Recent advances in phytase thermostability engineering towards potential application in the food and feed sectors

Abstract

This review comprehensively examines the advancements in engineering thermostable phytase through genetic modification and immobilization techniques, focusing on developments from the last seven years. Genetic modifications, especially protein engineering, have enhanced enzyme’s thermostability and functionality. Immobilization on various supports has further increased thermostability, with 50–60 % activity retention at higher temperature (more than 50 °C). In the food industry, phytase is used in flour processing and bread making, reducing phytate content by around 70 %, thereby improving nutritional value and mineral bioavailability. In the feed industry, it serves as a poultry feed additive, breaking down phytates to enhance nutrient availability and feed efficiency. The enzyme’s robustness at high temperatures makes it valuable in feed processing. The integration of microbial production of phytase with genetically engineered strains followed by carrier free immobilization represents a synergistic approach to fortify enzyme structure and improve thermal stability. These advancement in the development of phytase enzyme capable of withstanding high temperatures, thereby pivotal for industrial utilization.

Introduction

Phytase, a versatile enzyme, is crucial in nutrition, human health, and environmental protection. Found in plants, bacteria, and fungi, it catalyzes myo-inositol hexakisphosphate’s hydrolysis, generating ortho-phosphate groups, thereby breaking down myo-inositol phosphates, myo-inositol, and inorganic phosphates (Duru Kamaci and Peksel, 2020). One of the most well-known properties of phytases is their potential to enhance animal and human nutrition by degrading phytate inositol hexaphosphate, which releases nutrients that would otherwise be inaccessible to intestinal absorption (Handa et al., 2020). Phytases have been fundamental components in the animal-feed formulations for pigs, poultry, and certain fish species. The animal feed enzymes industry, having garnered a revenue of USD 1.1 billion in 2016, is projected to exceed USD 2 billion by 2024. Notably, Natuphos, the first commercially available phytase product, marked a significant milestone in enzymatic applications within this field, leading to many more innovations in recent years (Patel et al., 2023; Song et al., 2019). Integrated into poultry diets for more than two decades, phytases are anticipated to sustain commercial growth (Mrudula Vasudevan et al., 2019).

Phytase supplementation offers significant advantages in the feed and food sectors, but its benefits are often limited by reduced enzyme activity at high processing temperatures. This limitation allows researchers to improve phytase performance through genetic engineering and immobilization techniques (Pragya et al., 2023a). The prevalence of phytate enzyme utilization is more pronounced in the food industry, primarily attributed to its distinctive capacity for chelation with essential mineral molecules inherent in the human body, including Fe, Ca, and Zn (Philippi et al., 2024). This chelation process markedly mitigates mineral deficiencies within the human system, concurrently elevating the bioavailability of specific minerals. This, in turn, substantiates its instrumental role in enhancing the health of individuals experiencing deficiencies in certain minerals (Handa et al., 2020).

In the feed industry, thermostability becomes paramount, as enzymes subjected to high temperatures play a crucial role in inhibiting the proliferation of pathogenic microbes like Salmonella, influencing their overall efficacy (Davies and Wales, 2013). Thermostability proves to be of paramount importance in the context of pelletized feed, given its exposure to steam temperatures as elevated as 95 °C (Speight, 2016). Such high temperatures risk denaturing or diminishing the enzymatic activity of phytases. Enzymes displaying heightened activity at lower temperatures might not yield the anticipated results in the feed if administered before the pelletization process (Nezhad et al., 2020). In the food industry, thermostable phytase plays a crucial role by improving the bioavailability of essential nutrients and minerals in plant-based foods. Its stability at high temperatures ensures it remains effective during food processing, enhancing nutritional value and better overall health benefits in the final food products. This makes thermostable phytase a valuable additive in the production of nutrient-rich fortified foods.

The development of thermostable phytases has been achieved through four primary approaches such as harnessing natural thermotolerant phytase from microorganism (Reddy et al., 2015), recombinantly expressing phytase genes (Ushasree et al., 2017), employing protein engineering techniques on established phytases (Chen et al., 2015), and incorporating a thermoprotective coating (Pragya et al., 2023a). In regard to the natural production of thermostable phytase enzymes, it involves exploiting thermophilic microorganisms that inherently produce enzymes capable of retaining catalytic activity and stability at elevated temperatures. Regarding natural thermotolerant phytase, several strains of fungi, yeast, and bacteria have been identified for the production of phytase, specifically for application in the feed and food sectors (Neira-Vielma et al., 2018). The commercial availability of the phytase and its microbial origin were represented in Table 1. Numerous scientific reports have been published about microbial phytases derived from filamentous fungi and bacteria such as Mucor racemosus (Alves et al., 2016), Penicillium (Kaur et al., 2017), Rhizopus (de Oliveira Ornela and Guimarães, 2019), Aspergillus (Saxena et al., 2020), Thermomyces (Jatuwong et al., 2020), Trichoderma (Pirzado et al., 2024), Bacillus subtilis (Trivedi et al., 2022), Escherichia coli (McKinney et al., 2015).

Table 1.

Commercial availability of phytase with their source of production

Country	Company name	Micro-organism	Trade name	Patent number
USA	Agrivida Inc	Peniophora lycii	GRAINZYME	US20220095647A1
	International N&h Denmark Aps	Buttiauxella	–	US8735123B2
	Alltech	Aspergillus niger	Allzyme phytase	US9879238B2
Germany	BASF	Aspergillus niger PHYA	NATUPHOS	US10093909B2
	AB Enzymes	Aspergillus awamori	Finase
Finland	Finnfeeds International	Aspergillus awamori	Avizyme
	Roal	Aspergillus awamori	Finase
	Alko Biotechnology	Aspergillus oryzae	SP, SF, TP
Denmark	Novozymes	Aspergillus oryzae	Ronozyme, Roxazyme	EP3316699B1
Mexico	Fermic	Aspergillus oryzae	Phyzyme

For instance, Geobacillus sp. TF16 exhibited maximum phytase activity 96 h after being cultured in a medium with white flour, with the enzyme’s optimal temperature determined to be 85 °C at pH 4.0, maintaining 72 % of its activity at 95 °C (Dokuzparmak et al., 2017). In another study, Bacillus subtilis B.S.46, an extracellular alkali-thermostable phytase-producing bacterium, was isolated, and the enzyme retained 83 % and 60 % of its activity at 60 °C after 90 and 120 min, respectively (Rocky-Salimi et al., 2016). Phytase from Bacillus tequilensis Dm018 exhibited approximately 70 % activity at 70 °C and maintained 100 % stability up to 60 °C (Badoei-Dalfard et al., 2019). Similarly, endogenous thermostable phytase has been synthesized and reviewed for its potential applications and advantages (Mrudula Vasudevan et al., 2019). This comprehensive review focuses on the synthesis of thermostable phytase using advanced techniques, including strain improvement and immobilization methodologies. Furthermore, the application of phytase is extensively examined in the food industry, particularly in bread-making and flour processing, as well as in the feed industry for poultry and swine nutrition.

