NOVEL THERMOPHILE-DERIVED KERATINASE AND USE THEREOF

Abstract

The present invention relates to a novel thermophile-derived keratinase having keratin decomposition ability. Further, the present invention relates to a polynucleotide encoding the keratinase, a recombinant vector containing the same, host cells transformed by using the recombinant vector, and a method for preparing a keratinase including a step of culturing the host cells. Further, the present invention relates to a composition for decomposing keratin containing the enzyme; and a method for decomposing keratin by using the same. Further, the present invention relates to a keratin decomposed product decomposed by the enzyme.

The keratinase according to the present invention rapidly and effectively decomposes hardly-decomposable keratin, and thus it is expected that the keratinase can be used for the effective treatment and the high value-added resource recovery of agricultural and livestock waste, which causes environmental problems (for example, a novel material for enzyme cosmetics), and can be used in an innovative enzymatic bioconversion technique utilizing various decomposition enzyme groups.

Claims

1. Thermophile-derived keratin degrading enzymes which contain at least more than one selected from a group consisting of: a cysteine desulfurase (CDS); a thermostable carboxypeptidase 1 (CBP); a thioredoxin-disulfide reductase (CDR); and an iron-sulfur assembly scaffold protein (SufE).

2. The thermophile-derived keratin degrading enzymes of claim 1, wherein the cysteine desulfurase (CDS) consists of an amino acid sequence of SEQ ID NO: 1; the thermostable carboxypeptidase 1 (CBP) consists of an amino acid sequence of SEQ ID NO: 3; The thioredoxin-disulfide reductase (CDR) consists of an amino acid sequence of SEQ ID NO: 5; and the iron-sulfur assembly scaffold protein (SufE) consists of an amino acid sequence of SEQ ID NO: 7.

3. The thermophile-derived keratin degrading enzymes of claim 1, wherein the CDS, CBP, CDR, and SufE are encoded by a polynucleotide consisting of a base sequence of SEQ ID NO: 2; a base sequence of SEQ ID NO: 4; a base sequence of SEQ ID NO: 6; and a base sequence of SEQ ID NO: 8, respectively.

4. The thermophile-derived keratin degrading enzymes of claim 1, wherein the thermophilic bacteria are thermotogales order.

5. The thermophile-derived keratin degrading enzymes of claim 4, wherein the thermotogales order includes Caldotoga, Mesotoga, Thermopallium, Thermotoga, Fervidobacterium, Thermosipho, Kosmotoga, Thermococcoides, Mariniloga, Geotoga, or Petrotoga genus.

6. Polynucleotide encoding at least one keratin degrading enzymes selected from a group consisting of: a cysteine desulfurase (CDS) consisting of an amino acid sequence of SEQ ID NO: 1; a thermostable carboxypeptidase 1 (CBP) consisting of an amino acid sequence of SEQ ID NO: 3; a thioredoxin-disulfide reductase (CDR) consisting of an amino acid sequence of SEQ ID NO: 5: and an iron-sulfur assembly scaffold protein (SufE) consisting of an amino acid sequence of SEQ ID NO: 7.

7. The polynucleotide of claim 6, wherein the polynucleotides encoding the CDS, CBP, CDR, and SufE are polynucleotides consisting of: a base sequence of SEQ ID NO: 2; a base sequence of SEQ ID NO: 4; a base sequence of SEQ ID NO: 6; and a base sequence of SEQ ID NO: 8, respectively.

8. A recombinant vector comprising the polynucleotide of claim 7.

9. Host cells transformed by the recombinant vector of claim 8.

10. A method for preparing keratin degrading enzymes, comprising a step of culturing the host cells of claim 9.

11. A composition for keratin decomposition comprising at least one thermophile-derived enzyme selected from a group consisting of: a cysteine desulfurase (CDS); a thermostable carboxypeptidase 1 (CBP); a thioredoxin-disulfide reductase (CDR); and an iron-sulfur assembly scaffold protein (SufE).

