Fish feed protease mutant with improved enzyme activity and preparation method thereof

Abstract

The invention discloses a fish feed protease mutant with improved enzyme activity and a preparation method thereof, and belongs to the field of enzyme engineering. Error-prone PCR mutation is carried out on the basis of fish feed protease to obtain a mutant library, eight mutants (ER12, ER5, ER8, ER10, ER9, ER14, ER6 and ER4) with the protease activity remarkably improved are obtained through primary screening and secondary screening, and ER5 (the mutation site is N223S) and ER12 (the mutation site is S222P) are named as E0TYP4-2 and E0TYP4-3; the protease activity of the mutant E0TYP4-3 is about 2.26 times that of a wild type, and the protease activity of E0TYP4-2 is about 2.11 times that of the wild type. The enzyme activity of the mutant is greatly improved, and the mutant is more suitable for being used as a feed additive than a wild type, and is beneficial to wide application of fish 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 enhanced enzyme activity and its preparation method. Background Technology

[0002] Proteases account for 60-65% of the global industrial market and are widely used in industries such as detergents, pharmaceuticals, leather, food, and agriculture. Bacillus subtilis, as a major producer of these proteases, is used not only in the production of specialty chemicals but also extensively in the production of industrial enzymes. Furthermore, proteases play an important role in biopharmaceutical products, such as in contact lens cleaners and enzymatic detergents.

[0003] In fish farming, 60% of the protein in feed is wasted. Using proteases in feed can improve feed utilization, so improving the enzyme activity of fish feed proteases is of great practical significance to the feed industry. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a fish feed protease mutant with enhanced enzyme activity and its preparation method. This mutant exhibits significantly improved enzyme activity, which is beneficial for its widespread application in the feed industry.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides a mutant fish feed protease with enhanced enzyme activity, wherein the amino acid sequence of the mutant is obtained by mutating SEQ ID NO: 1 through any of the following mutations: At least one of S222P, N223S, S236P, D354G, T359I, V150A, V132I, G237A, and D226Y; wherein, S222P, i.e., the 222nd amino acid, is mutated from serine (S) to proline (P), and the others are similar.

[0006] Furthermore, a fish feed protease mutant with enhanced enzyme activity, wherein the amino acid sequence of the mutant is obtained by mutating SEQ ID NO: 1 through any of the following mutations: S222P, N223S, S236P, D354G / T359I, V150A, V132I, G237A or D226Y.

[0007] Preferably, a fish feed protease mutant ER12 (S222P) with enhanced enzyme activity has the amino acid sequence shown in SEQ ID NO: 3, and the nucleotide sequence encoding the mature peptide of the mutant S222P is shown in SEQ ID NO: 4.

[0008] Preferably, a fish feed protease mutant ER5 (N223S) with enhanced enzyme activity has the amino acid sequence shown in SEQ ID NO: 5, and the nucleotide sequence encoding the mature peptide of the mutant N223S is shown in SEQ ID NO: 6.

[0009] Preferably, the gene sequence encoding the amino acid sequence shown in SEQ ID NO: 30-381aa in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

[0010] The coding gene of one of the mutants.

[0011] The aforementioned mutant-related biological materials are any one or more combinations of the following biological materials: (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).

[0012] Furthermore, the starting vector for the recombinant expression vector described in (b) and (c) is a pUB series vector, etc.; preferably, the pUB110 vector.

[0013] 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.

[0014] An immobilized enzyme containing the above-mentioned mutant.

[0015] The above-mentioned mutants, encoding genes, mutant-related biological materials, or immobilized enzymes are used in the preparation of fish feed protease mutants with enhanced enzyme activity.

[0016] The application of the aforementioned mutants, encoding genes, mutant-related biomaterials, or immobilized enzymes in the degradation of feed proteins.

[0017] The above-mentioned mutants, coding genes, mutant-related biological materials, or immobilized enzymes are used in the feed industry.

[0018] A method for obtaining the above-mentioned mutant includes the following steps: By designing primers containing mutation sites to perform site-directed mutagenesis 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 enhanced enzyme activity was obtained.

[0019] Furthermore, primers containing mutation sites were designed to introduce mutations 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 enhanced enzyme activity.

