A keratinase mutant without collagen hydrolysis activity

Abstract

The invention discloses a keratinase mutant without collagen hydrolysis activity, belonging to the technical field of genetic engineering and enzyme engineering. The invention provides a keratinase mutant without collagen hydrolysis activity, wherein the mutant is a keratinase mutant whose amino acid sequence is mutated to isoleucine at position 221 of the keratinase shown in SEQ ID NO.1, and is named KerZ1 / M221I. The results of enzyme activity assay show that the keratinase mutant KerZ1 / M221I retains 59235U / mL of keratin hydrolysis activity and has no collagen hydrolysis activity, so the keratinase mutant can avoid collagenase damage during application and improve the quality of leather.

Description

Technical Field

[0001] The invention relates to a keratinase mutant without collagen hydrolysis activity, belonging to the technical field of gene engineering and enzyme engineering. Background Art

[0002] Keratinase is a type of hydrolase that catalyzes the decomposition of keratin and is widely used in the detergent, cosmetics, pharmaceutical, and leather industries. The wide range of applications of keratinase also reflects its poor substrate specificity. It not only has a hydrolytic effect on keratin, but also has a high hydrolytic activity on other proteins, especially collagen. This also limits its application in cosmetics, leather, and other fields. For example, in depilatory cream products, the collagen hydrolysis activity of keratinase can cause skin damage, and the same problem exists in leather depilation applications. Therefore, removing the collagen activity of keratinase is of great significance to realizing the commercial value of keratinase.

[0003] The research team has previously constructed a recombinant keratinase strain, which can ferment a keratinase product with a keratinase hydrolysis activity of more than 426.60KU / mL in a 15L fermenter (the keratinase is recorded in the literature of Biotransformation of keratin waste to amino acids and active peptides based on cell free catalysis), which is the highest level of recombinant keratinase expression reported in the literature. However, the keratinase also has a high collagen hydrolysis activity. The present invention screened a keratinase mutant with completely removed collagen hydrolysis activity by saturating the amino acid sites of the parent enzyme. Summary of the invention

[0004] The present invention provides a keratinase mutant without collagen hydrolysis activity. The mutant is obtained by mutating the methionine at position 221 of the keratinase with an amino acid sequence as shown in SEQ ID NO.1 to isoleucine, and is named KerZ1 / M221I.

[0005] In one embodiment of the present invention, the nucleotide sequence encoding the parent enzyme keratinase is shown as SEQ ID NO.2.

[0006] In one embodiment of the present invention, the amino acid sequence of the keratinase mutant KerZ1 / M221I is shown in SEQ ID NO.3.

[0007] In one embodiment of the present invention, the nucleotide sequence encoding the keratinase mutant KerZ1 / M221I is shown as SEQ ID NO.4.

[0008] The present invention also provides a gene encoding the keratinase mutant KerZ1 / M221I.

[0009] The invention also provides a recombinant vector carrying the gene.

[0010] In one embodiment of the present invention, pP43NMK is used as the expression vector.

[0011] In one embodiment of the present invention, the vector of the recombinant plasmid is pP43NMK plasmid.

[0012] The present invention also provides a recombinant cell expressing the keratinase mutant KerZ1 / M221I, or carrying the gene, or carrying the recombinant vector.

[0013] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as host cells.

[0014] In one embodiment of the present invention, the host cell is Bacillus subtilis WB600.

[0015] The invention provides a recombinant Bacillus subtilis. The recombinant Bacillus subtilis uses Bacillus subtilis WB600 as a host cell and a pP43NMK plasmid as an expression vector, and expresses a keratinase mutant KerZ1 / M221I without collagen hydrolysis activity.

[0016] The present invention also provides a method for removing the collagen hydrolyzing activity of keratinase, which comprises mutating the methionine at position 221 of the keratinase whose amino acid sequence is shown in SEQ ID NO.1 to isoleucine.

[0017] The present invention also provides a product of degraded keratin, wherein the degraded keratin contains the keratin mutant KerZ1 / M221I.

[0018] In one embodiment of the present invention, the product for degrading keratin includes: depilatory cream and leather depilatory agent.

[0019] The present invention also provides a method for degrading keratin. The method uses a substance containing keratin as a substrate and utilizes the keratinase mutant KerZ1 / M221I or the above recombinant cell or the above recombinant Bacillus subtilis to degrade keratin.

[0020] In one embodiment of the present invention, the keratin-containing substance includes hair, elastin, scales, and fibers.

[0021] In one embodiment of the present invention, the method for constructing the recombinant cell is to transfer the recombinant expression vector carrying the gene encoding the mutant enzyme into the host cell by electroporation or chemical transformation.

