Production of Phytase by Penicillium oxalicum in Solid State Fermentation Exhibiting Hydrolysis of Phytates and Haloperoxidase Activity

Abstract

Penicillium oxalicum PBG30 produced an extracellular phytase in solid-state fermentation and maximum production was obtained (200 ± 6.01 U/g DMR) at 30 °C after 5 days. The optimal temperature and pH for enzyme were 70 °C and 3.0, respectively. The phytase is thermostable with T_1/2 of 1 h at 70 °C and showed broad-substrate specificity with K_m and V_max values of 4.42 mM and 909.1 U/ml, respectively with calcium phytate. Phytase activity was enhanced in the existence of metal ions and surfactants and retarded by SDS, EDTA, sodium molybdate, DTT, and ß-ME. Phytase exhibited resistance against trypsin and pepsin with better storage stability at 4 °C and −20 °C. Insoluble phytates (metal and protein) were efficiently hydrolyzed by fungal phytase showing liberation of inorganic phosphate in a time-dependent manner. Also, the phytic acid reduction was observed in phytase-treated fish feed with the lowest phytic acid occurring in a diet supplemented with 1500 FTU/kg dose of phytase. Furthermore, phytase was converted into vanadium-dependent peroxidase. Fungal phytase, due to its thermostability, protease resistance, broad substrate specificity, and ability to hydrolyze phytate forms, has the potential to serve as an additive for improving nutrient digestibility in the food and feed industry.

Introduction

Phytase is a well-known feed enzyme in today’s world that hydrolyses the phytic acid into lower esters of myo-inositol and inorganic phosphate. It also releases other phytate-associated nutrients necessary for the organisms [1]. Phytic acid or phytate is a precursor of organic phosphorus and is predominantly present in the plant’s seeds. Phytate, the complex form of phytic acid binds metal ions, amino acids, proteins, digestive enzymes, and other biomolecules. Due to the chelation property, phytate acts as an anti-nutritive factor [2]. Phytic acid can be reduced by physical and chemical methods which decreases the nutritive value, hence enzymatic action (phytase) is required for the degradation of phytic acid [3]. In ruminant animals, phytase is found in the microbes of the gut region while it is absent in monogastric animals [4]. Since monogastric animals lack phytase in the digestive tract, phytate molecules accumulate inside the body and are excreted out in the soil via fecal matter [1]. In the soil, various phytase-containing microbes are present that break phytate molecules and liberate phosphorus which goes to water bodies by run-off or leaching [5]. The released phosphorus forms algal blooms that lead to eutrophication and phosphorus pollution in water. Therefore, supplementation of phytase in monogastric animals as a feed additive is requisite for their growth and development. Phytase ameliorates the nutritive value of feed and also reduces the level of phosphorus in the environment thus minimizing water pollution. It also limits the need for inorganic phosphorus and therefore restricts the load on non-renewable rock phosphate [4]. Although phytase enzymes have been reported from many microorganisms (bacteria, fungi, yeasts) they are still lacking in fulfilling the properties of an ideal phytase [6]. Thermostable, protease-resistant, broad pH range, substrate-specificity, long shelf life, and cost-effectiveness are the properties of an ideal phytase. The commercially available phytases either lack one or more features of an ideal phytase [4]. Therefore, the necessity of an ideal phytase in the industries is continuously increasing.

Solid-state fermentation (SSF) is the most widely adopted cost-effective method that uses agricultural waste as a substrate and reduces its accumulation. SSF is a simple process to operate and maintain for fungal enzyme production. It requires less water, minimal oxygen supply and less energy, while producing higher yields compared to other fermentation methods. The parameters used in the SSF significantly influence microbial growth and enzyme production, making the selection of appropriate physio-chemical factors a critical aspect [7]. Purification and characterization are important aspects that demonstrate the properties of phytase enzyme and thus need to be studied. Purification includes precipitation and dialysis followed by chromatography techniques [8]. Purification and biochemical characterization of phytase from various fungal species have been reported in the literature [9–15].

Phytic acid or phytate complexes are commonly present in plant-based food and feed ingredients which are insoluble in the GIT pH of monogastric animals. Consequently, monogastric animals are unable to utilize certain nutrients, resulting in nutrient deficiencies that can lead to malnutrition and severe health complications. The addition of phytase to the diet releases bounded nutrients and ameliorate the nutritional quality of the food and feedstuffs [16]. The application of phytase in lowering phytic acid amount has been studied in poultry feed ingredients [15], and wheat flour [12, 15, 17]. Also, the liberation of inorganic phosphate, reducing sugar and protein content was observed along with the reduction of phytic acid [16–18].

Phytase can act as haloperoxidase when vanadate is incorporated with phytase. Vanadium is an analog of phosphate molecules and an inhibitor of acid phosphatase. It binds to the active site of histidine acid phosphatase (HAP)-phytase and converts it into peroxidase. Vanadium haloperoxidase is similar in structure to phytase except for the presence of vanadate instead of phosphate. This semi-synthetic haloperoxidase is involved in various catalytic oxidative reactions [19, 20].