[0020] The present invention has the following advantages and effects compared with the prior art: (1) The single-point mutant of fish feed protease provided by the present invention generally has higher enzyme activity than wild type, which indicates that the above single-point mutation leads to a significant increase in enzyme activity of fish feed protease, making it more suitable as a feed additive than wild type, which is conducive to the widespread application of fish feed protease in feed, and thus has a broad market prospect.

[0021] (2) This invention uses fish feed protease as a basis to perform error-prone PCR mutation to obtain a mutant library. Through primary and secondary screening, eight mutants with significantly increased protease activity (ER12, ER5, ER8, ER10, ER9, ER14, ER6, ER4) were obtained. ER5 and ER12 were named E0TYP4-2 and E0TYP4-3, respectively. The mutation site for ER5 is N223S, and the mutation site for ER12 is S222P. The protease activity of the mutant E0TYP4-3 obtained in this invention is approximately 2.26 times that of the wild type, and the protease activity of E0TYP4-2 is approximately 2.11 times that of the wild type. Attached Figure Description

[0022] Figure 1 This is a screening diagram of 16 mutants on milk agar plates.

[0023] Figure 2 This is a graph showing the enzyme activity of 16 mutants; Note: Compared to WT, * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001. Detailed Implementation

[0024] 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.

[0025] Example 1: Obtaining a high-enzyme-activity mutant of fish feed protease A mutant library was obtained using error-prone PCR, and primary and secondary screenings were performed to identify mutants with enhanced enzyme activity. 1) See the error-prone PCR procedure. Figure 1 With plasmid pUB110-P 43 Using -NprE-E0TYP4 (the target protein is the mature peptide shown in SEQ ID NO: 1, segments 30-381aa) as a template, high-fidelity enzyme PCR was performed with primers F1 and R1 to obtain the plasmid framework. Low-fidelity enzyme PCR was then performed with primers F2 and R2 to amplify the E0TYP4 fish feed protease gene. Finally, 1 μL of [unspecified ingredient] was added. Dpn After digestion with enzyme I, the PCR product was recovered by gel extraction. 5 μL of plasmid framework and gene were added to PCR tubes, and a POE-PCR reaction was performed to form a multimeric plasmid. 5 μL of the PCR reaction solution was transformed into Bacillus subtilis SCK6 and plated on 2% milk agar plates (2% (w / v) skim milk powder + 2% (w / v) agar) containing 50 μg / mL kanamycin for 48 hours. Among these, pUB110-P... 43 -NprE-E0TYP4 is disclosed in the literature "CN117721057A, an engineered bacterium heterologously expressing the E0TYP4 gene and its application in the production of fish feed protease".

[0026] F1: 5'-TACAAGCAGCTGCCCAATAAGAATTCCTTAAGGAACGTACAGACGGC-3'; R1: 5'-TTCTGTACTGCTTTTTCCGGCACCTTCAGCAGCCTGAACACC-3'; F2: 5'-GGTGTTCAGGCTGCTGAAGGTGCCGGAAAAAGCAGTACAGAA-3'; R2: 5'-CTGTACGTTCCTTAAGGAATTCTTATTGGGCAGCTGCTTGTACG-3'; 2) Using plasmid pUB110-P 43 -NprE-E0TYP4 Bacillus subtilis SCK6 was used as the wild-type (WT) control. The clear zone was observed, and single colonies with clear zones larger than the control group were selected for secondary screening. The results of the secondary screening are shown below. Figure 1 Finally, 15 strains with a clear zone larger than the control group and 1 strain with a clear zone smaller than the control group were selected for sequencing to detect the mutation location.

[0027] Example 2: Determination of mutant enzyme activity 1) The mutants obtained from screening were transferred to 5 mL of LB medium (50 μg / mL kanamycin) and cultured overnight at 37℃ and 200 rpm with shaking. Then, 1% (v / v) of the inoculum was transferred to 100 mL of LB medium (50 μg / mL kanamycin) and cultured at 37℃ and 200 rpm with shaking for 60 h. The cells were removed by centrifugation at 12,000 rpm for 10 minutes to obtain the fermentation supernatant containing the fish feed protease mutant, and the enzyme activity was determined.

[0028] 2) Dilute the supernatant 10-fold, then mix 100 μL of the diluted supernatant with 50 μL of 1% w / v azocasein, followed by the addition of 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, mix 250 μL of the centrifuged supernatant 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 at pH 7.0 and 40°C. 450 The amount of enzyme required to increase the value by 0.001.

