Mutant for improving cutinase yield and preparation method thereof

Abstract

The invention relates to the technical field of enzyme engineering and recombinant protein expression, and discloses a mutant for improving cutinase yield and a preparation method thereof. The mutant protein is based on an amino acid sequence as shown in SEQ ID NO.1 and has G93Q or H209F single-point substitution, and the site number takes first methionine in the SEQ ID NO.1 as the first site. The invention also provides a nucleic acid molecule for coding the mutant, a recombinant expression vector and a recombinant host cell, and a method for adding an inducer to induce expression when OD600 is 0.6-1.2 in a culture process so as to obtain a culture containing the mutant. Embodiments show that the enzyme activity per unit volume of the mutant culture is higher than that of a control group.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and recombinant protein expression technology, specifically to a single-point mutant protein, encoding nucleic acid, recombinant expression vector, recombinant host cell, and preparation method thereof, which improves the enzyme activity (U / mL) of keratinase Est119SGIK per unit volume of culture in a recombinant expression system through site-directed mutation at a specific site. Background Technology

[0002] Polyethylene terephthalate (PET) and other synthetic polyester materials are widely used in packaging, textiles, and other fields. Due to their stable chemical structure, polyester materials have a low degradation rate in the natural environment, and related research has been carried out on the biological treatment and recycling of polyester materials.

[0003] Polyester hydrolases (such as keratinases) can catalyze the hydrolysis of ester bonds in polyester materials, achieving polyester hydrolysis under relatively mild conditions. Therefore, they can be used in the biodegradation or recycling processes of polyester materials. For example, keratinase / polyesterase Est119 from Thermobifida alba AHK119 has been reported in the literature, and its enzymatic properties and structure have been studied.

[0004] Existing research on the modification and screening of polyester hydrolases / keratinases typically focuses on indicators such as catalytic efficiency, substrate suitability, and stability. Furthermore, existing literature and patents disclose mutants of Thermobifida keratinases (including Est119-related enzymes) and their preparation methods. However, in engineering applications and scale-up preparation, enzyme expression / fermentation yield may still be limited, especially in recombinant expression systems using *E. coli* as the host. Insufficient expression yield can affect the enzyme activity (U / mL) per unit volume of culture, thus impacting enzyme supply capacity and production economics. Moreover, the effects of protein point mutations on recombinant expression yield / solubility and enzyme activity per unit volume of culture are often highly uncertain. Existing techniques typically cannot directly deduce specific mutation sites and replacement methods that can significantly improve expression / enzyme supply capacity solely from the teachings of catalytic performance modification.

[0005] Therefore, it is necessary to provide a protein engineering scheme for the keratinase Est119SGIK and a corresponding recombinant expression preparation method to improve the enzyme activity (U / mL) per unit volume of culture in the recombinant induced expression system. Summary of the Invention

[0006] Technical problems to be solved This invention provides a single-point mutant protein capable of increasing the enzyme activity (U / mL) of keratinase Est119SGIK per unit volume of culture in a recombinant-induced expression system, along with its corresponding encoding nucleic acid, recombinant expression vector, recombinant host cell, and preparation method, to meet the enzyme supply requirements for subsequent enzyme preparation and application.

[0007] Technical solution To address the aforementioned technical problems, the present invention provides the following combinable technical solution modules; each module can be implemented individually or in combination as needed.

[0008] All the above-mentioned technical solutions are based on the G93Q or H209F single-point mutant protein and jointly solve the technical problem of improving the enzyme activity (U / mL) of keratinase Est119SGIK per unit volume of culture in the recombinant expression system. They belong to the same inventive concept.

[0009] Mutant protein module: Provides a single-point mutant protein of keratinase Est119SGIK, the amino acid sequence of which is shown in SEQ ID NO.1; the single-point mutant is G93Q or H209F.

[0010] Nucleic acid encoding module: Provides a nucleic acid molecule encoding the above-mentioned single-point mutant protein. The nucleic acid molecule may be cDNA, genomic DNA, codon-optimized synthetic nucleic acid, or a complementary sequence thereof; degenerate variants without altering the encoding amino acid sequence are also included.

[0011] Expression vector module: Provides a recombinant expression vector containing the nucleic acid molecule. In some embodiments, the expression vector is a prokaryotic expression vector, such as the pET series vectors (e.g., pET-24a(+)).

[0012] Host cell module: Provides a recombinant host cell containing the recombinant expression vector and capable of expressing the single-point mutant protein. The host cell may be Escherichia coli, Bacillus, yeast, or filamentous fungi, etc.; in some embodiments, the host cell is Escherichia coli (e.g., BL21 Star (DE3)).