The present study deals with the production of extracellular phytase obtained from Penicillium oxalicum PBG30 using agricultural waste during the SSF method followed by the biochemical characterization to determine the properties of phytase. The involvement of phytase in hydrolysing insoluble metal- and protein-phytate complexes commonly found in food and feed ingredients as well as its role in degrading phytic acid in the fish feed, was studied to assess its potential as an additive in the food and feed industry. Additionally, the conversion of phytase enzyme to semi-synthetic haloperoxidase using vanadium was examined.

Materials and Methods

Culture Conditions

Penicillium oxalicum PBG30 was cultured on the potato dextrose agar (PDA) media at 30 °C and maintained at 4 °C on PDA slants, and also at − 20 °C in glycerol stock. The fungal spores were scrapped from 72 h old culture via a sterile loop by adding 25 ml normal saline solution containing 0.1% Tween 80 and the spore count was estimated by a hemocytometer and 1 ml of spore suspension (7.7 × 10⁷ CFU/ml) was used as inoculum for the production of phytase [18, 21].

Phytase Production in SSF

Ten gram of wheat bran was used with 20 ml production medium of pH 7.0 consisting 0.5% Urea, 0.1% MgSO₄.7H₂O, 0.1% KCl and 0.1% FeSO₄.7H₂O [22]. The medium was autoclaved for 20 min at 121 °C and then allowed to cool at room temperature. One ml of spore suspension was added and incubated for 5 days at 30 °C. The culture filtrate was extracted and centrifuged at 10,000 rpm for 10 min at 4 °C. The phytase activity was calculated from the supernatant using phytase assay [18, 21].

Phytase Assay

Phytase activity was calculated by estimating the liberation of inorganic phosphate from the calcium phytate substrate at 70 °C with 0.1 M sodium acetate buffer (pH 3.0) through the Fiske and Subbarow [23] method [18]. Each 1.0 ml reaction mixture contained 0.5 ml substrate dissolved in buffer and 0.5 ml enzyme diluted with buffer. Blank and control (substrate and enzyme) were also prepared during the experiment and performed in triplicate. The substrate control consisted of 0.5 mL substrate with 0.5 mL buffer, while the enzyme control consisted of 0.5 mL enzyme with 0.5 mL buffer. The tubes were incubated for 5 min at 70 °C in a water bath. Following this, 1.0 mL of TCA, 1.0 mL of distilled water, and 0.5 mL of 9 N H₂SO₄ were added, and the tubes were incubated for additional 10 min at room temperature. After incubation, 0.5 ml of ammonium molybdate (6%) was added and the tubes were kept for 15 min in the dark. Then, 0.5 mL of freshly prepared FeSO₄ was added and the tubes were kept for 30 min in the dark. The absorbance was measured at 660 nm [23]. Phytase activity was calculated using the regression equation derived from the standard curve of KH₂PO₄ [25]. One unit indicates the amount of phytase used to liberate 1 nmol of inorganic phosphorus per sec under assay conditions [24]. The phytase activity is expressed as a unit per gram of dry mouldy residue (U/g DMR).

Biochemical Characterization of Phytase

For characterization, the enzyme was concentrated using ammonium sulphate precipitation method. The crude enzyme solution was placed in a beaker and stirred using a magnetic stirrer. Ammonium salt was added slowly to the extract until it reached 80% saturation. The entire procedure was conducted at 4 °C, followed by overnight incubation to allow the precipitated enzyme to settle. The sample was centrifuged at 10,000 rpm for 10 min at 4 °C and the pellet was re-suspended in 0.1 M sodium acetate buffer (pH 3.0). Dialysis was then carried out at 4 °C. A suitable length of dialysis membrane was cut and pre-treated to activate it. After cooling, the membrane was filled with the precipitated enzyme and tied at both ends. The dialysis bag was submerged in 20 mM sodium acetate buffer (pH 3.0) and kept on a magnetic stirrer at 4 °C. The buffer was changed at least three times. After dialysis, the sample was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was collected and the pellet discarded. The clear supernatant was used for characterization studies [9].

Effect of Temperature on the Activity and Stability of Phytase

To determine the optimum value of temperature, a phytase assay was carried out from 30 °C to 80 °C in 0.1 M sodium acetate buffer (pH 3.0) with 1 mM calcium phytate. To investigate the thermostability of phytase, the enzyme was incubated at 70° and 80 °C in a buffer of pH 3.0 for 2 h. Samples were collected at regular intervals and the assay was performed at 70 °C (pH 3.0) [9, 18].

Impact of pH on the Activity and Stability of Phytase

To evaluate the optimum value of pH, phytase enzyme activity was studied at 70 °C with a buffer of different pH range 2.5—8.0 (0.1 M glycine–HCl buffer pH 2.5, 0.1 M sodium acetate buffer pH 3.0—6.0, 0.1 M tris–HCl buffer pH 7.0—8.0). For assessing the pH stability of phytase, the enzyme was dissolved in pH (3.0, 5.0) and incubated at 70 °C. Samples were collected at 0, 5, 10, 30, 60, and 120 min interval and the assay was performed at 70 °C (pH 3.0) [9, 18].