[0022] The present invention provides an application of the keratinase mutant KerZ1 / M221I, the gene, the expression vector or the host cell in animal husbandry, feed industry, leather industry, cosmetics industry and pharmaceutical industry.

[0023] The present invention also claims to protect the use of the above keratinase mutant KerZ1 / M221I, the above gene, the above expression vector or the above host cell in degrading skin, hair, casein, elastin, wool, nails, scales, fibers, and hair keratin.

[0024] Beneficial Effects

[0025] The present invention provides a keratinase mutant KerZ1 / M221I, and the result of the collagen hydrolysis activity measurement of the mutant shows that the keratinase mutant KerZ1 / M221I of the present invention has no collagen hydrolysis activity, so the keratinase mutant can avoid collagenase damage when applied. For example, in the leather industry, enzymatic dehairing is used. Since keratinase is a serine protease and has basically no substrate specificity, it is easy to indiscriminately hydrolyze collagen on the surface of leather during application, causing leather damage and reducing the quality of leather. However, the keratinase mutant KerZ1 / M221I of the present invention has no collagen hydrolysis activity and will not cause any damage to the leather. Therefore, the keratinase mutant KerZ1 / M221I without collagen activity provided by the present invention has more application value and potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Comparison of keratinase hydrolysis activity and collagen hydrolysis activity between different mutants and control bacteria.

[0027] Figure 2 The SDS-PAGE gel electrophoresis of the supernatant of the fermentation of the keratinase parent and the mutant KerZ1 / M221I in Bacillus subtilis; M represents the protein molecular weight standard, different lanes represent different keratins, and the arrow indicates the position of the target protein band. DETAILED DESCRIPTION

[0028] The Escherichia coli JM109 involved in the following examples was purchased from Beina Biotechnology; the pP43NMK plasmid involved in the following examples was purchased from Fenghui Biotechnology; the Bacillus subtilis WB600 involved in the following examples is recorded in the patent application text with publication number CN102492645A.

[0029] The culture medium involved in the following examples is as follows:

[0030] LB liquid medium: yeast powder 5g·L 1 , Tryptone 10 g·L 1 、NaCl 10g·L 1 .

[0031] LB solid medium: yeast powder 5g L 1 , Tryptone 10 g·L 1 、NaCl 10g·L 1 , agar powder 20g·L 1 .

[0032] Bacillus subtilis fermentation medium: peptone 20g·L 1 , Yeast powder 10g·L 1 , sucrose 20g·L 1 、KH2PO43g·L 1 、Na2HPO4 6g·L 1 、MgSO4 0.3g·L 1 .

[0033] The detection methods involved in the following embodiments are as follows:

[0034] Keratin hydrolysis activity assay method: Take 50 μL of appropriately diluted fermentation supernatant, add 150 μL of 50 mM Gly / NaOH solution as buffer and 100 μL of 2.5% water-soluble keratin (purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., product code: K0043) as substrate, mix well and react at 60°C for 20 min; add 200 μL 4% (w / v) trichloroacetic acid (TCA) to terminate the reaction, centrifuge at room temperature at 8000 r / min for 3 min. Take 200 μL of supernatant, add 1 mL 4% (w / v) Na2CO3 and 200 μL of Folin phenol reagent, mix well and color at 50°C for 10 min, use 0.5 cm quartz cuvette to measure the absorbance of the supernatant at 660 nm; the experimental group is 3 parallels, the blank control is to add the reaction terminator TCA before adding the substrate, and the rest of the operation is the same as above.

[0035] Enzyme activity unit definition: At 60℃, pH 10, OD 660 The amount of enzyme required for each increase of 0.001 is one enzyme activity unit (U).

[0036] Collagen hydrolysis activity assay method: 1 mg gelatin, 0.5 mL Tris-HCl as buffer solution, 0.1 mL appropriately diluted fermentation supernatant, react at 60°C for 30 min, add 0.5 mL 10% trichloroacetic acid to terminate the reaction. The amount of amino acids released by the reaction was determined by the ninhydrin colorimetric method, and the enzyme activity was calculated against the glycine standard curve; the experimental group was paralleled 3 times, and the blank control was to add the reaction terminator TCA before adding the substrate, and the rest of the operation was the same as above.

[0037] The enzyme activity unit is defined as: at 60°C and pH 7.5, the amount of enzyme that hydrolyzes collagen per minute to produce 1 μg of glycine is 1 enzyme activity unit (U).

[0038] Example 1: Construction of keratinase mutants

[0039] 1. Construction of mutants

[0040] (1) The construction of the pP43NMK-ker recombinant vector is described in US20210079371A1.