[0013] Construction Method Module: Provides a method for constructing the recombinant host cell, including: introducing the target mutation by site-directed mutagenesis PCR, removing the unmutated template by DpnI digestion, transforming and screening positive clones and sequencing verification, and transforming the verified recombinant expression vector into the expression host.

[0014] Preparation method module: Provides a method for preparing the single-point mutant protein, comprising: culturing the recombinant host cells and, after the cells reach a preset growth stage, adding an inducer to induce expression, thereby obtaining a culture containing the single-point mutant protein; in some embodiments, OD during induction... 600 The concentration is 0.6–1.2 (preferably 0.8–1.0), and the inducing agent is IPTG (preferably a final concentration of 0.025 mM).

[0015] Beneficial effects Compared with the prior art, the present invention has at least the following beneficial effects: Under the same vector / host, culture and induction conditions, and enzyme activity detection conditions, the G93Q or H209F single-point mutants can increase the enzyme activity (U / mL) per unit volume of culture.

[0016] The examples show that, under shake-flask induced expression conditions, the enzyme activity of the control group culture was 40 U / mL; the enzyme activity of the G93Q mutant culture was 82.36 U / mL; and the enzyme activity of the H209F mutant culture was 76.42 U / mL (see...). Figure 1 ).

[0017] Under the conditions described in the above examples, the enzyme activities of the cultures of G93Q and H209F mutants were 2.06 times and 1.91 times that of the control group, respectively.

[0018] The above results indicate that the single-site substitution at the specific site can significantly improve the enzyme supply capacity of the keratinase Est119SGIK under the same expression system and induction conditions, which is a technical effect that is hard to expect in the field. Attached Figure Description

[0019] Figure 1 This is a comparison of enzyme activity (U / mL) in cultures of the control group Est119SGIK and single-point mutants G93Q and H209F under shake-flask induction expression conditions. Detailed Implementation

[0020] Terms and Definitions Keratinase Est119SGIK: refers to a protein with an amino acid sequence as shown in SEQ ID NO.1.

[0021] Site numbering: Unless otherwise specified, the amino acid site numbering is based on the first methionine in SEQ ID NO.1.

[0022] Control group: refers to recombinant host cell samples expressing the Est119SGIK protein shown in SEQ ID NO.1, used for comparison under the same culture and detection conditions as the mutant.

[0023] Yield / Expression Yield: In this application, enzyme activity (U / mL) per unit volume of culture is used to characterize the enzyme activity. When quantifying soluble protein, the amount of soluble protein (mg / L) can also be used as a supplementary characterization indicator. The determination method and the definition of enzyme activity units are detailed in the specific implementation method.

[0024] Cultures include fermentation broth after induced expression, its centrifuged supernatant, cell lysate, or soluble protein components; the specific sample type is subject to the sample preparation method in the examples.

[0025] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make appropriate modifications or substitutions to the specific conditions of the embodiments without departing from the spirit of the present invention.

[0026] Example 1: Construction of a single-point mutant of keratinase Est119SGIK Template and Vector: Site-directed mutagenesis is performed using an expression vector containing a nucleic acid fragment encoding the keratinase Est119SGIK shown in SEQ ID NO.1 as a template. In some embodiments, the nucleic acid fragment encoding Est119SGIK is cloned into a pET-24a(+) vector to obtain a template expression vector: for example, NdeI and XhoI restriction endonuclease sites are introduced at the 5' and 3' ends of the nucleic acid fragment, respectively, and after double digestion and ligation, it is directionally cloned into the multiple cloning site region of pET-24a(+); this vector can fuse a 6×His tag (His-tag) to the C-terminus of the target protein. In some embodiments, the prokaryotic expression vector includes a promoter capable of driving transcription of the target gene and its inducible regulatory elements, a transcription terminator, an resistance marker for selection, and a replication origin for replication in the host cell.

[0027] Template plasmid preparation: The strain containing the template plasmid was inoculated into LB liquid medium and cultured at 37℃ and 180-220 rpm for 10-16 h with shaking; plasmid DNA was extracted using a plasmid mini-extraction kit for later use.

[0028] Site-directed mutagenesis PCR: High-fidelity DNA polymerase is used for amplification to introduce the target mutation. Primer sequences for introducing the G93Q mutation are shown in SEQ ID NO.2 and SEQ ID NO.3; primer sequences for introducing the H209F mutation are shown in SEQ ID NO.4 and SEQ ID NO.5 (see Table 1). The PCR amplification program may include: 98℃ pre-denaturation for 2–5 min; 98℃ denaturation for 10–15 s, 55℃ annealing for 20–40 s, 72℃ extension for 1–3 min, for 25–35 cycles; 72℃ extension for 5–10 min. The total PCR volume can be 25–50 μL; the template plasmid volume can be 10–100 ng; and the final primer concentration can be 0.2–0.5 μM.