Determination of Substrate Specificity and Kinetic Constants

The specificity of phytase towards different substrates, viz, sodium phytate, calcium phytate, glucose-6-phosphate, p-nitrophenyl phosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) was examined. The phytase assay was performed at 70 °C with a pH 3.0 and 1 mM substrate concentration, using calcium phytate as the substrate, with its activity considered as 100%. The kinetic parameters (K_m and V_max) were evaluated from the Lineweaver–Burk plot by using different concentrations of calcium phytate (0.1—2 mM) and the assay was conducted at 70 °C (pH 3.0) [9, 18].

Effect of Organic Solvents, Detergents, Metal ions, and Inhibitors on Phytase Activity

The experiments were performed to determine the influence of organic solvents (ethanol, methanol, butanol, propan-2-ol, acetone, DMSO) and detergents (SDS, Tween-20, Tween-60, Tween-80, Triton-X) on phytase activity. In the reaction mixture, different concentrations of organic solvents (2 and 5%) and detergents (0.1 and 0.5%) were added [9].

Similarly, the effect of metal ions and inhibitors on phytase activity was analyzed by providing different metal ions (Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Fe²⁺, Na⁺) and inhibitors (sodium azide, EDTA, sodium molybdate, ß-ME, DTT) with 1 mM and 5 mM concentrations in the reaction mixture.

The enzymatic assay was performed along with the control. The control value is taken as 100% having no additional chemical [9, 26].

Effect of Proteolytic Enzymes and Storage Conditions on Phytase Activity

The phytase enzyme was treated with 1% (w/v) trypsin and pepsin for 2 h at 37 °C. The samples were taken at the desired interval (0, 30, 60, 120 min) and a phytase assay was performed [18, 25]

Phytase was kept for 6 months in different conditions (room temperature, 4 °C, −20 °C), and activity was determined at the desired interval. Activity on the 0 th day is considered 100% [9].

Hydrolysis of Insoluble Phytates

Metal phytates (Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Cu²⁺, Zn²⁺) were formed by mixing each metal ion along with sodium phytate. 100 mM stock solution of metal ions (CaCl₂·2H₂O, CoCl₂·2H₂O, FeSO₄·7H₂O, MgCl₂·6H₂O, MnCl₂·4H₂O, ZnCl₂·7H₂O) and 10 mM of sodium phytate were prepared in distilled water separately. Both were mixed in equal volume and kept at 4 °C for overnight incubation. The precipitated salts were centrifuged at 10,000 g for 5 min at 4 °C. The supernatant was discarded and the pellet was washed three times with distilled water, and finally suspended in 5 ml of 0.1 M Na-acetate buffer (pH 3.0). Each phytate salt was treated with 20 U of phytase and incubated at 37 and 50 °C. Samples were taken at different intervals (3, 6, 12, 24 h) and salts were pelleted by centrifuge. The supernatant was used to determine the amount of released inorganic phosphate at 600 nm [25]. The protein-phytate complex was made by adding an equal amount of lysozyme (2.5 mM) in distilled water and sodium phytate (3 mM) in distilled water and kept overnight at 4ºC. Centrifuge the solution at 10,000 g for 5 min at 4ºC and discard the supernatant. Washed the pellet three times with distilled water, dissolved in buffer and 20 U of phytase was added into it. The reaction was incubated at 30ºC and samples were collected at defined intervals (0, 5, 10, 30, 60, 120, 360 min). Centrifuge the sample and inorganic phosphate was estimated in the supernatant at 600 nm [25].

Phytic Acid Reduction in Fish Feed Using Fungal Phytase

The formulation of the feed sample is mentioned in Table 1 containing 35% protein in the diet. Different doses of phytase were added to the feed ingredients (500 FTU/kg, 1000 FTU/kg, 1500 FTU/kg). The pellet of different diets was formed with the help of pelletizer. The determination of phytic acid in the phytase-treated and untreated feed pellet was performed by Gao et al. [27] method. Firstly, the sample was treated with 10 ml of 2.4% HCl and kept on a shaker for 16 h at 220 rpm. Centrifuge the sample for 20 min with 10,000 rpm at 10 °C and the obtained supernatant was shifted to falcon consisting of 1 g NaCl. Again, the mixture was shaken at 350 rpm for 20 min so that salts dissolved completely and settled by keeping it at 4 °C for 60 min or −20ºC for 20 min. After that, centrifugation was done at 10,000 rpm for 20 min. The supernatant was used to estimate the phytic acid content by adding 500 μl Wade’s reagent (containing 0.03% ferric chloride and 0.3% sulfosalicylic acid) and kept for 20 min incubation. The absorbance was taken at 500 nm [20, 28]. The quantity of phytic acid in the diet was calculated through the regression equation of the standard curve of sodium phytate.

Table 1.

Composition of the control and experimental diets

Feed ingredients (g)	Diet I (control)	Diet II	Diet III	Diet IV
Soybean flour	181.57	181.57	181.57	181.57
Wheat flour	118.40	118.40	118.40	118.40
Fish oil	3.00	3.00	3.00	3.00
Vitamins and minerals	1.20	1.20	1.20	1.20
Phytase	-	500 FTU/kg	1000 FTU/kg	1500 FTU/kg

Transformation of Phytase into Haloperoxidase

Phytase was mixed with ammonium metavanadate (5 and 10 μM) and incubated at 30 °C for 4 h with 100 rpm. H₂O₂ and guaiacol were added to the solution and absorbance was recorded at 470 nm for each minute up to 10 min [20]. The relative activity of phytase was also calculated during the experiment.