Genetic engineering in microbial phytases: designing thermostable phytases

To begin with, genetic modification techniques aimed at enhancing phytase thermostability are explored. These modifications, including site-directed mutagenesis and protein engineering, have demonstrated significant success in improving enzyme resilience at elevated temperatures. Phytases are a class of digestive enzymes applied as a feed additive for monogastric animals, which lack the enzyme to release phosphate in their gastrointestinal tract (Henninger et al., 2023). In addition to nutrition, phytase also plays an essential role in human health and environmental protection (Gharibzahedi and Smith, 2021). Earlier, phytases utilized in feed production were conventionally sourced from microorganisms, yet proved unsuitable for the conditions inherent in feed manufacturing. Temperature is one of the crucial factors for the process conditions adopted in the feed industry, where the demand for thermostable phytases significantly increased (Kumari and Roy, 2023b). Thermostability engineering of microbial phytase entails structural modifications to augment the enzyme’s resistance to elevated temperatures. This process aims to develop phytases with sustained catalytic efficiency under high-temperature conditions, which is essential for their applications in the food and feed industries.

Phytases encounter high-temperature conditions (up to 80 °C) during the feed extrusion process in the feed industry, which leads to denaturation and loss of enzymatic activity. Therefore, phytases that can retain their catalytic activity at elevated temperatures are crucial for consideration in the feed industry (Vohra and Satyanarayana, 2002). Recent studies have unveiled structural features enabling microbial phytases to withstand industrial conditions with elevated temperatures (Yao et al., 2012). Various methods were used to analyze this, which includes the crystallization of phytases from different microbial sources, comparison of mesophilic and thermophilic phytases, and random amino acid alterations on the enzyme structure (Rebello et al., 2017; Shivange and Schwaneberg, 2017). Additionally, the quantities of isolated phytase from these microorganisms were inadequate for industrial applications (Lei et al., 2013). Genetic engineering techniques were subsequently employed to address these limitations, aiming to generate substantial quantities of enzymes required for industrial demands (Fig. 1). The thermostable phytase produced from different microbes is noted down in Table 2.

Fig. 1.

Development of thermostable phytase by various biotechnological approaches

Table 2.

Thermostable phytases derived from different microbial sources and its use in various applications

Microbial source of enzyme	Wild/Mutant/Recombinant	Method adopted	Thermal stability	Application	Reference
Yersinia intermedia	Mutant	N- glycosylation engineering according to the motif N-X-T/S	7 single mutants retained > 65 % residual activity above 50 °C Triple mutant M14 retained 75 % initial activity at 100 °C for 10 min	Feed supplement	(Wang et al., 2022)
Escherichia coli	Mutant	Saturation mutagenesis- substitution of Cysteine at position 75	Mutants designated as D7, E3, F8, F9 and G10 retained residual activity after heating at 99 °C for 60 min	Feed industry	(Xing et al., 2023)
Lactobacillus plantarum	Mutant	Disulfide bond engineering and C terminal truncation in 233 aa phytase gene(LpPHY233)	5.6 times higher specific activity and 15 °C higher thermal stability than parent strain	Agriculture and feed industry	(Yang et al., 2022b)
Penicillium polonicum MF82	Wild		81 % of initial activity retained at 80 °C	Feed industry	(Kalkan et al., 2020)
Bacillus amyloliqueifaciens ATCC 23350/DSM7 and Bacillus licheniforms ATCC 14580	Recombinant	Multifunctional xylanase-phytase domains on a single polypeptide fused by helical linker	Retained 64.2 % xylanase and 59.2 % phytase activity at 100 °C	Feed industry	(Patel et al., 2023)
E. coli	Mutant	Cloning and integration into P. pastoris genome, co-expression of PDI using a bi-directional promoter	Retained 50 % activity for 65, 75 and 85 °C after 20 min	–	(Navone et al., 2021a)
Phaselous vulgaris cv	Recombinant	Phytase gene expression and the glyphosate resistance cassettes through Agrobacterium-mediated transformation	Thermostability at 70 °C	Feed additive	(Zhao et al., 2019)
Yersinia frederiksenii	Recombinat	Plasmid construction and transformation of P. pastoris of pPICZαA-PPHO89 gene	Thermally stable at 40–50 °C	Food and feed processing industry	(Xie et al., 2020)
P. pastoris GS115	Mutant	Site-Directed Saturation Mutagenesis with phyA-C3 gene	Maintaining the thermal stability at 80 °C		(Gordeeva et al., 2019)
Streptomyces sp. (NCIM 5533)	Wild	–	Thermally stable at 70 °C with activity of 0.04 IU ml−1	Plant growth promotors	(Puppala et al., 2019a)
Aspergillus tubingensis TEM 37	Wild	–	Retaining 100 % of activity at 80 °C	Feed industry	(Çalışkan-Özdemir et al., 2021)
Bacillus tequilensis Dm018	Wild	–	Retaning 70 % of activity at 60 °C	Animal feed preservatives	(Badoei-Dalfard et al., 2019)
Streptococcus thermophilus	Wild	–	Thermally stable at 70 °C with enzyme activity of 9.90 U	Feed industry	(Priyodip and Balaji, 2020)
Aspergillus niger NT7	Wild	–	Thermally stable at 60 °C	Feed supplement	(Kumari and Bansal, 2021)
E. coli	Mutant	Construction of disulfide mutants Pichia pastoris expression	Thermostability exhibited between 70–95 °C	Feed additive	CN112204136B
Buttiauxella sp	Wild	–	Retaining 50% of activity till 65 °C	Phytic acid degradation in wheat for animal feed	EP3118309B1
E. coli	Mutant	Nucleic acid expression of phytase and monitoring Expression of an mRNA Transcript	Thermally stable between 70 and 100 °C	Ethanol production	US8936924B2
Trichoderma reseei		Expression of phytase genes through pI1c-AFP vector	Retaining its activity at 85 °C	Feed additive	US20230009832A1
Commercial	Wild	–	Thermostable	Sodium starch liquefaction	WO2021216542A2
Aspergillus sp	Mutant	Direct transformation and expression of polypeptide sequence	Thermally stable till 85 °C	Feed industry	WO2018004627A1
Pichia pastoris	Mutant	Protein engineering method through site-directed saturation mutation	Increased 27 % of activity at 85 °C	Feed industry	CN113025593B
E. coli	Mutant	Sequence of the appA gene by mutagenesis of a parent polynucleotide	Retaining its activity at 70 °C	Animal feed	EP2553091B1

