Phytase mutants and uses thereof

Abstract

The application relates to the technical field of enzyme engineering, and particularly discloses a phytase mutant and application thereof. The phytase mutant T273K with improved heat resistance is obtained through rational design and site-directed mutagenesis based on a Tuberinflata phytase. The mutant has obvious improvement in heat stability and catalytic capacity relative to the mutant before the mutation; the relative enzyme activity is 69.4% after incubation at 60 DEG C for 90 min, which is 20.4% higher than that of WT; the specific activity is about 77.78% higher than that of WT, the catalytic efficiency k cat / K m is 50.56% higher than that of WT; and the enzyme activity retention rate reaches 33.8% under 10% salt. The phytase mutant T273K has the comprehensive characteristics of salt resistance and heat resistance, can better play the effect of enzymatic hydrolysis of phytic acid in the food processing process, can significantly reduce the turbidity caused by phytic acid in liquid foods such as soy sauce, vinegar and cooking wine, thereby improving the product quality, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and in particular relates to a plant acidase mutant and its application. Background Technology

[0002] In the natural plant system, phytic acid mainly exists in the form of its salt derivatives (such as calcium and magnesium phytate), and is widely distributed in the seed tissues of high-fiber plants such as cereals, legumes, and nuts as a key form of phosphorus storage. Due to its unique spatial topology formed by six phosphate groups, this molecule exhibits a specific chelating ability with dietary minerals and proteins. Sufficient research evidence has shown that phytates reduce the nutritional value of food by inhibiting the bioavailability of trace elements (calcium, magnesium, zinc, iron, etc.), interfering with the hydrolysis of food proteins, and hindering the function of digestive enzymes. Given the central role of metal ions and biomolecules in the nutritional metabolic network, phytic acid and its derivatives have been established as classic anti-nutritional factors since the 1920s.

[0003] Phytase, a class of specific hydrolases widely found in plants, bacteria, and fungi, catalyzes the dephosphorylation of phytate to produce orthophosphate, inositol phosphate isomers, and free inositol. The multidimensional physiological functions of this enzyme demonstrate significant scientific value in nutritional fortification strategies, public health interventions, and ecosystem sustainability. Its core mechanism involves degrading phytate-mineral complexes, releasing chelated essential elements such as phosphorus, calcium, zinc, and iron, thereby significantly improving the bioavailability of plant-derived nutrients in monogastric animals and humans. In livestock production systems, phytase has become an indispensable biological additive in the formulation of diets for pigs, poultry, and certain aquatic species.

[0004] The potential applications of phytase in human food systems are of equal strategic value to the mature practices in animal feed. This importance stems from the metal chelating effect of phytic acid (inositol hexaphosphate)—its phenolic hydroxyl groups react with Fe... 2 ⁺、Zn 2 ⁺ and Ca 2Phytase forms insoluble complexes with minerals such as ⁺, which may lead to or exacerbate nutrient antagonistic deficiencies in humans. Its chelation with large protein molecules can also reduce the nutritional value of food. Existing research shows that phytase has several successful applications in the food industry: adding immobilized phytase to wheat and millet flour to develop low-phytic acid functional foods and reduce the use of mineral fortifiers; introducing phytase into bread making to effectively reduce phytic acid content and improve nutritional quality, while simultaneously activating endogenous α-amylase by improving calcium utilization. However, in food processing, phytase is often used as a processing aid and is inactivated after product manufacturing. For comprehensive considerations such as flavor preservation, microbial control, and processing efficiency, enzyme treatment temperatures in food processing are generally between 50-70℃. Besides the challenge of high temperatures to enzyme stability, different foods often contain environmental factors such as high salt, high sugar, and alcohol content, which affect the enzyme's effectiveness. The stability of naturally derived phytases is often affected by factors such as high temperature, high salt, and alcohol content, which limits their application in the food industry. Fungal phytases suitable for food applications have certain advantages in dealing with alcohol and high osmotic pressure, but they have inherent deficiencies in terms of thermal stability. Improving the stability of phytases is an urgent need. Summary of the Invention

[0005] The purpose of this invention is to overcome the aforementioned problems in the prior art and provide a phytase mutant with significantly improved heat resistance, which is beneficial for promoting its widespread application in the food processing field.

