A keratinase variant with enhanced hydrolysis ability by fusing substrate binding domain

Abstract

The present invention discloses a keratinase variant that improves hydrolysis ability by fusing a substrate binding domain, and belongs to the field of genetic engineering and enzyme engineering technology. The present invention uses PCR and Gibson connection technology to successfully fuse and express a substrate binding domain from Trichoderma reesei at the C-terminus of keratinase KerZ1 through a connecting peptide to obtain a keratinase variant KerZ1 / CBM‑L8. The enzyme activity assay results show that the enzyme activity of the keratinase variant KerZ1 / CBM‑L8 is only 41.5% of that of KerZ1, but the feather hydrolysis experiment results show that the average enzyme activity of the keratinase variant KerZ1 / CBM‑L8 is 2.71 times that of KerZ1, which has more application value and potential.

Description

Technical Field

[0001] The invention relates to a keratinase variant with improved hydrolysis ability by fusing a substrate binding functional domain, and belongs to the technical field of genetic engineering and enzyme engineering. Background Art

[0002] Keratinase is a type of hydrolase that can catalyze the decomposition of keratin, and is widely used in feed, cosmetics, pharmaceuticals, leather and other industries. However, feathers, wool and the like are all highly hydrophobic substrates, and the binding ability of the enzyme and the substrate is particularly important during the hydrolysis process. Therefore, improving the binding ability of keratinase and the substrate is of great significance to improving the hydrolysis ability of keratinase. In order to improve the hydrolysis ability of keratinase, the present invention improves the collision probability of keratinase and the substrate by fusing the substrate binding functional domain and thus improves its hydrolysis ability.

[0003] Although the inventor's research group has previously constructed a recombinant keratinase strain that can ferment a keratinase product with a keratin hydrolysis activity of more than 426.60 KU / mL in a 15L fermenter, which is the highest level of recombinant keratinase expression reported in the literature, this keratinase did not show significant advantages in the application experiment of feather hydrolysis.

[0004] In practical industrial applications, keratinase, which greatly improves the application value of enzyme preparations in the recycling of keratin waste such as feather hydrolysis, has become a research hotspot. Summary of the invention

[0005] The object of the present invention is to provide a keratinase variant with improved hydrolysis ability by fusing a substrate binding functional domain.

[0006] The present invention provides a keratinase variant with improved hydrolysis ability by fusing a substrate binding domain. The variant is obtained by fusion expression of a keratinase with an amino acid sequence as shown in SEQ ID NO.1 and a substrate binding domain with an amino acid sequence as shown in SEQ ID NO.3, and is named KerZ1 / CBM-L8.

[0007] In one embodiment of the present invention, the keratinase variant is obtained by fusion expression of the keratinase with an amino acid sequence as shown in SEQ ID NO.1 and the substrate binding domain with an amino acid sequence as shown in SEQ ID NO.3 via a connecting peptide GGGGSGGGGSGGGGS.

[0008] In one embodiment of the present invention, the nucleotide sequence encoding the substrate binding domain is shown as SEQ ID NO.4.

[0009] In one embodiment of the present invention, the nucleotide sequence encoding the parent enzyme keratinase is shown as SEQ ID NO.2.

[0010] In one embodiment of the present invention, the amino acid sequence of the keratinase variant KerZ1 / CBM-L8 is shown in SEQ ID NO.9.

[0011] In one embodiment of the present invention, the nucleotide sequence encoding the keratinase variant KerZ1 / CBM-L8 is shown as SEQ ID NO.10.

[0012] The present invention also provides a gene encoding the keratinase variant KerZ1 / CBM-L8.

[0013] The invention also provides a recombinant vector carrying the gene.

[0014] In one embodiment of the present invention, the recombinant vector uses pP43NMK as an expression vector.

[0015] The present invention also provides a recombinant cell expressing the keratinase variant KerZ1 / CBM-L8, or carrying the gene, or carrying the recombinant vector.

[0016] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as host cells.

[0017] In one embodiment of the present invention, the host cell is Bacillus subtilis WB600.