[0029] DpnI digestion: Add DpnI restriction endonuclease to the PCR product and react at 37°C for 1.0–2.0 h to digest the unmutated methylated template DNA and obtain the digestion product. For example, take 8–10 μL of PCR product, add 1–10 U of DpnI, and make up to 10–20 μL of reaction system.

[0030] Transformation and screening: The digestion products are transformed into competent Escherichia coli (e.g., E. coli JM109) cells, the cloning host; positive clones are screened on LB solid medium plates containing the appropriate antibiotics. In some embodiments, the heat shock conditions for chemocompetent cells are: ice bath for 20–30 min, heat shock at 42°C for 45–90 s, ice bath for 2–5 min, recovery with SOC or LB for 30–60 min, and then plated for culture.

[0031] Sequencing verification: Positive clones were selected for plasmid extraction, and Sanger sequencing was used to sequence and verify the region containing the mutation site, confirming that the target mutation was correct and that there were no unexpected mutations. T7 universal primers or universal primers flanking the vector can be used for sequencing, and the sequencing coverage area should cover at least 200 bp on both sides of the mutation site.

[0032] Culture medium composition: LB medium can be prepared according to the following formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride; solid culture medium can be prepared by adding 15 g / L agar powder.

[0033] Table 1. Mutation sites and site-directed mutagenesis primers (5'→3') Example 2: Induction of mutant expression and evaluation of enzyme activity per unit volume (shake flask) Construction of expression strain: The mutant plasmid verified by sequencing in Example 1 was transformed into expression host cells to obtain a recombinant expression strain. In some embodiments, the expression host is *Escherichia coli* BL21 Star (DE3). The control strain was a strain obtained by transforming the expression vector containing the encoding Est119SGIK shown in SEQ ID NO.1.

[0034] Shake-flask culture and induction of expression: After resuscitation culture in LB liquid medium containing kanamycin, the recombinant expression strain is transferred to TB liquid medium containing kanamycin at an inoculation rate of 3%–10% (e.g., 5%) for scale-up culture. Shake flasks can be 250 mL in size, with a liquid volume of 30–80 mL (e.g., 50 mL), and a shaking speed of 180–220 rpm (e.g., 200 rpm). The temperature during the scale-up culture phase can be 30–37℃ (e.g., 37℃). When the bacterial culture OD... 600 When the concentration reaches 0.6–1.2 (preferably 0.8–1.0), the temperature is lowered to 16–30°C (e.g., 25°C), and IPTG is added to induce expression. The final IPTG concentration can be 0.01–0.10 mM (preferably 0.025 mM), and the induction time can be 12–36 h (e.g., 24 h). The final kanamycin concentration can be 20–50 μg / mL (e.g., 30 μg / mL).

[0035] Culture medium composition: TB culture medium can be prepared according to the following formula: tryptone 12 g / L, yeast extract 24 g / L, glycerol 5 g / L, dipotassium hydrogen phosphate trihydrate 16.43 g / L, potassium dihydrogen phosphate 2.31 g / L.

[0036] Sample preparation: After induction, culture samples were taken for enzyme activity assay. In this example, the supernatant was collected by centrifugation at 4°C and 8000×g for 5–10 min and used as the test sample. The sample was then diluted with pH 8.0, 100 mM phosphate buffer (e.g., diluted 10 times) before measurement to reduce the influence of cell scattering on absorbance.

[0037] Enzyme activity assay: Using p-nitrophenylbutyrate (pNPB) as the substrate (the substrate solution can be prepared with isopropanol or acetonitrile), the enzyme activity of the sample was determined spectrophotometrically. The reaction buffer can be 10 mM Tris-HCl (pH 8.0) or 100 mM phosphate buffer (pH 8.0); the reaction temperature is 60℃; the detection wavelength is 405 nm. In an example system, the total reaction volume is 1.50 mL: 30 μL of the sample to be tested is added to 1.44 mL of buffer preheated to 60℃, mixed and equilibrated, and then 30 μL of pNPB substrate solution (final pNPB concentration of 0.2–1.0 mM, e.g., 0.5 mM) is added to start the reaction. Immediately afterward, the absorbance change is continuously recorded at 405 nm for 1 min (e.g., every 5 s). A blank control can be obtained by replacing the sample with an equal volume of buffer and measured under the same conditions.

