Fish feed protease mutant with improved thermal stability and preparation method thereof

Abstract

The invention discloses a fish feed protease mutant with improved thermal stability and a preparation method thereof, and belongs to the field of enzyme engineering. The amino acid sequence of the mutant E0TYP4-4 is as shown in SEQ ID NO: 3, and the relative residual enzyme activity of the mutant E0TYP4-4 is 88.19% and 79.51% after the mutant E0TYP4-4 is treated at 60 DEG C and 70 DEG C; under the condition of 40 DEG C, the amino acid release content of the mutant E0TYP4-4 reaches 116.12 + / -4.96 [mu] mol / mg protein within 60 minutes; after heat treatment, the release contents of amino acids at 60 DEG C and 70 DEG C are respectively 91.6% and 87.6% of those before heat treatment. Therefore, the heat resistance of the feed protease caused by the mutation is greatly improved, and the feed protease is more suitable for being used as a feed additive than a wild type, and is beneficial to wide application of the feed protease in feed, so that the market prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, and specifically relates to a fish feed protease mutant with improved thermal stability and its preparation method. Background Technology

[0002] Feed proteases are widely distributed in animals, plants, and microorganisms. Industrially, they are mainly produced using *Aspergillus niger* or *Penicillium*, but this process often suffers from low enzyme activity, poor stability, contamination by other proteins, and cumbersome separation and purification. Currently, pellet production involves a brief high-temperature spray formulation stage, and feed proteases derived from wild *Bacillus subtilis* have poor thermostability; their aqueous solution retains less than 40% of its enzyme activity after incubation at 65°C for 5 minutes, limiting their application in pelleted feed. Therefore, improving the thermostability of feed proteases is of significant practical importance for current feed proteases. Summary of the Invention

[0003] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a fish feed protease mutant with improved thermal stability and its preparation method. This mutant exhibits significantly enhanced heat resistance, which is beneficial for its widespread application in the feed industry.

[0004] The objective of this invention is achieved through the following technical solution: The present invention provides a fish feed protease mutant with improved thermal stability, the amino acid sequence of which is shown in SEQ ID NO: 3, or as shown in SEQ ID NO: 30 to 381aa.

[0005] The encoding gene of a fish feed protease mutant with improved thermal stability.

[0006] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the thermostable fish feed protease mutant is shown in SEQ ID NO: 4.

[0007] The aforementioned biomaterials related to fish feed protease mutants with improved thermal stability are any one or more combinations of the following biomaterials: (a) An expression cassette containing the above-mentioned encoded genes; (b) Recombinant expression vectors containing the above-mentioned coding genes; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant bacteria containing the above-mentioned encoding genes; (e) Recombinant bacteria containing the expression cassette described in (a); (f) Recombinant bacteria containing the recombinant expression vector described in (b) or (c).

[0008] Furthermore, the starting vector for the recombinant expression vectors described in (b) and (c) is a pUB series vector or an Escherichia coli-Bacillus subtilis shuttle plasmid vector, etc.; preferably, it is a pUB110 vector or a pAc1 vector.

[0009] Furthermore, the host bacteria corresponding to the recombinant bacteria mentioned in (d), (e), and (f) are selected from prokaryotes or yeast, etc.; the prokaryotes include Bacillus spp. ( Bacillus Bacteria such as Bacillus subtilis. More specifically, the prokaryotes are Bacillus subtilis (…). Bacillus subtilis Specifically, it could be Bacillus subtilis SCK6.

[0010] An immobilized enzyme containing the aforementioned fish feed protease mutant with improved thermal stability.

[0011] The above-mentioned mutants, encoding genes, mutant-related biological materials, or immobilized enzymes are used in the preparation of fish feed protease mutants with improved thermal stability.

[0012] The application of the aforementioned mutants, encoding genes, mutant-related biological materials, or immobilized enzymes in the degradation of feed proteins.

[0013] Preferably, the feed includes fish feed, etc.

[0014] The above-mentioned mutants, coding genes, mutant-related biological materials, or immobilized enzymes are used in the feed industry.

[0015] A method for obtaining the above-mentioned mutant includes the following steps: By designing primers containing mutation sites to perform site-directed mutation G272S on the gene encoding the fish feed protease with the amino acid sequence shown in SEQ ID NO: 1, a fish feed protease mutant with improved thermostability was obtained.

[0016] Furthermore, primers containing the mutation site were designed to introduce the mutation G272S into the gene encoding the fish feed protease with the amino acid sequence shown in SEQ ID NO: 1. After correct sequencing, the gene was transformed into Bacillus subtilis SCK6 for expression, resulting in a fish feed protease mutant with improved thermostability.

