High-temperature-resistant neutral phytase mutant and application thereof

Abstract

The application relates to the technical field of genetic engineering and protein engineering, and particularly provides a high-temperature-resistant neutral phytase mutant and application thereof. The heat resistance of the mutant is significantly enhanced. The phytase mutant containing a V405P single-point mutation has the strongest heat resistance, and the enzyme activity residual rate is as high as 88.68% after being treated at 90 DEG C for 5 min, unexpected technical effects are achieved. The mutant can be widely applied to aquatic feed.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and protein engineering technology, and specifically relates to a heat-resistant neutral phytase mutant and its applications. Technical Background

[0002] Phytic acid (inositol hexaphosphate) is the main storage form of phosphorus in plant-based feed ingredients. However, the phosphorus element is chelated within the phytic acid molecule. Monogastric animals (such as pigs and poultry) and most farmed fish lack endogenous phytase, making it difficult to effectively decompose and utilize phytic acid phosphorus. To meet the growth needs of animals, inorganic phosphorus must be added to the feed, which not only significantly increases feed costs but also leads to a large amount of unused phosphorus being discharged into the environment with feces, causing excessive total phosphorus in aquaculture wastewater and eutrophication of water bodies. Therefore, improving the utilization rate of phytic acid phosphorus in feed and reducing phosphorus emissions at the source has become one of the core issues in the green transformation of modern aquaculture.

[0003] Phytase can catalyze the hydrolysis of phytic acid into inorganic phosphorus and inositol derivatives, making it an effective biological agent for solving the aforementioned problems. However, traditional phytases are mostly acidic phytases, with an optimal pH range of 2.5-5.0, primarily suitable for the acidic environment of the stomach. In contrast, the intestinal physiological environment of freshwater fish such as cyprinids, which are mainly farmed in my country, is neutral or slightly alkaline (pH 6.5-7.5). Under these conditions, the activity of acidic phytases decreases significantly, limiting their application effectiveness. In comparison, neutral phytase (optimal pH 6.5-7.5) is highly compatible with the intestinal environment of freshwater fish, demonstrating superior application potential.

[0004] Despite the significant advantages of neutral phytase in terms of enzymatic properties, its industrial application in aquatic feed still faces a key technological bottleneck—insufficient heat resistance. Modern aquatic feed production generally employs high-temperature pelleting or extrusion processes (conditioning temperatures are typically 75-95℃, with some extrusion processes exceeding 100℃) to achieve sterilization, cooking, and improvement of feed physical properties. However, most reported neutral phytases are derived from mesophilic microorganisms, and their enzyme proteins have limited thermal stability, making them highly susceptible to irreversible denaturation and inactivation during high-temperature pelleting. Although post-coating technology can mitigate high temperatures to some extent, this method suffers from practical drawbacks such as high equipment investment, poor coating uniformity, and easy enzyme degradation during storage and transportation, making it difficult to widely promote in large-scale feed production.

[0005] Therefore, the significant loss of enzyme activity caused by high-temperature pelleting is the core technical obstacle restricting the practical application of neutral phytase in aquatic feed. Once the enzyme is inactivated during pelleting, it cannot effectively degrade phytic acid phosphorus after the feed enters the water, resulting in animals ingesting far less active enzyme than the designed dosage. This makes it difficult to achieve the expected goals of phosphorus reduction and cost reduction. Therefore, developing a novel neutral phytase that retains its neutral enzyme activity characteristics while possessing excellent heat resistance, and is directly compatible with existing high-temperature pelleting processes, is of significant industrial value and urgent practical importance for promoting the green and sustainable development of aquaculture. Summary of the Invention

[0006] This invention addresses the problems of existing technologies by providing a heat-resistant neutral phytase mutant and its applications. The mutant exhibits significantly improved heat resistance, which facilitates its widespread use in aquatic feed.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention relates to a phytoacidase mutant comprising an amino acid sequence having at least 90% identity with SEQ ID NO:1, and comprising an amino acid substitution at at least one position selected from the group consisting of: 41, 84, 203, 221, 222, 272, 350, 361, 402, 405 compared with SEQ ID NO:1.

[0008] In some embodiments of the present invention, the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO:1.

[0009] In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity with SEQ ID NO:1.

[0010] In some embodiments of the present invention, the mutant comprises a substitution of at least one amino acid from the following group: V41D, R84M, A203S, R221Y, L222P, Y272V, A350P, S361L, R402Y, V405P.

[0011] The present invention also relates to DNA molecules encoding the above-mentioned phytase mutants.

[0012] The present invention also relates to recombinant expression vectors comprising the above-described DNA molecules.

[0013] The present invention also relates to a host cell comprising the above-described recombinant expression vector.