Protein engineering

Protein engineering plays a crucial role in enhancing the thermostability of enzymes including phytase. Various methodologies are employed to achieve this objective, each leveraging distinct principles and techniques to increase protein stability (Herrmann et al., 2019). One prevalent approach is rational design, which involves making precise and intentional modifications to the amino acid sequence of the enzyme based on detailed structural and functional information. This method employs techniques such as site-directed mutagenesis and computational modeling to facilitate targeted alterations. By understanding the enzyme’s structure at a molecular level, researchers can identify specific amino acids to modify, thereby improving protein stability (Zhou et al., 2022). Site-directed mutagenesis was employed to induce mutations in the phyA-C3 gene, which is responsible for encoding PhyA-Cf3 phytase. The mutated phytase retains 60 % of its initial activity at 60 °C and 38 % at 80 °C after 10 min, whereas the native enzyme retains only 3 % of its activity under the same conditions (Gordeeva et al., 2019). The expression of E. coli AppA phytase in P. pastoris was investigated by using bidirectional promoters (BDPs) to co-express AppA. The AppA (PHpFMD) strain exhibited the highest AppA phytase production, demonstrating a 1.53 ± 0.01-fold increase after 132 h compared to the AppA (PAOX1) strain (Navone et al., 2021b).

Directed evolution is another highly effective strategy for augmenting enzyme thermostability (Li et al., 2019). This method involves generating a vast library of enzyme variants through processes such as error-prone PCR and DNA shuffling. The resulting variants are subjected to high-throughput screening to isolate rare variants that exhibit enhanced thermostability. Directed evolution mimics the natural selection process, allowing for the identification of superior enzyme variants without requiring prior structural knowledge (Nezhad et al., 2022). In a different study, a mutant library of E.coli AppA phytase was generated using epPCR which was followed by the saturation mutagenesis technique to enhance the thermostability while maintaining the catalytic efficiency of the respective phytase. Five mutants screened from this study including D7, E8, F8, F9, and G10 obtained 9.6 %, 10.6 %, 11.5 %, 11.6 %, and 12.2 % respectively, achieving higher residual activity than the parent strain after heat treatment for 60 min at 99 °C. The substitution of the cysteine residue at position 75 was the primary factor for the improved thermostability of mutants generated through saturation mutagenesis (Xing et al., 2023).

Consensus design represents an additional effective technique for enhancing enzyme stability. This approach involves integrating the most frequent amino acids found at each position within a protein family that shares similar three-dimensional structures. By capitalizing on the stability characteristics observed among homologous proteins, consensus design can lead to the development of more stable enzymes (Krishnan et al., 2023). A study predicted the 3D structure of the N-terminal domain and substituted amino acid residues within the active site. The E263A mutant showed significant enzymatic activity changes, highlighting the importance of residue 263 and replacing E263 with alanine-enhanced activity, increasing by 25.89 % at 40 °C and surging by 39.22 % at 45 °C. This indicates the crucial role of residue 263 in enzyme function (Lee et al., 2020).

The construction of chimeric proteins is another strategy employed to increase enzyme stability. This technique involves amalgamating various domains or motifs from different proteins to synthesize novel enzymes with improved stability. Domain swapping or the fusion of a thermostable domain into the target enzyme are common methods used in this approach, resulting in chimeric proteins with enhanced thermostability (Herrmann et al., 2021). Engineering the hydrophobic core of a protein is also a viable strategy to bolster its stability. This involves augmenting the hydrophobic interactions within the protein’s interior by substituting specific residues with more hydrophobic amino acids and designing the core to be optimally packed.

Enhancing the hydrophobic core can significantly increase the overall stability of the enzyme, making it more resistant to thermal denaturation (Gordeeva et al., 2019). In a recent study, the xylanase-phytase fusion protein was generated from a single promoter where the catalytic activity of the protein improved at optimum temperature and pH. These fusion proteins containing phytases are thermally stable up to 124 °C, higher than the wild-type phytase, which was stable only up to 113.5 °C (Patel et al., 2021). A helical linker was introduced into the XP chimeric protein in phytase to enhance its structural flexibility. Multifunctional xylanase-phytase domains, fused by the linker polypeptide, retained 64.2 % xylanase and 59.2 % phytase activity at 100 °C (Patel et al., 2023).

In conclusion, the protein engineering of phytase represents a significant advancement in enhancing its stability, activity, and overall efficacy. Methodologies such as site-directed mutagenesis, directed evolution, and computational design have successfully reported numerous improvements in the thermostability and catalytic efficiency of phytase enzymes. These engineered enzymes demonstrate superior performance in food and feed applications, ensuring better nutritional value delivery and promoting environmental sustainability.

Disulfide bonds engineering

Disulfide bond engineering enhances the thermostability of phytase by introducing covalent linkages between cysteine residues, which stabilize the tertiary and quaternary structures of the enzyme, thus increasing their resistance to thermal denaturation. This modification helps to maintain the enzyme active conformation at higher temperatures, reducing the likelihood of unfolding and inactivation. The strategic placement of disulfide bonds can significantly improve the overall structural rigidity of enzymes and functional resilience in industrial processes which requiring elevated temperatures. Engineering disulfide bonds, via methods like site-directed mutagenesis, in phytases plays a pivotal role in shaping protein stability, fostering the development of robust and thermally resilient phytases (Zhang et al., 2020).