[0018] The present invention also provides a method for improving the protein hydrolysis ability of keratinase, wherein the method comprises fusing and expressing the keratinase having an amino acid sequence as shown in SEQ ID NO.1 and the substrate binding domain having an amino acid sequence as shown in SEQ ID NO.3 through a connecting peptide GGGGSGGGGSGGGGS.

[0019] The present invention also provides a product for degrading feather keratin, wherein the product contains the keratinase variant KerZ1 / CBM-L8 or the recombinant cell.

[0020] In one embodiment of the present invention, the product for degrading feather keratin includes, but is not limited to, a depilatory cream, a feather hydrolase preparation or a leather depilatory.

[0021] The present invention also provides a method for degrading feather keratin. The method uses feathers as substrates and utilizes the keratinase variant KerZ1 / CBM-L8 to degrade substances containing keratin.

[0022] In one embodiment of the present invention, the keratin-containing substance includes poultry feathers, animal fur, and the like.

[0023] The present invention also provides a method for constructing the above-mentioned recombinant cell, wherein the method for constructing the recombinant cell is to transfer a recombinant expression vector carrying a gene encoding the mutant enzyme into a host cell by electroporation or chemical transformation.

[0024] The present invention provides an application of the mutant, gene, expression vector or host cell in animal husbandry, feed industry and leather industry.

[0025] The present invention also claims to protect the use of the mutant, gene, expression vector or host cell in degrading feathers, hair, casein, elastin, wool, nails, and scale keratin.

[0026] Beneficial Effects

[0027] The present invention provides a keratinase variant KerZ1 / CBM-L8, and the feather hydrolysis assay results of the variant show that the keratinase variant KerZ1 / CBM-L8 of the present invention has a higher feather hydrolysis efficiency than the original enzyme KerZ1, so the keratinase variant can greatly improve the application value of the enzyme preparation in the recycling of keratin waste such as feather hydrolysis. Although the variant has a lower enzyme activity, the feather hydrolysis efficiency is greatly improved, so the method of fusing the substrate binding functional domain can improve the binding ability of the keratinase to the hydrophobic substrate and thus improve its hydrolysis efficiency. Therefore, the keratinase variant KerZ1 / CBM-L8 provided by the present invention by fusing the substrate binding functional domain has more application value and potential.

[0028] (1) The present invention uses PCR and Gibson ligation technology to successfully fuse and express the substrate binding functional domain from Trichoderma reesei at the C-terminus of keratinase KerZ1 through a connecting peptide.

[0029] (2) Although the enzyme activity of KerZ1 / CBM-L8 in shake flask fermentation was only 41.5% of that of KerZ1, the results of the feather hydrolysis experiment showed that the average enzyme activity of the keratinase variant KerZ1 / CBM-L8 was 2.71 times that of KerZ1 in feather hydrolysis, which has greater application value and potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Preliminary screening of keratinase variants for different types of linker peptide combinations.

[0031] Figure 2 Secondary screening of keratinase variants for different types of linker peptide combinations.

[0032] Figure 3The figure shows the SDS-PAGE gel electrophoresis of keratinase KerZ1 and keratinase KerZ1 / CBM; wherein M represents the protein molecular weight standard, different lanes represent different keratinase, and arrows indicate the position of the target protein band.

[0033] Figure 4 The diagram shows the hydrolysis effects of different keratinase enzymes. DETAILED DESCRIPTION

[0034] 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.

[0035] The culture medium involved in the following examples is as follows:

[0036] LB liquid medium: yeast powder 5g·L -1 , Tryptone 10 g·L -1 、NaCl 10g·L -1 .

[0037] LB solid medium: yeast powder 5g L -1 , Tryptone 10 g·L -1 、NaCl 10g·L -1 , agar powder 20g·L -1 .

[0038] Fermentation medium: peptone 20 g·L -1 , Yeast powder 10g·L -1 , sucrose 20g·L -1 、KH2PO4 3g·L -1 、Na2HPO4 6g·L -1 、MgSO4 0.3g·L -1 .