[0038] Enzyme activity unit definition and calculation: 1 U is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol (pNP) per minute at 60℃ and pH 8.0. A405 can be converted to pNP production using a pNP standard curve; sample enzyme activity (U / mL) can be calculated based on production amount, reaction time, sample volume, and dilution factor.

[0039] Results: Under the above conditions of shake-flask induction expression and enzyme activity assay, the enzyme activity of the control group culture was 40 U / mL; the enzyme activity of the G93Q mutant culture was 82.36 U / mL; and the enzyme activity of the H209F mutant culture was 76.42 U / mL. Figure 1 As shown, the enzyme activity of cultures of G93Q and H209F mutants was higher than that of the control group.

[0040] Quality control and characterization Plasmid and mutation verification: The positive plasmids obtained from the cloning host are identified by restriction enzyme digestion and Sanger sequencing; the sequencing results should cover a region of at least 200 bp on both sides of the mutation site.

[0041] Expression confirmation: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis can be performed on samples before and after induction to compare the target protein bands; if the vector contains a fusion tag (e.g., His-tag), Western blot can be used for further verification.

[0042] Protein quantification and comparability: The protein concentration of the sample can be determined using the BCA (bisquinolinic acid) or Bradford (Coomassie brilliant blue) method, and enzyme activity can be measured at the same dilution factor to ensure comparability.

[0043] Blank and negative control: In enzyme activity assays, enzyme-free blank, substrate-free blank, and control group samples can be set to exclude the influence of non-enzymatic hydrolysis or background absorption.

[0044] The present invention has been described above with reference to specific embodiments, but the present invention is not limited to the above embodiments. Those skilled in the art can make modifications or substitutions to the above embodiments without departing from the technical solution and scope defined by the claims, and all such modifications or substitutions should fall within the protection scope of the present invention, which is defined by the claims.

[0045] Furthermore, the technical features of the various embodiments in this application can be combined with each other without contradiction.

Claims

1. A keratinase single-point mutant protein based on the amino acid sequence shown in SEQ ID NO.1, characterized in that, The single-point mutant protein is subjected to any of the following amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO.1: replacing the 93rd amino acid residue G with Q to obtain the G93Q mutant, or replacing the 209th amino acid residue H with F to obtain the H209F mutant; the site numbering is based on the first methionine in SEQ ID NO.1 as position 1.

2. The single-point mutant protein according to claim 1, characterized in that, The single-point mutant is the G93Q mutant, and its amino acid sequence is shown in SEQ ID NO.

6.

3. The single-point mutant protein according to claim 1, characterized in that, The single-point mutant is the H209F mutant, whose amino acid sequence is shown in SEQ ID NO.

7.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the single-point mutant protein as described in any one of claims 1-3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule of claim 4 and is used to express the single-point mutant protein of any one of claims 1-3 in host cells.

6. A recombinant host cell, characterized in that, The recombinant host cell is *Escherichia coli*, containing the recombinant expression vector of claim 5, and is used to express the single-point mutant protein of any one of claims 1-3.

7. The recombinant host cell according to claim 6, characterized in that, The *Escherichia coli* strain is BL21 Star(DE3); the recombinant expression vector is pET-24a(+).

8. A method for constructing the recombinant host cell of claim 6 or 7, characterized in that, Includes the following steps: Using an expression vector containing the keratinase Est119SGIK shown in SEQ ID NO.1 as a template, the G93Q mutation or H209F mutation was introduced by site-directed mutagenesis PCR to obtain the PCR product; The PCR product was digested with DpnI to digest the unmutated template DNA, yielding the digested product. The digestion product was transformed into a cloning host cell, positive clones were screened and sequenced to verify the results, and a recombinant expression vector containing the mutation was obtained. The recombinant expression vector, which has been verified to be correct by sequencing, is transformed into expression host cells to obtain recombinant host cells capable of expressing the single-point mutant protein.

9. A method for preparing the single-point mutant protein according to any one of claims 1-3, characterized in that, include: The recombinant host cells described in claim 6 or 7 are inoculated into a culture medium and cultured until the bacterial cells grow to the OD value. 600 When the optical density at 600 nm is 0.6 to 1.2, an inducer is added to induce expression, and a culture containing the single-point mutant protein is obtained.

10. The method according to claim 9, characterized in that, The inducer was isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.025 mM; the induction temperature was 25°C, and the induction time was 24 h; the OD during induction was... 600 The optical density at 600 nm is 0.8–1.0; the culture includes first reviving and culturing in LB medium, and then transferring to TB medium for culture.

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