Recombinant phytase produced by expressing the highly active 233-amino acid phytase gene (LpPHY233) isolated from Lactobacillus plantarum in E. coli exhibited increased residual activity compared to that expressed in L. plantarum. Catalytic efficiency and thermal stability of LpPHY233 were also enhanced by developing mutants through disulfide bond engineering and C terminal truncation. Mutants showed increased catalytic activity (5.6 % higher than LpPHY233) and the optimum temperature also increased (15 °C higher than LpPHY233) compared to the native phytase (Yang et al., 2022a). The strategy of manipulating disulfide bonds that enhances the ability of phytase to resist thermal degradation, making it suitable for applications in high-temperature environments.

A thermostable version of the E. coli AppA phytase, known as ApV1, which includes an additional non-sequential disulfide bond, maintained around 50 % of its activity after 20-min incubations at temperatures of 65, 75, and 85 °C. In contrast, the wild-type enzyme was nearly completely inactivated under the same conditions (Navone et al., 2021a). The PhyBL phytase gene from Bacillus licheniformis WHU was isolated, cloned, and expressed in Escherichia coli. This enzyme exhibited peak activity at pH 7.0 and maintained over 40 % of its activity within a temperature range of 35–65 °C, also its thermostability and resistance to proteolysis were improved through the introduction of disulfide bonds (Zhang et al., 2020).

Hydrogen bonds also play a critical role in the thermostability of proteins, particularly enzymes like thermostable phytases. In the context of protein structure, hydrogen bonds contribute to the stabilization of secondary and tertiary structures by holding together different parts of the polypeptide chain (Li et al., 2019). The presence of multiple hydrogen bonds can significantly increase the melting temperature of a protein, enhancing its resistance to denaturation at high temperatures. This property is particularly advantageous for enzymes used in industrial processes, where high thermal stability ensures prolonged activity and efficacy (Nezhad et al., 2022). In conclusion, the incorporation of disulfide bonds as well as hydrogen bonds significantly enhance the thermostability of phytase, making it a more robust and reliable enzyme under high-temperature conditions. This increased stability ensures that phytase retains its activity and efficacy, offering substantial benefits for its various industrial applications.

Engineering the glycosylation pattern of phytase

Proteins undergo glycosylation, a key structural modification in biological systems, to enhance diversity. This process targets serine/threonine (O-linked) and asparagine (N-linked via Asn-X-Thr/Ser) residues with diverse glycans. Notably, glycans play a crucial role in significantly improving glycoprotein stability. With increasing glycosylation, the protein structure becomes stiffer, and effective distance between protein electrostatics expands, contributing to enhanced overall stability (Gordeeva et al., 2023). A study reveals that a novel phytase from Cronobacter turicensis, characterized by glycosylation exhibits strong resistance to pepsin, trypsin, and acidic pH, and enhanced thermostability, demonstrating that N-glycosylation in P. pastoris enhances its biochemical properties (Gordeeva et al., 2023).

N-glycosylation engineering done on the phytase APPA from Yersinia intermedia produced 7 single mutants with improved thermal stability compared to the wild type. Additionally, the triple mutant M14 developed from the same study showed unchanged catalytic efficiency along with 75% retention in its initial activity after the heat treatment for 10 min at 100 °C (Wang et al., 2022).

Another study uses Metadynamics (METADY) simulations to examine Aspergillus nidulans phytase, focusing on pH and glycosylation effects on stability and activity. Strategic combinations of pH and glycosylation shift the enzyme’s conformations from metastable to stable. Key segments (H2, H5-7, L8, L10, L12, L17) are crucial in these changes. Glycosylation stabilized the structure and enhances substrate docking, with the best phytate receptivity at pH 6.5 for unglycosylated and pH 4.5 for glycosylated forms (De Souza et al., 2024). Engineering the glycosylation pattern of phytase not only markedly improves its stability and activity in acidic environments but also enhances its overall structural integrity and substrate affinity. Optimal glycosylation shifts the conformational states of enzyme, resulting in superior performance across various pH values (Wang et al., 2022). These findings provide valuable insights into the rational design of more efficient phytase enzymes for use in animal feed and human dietary applications. Furthermore, they highlight the potential for biotechnological advancements through the optimization of glycosylation patterns (Herrmann et al., 2021).

In conclusion, genetic modification has proven to be a powerful tool in the strain improvement of microorganisms for the production of thermostable phytase. These modifications, including the introduction of disulfide bonds, glycosylation motifs, and targeted amino acid substitutions, have resulted in significant improvements in enzyme performance under high-temperature conditions. The ability to produce thermostable phytase addresses the challenges posed by industrial processes and opens new avenues for its application in the food and feed industries, where high thermal resistance is crucial. Furthermore, on the downside, the promise of genetic engineering in phytase enables the improvement of the food and feed industries in pursuit of improving nutritional and environmental sustainability. However, its application in these industries faces several challenges, including regulatory issues, public perception, and potential ecological impact. These limitations must be overcome by implementing appropriately strong regulatory regimes that guarantee the safety and efficacy of genetically modified phytase.

Matrices for immobilization of thermostable phytases

Free form of enzymes has higher catalytic activity but, they lack stability and cannot be reused. However, immobilized enzymes are more stable, reusable, and suitable for continuous processing, and lowering operational costs. Therefore, immobilization of phytase enzymes is a promising strategy to enhance their thermostability, help to maintain their native conformation, and ensure prolonged activity under elevated temperatures (He et al., 2023). Furthermore, immobilization promotes the interactions between enzyme moieties and the supporting matrix, thereby reinforcing the enzyme’s structure through either non-covalent interactions or chemical bonding mechanisms (Pragya et al., 2023a). Moreover, Immobilization removes inhibitory by-products, enhancing enzyme stability and functionality, making them more effective in high-temperature industrial applications. (Filippovich et al., 2023).

Various immobilization techniques include enzyme immobilization by adsorption (Lahiji et al., 2021), covalent bonding (Naghshbandi et al., 2018), entrapment (Zahid et al., 2023), cross-linking (Venkataraman and Vaidyanathan, 2023), and encapsulation (Weng et al., 2024) were utilized to improve thermostable phytase by enhancing stability and performance across different temperatures. Support materials for enzyme immobilization encompass a diverse range, including organic matrices, inorganic materials, and nanomaterials, each contributing distinct advantages to enhance enzyme stability and catalytic efficiency.