[0039] The detection methods involved in the following embodiments are as follows:

[0040] Keratin hydrolysis activity assay method:

[0041] Take 50 μL of appropriately diluted fermentation supernatant, add 150 μL of 50 mM Gly / NaOH solution as a buffer and 100 μL of 2.5% water-soluble keratin (purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., product code: K0043) as a substrate, mix well and react at 60°C for 20 min; add 200 μL of 4% (w / v) trichloroacetic acid (TCA) to terminate the reaction, and centrifuge at 8000 r / min at room temperature for 3 min.

[0042] 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, and use a 0.5 cm quartz cuvette to measure the absorbance of the supernatant at 660 nm; the experimental group has 3 parallels, the blank control is to add the reaction terminator TCA before adding the substrate, and the rest of the operations are the same as above.

[0043] 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).

[0044] Feather hydrolysis efficiency determination method:

[0045] Take 0.2g chicken feathers, 20mL fermentation supernatant, 0.2mg sodium sulfite, keep warm at 60℃ for 4h, filter with filter paper of known mass to get the unhydrolyzed feather residue, and dry at 105℃ to constant weight. The amount of hydrolyzed feathers is calculated by the mass of the feather residues, and then the feather hydrolysis capacity of the average enzyme activity is calculated.

[0046] The calculation formula for feather hydrolysis efficiency is: Feather hydrolysis efficiency (g / U) = amount of hydrolyzed feather (g) / enzyme activity (U).

[0047] Example 1: Construction of keratinase variants

[0048] 1. Construction of fusion expression plasmid

[0049] (1) The construction method of the pP43NMK-ker recombinant vector is described in the patent publication number US20210079371A1, and the nucleotide sequence of the keratinase gene is shown in SEQ ID NO.2.

[0050] (2) Construction of pP43NMK-ker / CBM recombinant vector

[0051] The specific steps are as follows:

[0052] The gene of the substrate binding domain CBM from Trichoderma reesei (nucleotide sequence such as SEQ ID NO.4) was chemically synthesized, and the obtained gene was connected to the pP43NMK-ker plasmid using Gibson ligation technology (the substrate binding domain CBM was connected to the C-terminus of keratinase KerZ1) to obtain a ligation product.

[0053] The Gibson ligation reaction system is: 5×Reaction buffer 100μL, T5 exonuclease (10U / μL) 0.31μL, Phusion polymerase (2U / μL) 6.25μL, Taq ligase (40U / μL) 50μL, H2O 218.44μL.

[0054] The ligation product was transformed into E. coli JM109, and the transformation product was spread on a plate supplemented with ampicillin (final concentration 100 mg·L 1 ) in LB solid medium, cultured at 37°C for 8-10 h, and then supplemented with ampicillin (final concentration 100 mg·L 1 ) LB liquid medium, wash the single colony on the LB solid culture plate and transfer it to a 15mL shaking tube, culture it at 37℃, 220r / min for 3-4h, extract the plasmid, and send it to the company for sequencing verification. If the verification is correct, the recombinant plasmid pP43NMK-ker / CBM is obtained (electrophoresis diagram as shown in Figure 3 shown).

[0055] (3) Construction of keratinase recombinant plasmids with different types of linker peptide combinations

[0056] Primers (as shown in Table 2) were designed according to the sequences of different types of connecting peptides (as shown in Table 1), and PCR amplification was performed on the recombinant plasmid pP43NMK-ker / CBM carrying the substrate binding functional domain to amplify the connecting peptide sequence.

[0057] Table 1: Amino acid sequences of different types of linker peptides

[0058]

[0059]

[0060] Table 2: Primers

[0061]

[0062] 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;

[0063] 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.