[0014] When the above plasmids were transferred into host cells, the heat resistance of the recombinant phytase mutant was significantly improved.

[0015] In some embodiments of the present invention, the host cell is Pichia pastoris (Pichia pastoris). Pichia pastoris ).

[0016] The present invention also provides the application of the above-mentioned phytase mutant in the field of feed.

[0017] Based on the phytase PHY-M12, this invention provides phytase mutants containing single mutation sites of V41D, R84M, A203S, R221Y, L222P, Y272V, A350P, S361L, R402Y, and V405P. After treatment at 90℃ for 5 minutes, these mutants exhibited increased enzyme activity residual rates of 10.02%–42.38%, demonstrating significantly enhanced heat resistance. Among them, the phytase mutant containing the V405P single-point mutation exhibited the strongest heat resistance, achieving an enzyme activity residual rate as high as 88.68% after treatment at 90℃ for 5 minutes, achieving unexpected technical results.

[0018] In summary, the phytase mutant provided by this invention has stronger heat resistance and can be widely used in aquatic extruded feed, thereby helping to reduce the addition of inorganic phosphates in the feed, reduce breeding costs, and effectively reduce the pollution of aquatic animal fecal phosphorus to the environment. Detailed Implementation

[0019] This invention discloses a neutral phytase mutant, its preparation method and application, the DNA molecule encoding the phytase mutant, the vector, and the host cell. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0020] This invention utilizes conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in *MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed.* (Sambrook, 2001) and *CURRENTPROTOCOLS IN MOLECULAR BIOLOGY* (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can utilize other conventional methods, experimental protocols, and reagents based on the technical solutions described in this invention, without being limited to the specific embodiments of this invention.

[0021] For example, the following experimental materials and reagents may be used in this invention: Strains and vectors: Escherichia coli DH5α, Pichia pastoris GS115, vector pPIC9k, Amp, and G418 were purchased from Invitrogen.

[0022] Enzymes and kits: PCR enzymes and ligases were purchased from Takara, restriction endonucleases from Fermentas, plasmid extraction kits and gel purification and recovery kits from Omega, and GeneMorph II random mutagenesis kits from Beijing Bomais Biotechnology Co., Ltd.

[0023] Culture medium formulation: Escherichia coli culture medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0; Yeast medium (YPD medium): 1% yeast extract, 2% peptone, 2% glucose; Yeast selection medium (MD medium): 2% peptone, 2% agarose; BMGY medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 × 10⁻⁶ -5 1% Biotin, 1% Glycerin; BMMY medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 × 10⁻⁶ -5 % Biotin, 0.5% Methanol; LB-AMP medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 100 μg / mL ampicillin, pH 7.0; LB-AMP plates: 0.5% yeast extract, 1% peptone, 1% NaCl, 1.5% agar, 100 μg / mL ampicillin, pH 7.0.

[0024] The present invention will be further illustrated below with reference to the embodiments: Example 1 Construction of recombinant plasmid The amino acid sequence of phytase PHY-M12 is SEQ ID NO: 1, and the encoding nucleotide sequence is SEQ ID NO: 2. Its optimal reaction pH is 6.5, and it can maintain more than 80% of its enzyme activity within the pH range of 6.0-7.0. The phytase PHY-M12 gene was optimized according to the codon preference of Pichia pastoris. The optimized nucleotide sequence was synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0025] The phytase gene was digested with restriction endonucleases EcoRI and Not I (Fermentas); simultaneously, plasmid pPIC9K was digested with the same restriction endonucleases. The digestion products were purified using a gel purification kit, and the two digestion products were ligated using T4 DNA ligase (Fermentas). The ligation products were transformed into DH5α Escherichia coli (Invitrogen) and selected using ampicillin. To ensure accuracy, several clones were sequenced (Sangon).

[0026] The plasmid was purified from the correctly sequenced E. coli clone using the Plasmid Mini-Preparation Kit (Omega) to obtain the recombinant plasmid.

[0027] Example 2 Screening of high-temperature resistant mutants To further improve the thermostability of phytase PHY-M12, the applicant conducted extensive mutation screening on the enzyme using directed evolution technology.

[0028] Primer sequences were designed, and the PHY-M12 gene was used as a template. PCR amplification was performed using the GeneMorph II random mutagenesis PCR kit (Bomais). The PCR product was recovered from the gel, digested with EcoRI and NotI, and then ligated into the pET21a vector digested with the same enzymes. The resulting product was transformed into E. coli BL21(DE3), plated on LB+Amp plates, and incubated upside down at 37°C. After the transformants appeared, they were picked one by one into a 96-well plate with a toothpick. 150 μl of LB+Amp medium containing 0.1 mM IPTG was added to each well. The plate was incubated at 37°C and 220 rpm for about 6 h. The supernatant was discarded by centrifugation, and the cells were resuspended in buffer. The cells were repeatedly frozen and thawed to obtain E. coli cell lysate containing phytase.