[0041] (2) Construction of mutants

[0042] The invention uses a keratinase with an amino acid sequence of SEQ ID NO.2 as a parent enzyme, and selects eight amino acid sites (threonine at position 33, glycine at position 62, tyrosine at position 208, proline at position 209, leucine at position 216, aspartic acid at position 217, threonine at position 219, and methionine at position 221) thereof for saturation mutation modification.

[0043] According to the sequence of keratinase (nucleotide sequence as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.1), mutation primers (as shown in Table 1) were designed respectively to perform site-directed saturation mutagenesis on the keratinase gene on the pP43NMK plasmid.

[0044] Table 1 Primers

[0045]

[0046] The PCR reaction system was as follows: PrimeSTAR Max Premix (2×) 25 μL, 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, template DNA 1 μL, and double distilled water was added to 50 μL;

[0047] The amplification conditions of PCR products were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 2 min, 30 cycles; and finally incubation at 72°C for 10 min.

[0048] The PCR amplification product was detected by 1% agarose gel electrophoresis. After confirming that the size of the amplification product was correct, 0.5 μL of methylated template digestion enzyme (DpnI) was added to 10 μL of the amplification product, mixed well, and reacted at 37°C for 1.5 hours. The amplification product treated with DpnI was transformed into the competent E. coli JM109, and the transformation product was spread on LB solid medium containing ampicillin resistance and cultured at 37°C for 8 to 10 hours. The single colonies on the LB solid were transferred to the shaking tube using LB liquid medium, and the plasmid was extracted after culturing at 37°C and 220rpm for 2 to 3 hours, and the recombinant plasmid containing different mutant genes was obtained.

[0049] Example 2: Construction of recombinant plasminogen activator and expression of keratinase

[0050] The specific steps are as follows:

[0051] (1) The recombinant plasmids obtained in Example 1 were transformed into competent Bacillus subtilis WB600, and the transformation products were spread on LB solid medium containing kanamycin resistance, and cultured at 37° C. for 10 to 12 hours to prepare recombinant strains. The single colonies grown on the plate medium were inoculated into a 96-deep-well plate containing LB liquid medium containing kanamycin resistance, and cultured at 37° C. and 220 rpm for 10 to 12 hours to prepare seed solutions;

[0052] (2) The seed solution obtained in step (1) was transferred to a 96-deep-well plate containing a Bacillus subtilis fermentation medium at a ratio of 5%, and cultured at 37° C. and 220 rpm for 24 h to obtain a fermentation broth containing each mutant enzyme.

[0053] (3) The fermentation broth was centrifuged at 5000 rpm for 10 min at 4°C to obtain the fermentation supernatant and the crude enzyme solution of each mutant enzyme.

[0054] (4) The crude enzyme solutions of the mutants obtained in step (3) were subjected to SDS-PAGE gel electrophoresis. The results were as follows: Figure 2 As shown (only the effects of the beneficial mutants of the present application are shown).

[0055] The results showed that the recombinant strain WB600 / pP43NMK-KerZ1 / M221I could produce keratinase with the same molecular weight and concentration as the original bacteria.

[0056] (5) The keratin hydrolysis activity and collagen hydrolysis activity of the crude enzyme solution of each mutant were tested respectively. The results are shown in Table 2 and Figure 1 As shown:

[0057] Table 2: Keratin hydrolysis activity and collagen hydrolysis activity in crude enzyme solution of each mutant

[0058]

[0059]

[0060] The results showed that the mutant KerZ1 / M221I completely eliminated the collagen hydrolysis activity while retaining 59235U / mL keratin hydrolysis activity. This characteristic of the mutant is conducive to its application in depilatory creams and enzymatic leather making.

[0061] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A keratinase mutant without collagen hydrolysis activity, characterized in that: The mutant is a mutant in which the methionine at position 221 of the keratinase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to isoleucine.

2. A gene encoding the mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that pP43NMK was used as the expression vector.

5. A recombinant cell expressing the mutant according to claim 1, or carrying the gene according to claim 2, or carrying the recombinant vector according to claim 3 or 4.

6. The recombinant cell according to claim 5, characterized in that The recombinant cell uses bacteria or fungi as host cells.

7. A method for removing the collagen hydrolyzing activity of keratinase, characterized in that: The method comprises the following steps: mutating the methionine at position 221 of the keratinase of the amino acid sequence as shown in SEQ ID NO. 1 into isoleucine.

8. A product for degrading keratin, characterized in that: The keratin-degrading product contains the keratin mutant according to claim 1.

9. A method for degrading keratin, characterized in that: The mutant according to claim 1 or the recombinant cell according to claim 5 or 6 is added to a reaction system containing keratin to degrade keratin.

10. The method according to claim 9, characterized in that The keratin-containing reaction system contains substances including hair, elastin, scales or fibers.

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