[0064] The PCR amplification product was detected by 1% agarose gel electrophoresis. After confirming that the amplification product was of the correct size, the amplification product was transformed into Escherichia coli JM109 and the transformation product was spread on a plate supplemented with ampicillin (final concentration 100 mg / L 1 ) on LB solid medium and cultured at 37°C for 8-10 h. The single colony grown on the plate was inoculated into a plate supplemented with ampicillin (final concentration 100 mg·L 1 ) in LB liquid culture medium, cultured at 37°C and 220r / min for 3-4h, then the plasmid was extracted and sent to the company for sequencing verification. If the verification was correct, the keratinase recombinant plasmids pP43NMK-KerZ1 / CBM-L1, pP43NMK-KerZ1 / CBM-L2, pP43NMK-KerZ1 / CBM-L3, pP43NMK-KerZ1 / CBM-L4, pP43NMK-KerZ1 / CBM-L5, pP43NMK-KerZ1 / CBM-L6 with different types of connecting peptide combinations were obtained. erZ1 / CBM-L6, pP43NMK-KerZ1 / CBM-L7, pP43NMK-KerZ1 / CBM-L8, pP43NMK-KerZ1 / CBM-L9, pP43NMK-KerZ1 / CBM -L10, pP43NMK-KerZ1 / CBM-L11, pP43NMK-KerZ1 / CBM-L12, pP43NMK-KerZ1 / CBM-L13, pP43NMK-KerZ1 / CBM-L14.

[0065] Example 2: Construction of recombinant plasminogen activator and expression of keratinase

[0066] The specific steps are as follows:

[0067] (1) The recombinant plasmids pP43NMK-KerZ1 / CBM-L1, pP43NMK-KerZ1 / CBM-L2, pP43NMK-KerZ1 / CBM-L3, pP43NMK-KerZ1 / CBM-L4, pP43NMK-KerZ1 / CBM-L5, pP43NMK-KerZ1 / CBM-L6, pP43NMK-KerZ1 / CBM-L7, and pP43NMK-KerZ1 / CBM-L8 obtained in Example 1 were added to the culture medium. NMK-KerZ1 / CBM-L8, pP43NMK-KerZ1 / CBM-L9, pP43NMK-KerZ1 / CBM-L10, pP43NMK-KerZ1 / CBM-L11, pP43NMK-KerZ1 / CBM-L12, pP43NMK-KerZ1 / CBM-L13, and pP43NMK-KerZ1 / CBM-L14 were transformed into Bacillus subtilis. subtilis) WB600 competent cell, and the transformation products were respectively spread on LB solid medium containing kanamycin resistance, and cultured at 37°C for 10-12 hours to prepare recombinant strains KerZ1 / CBM-L1, KerZ1 / CBM-L2, KerZ1 / CBM-L3, KerZ1 / CBM-L4, KerZ1 / CBM-L5, KerZ1 / CBM-L6, KerZ1 / CBM-L7, KerZ1 / CBM-L8, KerZ1 / CBM-L9, KerZ1 / CBM-L10, KerZ1 / CBM-L11, KerZ1 / CBM-L12, KerZ1 / CBM-L13, and KerZ1 / CBM-L14, respectively.

[0068] The single colonies grown on the plate culture medium were inoculated into LB liquid culture medium containing kanamycin, and cultured at 37° C. and 220 rpm for 10 to 12 hours to prepare seed solutions;

[0069] (2) The seed solution obtained in step (1) was transferred to the fermentation medium at a ratio of 5%, and cultured at 37° C. and 220 rpm for 24 h to obtain a fermentation liquid.

[0070] (3) The fermentation broth was centrifuged at 5000 rpm for 10 min at 4°C to obtain the fermentation supernatant.

[0071] According to the above method, pP43NMK-ker was transformed into Bacillus subtilis WB600 to prepare the recombinant strain KerZ1.

[0072] (4) The keratinase activity in the supernatant of step (3) was detected respectively. The results are shown in Table 3.