[0006] To achieve the above objectives, the inventors conducted in-depth research and, through repeated studies and demonstrations, obtained the solution of this invention, as detailed below:

[0007] In a first aspect, the present invention provides a phytase mutant, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0008] Secondly, the present invention provides a gene that encodes the aforementioned phytase mutant.

[0009] Thirdly, the present invention provides a recombinant expression vector carrying a gene encoding the above-mentioned phytase mutant.

[0010] The vector backbone of the recombinant vector can be selected by those skilled in the art according to implementation needs, and can be any expression-capable vector disclosed or not disclosed in the art, including but not limited to eukaryotic vectors and prokaryotic vectors. The protease gene of the present invention is inserted into a suitable site on the expression vector, so that its nucleotide sequence is operably linked to the expression regulatory sequence.

[0011] Fourthly, this application provides a recombinant host cell that expresses the above-mentioned phytase mutant or is transformed / transfected with the above-mentioned recombinant expression vector.

[0012] The host cell can be any cell type with expression capability that is disclosed or not disclosed in the art, including but not limited to eukaryotic cells or prokaryotic cells, preferably Pichia pastoris.

[0013] Fifthly, embodiments of this application provide the application of the above-mentioned recombinant expression vector or recombinant host cell in the production of phytase.

[0014] Sixthly, embodiments of this application provide the application of the above-mentioned phytase mutant in phytic acid degradation.

[0015] Seventhly, embodiments of this application provide the application of the above-mentioned phytase mutant in food processing; specifically, it can be used for enzymatic hydrolysis of phytic acid.

[0016] The food products mentioned are liquid foods such as soy sauce, vinegar, and cooking wine.

[0017] Eighthly, embodiments of this application provide an enzyme preparation comprising the above-mentioned phytase mutant.

[0018] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0019] 1. Based on Aspergillus tabineum phytase, this invention obtains a mutant T273K with improved heat resistance through rational design and site-directed mutagenesis screening. This mutant has significantly improved thermal stability and overall catalytic ability for proteins compared to the original mutant. It can better exert the effect of enzymatic hydrolysis of phytic acid in food processing and has broad application prospects in the food processing field.

[0020] 2. The phytase mutant T273K provided by this invention retains 69.4% of its relative enzyme activity after incubation at 60℃ for 90 min, which is 20.4% higher than that of WT. Its half-life at 60℃ is 25.72 min, which is about 40.43% longer than that of WT, and its half-life at 80℃ is 1.89 min, which is about 1 times longer than that of WT. Its half-inactivation temperature is 63.04℃, which is 12.83℃ higher than that of WT, and it can meet the application requirements of high-temperature scenarios in food processing.

[0021] 3. The phytase mutant T273K provided by this invention has a specific activity that is approximately 77.78% higher than that of WT, and a catalytic efficiency k cat / K m Compared to WT, it increased by 50.56%, showing a significant improvement in the overall catalytic ability of proteins.

[0022] 4. The phytase mutant T273K provided by this invention exhibits significant advantages in salt tolerance, with an enzyme activity retention rate of 33.8% at 10% salt concentration, which can meet the application requirements of high-salt scenarios in food processing.