Inorganic materials

Inorganic supports are ideal for enzyme immobilization due to their thermal stability, mechanical resistance, and robustness. They protect enzymes from denaturation under high temperatures and harsh conditions, ensuring longevity and durability. These properties enhance the operational stability and catalytic efficiency of enzymes, leading to more reliable and sustainable industrial processes (Sharma et al., 2022). Inorganic supports includes hydroxyapatite and metal ions are highly resistant to microbial growth and are characterized by their stiffness and porosity. Commercially available inorganic carriers with various pore sizes optimize protein loading, enhancing the efficiency of the immobilization process (Zucca and Sanjust, 2014).

Lopes and co-researchers conducted a study on the immobilization of phytase in iron-modified zeolite, employing a 1 mM concentration of iron for fabrication. The immobilization process resulted in a 73 % immobilization yield and a notable 131 % activity recovery under pH 7.0 conditions with a 2 h incubation period (Lopes et al., 2021b). In a subsequent study by the same group, zeolite was engineered with nickel, and phytase was immobilized onto this modified substrate. The immobilized enzyme demonstrated thermostability, achieving a 66 % immobilization yield and 151 % activity recovery. This highlights the zeolite-nickel composite’s effectiveness for enhanced phytase immobilization and thermal resilience (Lopes et al., 2021a). Inorganic hydroxyapatite and zeolite have been employed as pivotal components and most of the inorganic materials are employed in the nanoform, establishing a robust foundation for various applications. Immobilization of phytase with polyvinyl alcohol and chitosan with the optimum condition of pH 5.0 and temperature 45 °C exhibiting the maximum velocity of 31.25 U/mg utilized in animal feed, agriculture, and food industry (Duru Kamaci and Peksel, 2020). Among the various inorganic supports, metal ion-based supports have demonstrated significant thermostability of phytase. This evidence underscores the potential of thermostable phytase for improving the efficiency and durability of processes in the animal feed, agriculture, and food industries.

Organic materials

Organic supports provide a biocompatible matrix that enhances enzyme stability and functionality, crucial for diverse biotechnological applications. Organic supports which include polysaccharides (Sharmeen et al., 2019), acrylic and vinylic polymers (Zahid et al., 2023) as well as modified celluloses (Jalili Tabaii et al., 2018), dextran (Henninger et al., 2023), chitosan (Onem et al., 2016), and polyvinyl alcohol (Kamaci and Peksel, 2020) were used for the immobilization. Phytase was encapsulated within a core–shell hydrogel bead system featuring a chitosan-phytase core. Remarkably, approximately 70 % of the catalytic activity was demonstrated to be retained even following exposure to elevated temperatures, specifically heating at 90 °C (Yang et al., 2024).

In another study, alginate was selected as the organic matrix for immobilizing phytase using the spray drying method to enhance the enzyme’s tolerance to high temperatures. Remarkably, the immobilized enzyme demonstrated significant thermostability, retaining 58 % of its activity even at 95 °C. This approach highlights alginate’s promise as a robust organic support for improving the functional characteristics of immobilized enzymes (Weng et al., 2022b). Furthermore, the immobilization of phytase onto glutaraldehyde-activated chitosan microsphere with optimum pH 4.0 and 40 °C achieved the maximum yield of 77.48 %. This immobilized phytase with chitosan utilized in the additives in the food and feed industry (Belho and Ambasht, 2021). Additionally, for the degradation of phytate content in animal feed, cross-linked enzyme aggregates of phytase achieved 80 % phosphate liberation (Henninger et al., 2023).

In conclusion, using organic supports for phytase immobilization significantly enhances enzyme thermostability and functionality, especially in alginate, crucial for high-heat processes in the food and feed industry. This method extends enzyme lifespan, reduces operational costs, and promotes sustainability. The versatility of organic supports allows for customization, optimizing enzyme activity for specific applications, thereby broadening the scope of phytase use and advancing efficiency and innovation in the industry.

Nanomaterials

Nanomaterials are ideal for enzyme immobilization due to their unique physicochemical characteristics, including chemical inertness, structure, biocompatibility, thermal and mechanical capabilities, expansive surface area, and electrical and magnetic properties. For the loading of enzymes and minimal sustainability for mass transfer of the substrate to the catalyst, nanomaterials provide large surfaces. Currently, nanomaterials are employed for immobilizing certain enzymes, resulting in enhanced stability and superior performance of the immobilized enzyme (Xin et al., 2020). They boost the ratio between surface area and volume. In the immobilization approach, nanoparticles, nanofiber (Harati et al., 2017), nanotubes (Lahiji et al., 2021), and nanoparticles (Greiner et al., 2013), porous and polymeric nanoparticles (Coutinho et al., 2020) were employed.

In a study, hydroxyapatite nanoparticles effectively immobilized phytase (Natuphos®) under optimal conditions of 20 mM and pH 5.0. This resulted in a 100 % immobilization yield and recovery activity, reaching 2086 ± 41 IU × g⁻¹ support. The findings emphasize hydroxyapatite’s potential for enhancing enzyme thermostability and catalytic performance (Coutinho et al., 2020). Polyvinyl alcohol (PVA) and chitosan were electrospun into nanofibers for phytase immobilization, employing a ratio of 90:10 for PVA and chitosan, along with a 1.5 % enzyme load. The resulting nanofibers exhibited an average diameter of 65.3 ± 26.0 nm. Notably, retention of enzyme activity even at 90 °C demonstrated enhanced thermal stability of the immobilized phytase (Duru Kamaci and Peksel, 2020).

The adsorption of phytase from Yersinia intermedia onto amino-functionalized multi-walled carbon nanotubes significantly enhanced its catalytic activity and thermostability. The optimal immobilization conditions were determined to be 4 h at 50 rpm at 20 °C, resulting in an immobilization efficacy of 55.6 % (Lahiji et al., 2021). Rather than maintaining enzyme stability, nanoparticles can also be easily separated from the reaction mixture and their chaperon-like property prevents the possibility of product contamination (Weng et al., 2022a).

Chitosan-coated zinc nanoparticles with the activity of 25,186 U/mL utilized in improvement of the human and animal health along with probiotic bacteria (Chatraei and Emtiazi, 2019). Similarly, zinc oxide coated nanoparticles with phytase exhibiting thermal stability of 80 °C utilized in the application of agriculture for tomato seedling growth (Dikbaş et al., 2024). Additionally, magnetic nanoparticle-coated phytase with catalytic efficiency of 114.03 M⁻¹ S⁻¹ was utilized to prevent the eutrophication in water bodies (Dave and Modi, 2019)_.