[0073] Table 3: Keratinase activity expressed by strains containing different linker peptides

[0074]

[0075] Example 3: Determination of Feather Hydrolysis Efficiency of Keratinase Variants

[0076] The specific steps are as follows:

[0077] 1. Preliminary screening of feather hydrolysis ability:

[0078] (1) The keratinase variants obtained in Example 2 were divided into three groups according to the length of the connecting peptide (respectively: n=1 group, n=2 group, n=3 group) for hydrolysis experiments:

[0079] The fermentation supernatant obtained in Example 2 was diluted to the same keratinase activity, and 10 mL of the enzyme solution with the same enzyme activity was added to a 50 mL centrifuge tube containing 0.1 g chicken feathers and 0.1 g anhydrous sodium sulfite to obtain a reaction system;

[0080] Wherein: n = 1 group (enzyme activity added: 58810U): KerZ1 / CBM-L1, KerZ1 / CBM-L2;

[0081] n = 2 groups (enzyme activity added: 58550 U): KerZ1 / CBM-L3, KerZ1 / CBM-L4, KerZ1 / CBM-L5, KerZ1 / CBM-L6;

[0082] n=3 groups (enzyme activity added: 57477U): KerZ1 / CBM-L7, KerZ1 / CBM-L8, KerZ1 / CBM-L9, KerZ1 / CBM-L10, KerZ1 / CBM-L11, KerZ1 / CBM-L12, KerZ1 / CBM-L13, KerZ1 / CBM-L14.

[0083] (2) The reaction system was placed at 60°C for 4 hours, during which samples were taken every half an hour. Each time, 20 μL of the reaction solution was added to 980 μL of deionized water to terminate the reaction and dilute 50 times, mixed, and centrifuged at 8000 r / min for 3 minutes. 200 μL of the supernatant was taken, 1 mL of 4% (w / v) Na2CO3 and 200 μL of Folin phenol reagent were added, mixed, and color was developed at 50°C for 10 minutes, and the absorbance was measured at 660 nm using a 0.5 cm quartz cuvette;

[0084] There were 3 parallel experimental groups, the blank control was the original fermentation supernatant (KerZ1), and the rest of the operations were the same as above.

[0085] The results are as follows Figure 1As shown, the absorbance values ​​of the keratinase variants n=1 and n=2 after color development were only slightly improved compared with the control, and the maximum absorbance value after 4 hours of reaction was only 1.09 times that of the control, while the absorbance value of the n=3 group was greatly improved compared with the control, up to 1.34 times that of the control (KerZ1 / CBM-L7), so the variants of the n=3 group were screened a second time.

[0086] 2. Secondary screening of feather hydrolysis ability:

[0087] (1) Take 0.2 g chicken feathers, 20 mL of fermentation supernatant of n=3 groups of keratinase variants, and 0.2 mg of sodium sulfite, react at 60°C for 4 h, filter with filter paper of known mass to obtain unhydrolyzed feather residues, and dry at 105°C to constant weight. The amount of hydrolyzed feathers is calculated by the mass of the feather residues, and then the average enzyme activity is calculated to calculate the feather hydrolysis capacity. The hydrolysis effect is as follows: Figure 4 shown.

[0088] Calculation method of feather hydrolysis capacity:

[0089] Feather hydrolysis capacity (g / U) = hydrolyzed feather mass (g) / initial fermentation supernatant enzyme activity (U), the results are shown in Table 4 and Figure 2 shown.

[0090] Table 4: KerZ1 / CBM Linker rescreening

[0091]

[0092]

[0093] The results showed that the feather hydrolysis ability of the keratinase variants was significantly improved compared with the control, among which the feather hydrolysis ability of KerZ1 / CBM-L8 per unit enzyme activity was 2.71 times that of KerZ1, indicating that by fusing the substrate binding functional domain, the ability of keratinase to hydrolyze feathers has been greatly improved, and it has great application potential.

[0094] Example 4: Screening of keratinase variants fused with substrate binding domains from different sources

[0095] (1) According to the screening results of Example 3, when the linker is L8, that is, the amino acid sequence is GGGGSGGGGSGGGGS, the feather hydrolysis ability of the keratinase variant is the best. Therefore, L8 is used as the best linker to screen the substrate binding domain CBM1 derived from Aspergillus niger and the substrate binding domain CBM2 derived from Aspergillus terreus.