[0029] 30 μL of lysis buffer was transferred to two new 96-well plates. One plate was treated at 90℃ for 5 min. 30 μL of substrate was added to both plates, and the plates were reacted at 37℃ for 30 min. The reducing sugars produced were measured using the DNS method. Different mutants retained different activities after high-temperature treatment. The results showed that some mutations had no effect on the thermostability of phytase, while others even worsened its thermostability or enzyme activity. Additionally, some mutations, although improving the temperature tolerance of phytase, significantly altered its enzymatic properties, which did not meet the requirements. Finally, the following mutation sites were identified that significantly improved the thermostability of phytase without affecting its enzyme activity and original enzymatic properties: V41D, R84M, A203S, R221Y, L222P, Y272V, A350P, S361L, R402Y, and V405P.

[0030] Based on the above-mentioned phytase PHY-M12, this invention provides mutants containing a single mutation site of V41D, R84M, A203S, R221Y, L222P, Y272V, A350P, S361L, R402Y, or V405P.

[0031] Example 3: Expression of phytase in Pichia pastoris 3.1 Construction of expression vector Based on the codon preference of Pichia pastoris, the gene sequences of phytase PHY-M12 and its single-point mutant were optimized and synthesized by Shanghai Jierui Biotechnology Co., Ltd., with EcoRI and NotI restriction sites added at the 5' and 3' ends of the synthesized sequences, respectively.

[0032] Following the method described in Example 1, the gene sequences of the synthesized phytase PHY-M12 and its single-point mutant were digested with EcoRI and NotI, respectively, and then ligated with the pPIC-9K vector digested with the same enzymes overnight at 16°C. The ligation was then performed on E. coli DH5α, plated on LB+Amp plates, and incubated upside down at 37°C. After the transformants appeared, colony PCR was performed (reaction system: single clones picked from the template, rTaq DNA polymerase 0.5 μl, 10× Buffer 2.0 μL, dNTPs (2.5 mM) 2.0 μL, 5' AOX primer (10 mM): 0.5 μL, 3' AOX primer: 0.5 μL, ddH2O 14.5 μL, reaction program: 95°C pre-denaturation for 5 min, 30 cycles: 94°C for 30 sec, 55°C for 30 sec, 72°C for 2 min, 72°C for 10 min). The positive clone was verified, and the correct recombinant expression plasmid was obtained after sequencing.

[0033] 3.2 Construction of Pichia pastoris engineered strains 3.2.1 Preparation of competent yeast cells Pichia pastoris strain GS115 was activated on YPD plates and cultured at 30℃ for 48 h. Activated GS115 single clones were then inoculated into 6 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for approximately 12 h. The culture was then transferred to Erlenmeyer flasks containing 30 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for approximately 5 h. Cell density was measured using a UV spectrophotometer. Once the OD600 value was within the range of 1.1–1.3, 4 mL of cells were collected by centrifugation at 4℃ and 9000 rpm for 2 min, and transferred to sterile EP tubes. The supernatant was gently discarded, and the remaining supernatant was blotted dry with sterile filter paper. The cells were resuspended in 1 mL of pre-cooled sterile water, centrifuged at 4℃ and 9000 rpm for 2 min, and the supernatant was gently discarded. The cells were washed once more with 1 mL of sterile water, centrifuged at 4℃ and 9000 rpm for 2 min, and the supernatant was gently discarded. The cells were then resuspended in 1 mL of pre-cooled sorbitol (1... Resuspend the bacterial cells in sorbitol (1 mol / L); centrifuge at 4℃ and 9000 rpm for 2 min, gently discard the supernatant, and gently resuspend the bacterial cells in 100-150 μl of pre-cooled sorbitol (1 mol / L).

[0034] 3.2.2 Conversion and Screening The recombinant expression plasmids constructed in 3.1 were linearized with Sac I. After purification and recovery of the linearized fragments, they were transformed into Pichia pastoris GS115 by electroporation. Recombinant Pichia pastoris strains were screened on MD plates, and multi-copy transformants were then screened on YPD plates (0.5 mg / mL-8 mg / mL) containing different concentrations of genimycin.

[0035] The obtained transformants were transferred to BMGY medium and cultured at 30℃ and 250 rpm for 1 day with shaking. They were then transferred to BMMY medium and cultured at 30℃ and 250 rpm with shaking. 0.5% methanol was added daily to induce expression for 4 days. The cells were removed by centrifugation at 9000 rpm for 10 min to obtain fermentation supernatants containing phytase PHY-M12 and its single-point mutant, respectively.