Statistical Analysis

All the experiments were carried out in triplicate. The values were reported as the mean and standard error (mean ± S.E). The significance level was determined using a t-test and is expressed as a p-value. IBM SPSS Statistics software is used for the statistical analysis. Sigma plot (version 10.0) is used for creating the graphs.

Results and Discussion

Phytase Production in SSF

SSF is the commonly used approach nowadays for the production of enzymes. In this process, microbes are grown on a solid substrate in either the absence or a low amount of water [2]. The agricultural waste or residues are commonly utilized as a solid substrate that makes the production cost-effective and also resolves the issues that occur due to their accumulation [29]. SSF has been utilized by various fungi for the production of phytase [10, 16, 24, 30, 31]. The highest amount of phytase production was achieved (200.41 ± 6.01 U/g DMR) with 10 g wheat bran moistened with media (0.5% Urea, 0.1% MgSO₄.7H₂O, 0.1% KCl and 0.1% FeSO₄.7H₂O) in 1:2 ratio of pH 7.0, incubated at 30 °C for 5 days. The other fungal strains that produced phytase were A. oryzae (506.12 U/g DMR) [20], A. awamori NRC- F18 (437.195 ± 7.16 IU/g) [35], A. niger NT-7 (208.30 ± 0.22 U/g DMR) [16], P. adiposa (24.48 0.3 U/gds) [30]. These variations in phytase levels occurred due to the utilization of different fungi, substrates, production media, and fermentation conditions. Among the various substrates used for the SSF method, wheat bran is commonly used because it is rich in carbon, nitrogen, amino acids, and a high amount of phytic acid [32]. Due to the presence of phytate, it acts as an inducer for the production of phytase [33]. It is easily available, has a low cost, and provides support to the growth of fungi [16, 34]. The involvement of wheat bran in the production of phytase has been mentioned in various studies [16, 31, 35].

Biochemical Characterization of Phytase

Partial purification of P. oxalicum phytase was done by ammonium sulphate precipitation followed by the dialysis method. The crude sample expressed the lowest specific activity (0.31 U/mg) with a total enzyme activity of 381.57 U. After 80% ammonium sulphate precipitation, the sample showed 357.48 U activity with 1.23 U/mg specific activity. Further, the dialyzed sample exhibited 1.52 U/mg specific activity with total enzyme activity (211.04 U). The dialyzed enzyme was purified to homogeneity with 4.9-fold and a yield of 55.31%. Further, purification by column chromatography was omitted to make the process cost-effective. The phytase isolated from A. niger CFR 335 reported yields of 66.8% with a 4.1-fold and 71.9% with a 2.7 purification fold following the precipitation and dialysis methods, respectively [36]. Similarly, Saxena et al. [12] found 1.42-fold purification after the dialysis of A aculeatus APF1 phytase with a 25.54% yield. The phytase obtained from T. purpureogenus NSA20 was concentrated by precipitation and dialysis methods exhibiting yields of 39.8% and 11.9% with 2.6 and 5.9 purification folds, respectively [14]. Table 2 demonstrate the comparison of biochemical properties of phytases isolated from different fungi.

Table 2.

Comparison of properties of phytases isolated from various fungi

Fungal source	Optimum temperature (°C)	Optimum pH	Thermostability	Protease resistance after 120 min	Substrate specificity	References
Penicillium oxalicum PBG30	70	3.0	t1/2 60 min at 70 °C and 30% activity left after 1 h at 80 ºC	88% and 80% activity with pepsin and trypsin	sodium phytate, calcium phytate, p-nitrophenyl phosphate, ATP, ADP, Glucose-6-phosphate and AMP	Current study
P. oxalicum EUFR-3	40	7.0	5.35% activity left after 20 min at 80 °C	–	–	[38]
Aspergillus niger BIONCL8	40	2.1	30% activity left after 1 h of incubation at 80 °C	–	Sodium phytate	[15]
A. fumigatus	55	5.5	Retain > 50% activity after 2 h at 70 °C	–	Calcium phytate	[17]
A. niger NT7	60	2.6, 4.8	t1/2 ≤ 60 min at 70 °C	70.12% and 52.35% activity with pepsin and trypsin	Sodium phytate	[16]
A. tubingensis TEM 37	45	2.0, 5.5	Possess 100% activity after 3 h at 80 °C	Retain 88% and 98% activity with pepsin and trypsin	D-Glucose-6-phosphate, D-Fructose-6-phosphate, ATP, ADP, AMP, Sodium phytate	[13]
A. aculeatus APF1	50	3.0	Only 6–7% activity left after 5 min at 80 °C	Retain 74% and 73% activity with pepsin and trypsin	Calcium phytate	[12]
A. foetidus MTCC 11682	37	3.5, 5.5	56% activity after 30 min at 80 °C	–	–	[37]
A. fumigatus	40	6.0	20% and 14% activity left after 1 h at 70 °C and 80 °C	–	Sodium phytate	[11]
A. niger 7 A-1	56	5.3	9.3% activity left after 5 min at 80 ºC	–	Sodium phytate, Phenyl phosphate, Naphthyl phosphate, p-Nitrophenyl phosphate, Glycerol-2-phosphate, Glucose-1-phosphate, ADP and AMP	[10]
A. oryzae SBS50	50	5.0	t1/2 10 min at 80 °C	Retain 97% and 62% activity with pepsin and trypsin	sodium phytate, calcium phytate, potassium phytate, ADP, ATP, glucose-6-phosphate and p-nitrophenyl phosphate, β- glycerophosphate	[9]
Rhizopus oligosporus MTCC 1116	50	5.5	55% and 30% activity left after 5 min at 70 °C and 80 °C	–	Sodium phytate, glucose-6-phosphate, glucose-1-phosphate, disodium pyrophosphate, ATP and GTP	[39]

