High-temperature resistant neutral phytase mutants

Abstract

This invention relates to the fields of genetic engineering and protein engineering modification technology, specifically providing a heat-resistant neutral phytase mutant. The mutant exhibits significantly enhanced heat resistance and can be widely used in aquatic feed, thereby reducing the addition of inorganic phosphates in feed, lowering aquaculture costs, and effectively reducing environmental pollution from aquatic animal fecal phosphorus.

Description

Technical Field

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

[0002] Phytic acid is the main form of phosphorus storage in plant-based feed ingredients, but monogastric animals (including most farmed fish) cannot effectively utilize phytic acid phosphorus due to a lack of endogenous phytase. To meet nutritional requirements, inorganic phosphorus must be added to aquatic feed, which not only increases feed costs but also leads to a large amount of phosphorus being excreted into water bodies with feces, causing excessive total phosphorus in aquaculture wastewater and eutrophication. Therefore, improving feed phosphorus utilization and reducing phosphorus emissions at the source has become crucial for the green transformation of the aquaculture industry.

[0003] Phytase can hydrolyze phytic acid phosphorus into inorganic phosphorus and inositol derivatives. However, traditional acidic phytase (optimal pH 2.5-5.0) is mainly suitable for the acidic environment of the stomach, while the intestinal pH of most freshwater fish, such as cyprinids, which are mainly farmed in my country, is neutral or slightly alkaline, resulting in a significant decrease in the activity of acidic phytase under these conditions. In contrast, neutral phytase (optimal pH 6.5-7.5) is highly compatible with the intestinal physiological environment of freshwater fish and has greater application potential.

[0004] However, the current promotion of neutral phytase in aquaculture faces a more critical technical bottleneck—insufficient heat resistance. Aquatic feed production commonly employs high-temperature pelleting processes (conditioning temperatures typically range from 75-95℃, with some extruded feeds exceeding 100℃) to kill pathogens and improve feed physical properties. However, most existing neutral phytases are derived from mesophilic microorganisms, and their enzyme proteins have limited thermal stability, making them highly susceptible to denaturation and inactivation during high-temperature pelleting. Even with post-coating processes, issues such as high equipment investment, poor coating uniformity, and easy enzyme degradation during storage and transportation persist. The significant loss of enzyme activity due to high-temperature pelleting is the core pain point hindering the industrial application of neutral phytase in aquatic feed.

[0005] If the enzyme is inactivated during pelleting, it cannot effectively degrade phytate phosphorus after the feed enters the water. The actual amount of active enzyme ingested by fish is far lower than the designed addition amount, making it difficult to achieve the goals of phosphorus emission reduction and cost reduction and efficiency improvement. Therefore, neutral enzyme activity alone is not enough to meet production needs. It is also necessary to endow neutral phytase with excellent heat resistance so that it can maintain sufficient residual enzyme activity under high-temperature pelleting conditions.

[0006] In summary, developing novel neutral phytases that combine neutral enzyme activity and high thermal stability, and achieving direct compatibility with existing high-temperature granulation processes, is of significant industrial value and urgent practical importance for reducing costs and increasing efficiency in aquaculture, reducing phosphorus emissions, and promoting sustainable ecological development. Summary of the Invention

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] The present invention also relates to DNA molecules encoding the above-mentioned phytase mutants.

[0013] The present invention also relates to recombinant expression vectors comprising the above-described DNA molecules.

[0014] The present invention also relates to a host cell comprising the above-described recombinant expression vector.

[0015] When the above plasmids were transferred into host cells, the heat resistance of the recombinant phytase mutant was significantly improved.

[0016] In some embodiments of the present invention, the host cell is Pichia pastoris (Pichia pastoris). Pichia pastoris ).

[0017] The present invention also provides the application of the above-mentioned phytase mutant in the field of feed.

[0018] 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.

[0019] In summary, the phytase mutant provided by this invention has stronger heat resistance and can be widely used in aquatic feed, showing broad application prospects. Detailed Implementation

[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] The present invention will now be described in detail with reference to specific embodiments.

[0022] 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.

[0023] 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).

[0024] The plasmid was purified from the correctly sequenced E. coli clone using the Plasmid Mini-Preparation Kit (Omega) to obtain the recombinant plasmid.

[0025] 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.

[0026] Primer sequences were designed, and PCR amplification was performed using the PHY-M12 gene as a template with the above primers and 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 transformed product was then 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. After centrifugation, the supernatant was discarded, and the cells were resuspended in buffer. The cells were repeatedly frozen and thawed to obtain E. coli cell lysate containing phytase.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Following the method described in Example 1, the gene sequences of the synthesized phytase PHY-M12 and its single-point mutant were double-digested with EcoRI and NotI, respectively. Then, they were ligated with the similarly digested pPIC-9K vector overnight at 16°C and transformed into *E. coli* DH5α. The transformed bacteria were plated on LB+Amp plates (0.5% yeast extract, 1% peptone, 1% NaCl, 1.5% agar, 100 μg / mL ampicillin, pH 7.0) and incubated upside down at 37°C. After transformants appeared, colony PCR was performed (reaction system: single clones picked from 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℃ pre-denaturation for 5 min, 30 cycles: 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2 min, 72℃ for 10 min). Positive clones were verified, and the correct recombinant expression plasmid was obtained after sequencing confirmation.

[0031] 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 (1% yeast extract, 2% peptone, 2% glucose, 2% agarose) and cultured at 30℃ for 48 h. Afterward, activated GS115 single clones were inoculated into 6 mL of YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) and cultured at 30℃ and 220 rpm for approximately 12 h. The bacterial 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, the cells were centrifuged at 4℃ and 9000 rpm for 2 min, and 4 mL of cells were collected into 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 and centrifuged at 4℃ and 9000 rpm for 2 min. Centrifuge at 9000 rpm for 2 min at 4℃, gently discard the supernatant, wash once with 1 mL of sterile water, centrifuge at 9000 rpm for 2 min at 4℃, gently discard the supernatant, and resuspend the bacterial cells in 1 mL of pre-cooled sorbitol (1 mol / L); centrifuge at 9000 rpm for 2 min at 4℃, gently discard the supernatant, and gently resuspend the bacterial cells in 100-150 μl of pre-cooled sorbitol (1 mol / L).

[0032] 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.

[0033] The obtained transformants were transferred to BMGY medium (2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 × 10⁻⁶ mol / L) respectively. -5 In a medium containing 1% biotin and 1% glycerol, the culture was incubated at 30°C with shaking at 250 rpm for 1 day; then transferred to BMMY medium (2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 × 10⁻⁶ ppm). -5 The culture was carried out in a solution of 1% biotin and 0.5% methanol at 30°C 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 supernatant containing phytase PHY-M12 and its single-point mutant, respectively.

[0034] 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.

[0035] (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.

[0036] Enzyme activity calculation formula: U=(A-A0-0.0016)×F / (0.0415×30).

[0037] 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.

[0038] (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.

[0039] 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.

[0040] 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.

[0041] In summary, the phytase mutant provided by this invention has stronger heat resistance and can be widely used in the field of feed production, showing broad application prospects.

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: 41, 84, 203, 221, 222 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: V41D, R84M, A203S, R221Y, L222P.

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.