12. The composition for keratin decomposition of claim 11, further comprising: a thermophile-derived cell extract.

13. A method for keratin decomposition comprising, a step of treating keratin in at least one thermophile-derived enzyme selected from a group consisting of: a cysteine desulfurase (CDS); a thermostable carboxypeptidase 1 (CBP); a thioredoxin-disulfide reductase (CDR); and an iron-sulfur assembly scaffold protein (SufE).

14. The method for keratin decomposition of claim 13, wherein the keratin is derived from poultry feathers.

15. A keratin byproducts decomposed by the method for keratin decomposition of claim 13.

Description

DESCRIPTION OF DRAWINGS

[0078] FIG. 1 illustrates a result of analyzing genome information of F. islandicum AW-1 having keratin decomposition ability by using a RAST server.

[0079] FIG. 2 illustrates a cysteine desulfurase (CDS) screened as a keratin degrading enzymes group based on a result of comparing and analyzing protein function-based genomes.

[0080] FIG. 3 illustrates a thermostable carboxypeptidase 1 (CBP) screened as a keratin degrading enzymes group based on a result of comparing and analyzing amino acid sequence-based genomes.

[0081] FIG. 4 illustrates a thioredoxin-disulfide reductase (CDR) screened as a keratinase group based on a result of comparing and analyzing amino acid sequence-based genomes.

[0082] FIG. 5 illustrates an iron-sulfur assembly scaffold protein (SufE) screened as a keratinase group based on a result of comparing and analyzing amino acid sequence-based genomes.

[0083] FIG. 6 illustrates a schematic diagram of expression vectors of key proteins expected as a keratinase group.

[0084] FIG. 7 illustrates a biophysicochemical characteristic of a cysteine desulfurase (CDS) recombinant protein expected as a keratinase of F. islandicum AW-1.

[0085] FIG. 8 illustrates a biophysicochemical characteristic of a thermostable carboxypeptidase 1 (CBP) recombinant protein expected as a keratinase of F. islandicum AW-1.

[0086] FIG. 9 is a schematic diagram illustrating a method of extracting a whole cell extract for obtaining an enzyme group having keratin decomposition ability from F. islandicum AW-1.

[0087] FIG. 10 illustrates a result of verifying decomposition of a chicken feather wastes by using a whole cell extract and a recombinant protein of F. islandicum AW-1 having keratin decomposition ability.

[0088] FIG. 11 illustrates quantitative analysis of free amino acids produced by a decomposition experiment of the chicken feather wastes of FIG. 10.

[0089] FIG. 12 is a schematic diagram illustrating a method for extracting a whole cell extract extract from colonies in which F. islandicum AW-1 having keratin decomposition ability is anaerobically cultured in a medium added with 0.5% (v/v) glucose as a unique carbon source.

[0090] FIG. 13 illustrates a merge treatment effect for decomposition of the chicken feather wastes by selectively adding cysteine desulfurase (CDS) and iron-sulfur assembly scaffold protein (SufE) recombinant proteins in the whole cell extract of F. islandicum AW-1.

[0091] FIG. 14 is a graph illustrating a result of amino acids quantitative analysis of the decomposition effect of the chicken feather wastes by FIG. 13.

MODES OF THE INVENTION

[0092] Hereinafter, the present invention will be described in more detail through Examples. However, these Examples are to exemplify the present invention and the scope of the present invention is not limited to these Examples.

Experimental Example 1: Genome Analysis of Fervidobacterium Islandicum AW-1

[0093] Colonies were collected from a culture broth of F. islandicum AW-1 having keratin decomposition ability to extract a genomic DNA by using a genomic DNA extraction kit (Solgent), and then obtain a genome base sequence by using a single molecule real-time (SNRT) sequencing platform (PacBio RS II system).

[0094] The genome information was analyzed by using a hierarchical genome-assembly process (RS-HGAP) assembly protocol and a RAST server (http://rast.nmpdr.org/) in an SNRT analysis pipeline v.2.2.0. As a result, it was verified that a genome DNA size of F. islandicum AW-1 was 2.35 Mb and had 2,259 coding genes.