[0096] (2) Construction of recombinant plasmids of keratinase with substrate binding domains from different sources

[0097] According to the steps of Examples 1 and 2, KerZ1 / CBM1-L8 and KerZ1 / CBM2-L8 were constructed and expressed.

[0098] The specific implementation method is the same as steps (1) to (2) of Example 1 and Example 2, except that the genes of the substrate binding domain CBM derived from Trichoderma reesei are adjusted as follows: the substrate binding domain CBM1 gene derived from Aspergillus niger (the amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6) and the substrate binding domain CBM2 gene derived from Aspergillus terreus (the amino acid sequence is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8) are chemically synthesized, and the obtained genes are connected to the pP43NMK-ker plasmid using the Gibson connection technology (the substrate binding domain CBM is connected to the C-terminus of keratinase KerZ1) to prepare recombinant plasmids pP43NMK-ker / CBM1-L8 and pP43NMK-ker / CBM2-L8, and the primers involved are shown in Table 5.

[0099] Table 5 Primers

[0100]

[0101]

[0102] According to the above method, crude enzyme solution containing KerZ1 / CBM-L8, crude enzyme solution containing KerZ1 / CBM1-L8, and crude enzyme solution containing KerZ1 / CBM2-L8 were prepared respectively.

[0103] (2) Feather hydrolysis experiments were conducted on KerZ1 / CBM-L8, KerZ1 / CBM1-L8, and KerZ1 / CBM2-L8; the method was as follows: 0.2 g of chicken feathers were taken, and 20 mL of crude enzyme solution of KerZ1, crude enzyme solution of KerZ1 / CBM-L8, crude enzyme solution of KerZ1 / CBM1-L8, and crude enzyme solution of KerZ1 / CBM2-L8 with enzyme activities of 128565 U / mL, 49245 U / mL, 81560 U / mL, and 81750 U / mL were added to the feathers, and 0.2 mg of sodium sulfite was added to the feathers. After reacting at 60°C for 4 hours, the unhydrolyzed feather residues were filtered with filter paper of known mass, and dried at 105°C to constant weight. The amount of hydrolyzed feathers was calculated by the mass of the feather residues, and then the feather hydrolysis capacity of the average enzyme activity was calculated.

[0104] Calculation method of feather hydrolysis capacity:

[0105] Feather hydrolysis capacity (g / U) = hydrolyzed feather mass (g) / initial fermentation supernatant enzyme activity (U). The results are shown in Table 6.

[0106] Table 6: Hydrolysis capacity of different keratinase enzymes

[0107]

[0108] The results showed that the present invention successfully fused and expressed the substrate binding domain from Trichoderma reesei at the C-terminus of keratinase KerZ1 through a connecting peptide using PCR and Gibson ligation technology, and the obtained keratinase variant KerZ1 / CBM-L8 had the highest hydrolysis rate.

[0109] 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 variant, characterized in that The keratinase variant is obtained by fusion expression of the keratinase with an amino acid sequence as shown in SEQ ID NO.1 and the substrate binding domain with an amino acid sequence as shown in SEQ ID NO.3 via a connecting peptide GGGGSGGGGSGGGGS.

2. A gene encoding the keratinase variant 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 The recombinant vector uses pP43NMK as an expression vector.

5. A recombinant cell expressing the protease variant 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 improving the ability of keratinase to hydrolyze protein, characterized in that: The method comprises the following steps: fusing the keratinase having an amino acid sequence as shown in SEQ ID NO.1 and the substrate binding domain having an amino acid sequence as shown in SEQ ID NO.3 through a connecting peptide GGGGSGGGGSGGGGS.

8. A product for degrading keratin, characterized in that: The product contains the keratinase variant according to claim 1 or the recombinant cell according to claim 6 or 7.

9. A method for degrading keratin, characterized in that: The method uses a substance containing keratin as a substrate and utilizes the keratinase variant according to claim 1 to degrade keratin.

10. The method according to claim 9, characterized in that The keratin-containing material includes feathers, elastin, scales or fibers.

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