[0017] Furthermore, using plasmid pAc1-E0TYP4-3 as a template, primers containing mutation sites were designed to introduce the mutant G272S into the gene encoding fish feed protease. The mutant was then transformed into Bacillus subtilis SCK6, and plasmids were extracted from the transformants for sequencing.

[0018] The present invention has the following advantages and effects compared with the prior art: (1) The heat resistance of the fish feed protease mutant provided by this invention is generally higher than that of E0TYP4-3 (SEQ ID NO: 1), and the amino acid release content reaches 116.12±4.96 μmol / mg protein within 60 minutes at 40℃. After heat treatment, the amino acid release content is still 91.6% and 87.6% of the pre-heat treatment level at 60℃ and 70℃, respectively. This indicates that the above mutation leads to a significant improvement in the heat resistance of the feed protease, making it more suitable as a feed additive than the wild type, which is conducive to the widespread application of feed protease in feed, and therefore has broad market prospects.

[0019] (2) Based on the fish feed protease E0TYP4-3, the present invention carried out thermostability mutation improvement and screened to obtain a mutant E0TYP4-4 with significantly improved heat resistance to fish feed. The mutation site is G272S. The mutant E0TYP4-4 obtained by the present invention had a relative residual enzyme activity of 88.19% and 79.51% after treatment at 60℃ and 70℃, respectively, which is higher than that of E0TYP4-3 after treatment at 60℃ and 70℃ (82.05% and 75.61%). Attached Figure Description

[0020] Figure 1 It is an RSMF diagram based on GROMACS.

[0021] Figure 2 This is a graph showing the thermostability of enzyme activity; Note: Compared to E0TYP4-3, * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001.

[0022] Figure 3 This is a graph showing the hydrolysis capacity and thermal stability of fish feed; where E0TYP4-4 represents the amino acid release content before heat treatment; Note: Compared with E0TYP4-4 before heat treatment, * P <0.05,** P <0.01. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0024] In this invention, A, G, S, and T are abbreviations for alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr), respectively.

[0025] Example 1: Construction of a thermostable mutant of fish feed protease 1) Obtaining heat-resistant mutants through semi-rational design First, the three-dimensional structure of the fish feed protease E0TYP4-3 (amino acid sequence as shown in SEQ ID NO: 1, gene sequence of mature peptide as shown in SEQ ID NO: 2) was modeled using the AlphaFold3 de novo modeling website, and evaluated using Laplace plots. Second, the qualified E0TYP4-3 three-dimensional structure was submitted to the Hotspot Wizzard website to predict the change in binding free energy (ΔΔG = ΔG mutant - ΔG wild type) before and after mutation of the fish feed protease E0TYP4-3. When ΔΔG is greater than 0, it indicates that the mutation makes binding more unstable and the reaction less likely to occur; when ΔΔG is less than 0, it indicates that the mutation makes binding more stable and the reaction more likely to occur. Therefore, we selected the following mutants with binding free energy ΔΔG less than 0 (3 in total) as mutants to be verified: A257V, G272S, and G272T.

[0026] The RSMF plots based on GROMACS for E0TYP4-3, mutants A257V, G272S, and G272T are shown below. Figure 1 As shown.

[0027] 2) Obtain recombinant plasmids with thermally stable single-point mutations. (1) Cloning of recombinant plasmids Using the nucleotide sequence of the wild-type strain recombinant plasmid pAc1-E0TYP4-3 as a template, whole-plasmid PCR polymerase chain reaction was performed using primers containing the mutation site. The PCR reaction system consisted of 25 μL of 2×Phanta Max Master Mix, 1.5 μL each of primers P1-F / P1-R, P2-F / P2-R, and P3-F / P3-R, 50–100 ng of plasmid template, and ddH2O added to a final volume of 50 μL. The reaction conditions were: 95℃ for 3 min; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 3 min, 30 cycles; 72℃ for 5 min; and storage at 4℃.

[0028] P1-F: 5'-GCTCAAGCACAATCacaTATCCTGCAAAATATCCTTCTACGATT-3'; P1-R: 5'-GATAtgtGATTGTGCTTGAGCTTCCCG-3'; P2-F: 5'-TCGTTGTTgtaGCAGCCGGAAATGAAGGCAC-3'; P2-R: 5'-GGCTGCtacAACAACGACGATAACCGCTGGCAAC-3'; P3-F: 5'-GCACAATCagcTATCCTGCAAAATATCCTTCTACGATTG-3'; P3-R: 5'-GCAGGATAgctGATTGTGCTTGAGCTTCCCGAAG-3'; Note: Lowercase letters are for the replaced codons.