[0023] 5. The phytase mutant T273K provided by this invention has comprehensive characteristics such as salt resistance and heat resistance, which can better exert the effect of enzymatic hydrolysis of phytic acid in food processing, and can significantly reduce the turbidity caused by phytic acid in liquid foods such as soy sauce, vinegar, and cooking wine, thereby improving product quality. Attached Figure Description

[0024] Figure 1 Map of phytase expression plasmids;

[0025] Figure 2 Figure showing the results of the thermostability screening of phytase and its mutants;

[0026] Figure 3 Figure showing the optimal reaction temperature test results for the phytase mutant T273K;

[0027] Figure 4 Figure showing the optimal pH test results for the phytase mutant T273K;

[0028] Figure 5 Figure showing the results of the thermostability test for the phytase mutant T273K;

[0029] Figure 6 Figure showing the salt tolerance test results of phytase mutant T273K. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional food-grade reagents, methods and equipment in the art.

[0033] The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] This invention utilizes conventional techniques and methods used in the fields of genetic engineering and molecular biology. Those skilled in the art can use other conventional methods, experimental schemes, and reagents in the field based on the technical solutions described in this invention, without being limited to the specific embodiments of this invention.

[0035] Example 1 Construction of mutants

[0036] The phytase gene of a *Aspergillus tubingensis* strain obtained through laboratory screening was optimized by removing the signal peptide sequence and then optimizing it according to the codon bias of *Pichia pastoris*. The optimized nucleotide sequence was synthesized by BGI Genomics Co., Ltd. and constructed into the pPIC9 plasmid as an expression vector. This phytase was named WT, and its amino acid sequence is SEQ ID NO: 1. The expression plasmid map is shown below. Figure 1 As shown.

[0037] The PDB structure files of the above phytases were uploaded to Fireprot for thermostability mutation prediction. Based on the obtained data, the mutants were sorted and screened for FoldX energy and Rosetta energy, involving 5 sites, namely: D32F, D32W, N153F, D211Y, T273E, T273K, A416W, and A416Y.

[0038] Mutation primers were designed based on the phytase nucleotide sequence described above, as shown in Table 1. High-fidelity PCR was used to amplify the fragment, obtaining a linear fragment containing both the vector sequence and the mutant gene sequence. PCR conditions were set as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 90 s, 30 cycles; 72℃ final extension for 5 min; storage at 4℃. The PCR product was digested with dpn I at 37℃ for 60 min and then transformed into *E. coli* JM109 for plasmid replication. After transformation, several clones of each mutant were selected for sequencing. Plasmids were purified from *E. coli* clones with correct sequencing results to obtain recombinant plasmids of the corresponding mutants.

[0039] Table 1 Primers used to construct recombinant plasmids

[0040]

[0041] Example 2: Preparation and initial screening of engineered Pichia pastoris strains

[0042] 1. Construction of the expression strain: The recombinant plasmid of the correctly sequenced mutant was linearized using BglII enzyme. The linearized product was directly recovered using a DNA recovery kit. 10 μL of the recovered product was thoroughly mixed with 80 μL of Pichia pastoris GS115 competent cells. After electroporation at 1.5 kV, 200 μL of 1M sorbitol was immediately added to resuspend the cells. The mixture was incubated at 30 °C for 30 min and then evenly spread on MD solid plates (1% agarose, 1.34% YNB, 2% glucose, 0.04 mg / L biotin). The plates were incubated upside down at 30 °C for 2-3 days. Colonies that were correctly screened on MD plates were selected for PCR verification using the primers listed below. The correctly verified colonies were then induced for expression.

[0043] Upstream primer HSO578: 5'-ccaacagcacaaataacgggtta-3'

[0044] Downstream primer HSO579: 5'-tagaatctagcaagaccggtcttc-3'

[0045] 2. Small-scale fermentation validation: Colonies of the PCR-verified Pichia pastoris recombinant strain were selected and placed into 24-well deep-well plates containing 3 ml of sterilized BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.04 mg / L biotin, 1% glycerol). The plates were sealed with 6 layers of gauze. The plates were placed in a shaker at 30°C for 48 h of enrichment culture. Then, 2% methanol was added for induction culture, and the plates were continued to be incubated at 30°C for another 72 h. The supernatant was used for enzyme activity assay. Phytase activity was determined using the spectrophotometric method according to the national standard GB / T 18634-2009. Phytase activity is defined as the release of 1 μmol of inorganic phosphorus per minute from a 5.0 mmol / L sodium phytate solution at a temperature of 37°C and a pH of 5.5. This is one unit of phytase activity, denoted by U.