[0029] 3) After enzyme activity assays of 16 rescreened mutants, it was found that 8 mutants exhibited significantly enhanced enzyme activity (Table 1). Mutant ER12 showed the highest activity at 159.4 ± 7.21 U / mL, which was 2.26 times that of the wild type. Mutant ER5 followed closely with an activity of 149.3 ± 4.51 U / mL. In contrast, mutant ER11 showed decreased activity at 38.3 ± 6.81 U / mL, only 54.2% of the wild type. Figure 2 Sequencing analysis revealed an S222P mutation in ER12 and an N223S mutation in ER5. Analysis of the S222P and N223S mutations using Missense 3D software showed that the S222P mutation altered the original secondary structure, weakening hydrogen bonding, while the N223S mutation increased the hydrogen bond length with 219W, from 2.62 Å to 2.91 Å. Simultaneously, a new hydrogen bond with a length of 3.21 Å was introduced between 220A and 223S. This mutation weakened the hydrogen bond interaction with the 219W benzene ring, creating a new hydrogen bond with a length of 3.21 Å between 220A and 223S. Furthermore, the newly formed 220A-223S hydrogen bond provides structural support, facilitating substrate access to the catalytic site.

[0030] In summary, the enzyme activities of the eight mutants ER12, ER5, ER8, ER10, ER9, ER14, ER6, and ER4 were all higher than those of the wild type. ER5 and ER12 were named E0TYP4-2 and E0TYP4-3, respectively, as high-enzyme-activity mutants of fish feed protease.

[0031] Table 1. Enzyme activities of 8 mutants with significantly enhanced enzyme activity.

[0032] 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 mutant fish feed protease with enhanced enzyme activity, characterized in that: The amino acid sequence of the mutant is obtained by mutating SEQ ID NO: 1 through any of the following mutations: At least one of S222P, N223S, S236P, D354G, T359I, V150A, V132I, G237A and D226Y.

2. The fish feed protease mutant with enhanced enzyme activity according to claim 1, characterized in that: The amino acid sequence of the mutant is obtained by mutating SEQ ID NO: 1 through any of the following mutations: S222P, N223S, S236P, D354G / T359I, V150A, V132I, G237A or D226Y; The amino acid sequence of mutant S222P is shown in SEQ ID NO: 3; the amino acid sequence of mutant N223S is shown in SEQ ID NO:

5.

3. A gene encoding a fish feed protease mutant with enhanced enzyme activity as described in claim 1 or 2.

4. The gene according to claim 3, characterized in that: The nucleotide sequence of the gene encoding the mature peptide of mutant S222P is shown in SEQ ID NO: 4; the nucleotide sequence of the gene encoding the mature peptide of mutant N223S is shown in SEQ ID NO:

6.

5. Biomaterials related to the fish feed protease mutant with enhanced enzyme activity as described in claim 1 or 2, 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 3 or 4; (b) A recombinant expression vector containing the gene of claim 3 or 4; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant bacteria containing the gene described in claim 3 or 4; (e) Recombinant bacteria containing the expression cassette described in (a); (f) Recombinant bacteria containing the recombinant expression vector described in (b) or (c).

6. The biomaterial according to claim 5, characterized in that: The recombinant expression vectors described in (b) and (c) are pUB series vectors; The host bacteria corresponding to the recombinant bacteria described in (d), (e), and (f) are selected from prokaryotes or yeast.

7. An immobilized enzyme, characterized in that: A fish feed protease mutant containing the enhanced enzyme activity described in claim 1 or 2.

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

9. The application of the fish feed protease mutant with enhanced enzyme activity according to any one of claims 1 to 2, the gene according to any one of claims 3 to 4, the biomaterial according to any one of claims 5 to 6, or the immobilized enzyme according to claim 7 in the field of feed.

10. A method for producing a fish feed protease mutant with enhanced enzyme activity as described in any one of claims 1 to 2, characterized in that, The method includes the following steps: by designing primers containing mutation sites to perform site-directed mutagenesis on the gene encoding the fish feed protease with the amino acid sequence shown in SEQ ID NO: 1, and then expressing the gene, a fish feed protease mutant with enhanced enzyme activity as described in any one of claims 1 to 2 is obtained.

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