[0029] Among them, the recombinant plasmid pAc1-E0TYP4-3 is produced using homologous recombination, a method commonly used in molecular cloning. The nucleotide sequence (SEQ ID NO: 2) of the gene encoding the mature peptide of fish feed protease E0TYP4-3 (SEQ ID NO: 1) is ligated to the *E. coli*-*Bacillus subtilis* shuttle plasmid pAc1. Sal I restriction site and Hind The plasmid pAc1, which is an Escherichia coli-Bacillus subtilis shuttle plasmid, was constructed between the III restriction sites. The plasmid pAc1 was disclosed in the literature “CN120330230A, An Escherichia coli-Bacillus subtilis shuttle plasmid and its application”.

[0030] (2) Product recovery and template digestion Product recovery: Add 50 μL of sterile water to the PCR product, then add 100 μL of Buffer GDP, mix well, and centrifuge at 12000 rpm for 30 s; discard the filtrate, wash twice with 600 μL of Buffer DW2 (diluted with anhydrous ethanol); finally, elute with 30 μL of water and measure the concentration.

[0031] Template digestion: Add restriction endonuclease to the recovered product. Dpn I. Remove template plasmid. Digested PCR product, 600–1200 ng, 1 μL 10× buffer. Dpn 1 μL of enzyme I was added, and ddH2O was added to bring the final volume to 10 μL. The reaction conditions were 37 ℃ for 60 min.

[0032] (3) Transformation and validation of recombinant plasmids Transformation of recombinant plasmids: 10 μL of the digested product was transformed into Escherichia coli DH5α competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37 °C for 12–16 h.

[0033] Transformed clone verification: Randomly select 10-20 single colonies for colony PCR and sequencing verification.

[0034] 3) Fermentation and thermal stability determination of single-point mutants (1) Take 10 μL of the plasmid that has been successfully sequenced and transform it into 500 μL of Bacillus subtilis SCK6 competent cells, spread it on LB solid plates containing a final concentration of 50 μg / mL kanamycin, and incubate overnight at 37 ℃ for 12–16 h.

[0035] (2) Pick colonies, and after verifying that the colonies are correct by colony PCR, inoculate them into LB medium containing a final concentration of 50 μg / mL kanamycin. The liquid volume is 10 mL / 50 mL. Incubate at 37℃ on a shaker at 200 rpm for 12-16 h to obtain seed liquid.

[0036] (3) The seed culture was transferred to LB medium containing a final concentration of 50 μg / mL kanamycin at an inoculation rate of 1%, with a liquid volume of 100 mL / 250 mL. After culturing at 37℃ and 200 rpm for 60 h, the culture was centrifuged at 12000 rpm for 10 min at 4℃ to obtain the fermentation supernatant (i.e. crude enzyme solution) of each mutant.

[0037] (4) After diluting the supernatant 10-fold, mix 100 μL of the diluted supernatant with 50 μL of 1% w / v azocasein, and then add 50 μL of phosphate buffer (pH=7.0). Incubate the mixture at 40°C for 10 minutes, then add 250 μL of 0.4 M trichloroacetic acid (TCA) to terminate the reaction, and continue incubation for another 10 minutes. Subsequently, centrifuge at 12,000 × g for 5 minutes. Then, take 250 μL of the centrifuged supernatant and mix it with 50 μL of 2 M NaOH, and measure the absorbance at 450 nm. The standard enzyme unit is defined as: the amount of OD produced within one minute under conditions of pH 7.0 and temperature 40°C. 450 The amount of enzyme required to increase the value by 0.001.

[0038] (5) The relative residual enzyme activity of each mutant was obtained by comparing the enzyme activity after heat treatment with that before heat treatment. For the above three single-point mutants, the relative residual enzyme activity of their fermentation supernatant after heat treatment at 50℃, 60℃ and 70℃ for 30 min was measured. Figure 2The G272S mutant was compared with E0TYP4-3 to screen for single-point mutants with improved thermostability. The relative residual enzyme activity of the G272S mutant after treatment at 60℃ and 70℃ was 88.19% and 79.51%, respectively, significantly higher than that of E0TYP4-3 (82.05% and 75.61%), making it the best among the three mutants. The G272S mutant with improved thermostability was ultimately named E0TYP4-4.

[0039] Example 2: Preparation of fish feed, application of fish feed hydrolysis, and determination of heat resistance. 1) Preparation of fish feed and fish feed degrading enzyme solution Purchase common fish feed from the market, centrifuge the fermentation broth of mutant E0TYP4-4 obtained in Example 1, obtain the supernatant, dilute it, and prepare a fish feed protease solution.

[0040] 2) Determination of fish feed degradation capacity and heat resistance (1) The control group, blank group and experimental group were set up according to the amino acid content detection kit (purchased from ELISA, specification: 100T / 96S).