In conclusion, the utilization of chitosan based nanomaterials for phytase immobilization represents a highly efficacious strategy, offering remarkable stability and efficient catalytic performance. This innovation is particularly advantageous in the food and feed industry, where high-temperature processes are common. Nanoparticle-immobilized phytase improves nutrient availability and digestion while reducing the need for frequent enzyme replenishment, promoting sustainability and cost-effectiveness. Various strategies involved in the designing of thermostable phytase have been shown in Table 3.

Table 3.

Different support materials used for the immobilization of phytase with their yield and applications

Support material	Immobilizing conditions	Characteristics of immobilized phytase	Yield/Activity Recovery	Applications	References
Organic material
Chitosan microsphere	Glutaraldehyde concentration (0.5 v/v%) Chitosan (1.5 w/v%) Cross-linking time: 4 h	Optimum pH: 4.0, Optimum Temp:40 °C, Ea: 34.5 kJ/mol, Km: 0.62 mM, Vmax: 3.42 µmol/min, Thermally stable up to 50 °C	Yield: 77.48 %	Additive for the food/feed industry	(Belho and Ambasht, 2021)
Poly- (acrylamide-coacrylic acid) hydrogel	Ammonium persulfate (1.5 ml) concentration Enzyme (0.25 w/v%) Cross-linking time: 12 min	Optimum temperature: 50 °C Optimum pH: 7.5 Thermostable up to 70 °C and a wide pH	Activity: 9.12 ± 0.28 U/g	Helps in plant growth promoting activities	(Zahid et al., 2023)
Ca-alginate	–	Km: 0.66 mM; Vmax: 666.6 nmol/sec	Recovery: 70 %	Additive in food industry	(Pragya et al., 2023b)
Cross linked enzyme aggregates	pH:5.5 Glutaraldehyde concentration: 100 mM Cross-linking time:1 h	Optimum pH:2.2–3.5:Thermally stable between 60–90 °C	Residual activity: 425 %	Phytate degradation in animal feed with 80% of phosphate liberation	(Henninger et al., 2023)
Inorganic material and nanomaterials
Polyvinyl alcohol and, Chitosan	PVA-12%, Chitosan-2 %, Phytase concentration-2 %, stirring, Cross-linking time:2 h	Optimum pH: 5.0, Optimum Temperature:45 °C, Km: 13.43 mM, Vmax: 31.25 U/mg:Thermally stable up to 90 °C	–	Animal feed, agriculture, pharmaceutical, and food industry	(Duru Kamaci and Peksel, 2020)
Immobilized on biochar by physisorption	Biochar: 1 mg Phytase: 2 mg pH: 5.0 Crosslinker concentration: 1.5 %; Immobilization time: 24 h	Optimum pH: 5.0 Km: 38.2 mM; catalytic activity exhibited up to 60 °C	Recovery: 77–85 %	Utilized in agricultural industry for facilitating mineralization	(Li et al., 2021)
Hydroxyapatite Nanoparticle	Enzyme concentration: 0.05 g/ml; Adsorption time: 1 h; Temperature: 25 °C	Optimum pH: 5.0 Thermal resistance: 60 °C to 70 °C, Thermal Stability: 80 °C to 90 °C, Ionic Strength: 20 mM	Yield & Recovery: 100 %	Animal and Aquatic feed	(Coutinho et al., 2020)
Polyvinyl alcohol:chitosan nanofibers	Enzyme concentration of nanoparticle: 0.5–2 % Voltage: 23 kV	Optimum pH: 5 to 6; Optimum temperature: 45 °C to 65 °C Vmax: 53.76 to 31.25 U/mg;	High yield	Animal feed, Agriculture, Pharmaceutical and Food industry	(Kamaci and Peksel, 2020)
Zeolite-Ni	Concentration of nanoparticle: 0.05 g/mL, Room temperature; Enzyme concentration: 0.1 g/mL; Immobilization time: 2 h	Km: 0.46 to 13.43 mM; Thermally stable up to 60 °C Natuphos® E 10000 Genetically modified from A. niger concentration—0.1 g mL −1	Yield upto 73 % Recovery upto 131 %	Utilized in hydrolysis of phytate in animal feed and food industry	(Lopes et al., 2021a)
Zeolite-Fe	pH: < 7, phytase concentration: 0.1 g/mL; concentration of support: 0.05 g/mL; Adsorption time: 15 mins	Improvement of the catalytic activity, better performance in acid pH, Reusability; Thermal stability up to 60 °C	Yield—66 % Recovery—150 %	Hydrolysis of food ingredients and animal feed	(Lopes et al., 2021b)
Amino-multi-walled carbon nanotubes	Concentration of carbon nanotubes-0.5 mg mg; Enzyme concentration: 1 mg/mL; Immobilization time: 4 h pH-7.0, Room temperature	Derived from Yersinia intermedia Optimum. pH-5.0 and 5.7, Optimum. temperature—50 °C and 70 °C	High stability at pH-3.7 Initial catalytic activity retention 35 % and 17 %	Animal feed industry	(Lahiji et al., 2021)
Polyvinyl alcohol/Sodium alginate, Electrospun nanofiber	Polyvinyl alcohol (10% (v/v)), Sodium alginate 2 % (v:v), Phytase concentration:2%, 23 kV voltage at room temperature	Optimum pH-5–6, Optimum Temperature—45–55 °C, Km- 0.46–4.66 mM and Vmax- 53.76–18.38 U/mg	53.76–18.38 U/mg	Animal feed, agriculture and food industry	(Duru Kamaci and Peksel, 2021)
Aluminum oxide beads	pH 4.5; Enzyme concentration: 16.02 IU room temperature (25–27 °C); Glutaraldehyde concentration: 2.5 %; Cross-linking time: 1.5 h	Thermally stable at temperature of 50 °C pH -4.5	Yield-71.50 %	Increase the digestability of poultry feed sector	(Gampa et al., 2024)
Zinc oxide nanoparticle	Crosslinker concentration 0.5 % ZnO nanoparticle: 1 mg/mL Crosslinking time: 30 min	pH: 5.2 Temperature: 37 °C; Thermally stable up to 45 °C	Activity: 25,186 U/mL	Probiotic bacteria with immobilized phytase in food improved the health of human and animal	(Chatraei and Emtiazi, 2019)
Silica magnetic nanoparticles	Phytase concentration: 2 mg/mL pH:5.5 Adsorption time: 20 h	pH 3.0; Enzyme active up to 55 °C	Recovery: 34 %	Prolonged release of enzyme in gastrointestinal conditions of monogastric animals	(Xin et al., 2020)
Znc oxide nanoparticle	Concentration of nanoparticle 100 mg/L Phytase: concentration: 715 pH 5.5; adsorption time: 2 h	Optimum pH: 5.0 Optimum temperature: 70–80 °C	Recovery: 7.6 %	Beneficial impact on tomato seedling growth	(Dikbaş et al., 2024)
Magnetic nanoparticle	pH:4.5 Glutaraldehyde concentration: 0.3% Cross-linking time:12 h Enzyme concentration:100 U	Thermostability up to 50 °C; Catalytic efficiency: 114.03 M−1 S−1	–	Prevent eutrophication in water bodies	(Dave and Modi, 2019)