Effect of Temperature

During food and feed processing, enzymes have to face extreme temperatures, therefore they must be resistant to high temperature. The P. oxalicum phytase exhibited more than 80% activity at the temperature ranging between 55 to 80 °C (Fig. 1a), however, the optimal activity was recorded at 70 °C, showing its survival at high temperature. In other studies, the optimal activity of fungal phytases was observed between 30 and 65 °C [9, 12, 24, 28, 36, 41–45]. Ajith et al. (2019) found maximum activity of A. foetidus MTCC 11682 phytase at 37ºC [37]. However, the phytases obtained from A. niger BIONCL8 [15], P. oxalicum [38], and A. fumigatus exhibited optimal activity at 40ºC [11] while the phytases isolated from R. oligosporus MTCC 556 exhibited optimal activity at 50ºC [39], A. fumigatus at 55ºC [17], A. niger 7 A-1 at 56ºC [10] and A. niger NT7 at 60ºC [16].

Fig. 1.

a Effect of temperature range (30–80 °C) on phytase activity. b Thermostability studies at 70 and 80 °C

The thermostability of phytase is studied at 70° and 80 °C. The P. oxalicum phytase is found thermostable at 70 °C with half-life of 60 min, while at 80 °C the phytase lost 70% activity within 60 min (Fig. 1b). However, the studies carried out on the A. niger BIONCL8 showed that it lost 70% phytase activity [15], and A. fumigatus lost 86% phytase activity [11] within 1 h at 80 °C. In the case of A. niger 7 A-1, only 9.3% activity is left after 5 min of incubation at 80 ºC [10] and A. oryzae retains only 4.2% phytase activity after 1 h [40]. A reduction in the phytase activity of A. fumigatus was observed after 1 h at 70 ºC [11]. In another study, phytase obtained from A. fumigatus showed 90% activity after 20 min while exhibiting more than 50% at 2 h [17]. The thermostable half-life of A. oryzae phytase was found at 300 min at 50 °C [9], while at 70 ºC phytase lost its activity within 100 min [24]. In the case of P. oxalicum, > 70% of activity was lost within 10 min [38].

Effect of pH

pH is a critical parameter that influences enzyme activity by changing the ionic composition in its microenvironment. The phytase is active in the acidic range (2.5–5.0) and found optimum at pH 3.0 (sodium acetate buffer) during the assay. The stomachs of humans and animals are acidic because their pH range between 1.5–3.5, therefore, the phytases active at this acidic pH are considered useful in the food and feed industry. Likewise, A. aculeatus APF1 [12], and A. niger [41] exhibited the optimum pH at 3.0. In contrast, neutral pH is mentioned in P. oxalicum EUFR-3 [38]. As shown in Fig. 2a, phytase activity declined in alkaline conditions due to changes in the ionic state of amino acids. The reduction of phytase activity in alkaline pH was observed in case of R. oligosporus MTCC 556 [39], A. niger NT7 [16], A. niger BIONCL8 [15], A. niger 7 A-1 [10] and A. oryzae [9, 24].

Fig. 2.

a Effect of pH range (2.5–8.0) on phytase activity. b Effect of pH stability studies with pH 3.0 and 5.0

The stability of pH was checked with both pH 3.0 and 5.0. In pH 3.0, 50% phytase activity was retained for up to 60 min, while in pH 5.0, 50% activity was found within 40 min (Fig. 2b). In the case of A. oryzae, 80% phytase activity was reported with pH 3.0 and 5.0 for 45 min and 100 min respectively [9]. A. niger BIONCL8 phytase exhibited pH stability at 2.0 and 8.0. At pH 2.0, phytase showed 80% activity with incubation of 24 h, while at pH 8.0, it retained more than 50% activity [15].

Substrate Spectrum of Fungal Phytase

The phytase exhibited broad substrate specificity, a characteristic feature of an enzyme to be used for the animal feed. The highest specificity is observed with sodium phytate and considered as best substrate for P. oxalicum phytase. Other studies also demonstrated the maximum specificity of sodium phytate with phytases obtained from P. oxalicum KCTC6440 [41], A. flavus [28], R. oligosporus MTCC 556 [39], A. niger 7 A-1 [10]. The order of specificity is sodium phytate > calcium phytate > p-nitrophenyl phosphate > ATP > ADP > Glucose-6-phosphate > AMP (Fig. 3a).