[0041] Control group: Weigh 0.02g of fish feed into a 1.5mL centrifuge tube, add 800μL of water, and treat in a 40℃ metal bath for 60 minutes. Use a kit to measure the absorbance at 570 nm every 10 minutes. The difference between the measured absorbance and the absorbance at 0 minutes is recorded as A. standard .

[0042] Control group: Add 400 μL of fish feed protease solution and 400 μL of phosphate buffer (pH=7.0) to a 1.5 mL centrifuge tube, incubate in a 40℃ metal bath for 60 minutes, and measure the absorbance at 570 nm every 10 minutes using a kit. Record the difference between the measured absorbance and the absorbance at 0 minutes as A. blank .

[0043] Experimental group: Weigh 0.02g of fish feed, and add 400 μL of fish feed protease solution and 400 μL of phosphate buffer (pH=7.0) to a 1.5 mL centrifuge tube. Incubate in a metal bath at 40℃ for 60 minutes. Measure the absorbance at 570nm every 10 minutes using a kit. Record the difference between the measured absorbance and the absorbance at 0 minutes as A. test .

[0044] The heat resistance was determined by preheating the fermentation supernatant at 60°C and 70°C for 30 minutes, followed by enzymatic hydrolysis of the fish feed at 40°C for 60 minutes. The control group was treated at 40°C throughout the process. Amino acid release levels at sampling time points were compared with those of the control group.

[0045] The formula for calculating amino acid release (μmol / mg protein) is (A test -A blank ) / (A standard -A blank Protein content (mg / mL). The protein content refers to the protein content of 400 μL of fish feed protease solution.

[0046] (2) The results are as follows Figure 3 As shown, at 40℃, the amino acid release content of EOTYP4-4 reached 116.12±4.96 μmol / mg protein within 60 minutes. After heat treatment, the amino acid release content of EOTYP4-4 remained at 91.6% and 87.6% of the pre-heat treatment level at 60℃ and 70℃, respectively. EOTYP4-4 exhibits excellent feed hydrolysis performance and strong thermostability, making it a promising candidate enzyme for agricultural applications. These results demonstrate that the fish feed degrading enzyme of this invention has a significant effect on the degradation of protein in fish feed ingredients.

[0047] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fish feed protease mutant with improved thermal stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO: 3, or as shown in SEQ ID NO: 30 to 381aa.

2. The gene encoding the fish feed protease mutant with improved thermal stability as described in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

4. The biomaterial related to the fish feed protease mutant with improved thermal stability as described in claim 1, characterized in that, It can be any one or more combinations of the following biological materials: (a) An expression cassette containing the gene of claim 2 or 3; (b) A recombinant expression vector containing the gene of claim 2 or 3; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant bacteria containing the gene described in claim 2 or 3; (e) Recombinant bacteria containing the expression cassette described in (a); (f) Recombinant bacteria containing the recombinant expression vector described in (b) or (c).

5. The biomaterial according to claim 4, characterized in that: The starting vector for the recombinant expression vectors mentioned in (b) and (c) is a pUB series vector or an Escherichia coli-Bacillus subtilis shuttle plasmid vector; The host bacteria corresponding to the recombinant bacteria described in (d), (e), and (f) are selected from prokaryotes or yeast.

6. An immobilized enzyme, characterized in that: Contains the fish feed protease mutant with improved thermal stability as described in claim 1.

7. The application of the fish feed protease mutant with improved thermal stability according to claim 1, the gene according to any one of claims 2-3, the biomaterial according to any one of claims 4-5, or the immobilized enzyme according to claim 6, characterized in that, For one of the following applications: (1) Application in the preparation of fish feed protease mutants with improved thermal stability; (2) Application in the degradation of feed protein.

8. The application of the fish feed protease mutant with improved thermal stability as described in claim 1, the gene as described in any one of claims 2-3, the biomaterial as described in any one of claims 4-5, or the immobilized enzyme as described in claim 6 in the field of feed.

9. A method for obtaining the fish feed protease mutant with improved thermal stability as described in claim 1, characterized in that, Includes the following steps: By designing primers containing mutation sites to perform site-directed mutation G272S on the gene encoding the fish feed protease with the amino acid sequence shown in SEQ ID NO: 1, the fish feed protease mutant with improved thermostability as described in claim 1 was obtained.

10. The method according to claim 9, characterized in that: Primers containing the mutation site were designed to introduce the mutation G272S into the gene encoding the fish feed protease with the amino acid sequence shown in SEQ ID NO:

1. After correct sequencing, the gene was transformed into Bacillus subtilis SCK6 for expression, resulting in the fish feed protease mutant with improved thermostability as described in claim 1.

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