Thermostable phytase for food and feed applications

Phytases play a crucial role in enhancing animal nutrition by mitigating phosphorus excretion, contributing to environmental sustainability, and improving mineral bioavailability. Their incorporation into commercial diets for swine, poultry, and fish yields notable benefits, and they can be effectively utilized in both cereal-based diets and general feed formulations (Valente Junior et al., 2024). Particularly advantageous in animal feed applications is the use of thermotolerant phytases. Furthermore, integrating phytase into food processing positively impacts mineral and trace element bioavailability, as well as the cost dynamics of the manufacturing process, thereby enhancing both the yield and quality of end products (Fig. 2) (Handa et al., 2020). Practical advancements in bread making can be realized through the strategic inclusion of phytase during food processing, emphasizing its potential to make valuable contributions to the broader realm of food production. The application of phytase in food and feed has been jotted down in Table 4.

Fig. 2.

Application of thermostable phytase in food and feed industries

Table 4.

Application of phytase in food and feed sectors

Phytase producing microbes	Target food/feed	Optimal conditions				Phosphorus liberated/observed	Remarks	Reference
		Enzyme Units	pH	Temperature (°C)	Incubation period
Food processing
Aspergillus aculeatus	Wheat flour	2 U	2.5	37	2 h	89.98 %	Potential for dephytinizing grain based food products	(Saxena et al., 2020)
Aspergillus fumigatus	Wheat flour	10 U	5.5	65	30 min	78.7 %	Higher activity towards Ca-phytate	(Thakur et al., 2022)
Saccharomyces cerevisiae	Ragi and Soya	15 IU	6.5	28	2	–	Improved nutritioned food by making them bioavailable	(Puppala et al., 2019b)
Aspergillus oryzae SBS50	Wheat flour	506.12 U/g	5.0	30	24 h	10.42 mg/g	Enhancing the nutritional quality of flours; Utilized in food processing sectors	(Pragya et al., 2023b)
	Pearl millet flour					9.1 mg/g
	Gram flour					6.2 mg/g
	Rice flour					3.9 mg/g
Commercial	Soy milk	2.5 %	6.0	45	120 min	–	Improved bone quality, circulating Fe and Zn level	(Ma et al., 2023)
Animal feed
Aspergillus niger	Soybean meal	25 U/g of feed	5.0	60	50 min	92.5–98 %	Potential additive in the fish feed	(Coutinho et al., 2020)
Aspergillus niger NT7	Maize bran	10 U	4.8	50	24 h	1723.04 ± 45.32 µg/ml	Magnesium and iron exhibited over 100% mineral release Manganese and zinc demonstrated less than 50% release; Additionally reducing sugar, total protein as well as amino acids has been released	(Kumari and Bansal, 2023a)
Mucor indicus MTCC 6333	Wheat ran	150 U/L	7.0	60	12 h	28.72 mg/g	Phytase lowers the anti-nutritional factor in the insoluble dietary fiber	(Venkataraman and Vaidyanathan, 2023)
	Rice bran					52.08 mg/g
Aspergillus niger	Mixture of sorghum, maize, soya bean, kidney bean, wheat, and rice	3 U	2.1	30	12 h	48.14 to 82.14 %	Enhance nutrient bioavailability in non-ruminant animals	(Bhandari et al., 2023)
Saccharomyces cerevisiae	Chick pea and Animal mash feed	15 IU	6.5	28	2 h	–	High chelating property	(Puppala et al., 2019b)
Saccharomyces cerevisiae	Rice bran	550 FTU kg−1	5.5	55	2 h	2.73 ± 0.05 %	Enhancing fish performance through improved nutritional value	(Ries et al., 2020)
Aspergillus niger NT7	Wheat bran	10 U	4.6	50	72 h	2460 ± 102 µg/ml	Ideal additive in cattle feed Potential as a low-cost animal feed supplement	(Kumari and Bansal, 2021)
Aspergillus oryzae SBS50	Soy bean meal		4.5	55	1 h	81 %	Improve the digestibility of poultry feed	(Gampa et al., 2024)

Food processing

Thermostable phytase plays a crucial role in food processing by breaking down phytic acid, an indigestible form of phosphorus found in plant seeds and grains, into a digestible form. This enzyme’s stability at high temperatures makes it particularly valuable in industrial processes such as baking, brewing, and animal feed production (Thakur et al., 2022). By improving phosphorus and mineral bioavailability, thermostable phytase enhances the nutritional quality of food and feed, reduces the need for inorganic phosphorus supplements, and mitigates environmental pollution from phosphorus runoff. Its ability to remain active under heat-intensive conditions ensures its effectiveness throughout various stages of food processing, making it an essential tool for enhancing nutrient utilization and promoting sustainability in the food industry (Longin et al., 2023).

Flour processing and bread making

In flour processing, thermostable phytase plays a pivotal role by breaking down phytic acid, an anti-nutrient, into digestible forms of phosphorus and inositol. This enzymatic action enhances the bioavailability of essential minerals such as calcium, magnesium, and zinc, even under high-temperature conditions typical of baking. Consequently, the nutritional value of flour-based products is significantly improved, promoting better health outcomes (Atuna et al., 2022). In a study, the thermotolerant Aspergillus fumigatus phytase demonstrated effective dephytinization of wheat flour, with a recorded reduction of 78.7 % of phytate with the optimum temperature of 65 °C at pH 5.5. This observation provides valuable insights for potential applications at a commercial scale, opening avenues for multi-dimensional future biotechnological applications (Thakur et al., 2022).

