic benefits can serve as ideal candidates for phytase research. The full potential of yeast phytases has not, however, been exploited. This review focuses attention on the present status of knowledge on the production, characterization, molecular characteristics, and cloning and over-expression of yeast phytases. Several potential applications of the yeast phytases in feeds and foods, and in the synthesis of lower myo-inositol phosphates are also discussed.

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.

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 degrees C at the initial substrate-to-moisture ratio of 1:2.5 and aw of 0.95. Supplementation 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.

Aim: Formulation of an inexpensive cane molasses medium for improved cell-bound phytase production by Pichia anomala., Methods and Results: Cell-bound phytase production by Pichia anomala was compared in synthetic glucose-beef extract and cane molasses media. The yeast was cultivated in 250 ml flasks containing 50 ml of the medium, inoculated with a 12 h-old inoculum (3 x 10(6) CFU ml(-1)) and incubated at 25 degrees C for 24 h at 250 rev min(-1). Different cultural parameters were optimized in cane molasses medium in batch fermentation. The cell-bound phytase content increased significantly in cane molasses medium (176 U g(-1) dry biomass) when compared with the synthetic medium (100 U g(-1) dry biomass). In fed-batch fermentation, a marked increase in biomass (20 g l(-1)) and the phytase yield (3000 U l(-1)) were recorded in cane molasses medium. The cost of production in cane molasses medium was pound 0.006 per 1000 U, which is much lower when compared with that in synthetic medium (pound 0.25 per 1000 U)., Conclusions: An overall 86.6% enhancement in phytase yield was attained in optimized cane molasses medium using fed-batch fermentation when compared with that in synthetic medium. Furthermore, the production in cane molasses medium is cost-effective., Significance and Impact of the Study: Phytase yield was improved in cane molasses when compared with the synthetic medium, and the cost of production was also significantly reduced. This enzyme can find application in the animal feed industry for improving the nutritional status of feed and combating environmental pollution., (Copyright 2004 The Society for Applied Microbiology)

The review deals with phytase-producing microorganisms along with optimum conditions for its production. Various methods used for purifying phytases and their characteristics are discussed. Heterologous gene expression, cost-effective large-scale phytase production, and various biotechnological applications of the enzyme in animal feed and food industries are also discussed.