Fig. 3.

a Effect of different substrate on phytase activity. b Lineweaver–Burk plot for K_m and V_max

The K_m and V_max were measured from the Lineweaver–Burk plot (Fig. 3b) and found to be 4.42 mM and 909.1 U/ml respectively. The K_m represents the enzyme affinity towards the substrate while V_max is the value at which all the active sites of enzymes were occupied by the substrate. The K_m and V_max values for A. oryzae phytase were 1.14 mM and 58.82 U/ml [9], and A. aculeatus APF1 phytase were 3.21 mM and 3.78 U/mg [12] with calcium phytate as a substrate. In other studies, the K_m and V_max values of phytase with sodium phytate were 0.545 mM and 600 U/mg for P. oxalicum PJ3 [43], 0.48 mM and 672 U/mg for P. oxalicum KCTC6440 [42], 0.815 mM and 1092 U/mg for R. mucilaginosa [44], 3.35 mM and 1.27 U/mg for A. niger BIONCL8 [15].

Effect of Organic Solvents, Detergents, Metal Ions and Inhibitors

Organic solvents have a positive impact on phytase activity. It increases in the presence of ethanol, methanol, butanol, propan-2-ol (isopropyl alcohol), acetone, and DMSO (Fig. 4a). Ethanol showed the maximum stimulatory effect on both 2 and 5% concentrations. The response of hydrophobic residues to the activity of phytase is negligible. In the existence of ethanol, methanol, butanol, and acetone, phytase activity was enhanced in A. oryzae [9]. The inhibitory effect of ethanol, butanol, isopropanol and acetone was observed in A. niger BIONCL8 [15]. In A. aculeatus APF1 phytase, butanol reduced the activity, acetone and isopropanol slightly affected while ethanol did not influence activity [12]. The functional group and molecular structure of organic solvents are accountable for designing the artificial environment around the enzyme which affects the enzyme structure and its catalytic properties. Depending upon the type of conformational modifications, phytase activity is affected. It can either stimulate or inhibit the enzyme activity [9]. Limited studies are available on the effect of organic solvents on phytase. Due to the presence of organic solvents solubility of hydrophobic residues is enhanced, it avoids reactions with water molecules, makes enzymes more stable and reusable, and protects them from microbial contamination [13].

Fig. 4.

a Effect of different organic solvents on phytase activity. b Effect of different detergents on phytase activity

In our result, Tween 20, Tween-60 and Tween-80, Triton-X-100 increased the phytase activity, while SDS decreased the activity (Fig. 4b). SDS has been mentioned as an inhibitor for phytases obtained from P. oxalicum [38], A. oryzae [9], and A. fumigatus [11]. Contrasting to the result, Tween 20 showed the inhibitory action on A. foetidus MTCC 11682 phytase [37]. Detergents can also increase or decrease the enzyme activity depending upon the type of interacting molecule whether it is anionic, cationic, or non-ionic. It binds with protein molecules and changes the tertiary structure of protein [9]. SDS is an anionic detergent that is not specific and can attach anywhere to the protein and unfold the protein. It made conformational changes in the active site and thus inactivated the enzyme. Non-ionic detergents (Tweens and Triton X-100) do not bind on the surface of the protein and thus either stimulate or do not influence the activity of phytase by making conformational changes in the enzyme structure.

The involvement of metal ions in the regulation of enzyme catalysis is crucial. The influence of different metal ions was observed on phytase activity (Fig. 5a). Inhibition of phytase activity is possibly due to the association of metal and substrate complex which restricts the binding of substrate with enzyme or the modification of enzyme active site due to binding of ions [12], while enhancement in activity might be due to the requirement of metal ions in the activation of enzymes [44]. In the presence of Ca²⁺, Co²⁺, and Cu²⁺, a significant enhancement is shown in the phytase activity which might be due to an increase in interaction between substrate and enzyme. Similarly, the involvement of Ca²⁺ in stimulating the phytase activity of A. niger NT7 [16], A. fumigatus [11], A. oryzae [9] has been mentioned in the literature. Co²⁺ was also found to increase the phytase activity in A. niger NT7 [16], and A. oryzae [9] but in contrast, Cu²⁺ inhibited the phytase activity in A. niger NT7 [16], A. oryzae [9], and A. niger BIONCL8 [15]. Mg²⁺ represented a non-significant positive impact on the activity at both concentrations. A similar stimulatory effect of Mg²⁺ is shown in A. oryzae [9], and A. niger BIONCL8 [15]. At 1 mM concentration of Mn²⁺, phytase activity was stimulated moderately but inhibited at high concentrations. Mn²⁺ decrease the activity of phytase obtained from A. flavus [28], and A. niger BIONCL8 [15]. With both concentrations of Na⁺ ions, activity was low as compared to the control during the study. The inhibitory effect of NaCl was observed with P. oxalicum [38] and A. niger BIONCL8 [15]. Dissimilar to this, Na⁺ enhances the activity in R. mucilaginosa [44]. There is no such effect of metal ions on the activity of A. tubingensis TEM 37 phytase [13].