Phytase significantly enhances breadmaking by reducing phytate content, improving both nutritional value and bread quality while shortening fermentation time, and boosting baking efficiency and volume (Vilanculos et al., 2024). Additionally, phytase refines crumb texture, enhancing sensory appeal. As a versatile tool, it combines nutritional fortification with processing improvements, making it an invaluable ally in breadmaking and elevating overall product quality (Nevskaya et al., 2021).

In conclusion, the strategic application of phytase in the food industry proves indispensable, enhancing nutrient bioavailability and promoting sustainable practices through its pivotal role in phytate hydrolysis, ultimately contributing to the formulation of nutritionally enriched and economically viable food products.

Animal feed

Most of the available phosphorous content in nature is present in plants, and this plant-based feed remains undigested and poorly absorbed in animals because of a lack of phytases (Wise et al., 2024). Thus, the addition of phytases to the feed helps to improve the absorption of phosphorous from plant products (Madhavan et al., 2019). The incorporation of phytase in the diets of monogastric animals proves to be a cost-effective strategy in mitigating the excessive expenses associated with supplying inorganic phosphate (Mahendran et al., 2024). Soybean meal, a prominent component in animal feed and a vital non-animal protein source contains phytase that aids in the efficient breakdown of phosphorus. This not only enhances the nutritional profile of the feed but also contributes to prudent resource utilization, thereby promoting economic efficiency in animal husbandry practices (Lopes et al., 2021a).

Poultry

Poultry diets mainly consist of cereals, legumes, oilseed crops, and their by-products. Unfortunately, the primary phosphorus in these feeds is phytate, which is not easily accessible to poultry. This is because monogastric animals lack sufficient endogenous mucosal phytase to effectively break down dietary phytate. The thermostable ApV1 phytase can be used as a dietary supplement for swine and poultry to enhance the digestibility of phytic acid, thereby improving nutrient absorption and overall animal health (Chen et al., 2013; Navone et al., 2021a). In a study, the incorporation of fungal phytase from Trichoderma reesei into the diets of laying hens significantly enhances their digestion of essential nutrients, such as phosphorus and calcium. This improvement fosters optimal egg production and quality, thereby contributing to sustainable and cost-effective poultry farming practices (Pirzado et al., 2024).

A study on phytase supplementation in broiler hens using 8000 U/kg highlighted its thermostability and dephytinization efficiency. Results showed increased nutrient availability, with dry matter at 79.6%, organic matter at 89.3 %, and crude protein at 73.3 %. Thermostable phytase also improved energy utilization to 13.36 MJ/kg, reducing the need for additional energy-rich diet ingredients and enhancing overall farming productivity (Song et al., 2021).

In another study, the phytase derived from Penicillium oxalicum, employed in the dephytinization of feed, demonstrated an enhancement in the nutritional value by liberating inorganic phosphate and additional nutrients. Optimal results were attained at a temperature of 37 °C, utilizing 200 U of phytase in 0.5 g of feed over 48 h, yielding the highest levels of inorganic phosphate, reducing sugar, and soluble protein 33.986, 134.4, 115.52 mg/g feed, respectively (Priya et al., 2023).

Several studies were conducted to evaluate variations in phytase activity after pelleting for thermostability. In a study, the thermo-stability of two phytase products, Ronozyme NP and Phyzyme XP TP, was investigated. The results showed that more than 80 % of the enzyme activity was preserved after conditioning and pelleting up to 95 °C. The study concluded that the pelleting process in a feed mill, which usually doesn’t exceed 95 °C, won’t affect the activity of the studied enzymes (Vasudevan et al., 2019).

In a separate investigation, prompted by a European Commission directive, the European Food Safety Authority (EFSA) evaluated the safety and efficacy of 6-phytase (Nutrase P), derived from genetically modified Komagataella phaffii. The additive was assessed for use in fattening chickens, other poultry, and ornamental birds. The EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP), after reviewing long-term trials and recent studies, concluded that the additive is effective at 1500 FTU/kg in complete feed formulations for these birds (FEEDAP et al., 2022). In a similar vein, the EFSA Panel on Additives and Products or Substances used in Animal Feed assessed the safety level of HiPhorius for fish feed. The study found that a nominal concentration of 1000 FTU/kg of feed is suitable (FEEDAP et al., 2024). Henceforth, the significance of thermostable phytase as a feed additive lies in its ability to withstand high temperatures, ensuring sustained enzymatic activity during feed processing. This resilience contributes to optimal nutrient release and absorption in animal diets, ultimately enhancing overall feed efficiency. In conclusion, the incorporation of thermostable phytase proves pivotal for maximizing the nutritional value of feed formulations in various livestock production systems.

Future perspective

The development of thermostable phytase holds significant potential to revolutionize the food industry by enhancing product quality, promoting sustainability, and supporting the growing demand for nutritious, plant-based, and functional foods. Immobilization techniques have been particularly successful in maintaining enzyme activity under extreme operational conditions, thus prolonging their functional life and markedly reducing operational costs. Similarly, advancements in genetic engineering along with carrier free approach in immobilization technique have led to the production of phytase variants that are both efficient and robust. These perspectives suggest that thermostable phytase will continue to be a valuable enzyme in various industrial applications, contributing into more sustainable and efficient processes.

The addition of thermostable phytase to food and feed formulations greatly enhances the bioavailability of important nutrients, such as phosphorus, which is crucial for optimal growth in animals and reducing the environmental impact of phosphorus run-offs. This dual-purpose benefit not only promotes sustainable agricultural practices but also addresses essential nutritional deficiencies in both animal and human diets. In addition to the benefits provided by thermostable phytase, the integration of non-starch polysaccharidases (NSPases) into food and feed formulations offers a promising avenue for further enhancing nutrient bioavailability. The synergistic approach of thermostable phytase and NSPases in feed formulations could revolutionize the agricultural industry by optimizing feed conversion rates, and reducing the need for supplemental nutrients. Also, it enhance the nutrient bioavailability and improve the digestibility of plant-based foods. These enzymes also offer potential for creating sustainable, health-focused food products that address specific dietary needs while minimizing environmental impact. Continued research and innovation in this field are essential to fully realize these benefits.

Funding

No funding.

Declarations

Competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.