Experimental Example 2: Analysis of Comparing Protein Function-Based and Amino Acid Sequence-Based Genomes Using Genome Information of Fervidobacterium islandicum AW-1

[0095] A target enzyme group involved in keratin decomposition was selected by comparing and analyzing genome information of F. islandicum AW-1. To this end, proteins expected to be in the keratinase group, cysteine desulfurase (CDS), thermostable carboxypeptidase 1 (CBP), and thioredoxin-disulfide reductase (CDR) were selected by directly comparing and analyzing genome information with F. islandicum AW-1 having excellent keratin decomposition ability and strains corresponding to the same genus.

[0096] Particularly, in the case of the CDS, genomes of fervidobacterium islandicum AW-1 as a chicken feather wastes decomposition strain and F. nodosum Rt17-B1 as a strain without chicken feather wastes decomposition ability were compared and analyzed based on a protein function by using the RAST server (http://rast.nmpdr.org/). The result is illustrated in FIG. 2.

[0097] As illustrated in FIG. 2, cysteine desulfurase (CDS) which was present only in F. islandicum AW-1 and expected to be involved in decomposition of the chicken feather wastes was screened. The amino acid sequence information was designated as SEQ ID NO: 1 and the base sequence information was designated as SEQ ID NO: 2.

[0098] In the case of the thermostable carboxypeptidase 1 (CBP), comparison and analysis of amino acid sequence identities between proteases of F. islandicum AW-1 and proteases of F. nodosum Rt17-B1 as a strain without chicken feather wastes decomposition ability were performed by using a NCBI website-based blastp (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=b1asthome) program. The result was illustrated in FIG. 3.

[0099] As illustrated in FIG. 3, a protease thermostable carboxypeptidase 1 (CBP) which was expected to be involved in decomposition of the chicken feather wastes having low identity with the F. nodosum Rt17-B1 was finally screened.

[0100] The amino acid sequence information was designated as SEQ ID NO: 3 and the base sequence information was designated as SEQ ID NO: 4.

[0101] In the case of the thioredoxin-disulfide reductase (CDR), as a result of comparing and analyzing a genome with a F. pennivorans strain, which was known to have keratin decomposition ability in addition to the F. islandicum AW-1 by using a RAST server and a Bioedit analysis program, thioredoxin-disulfide reductase which had the highest amino acid identity and was expected to be involved in decomposition of a chicken feather wastes was screened as illustrated in FIG. 4.

[0102] The amino acid sequence information was designated as SEQ ID NO: 5 and the base sequence information was designated as SEQ ID NO: 6.

[0103] In the case of the iron-sulfur assembly scaffold protein (SufE), it was expected that a complex with the CDS was formed to increase keratin decomposition ability. As a result, an iron-sulfur assembly scaffold protein which was verified as present in a downstream of the CDS in a Suf operon through the RAST server was screened as illustrated in FIG. 5.

[0104] The amino acid sequence information was designated as SEQ ID NO: 7 and the base sequence information was designated as SEQ ID NO: 8.

Experimental Example 3: Preparation of Expression Vectors of CDS, CBP, and DSR Proteins and E. coli Transformants

[0105] Expression vectors of proteins which were expected as a keratinase group screened by comparing and analyzing protein function-based and amino acid sequence-based genomes of F. islandicum AW-1 was prepared.

TABLE-US-00001 TABLE 1 Sequence Primer Primer base No. No. Primer name sequence (5′-3′)  9 A F.aw1_cds_NdeI F CATATGCGCTCAACGG TGTTCTC 10 B F.aw1_cds_XhoI R CTCGAGTCATTCGAAC CACCTCC 11 C F.aw1_cbp_NdeI F CATATGGAAGAACTAA AAAGCTATTACAAACG 12 D F.aw1_cbp_XhoI R CTCGAGTTAAAGCTCT ATCTCATACACTTTG 13 E F.aw1_dsr_NdeI F CATATGAGCGGATTTG AATTCGACA 14 F F.aw1_dsr_XhoI R CTCGAGTTAGAAGTAT TTCTTTGCAGCG 15 G F.aw1_sufE_NdeI F GCGCATATGATATACT CTGAATTCATAATGG 16 H F.aw1_sufE_XhoI R CTCGAGTAATTCATTC TTTAAAGCAATCTCC