[0036] 3.3 Phytase activity assay (1) Definition of phytase activity unit Under conditions of 37°C and pH 6.5, the amount of enzyme required to release 1 μmol of inorganic phosphorus per minute from a sodium phytate solution with a concentration of 5 mg / ml is defined as one unit of enzyme activity, U.

[0037] (2) Enzyme activity assay method Take 4 ml of 7.5 mmol / L sodium phytate solution (prepared with pH 5.0 0.25 mol / L acetate buffer) and add it to a colorimetric tube. Equilibrate at 37°C for 5 min. Then add 2 ml of phytase enzyme solution appropriately diluted with pH 5.0 0.25 mol / L acetate buffer and equilibrated at 37°C. Mix well and incubate at 37°C for 30 min. After the reaction is complete, add 4 ml of stop solution (2 parts nitric acid solution (nitric acid:water = 1:2), 1 part 100 g / L ammonium molybdate solution, and 1 part 2.35 g / L ammonium vanadate solution), mix well to terminate the reaction. Then let it stand at room temperature for 10 min for color development, and measure the absorbance at 415 nm using a spectrophotometer.

[0038] Enzyme activity calculation formula: U=(A-A0-0.0016)×F / (0.0415×30).

[0039] In the formula: A is the absorbance of the sample; A0 is the absorbance of the blank sample; F is the total dilution factor of the actual sample solution before reaction; 30 is the enzymatic reaction time, min.

[0040] (3) Results of enzyme activity assay Enzyme activity was detected using the above method, and the results showed that the enzyme activity of the fermentation supernatant of the recombinant Pichia pastoris strain expressing phytase PHY-M12 and its single-point mutant was 830-960 U / mL.

[0041] Example 4: Analysis of the heat resistance of phytase mutants The fermentation supernatant of the recombinant Pichia pastoris strains expressing phytase PHY-M12 and its single-point mutant was diluted to approximately 20 U / mL with acetate-sodium acetate buffer at pH 5.5. After treatment at 90℃ for 5 min, the residual enzyme activity was measured. The enzyme activity of the untreated sample was taken as 100%, and the residual enzyme activity rate was calculated. The specific results are shown in Table 1.

[0042] Table 1. Analysis of the heat resistance of phytase mutants Phytase mutant Residual enzyme activity after treatment at 90℃ for 5 minutes PHY-M12 46.30% V41D 61.75% R84M 80.22% A203S 56.32% R221Y 85.80% L222P 67.15% Y272V 74.45% A350P 88.00% S361L 62.74% R402Y 84.83% V405P 88.68% As shown in Table 1, compared with phytase PHY-M12, the phytase mutants provided by this invention, containing single mutation sites of V41D, R84M, A203S, R221Y, L222P, Y272V, A350P, S361L, R402Y, and V405P respectively, exhibited significantly enhanced enzyme activity residual rates (10.02%-42.38%) after treatment at 90℃ for 5 minutes, demonstrating a substantial increase in heat resistance. Among these, the phytase mutant containing the V405P single-point mutation exhibited the strongest heat resistance, achieving an enzyme activity residual rate as high as 88.68% after treatment at 90℃ for 5 minutes, demonstrating unexpected technical effectiveness.

[0043] In summary, the phytase mutant provided by this invention has stronger heat resistance and can be widely used in aquatic extruded feed, thereby helping to reduce the addition of inorganic phosphates in the feed, reduce breeding costs, and effectively reduce the pollution of aquatic animal fecal phosphorus to the environment.

Claims

1. A phytase mutant, characterized in that, The mutant comprises an amino acid sequence having at least 90% identity with SEQ ID NO:1, and contains an amino acid substitution at at least one position selected from the group consisting of: 272, 350, 361, 402, 405 compared to SEQ ID NO:

1.

2. The phytase mutant as described in claim 1, characterized in that, The amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO:

1.

3. The phytase mutant as described in claim 2, characterized in that, The amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity with SEQ ID NO:

1.

4. The phytase mutant as described in claim 1, characterized in that, The mutant contains a substitution of at least one amino acid from the following group: Y272V, A350P, S361L, R402Y, V405P.

5. A DNA molecule encoding any of the phytase mutants of claims 1-4.

6. A recombinant expression vector comprising the DNA molecule of claim 5.

7. A host cell, characterized in that, The host cell comprises the recombinant expression vector of claim 6.

8. The host cell as described in claim 7, characterized in that, The host cell is Pichia pastoris ( Pichia pastoris ).

9. The use of any of the phytase mutants described in claims 1-4 in feed production.