Fig. 5.

a Effect of different metal ions on phytase activity. b Effect of different inhibitors on phytase activity

The effect of inhibitors on phytase activity was also analyzed. Except for sodium azide, rest inhibitors (EDTA, ß-ME, DTT, sodium molybdate) were involved in suppressing the phytase activity as shown in Fig. 5b. Maximum inhibition was shown with sodium molybdate. In the literature, EDTA was involved in inhibiting the activity of phytase isolated from P. oxalicum [38], and A. tubingensis TEM 37 [13] indicating the requirement of metal ions for the activity. Dissimilar to this, EDTA increases the phytase activity in A. niger BIONCL8 [15] and has no impact on the phytase activity of A. oryzae [9], and A. aculeatus APF1 [12]. ß-ME and DTT were also reported to decrease the phytase activity of A. niger UFV-1 [45], and P. oxalicum [38], depicting the importance of sulfhydryl groups in the catalysis of phytase [45]. Sodium molybdate and sodium azide were involved in lowering the phytase activity of A. oryzae [9], and A. tubingensis TEM 37 [13] respectively.

Effect of Proteases

Pepsin and trypsin are two important digestive enzymes present in animals and human beings. For the utilization in animal feed, phytase must have the ability to resist the action of both enzymes. As shown in Table 3, there is no significant reduction in phytase activity against trypsin and pepsin treatment indicating the enzyme is protease resistant. Pepsin showed 8 and 12% reductions in phytase activity after incubation of 1 and 2 h respectively while trypsin exhibited 10–20% reduction in phytase activity within 1–2 h. The phytase isolated from A. niger UFV-1 exhibited more than 90% activity in the presence of pepsin and trypsin treatment for 1 h [45]. Similarly, the phytase obtained from A. aculeatus APF1 and A. tubingensis TEM 37 exhibited 74% and 73% and 88 and 98% activity after 120 min of treatment with pepsin and trypsin respectively [12, 13].

Table 3.

Effect of proteases on phytase activity

Sample	Phytase activity (%)
	0 min	30 min	60 min	120 min
Control	100	95 ± 0.98	92 ± 0.99	89 ± 0.32
Pepsin treated	100	99 ± 0.77	92 ± 0.09	88 ± 0.18
Trypsin treated	100	90 ± 1.24	90 ± 1.40	80 ± 1.31

Storage Life

The stability of the enzyme depends upon the molecule and storage conditions [9]. As shown in Fig. 6, 43% of the phytase activity is exhibited at −20 °C and 38% at 4 ºC after 6 months, while 15% of the activity is left with phytase stored at room temperature. When the enzyme is stored in liquid, frozen, or powder form at different temperatures, it faces stress due to changing microenvironmental conditions and thus conformational modification occurs in the enzyme [9]. A similar study was observed in the shelf life of A. oryzae phytase and 4 ºC was found as the most suitable storage condition retaining 63% phytase activity after 6 months, while 56% activity was exhibited with the sample kept at −20 °C [9]. A. niger CFR 335 showed more than 80% phytase activity at room temperature after the incubation of 3 months [36]. P. adiposa phytase expressed a reduction in its activity after incubation of 24 days at room temperature, 32 days at −20 °C, and 42 days at 4 °C [30].

Fig. 6.

Effect of storage conditions (RT, 4 °C and −20 °C) on phytase activity

Hydrolysis of the Insoluble Phytates

Phytic acid is associated with various metal ions and is available in the complex form of insoluble metal phytate. These insoluble complexes are unable to be digested by monogastric animals and restrict the assimilation of P and other essential metal ions. In the case of plants, phytic acid is adsorbed on clays or found in the form of insoluble salts of Fe and Al (acidic soils) or salts of Ca (alkaline soils). These insoluble salt forms are unable to be utilized hence must be hydrolyse by phytase before their absorption [25]. In the case of humans and animals, these insoluble metal phytate complex leads to malnutrition conditions [46]. As shown in Fig. 7, P. oxalicum PBG30 is capable of hydrolyzing insoluble metal phytate complex at both 37 and 50 °C. It releases inorganic phosphorus from the metal ions complex which increases with time. All the metal complexes of Ca²⁺, Co²⁺, Mn²⁺, Cu²⁺, and Zn²⁺ showed efficient hydrolysis and release of inorganic phosphate except Mg²⁺, and Fe²⁺. Similarly, the hydrolysis of insoluble salt phytates by A. oryzae was reported [25]. They showed that hydrolysis action occurred at 50 °C was better than 25 °C and liberation of inorganic phosphate was enhanced with time. A. niger NT-7 phytase was also capable of releasing metal ions from the insoluble metal phytate complex and showed more liberation of Zn²⁺ > Ca²⁺ > Co²⁺ > Fe²⁺ > Fe³⁺ at 50 °C than 37 °C [46]. Phytate complexes of Ca, Mn, Mg, Zn, and Cd were hydrolysed more efficiently by A. niger phytase than the complexes of Al and Fe-phytates [47].

Fig. 7.

Hydrolysis of insoluble metal phytates by P. oxalicum PBG30 phytase a at 37 °C b 50 °C

Phytic acid also makes complexes with proteins, amino acids, and enzymes and restricts their utilization. Plant-based proteins are usually found as insoluble protein-phytate complex forms and are thus beyond the approach of monogastric animals. Due to the phytate-protein complex, turbidity occurred in the solution [46]. In the present study, a turbid solution was observed when sodium phytate was mixed with lysozyme. The release of inorganic phosphate was increased with time as shown in Fig. 8. Similar work was performed on the hydrolysis of phytate association with BSA and lysozyme by using the phytase of A. oryzae. Further, they described the time-dependent relation between inorganic phosphorus and the absorbance of the solution. Inorganic phosphorus was increased while absorbance was decreased with time [25]. Kumari and Bansal [46] also studied the relation of the absorbance of turbid solution with the release of inorganic phosphate from the phytate-lysozyme complex in the presence of A. niger NT-7.