[0046] 3. Screening of phytase mutants: The supernatant from the previous small-system fermentation was incubated at 60℃ for 30 min and then in an ice bath for 20 min. The residual phytase activity was then measured. The untreated activity of each mutant was taken as 100%. The heat resistance of the original phytase and the mutant was tested. The results are recorded in Table 2. Finally, a mutant T273K with significantly improved phytase heat stability was screened. The recombinant strain of Pichia pastoris GS115 / pPIC9-T273K was named Pichia pastoris T273K, and the recombinant strain of Pichia pastoris GS115 / pPIC9-WT was named Pichia pastoris WT.

[0047] Table 2. Residual activity of phytase and its mutants after incubation at 60℃ for 30 min.

[0048]

[0049] Mutants showing improved heat resistance in the initial screening were re-screened. Fermentation supernatants of *Pichia pastoris* WT and *Pichia pastoris* T273K were incubated at 4℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃ for 60 min, respectively, followed by a 20 min ice bath. The residual phytase activity was then measured, with 4℃ activity defined as 100%. The thermostability of phytase and the mutants was tested, and the results were recorded. Figure 2 middle.

[0050] like Figure 2 As shown, the residual phytase activity in the Pichia pastoris WT fermentation supernatant decreased to 20.78% after treatment at 80℃ for 1 hour; while the Pichia pastoris T273K fermentation supernatant exhibited significantly improved thermostability, with the residual phytase activity reaching 56.99% under the same treatment conditions, which is 2.74 times that of WT. The data indicate that the single-point mutation in T273K enhances the thermostability of the phytase molecule, and its phytase amino acid sequence is SEQ ID NO: 2.

[0051] Example 3 Enzymatic Characterization of Mutant T273K

[0052] Pichia pastoris WT and Pichia pastoris T273K were inoculated into BMGY medium shake flasks and cultured at 30°C with shaking for 48 h. Then, 2% methanol was added for induction culture, and the mixture was further cultured at 30°C with shaking for 72 h. The fermentation supernatant was then used for phytase purification. Ni-NTA was used to purify the phytase, and the purified product was used for phytase property studies. The final specific enzyme activity of the purified WT sample was 36.002 U / mg, and the specific enzyme activity of the purified T273K mutant sample was 64.013 U / mg.

[0053] 1. Optimal reaction temperature

[0054] Under constant reaction conditions and pH 5.50, the phytase activities of the T273K mutant and WT phytase were measured at different temperatures ranging from 30 to 80°C. The highest enzyme activity detected by each phytase during this experiment was taken as 100%, and the optimal temperature for the enzyme was determined. The results were recorded in [document name missing]. Figure 3 middle.

[0055] Depend on Figure 3The results showed that the optimal reaction temperature for both WT phytase and the T273K mutant remained consistent, with the highest enzyme activity at 45℃. Between 30℃ and 45℃, the overall relative enzyme activity increased with increasing temperature, with little change between 35℃ and 40℃, reaching its peak at 45℃. Between 45℃ and 55℃, the overall relative enzyme activity decreased slightly, and at 55℃, it was actually higher than at 50℃, with both exhibiting approximately 90% relative activity. However, between 55℃ and 80℃, the relative enzyme activity decreased rapidly. The T273K mutant retained 21.6% relative activity at 60℃, while WT phytase's relative activity at 60℃ was 4.58%. After 70℃, the relative activities of both decreased to zero.

[0056] 2. Optimal pH

[0057] Under constant reaction conditions and at 37°C, the T273K mutant and WT phytase were placed in buffer solutions of different pH values ​​(pH 2-10) to determine phytase activity. The highest detected enzyme activity for each phytase during this experiment was taken as 100%, and the optimal pH for the enzyme was determined. The results were recorded in [document name missing]. Figure 4 middle.