[0106] As shown in Table 1, in order to prepare expression vectors CDS, CBP, DSR, and SufE, first, forward primers and reverse primers containing respective restriction enzymes sequence (NdeI or XhoI) were used in pairs in PCR according to the preparation of the expression vectors.

[0107] A genomic DNA of the fervidobacterium islandicum AW-1 strain was used as a template for the amplification of each gene by the PCR method, and the reaction was performed at 98° C. for 5 min using Primestar HS DNA polymerase from Takara Corporation, performed at 98° C. for 30 s, 55° C. for 15 s, and 72° C. for 1 min by 30 cycles, and then performed at 72° C. for 10 min.

[0108] The amplified PCR product was electrophoresed and isolated and purified by using a gel extraction kit (QIAGEN), cloned by using a pTOP Blunt V2 cloning kit (Enzynomics), and then whether to insert a mutant of each gene was verified through DNA sequencing (Solgent).

[0109] A pET-28a (+) (Novagen) vector was treated with restriction enzymes NdeI and XhoI for the preparation of each gene expression vector after verifying that there was no mutant in three kinds of genes expected as a keratinase group obtained through the PCR. For the recovery of each gene cloned in the pTOP Blunt V2 vector, the restriction enzyme corresponding to the expression vector was treated and isolated and purified by using electrophoresis and the gel extraction kit (QIAGEN).

[0110] Four kinds of expression vectors pET28a-CDS, pET28a-CBP, pET28a-DSR, and pET28a-SufE were prepared by ligating the restriction enzyme-treated vector and the recovered genes by using a DNA ligation kit (Takara). A cleavage map of the expression vectors was illustrated in FIG. 6. For expression of protein genes expected as a keratinase group, E. coli transformants were prepared as follows. Each expression vector plasmid was introduced into an E. coli strain BL21 (DE3) (Enzynomics). In order to selectively culture the transformants, an LB solid medium containing kanamycin, was cultured at 37° C. Kanamycin is an antibiotic suitable for the antibiotic resistance gene contained in the expression vector. The final concentration of kanamycin was 100 μg/ml.

Experimental Example 4: Mass Expression and Purification of Proteins CDS, CBP, DSR, and SufE

[0111] For expression of protein genes expected as the keratinase group, transformants into which each expression vector was inserted was obtained and then used. Each transformant was pre-cultured and then 1% (5 ml) of pre-cultured cells were inoculated and cultured in 500 ml of a LB added with kanamycin (100 μg/ml) in two 2 L flasks. An isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM was added when absorbance (600 nm) was 0.4 to 0.6, expression of the CDS, CBP, DSR and SufE genes was induced at 37° C. for 6 h.

[0112] Cells induced by protein expression were centrifuged after a culture broth with absorbance (600 nm) 1 was transferred to a 1.5 ml E-tube and then only cultured colonies were recovered. The cells were suspended in 100 μl of a 1×SDS sample buffer and treated in boiled water for 5 min, modified and isolated by a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then stained with coomassie brilliant blue (CBB) R-250, and then protein expression levels were confirmed. The cultured cells were recovered by centrifugation and resuspended in 50 mM Tris-HCl (pH 7.0) containing 150 mM NaCl and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells were broken by sonication at 4° C. and the supernatant was recovered by centrifugation (10,000×g, 20 min).