Fig. 8.

Hydrolysis of lysozyme-phytate complex by P. oxalicum PBG30 phytase at 30 °C

Phytic Acid Reduction in Fish Feed

In the plant-based feed, phytic acid is present in major amounts. Phytase hydrolyzes the phytic acid and liberates inorganic phosphate along with associated minerals from the feed. The application of phytase in the formulation of fish feed is analyzed to determine the reduction of phytic acid in feed. The phytase-treated sample showed less amount of phytic acid as compared to the untreated sample. As shown in Table 4, the control diet (without the addition of phytase) showed 50.43 mg/g phytic acid which decreases as the dosage of phytase increases in the diet. The sample with 1500 FTU/kg exhibited minimum phytic acid. Similar to this, S. cerevisiae MTCC 5421 phytase is mixed with refined wheat flour and pearl millet flour before forming naan and rabadi, and the amount of phytic acid was measured in control food (without phytase) and phytase-treated food. Naan and rabadi prepared with phytase enzyme showed 95% and 100% reduction of phytic acid respectively [48]. In other reports, there was a reduction of phytic acid amount in bread (70%), tandoori (75%), and naan (62.5%) prepared from dough of wheat flour supplemented with recombinant phytase of Sporotrichum thermophile [49]. Also, tandoori and naan made from recombinant phytase of Pichia anomala showed a 67.5% and 23.2% decrease in phytic acid respectively [50].

Table 4.

Estimation and reduction of phytic acid in the diet after the addition of phytase

Sample	Phytic content (mg/g feed)	Reduction in phytic acid level (%)
Diet I (Control)	50.43 ± 1.15	0
Diet II (Control + 500 FTU/kg phytase)	33.00 ± 0.13	34.6
Diet III (Control + 1000 FTU/kg phytase)	30.37 ± 0.45	39.8
Diet IV (Control + 1500 FTU/kg phytase)	26.91 ± 0.83	46.6

Haloperoxidase Activity

Phytase acts like a haloperoxidase when reacted with ammonium metavanadate and enhances the peroxidase activity while decreasing its activity (Fig. 9). The mechanism behind that is vanadium ion gets incorporated in the active site of phytase and makes a semi-synthetic peroxidase. This peroxidase tends to catalyze the sulfoxidation of thioanisole. Structurally, vanadium chloroperoxidase resembles acid phosphatases and acts as a substitute for phosphate and binds to phosphoryl transfer enzyme, thus reaching the intermediate state of reaction and decreasing the phytase activity. Vanadate acts as an anion and binds with positively charged amino acids present in the active site of phytase. By interfering with the phosphoesterase activity, it changes the enzyme functionality and develops a vanadate-dependent haloperoxidase. Only HAP phytase behaves as haloperoxidase due to the similarity in their active site [50, 51]. Likewise, the semi-synthetic peroxidase behaviour of phytase was studied in S. thermophile [51], P. anomala [50], and A. oryzae [20]. The application of vanadate synthesizes peroxidase found in the formation of radiolabelled monoclonal antibodies, quantification of chloride amount in samples, diagnostic purposes, antimicrobial work, and immunological studies [50, 51].

Fig. 9.

Reduction of phytase activity a and increase in peroxidase activity b with time

Conclusion

Wheat bran is used for the production of Penicillium oxalicum phytase through the SSF method. After optimizing the conditions using the OVAT approach, phytase production was enhanced by 2.4-fold. The P. oxalicum PBG30 phytase exhibits thermostability, acidity tolerance, broad substrate specificity, and protease resistance. Its high thermostability protects the enzyme during the pelleting process in feed production. The acidic nature of phytase helps it to survive and function effectively in the gastrointestinal tract. Its resistance to proteases help in withstanding the action of digestive enzymes such as trypsin and pepsin, allowing it to efficiently degrade phytic acid. Due to these distinctive features, phytase can serve as an ideal additive in the food and feed industry. P. oxalicum PBG30 phytase showed the hydrolysis of metal-phytate and protein-phytate complexes and leading to the liberation of inorganic phosphate in a time-dependent manner. This indicates that P. oxalicum PBG30 phytase is effective in improving the nutritional content of food and feedstuffs. Additionally, the phytase plays a role in reducing phytate levels in fish feed, which helps decrease P levels in water bodies, thereby mitigating environmental pollution. The presence of haloperoxidase activity in vanadium-modified phytase suggests potential for clinical, immunological, and various biotechnological applications. However, further research is imperative to validate these possibilities.

Author’s Contributions

The original draft was prepared and edited by P and all the experiments were performed by her. Data were analyzed by P, BS, BG and JGS. The draft was critically revised and improved under the supervision of BS, BG, RKG and JGS. All authors read and approved the final manuscript.

Funding

The author (Ms. Priya) gratefully acknowledges the financial assistance as Junior/Senior research fellowship from the University Grants Commission (UGC), New Delhi, India.

Declarations

Conflict of interests

The authors declare no competing interests.