[0058] Depend on Figure 4 The results show that the optimal pH for WT phytase and the T273K mutant is consistent, with the relative enzyme activity reaching its highest point at pH=5.5.

[0059] 3. Temperature resistance (thermal stability):

[0060] WT phytase and T273K mutant samples were incubated in a 60℃ water bath for different times, and the residual phytase activity was measured. The phytase activity at 0 min incubation was taken as 100%. The relative residual phytase activity at different incubation times was calculated to test the enzyme's thermal stability. Figure 5 The results show that the overall enzyme activity retention rate of the T273K mutant is higher than that of WT phytase, indicating that the T273K mutant has better thermostability than WT phytase. WT phytase retained 49.0% of its relative enzyme activity after incubation at 60℃ for 90 min, while the T273K mutant retained 69.4% of its relative enzyme activity after the same incubation period, representing a 20.4% increase compared to WT phytase, demonstrating a significant improvement in thermostability.

[0061] 4. Determination of thermodynamic parameters

[0062] To further understand the thermal stability of the mutants, the thermodynamic parameters, including half-life and half-inactivation temperature, of WT phytase and T273K mutant were determined. Since 60℃ is commonly used in food processing and 80℃ is often used in feed processing, half-life measurements were conducted at 60℃ and 80℃ to determine the applicability range of this phytase.

[0063] Table 3. Thermodynamic parameters of phytase WT and T273K mutants

[0064]

[0065] As shown in Table 3, the half-life of WT phytase at 60℃ is 15.35 min, while that of the T273K mutant is 25.72 min, an extension of approximately 40.43%. The half-life of WT phytase at 80℃ is 0.98 min, while that of the T273K mutant is 1.89 min, an extension of approximately 100%, indicating that the T273K mutant has a greater advantage over WT phytase at high temperatures. The half-inactivation temperature of WT phytase is 50.21℃, while that of the T273K mutant is 63.04℃, an increase of 12.83℃. All these results indicate that the T273K mutant has significantly improved thermostability compared to WT phytase.

[0066] 5. Measurement of kinetic parameters

[0067] Different concentrations of sodium phytate (0.05-1.00 mM) were prepared as substrates, and phytase activity was measured at 37℃ and pH 5.5. Data processing was then performed, the Michaelis-Menten equation was fitted, and K was calculated. m and k cat value.

[0068] Table 4 Kinetic parameters of phytase WT and T273K mutants

[0069]

[0070] As shown in Table 4, the specific activity of the T273K mutant was approximately 77.78% higher than that of WT phytase; its K m The value is close to that of WT phytase, and the catalytic efficiency k cat / K m Compared to WT phytase, it increased by 50.56%. This indicates that the introduction of the T273K mutation has little effect on the enzyme's substrate binding ability, but it significantly improves the overall catalytic ability of the protein.

[0071] Example 4: Enzyme Preparation and Application Testing

[0072] Pichia pastoris T273K was inoculated into YPD medium to prepare seed culture, which was then inoculated into a 15L fermenter containing fermentation medium for fermentation culture and induced expression. First, a large number of cells were obtained by culturing in glycerol-fed medium, and then phytase expression was induced by switching to methanol-fed medium. After obtaining the fermentation broth, solid-liquid separation was performed using a plate and frame filter press, and then the broth was concentrated using a 10 KD filter membrane. 30% glycerol was added as a stabilizer to the concentrate, and the solution was sterilized by filtration through 0.22 uM PVDF to obtain crude enzyme solution.

[0073] Fermentation medium: 2.67% phosphate, 0.093% calcium sulfate, 1.82% potassium sulfate, 1.49% magnesium sulfate heptahydrate, 0.413% potassium hydroxide, 4% glycerol.

[0074] Feeding media: a. Glycerol fed medium: 50% w / v glycerol, with 12 mL PTM1 trace element solution per 1 L of glycerol. b. Methanol fed medium: 100% methanol, with 12 mL PTM1 trace element solution per 1 L of methanol.

[0075] 1. Analysis of the salt tolerance of mutants:

[0076] The salt tolerance of the prepared T273K mutant phytase crude enzyme solution was compared with that of commercially available E. coli-derived phytase. Substrate solutions and buffer solutions containing 0%, 5%, 10%, 15%, and 20% salt were prepared, and the enzyme activities of the T273K mutant phytase crude enzyme solution and E. coli phytase were measured at different salt concentrations. The enzyme activity measured at 0% salt concentration was taken as 100%, and the relative enzyme activity at each salt concentration was calculated. Figure 6 As shown, when the salt concentration is 5%, the relative enzyme activity of *E. coli* phytase is only 11.5%, while the relative enzyme activity of the T273K mutant is 62.4%; when the salt concentration is 10%, the relative enzyme activity of *E. coli* phytase is 6.1%, while the relative enzyme activity of the T273K mutant is 33.8%; when the salt concentration is 15%, the relative enzyme activity of *E. coli* phytase is 0.9%, while the relative enzyme activity of the T273K mutant is 13.3%. Therefore, the T273K mutant phytase exhibits a significant advantage in salt tolerance.

[0077] 2. Phytase Application Test

[0078] Phytase is widely found in various plant products, including soy sauce, vinegar, and cooking wine. The presence of phytic acid in these products often leads to precipitation or turbidity. Solid-state fermented mature vinegar mash was treated simultaneously with T273K mutant phytase and E. coli phytase at a concentration of 10 U / g. After treatment at 50°C for 6-8 hours, the enzyme was inactivated by boiling, and then stored at 4°C. The turbidity of different samples was periodically measured to characterize the phytase treatment effect. The results are recorded in Table 5.

[0079] Table 5: Turbidity changes in vinegar mash treated with different phytases

[0080]

[0081] As shown in Table 5, at different detection times, the turbidity of the T273K mutant treatment group was consistently lower than that of the untreated control group and significantly lower than that of the E. coli phytase treatment group. Solid-state fermented vinegar, besides having an extremely low pH, contains approximately 2% salt. The T273K mutant's comprehensive characteristics, including salt tolerance and heat resistance, make it more effective at enzymatically hydrolyzing phytic acid in vinegar mash, thus better reducing turbidity in the vinegar mash and demonstrating its potential in vinegar processing.

[0082] In summary, the technical solution of this invention utilizes phytase derived from Aspergillus tabineus to obtain the phytase mutant T273K through point mutation. This mutant exhibits significantly improved thermal stability and enhanced overall catalytic ability for proteins compared to the original mutant. It can better exert the effect of enzymatic hydrolysis of phytic acid during food processing and has broad application prospects in the food processing field.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A phytase mutant, characterized in that, The amino acid sequence of the phytase mutant is shown in SEQ ID NO.

2.

2. A gene characterized in that, The gene encodes the phytase mutant of claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector carries the gene described in claim 2.

4. A recombinant host cell, characterized in that, The recombinant host cell expression right of claim 1 is derived from the phytase mutant or transformation / transfection of the recombinant expression vector of claim 3.

5. The recombinant host cell as described in claim 4, characterized in that, The host cell is Pichia pastoris.

6. The use of the recombinant expression vector of claim 3 or the recombinant host cell of claim 4 in the production of phytase.

7. The application of the phytase mutant according to claim 1 in phytic acid degradation.

8. The application of the phytase mutant according to claim 1 in food processing.

9. The application as described in claim 8, characterized in that, The application is for the enzymatic hydrolysis of phytic acid.

10. An enzyme preparation, characterized in that, The enzyme preparation comprises the phytase mutant of claim 1.

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