[0113] The recovered supernatant was heat-treated (70° C. for 30 min), centrifuged (10,000×g, 20 min) and 0.45 μm-filtered to obtain a sample for His-tag purification. As a next step, in order to purify each expression protein, the expression protein was isolated and purified by using Ni.sup.2+-NTA agarose beads (Novagen). The expression protein containing a 6-histidine residue in an N-terminus was bound through the Ni.sup.2+-NTA agarose beads equilibrated with a binding buffer (0.5 M NaCl, 20 mM Tris-HCl (pH 7.9), 5 mM imidazole), nonspecific binding proteins were removed by using a washing buffer (0.5 M NaCl, 20 mM Tris-HCl (pH 7.9), 60 mM imidazole), and then finally, target proteins bound to beads were eluted with an elution buffer (0.5 M NaCl, 20 mM Tris-HCl (pH 7.9), 250 mM imidazole) and the expressed proteins were His-tag-purified.

[0114] Since all of the purified proteins were His-tagged at the N-terminus, thrombin (1.6 unit/μl) as a protease was treated to recover the protein in which the tag was removed. After thrombin treatment, gel filtration was performed using a HiLoad 16/60 superdex 200 prep grade column (GE healthcare) and finally, the proteins were recovered and used in a decomposition experiment for the chicken feather wastes. In the case of the CBP, after the His-tag purification, except for the process of treating the protease, the gel filteration was directly performed and finally used for the decomposition experiment for the chicken feather wastes.

Experimental Example 5: Analysis of Biochemical Characteristics of Proteins CDS and CBP

[0115] As illustrated in FIGS. 7 and 8, characteristics of recombinant proteins CDS and CBP of F. islandicum AW-1 strains were analyzed. In the case of the CDS, an optimal enzyme addition amount, a substrate mass and an optimal reaction time were determined by using a methylene blue assay, and then maximum enzyme activity was shown at 90° C. and pH 8.0, respectively, as illustrated in FIG. 7. As the result of verifying the kinetics of the enzyme, it was verified that V.sub.max was 1135.0±11.0 (Unit/mg) and K.sub.m was 75.0±1.7 μmol. In the case of the CBP, an optimal enzyme addition amount, a substrate mass and an optimal reaction time were determined by using a ninhydrin assay, and then maximum enzyme activity was shown at 80° C. and pH 7.0, respectively, as illustrated in FIG. 8. Further, considering that metal ions affected CBP activity, it was confirmed that the activity of the CBP was increased four times or more under presence of cobalt ion (Co.sup.2+).

Experimental Example 6: Protein Fractionation of Fervidobacterium Islandicum AW-1 Strain

[0116] As shown in FIG. 9, cells of F. islandicum AW-1 having keratin decomposition ability were recovered from a culture broth (4 liters) containing the chicken feather wastes and grinded, and then a whole cell extract (Table 2) was obtained through protein fractionation and used for the decomposition experiment of the chicken feather wastes.

TABLE-US-00002 TABLE 2 Protein conc. (mg/ml) Volume (ml) Total amount (mg) Whole cell extract 3.96 5 19.81 Pellet 2.39 5 11.95 Cytosol 2.33 4.3 9.997 Membrane protein 5.86 1 5.86 Solubilized 1.91 1.1 2.10 membrane Pellet membrane 3.68 1 3.68

Experimental Example 7: Decomposition Experiment of Chicken Feather Wastes Using Whole Cell Extract and Recombinant Protein

[0117] The decomposition experiment of the chicken feather wastes was performed as follows by using the whole cell extract and the recombinant proteins CDS, CBP, and CDR of F. islandicum AW-1. The chicken feather wastes (10 mg) was added in a Hungate tube and 5 ml of a 50 mM HEPES buffer (pH 8.0) containing 0.05 mg of resazurin was dispensed. After replacing nitrogen gas for 10 min, a tube was sealed with a rubber stopper and an aluminum seal, and sterilized at 121° C. for 20 min, and then after cooling, 10 μl of Na.sub.2S was added to each sterilized tube. The whole cell extract and the recombinant proteins CDS, CBP and DSR were diluted with a 50 mM HEPES buffer (pH 8.0) and then 0.2 mg was used. Dithiothreitol (DTT), a reducing agent, was used to be a final 10 mM. Each enzyme reaction (see Table 3) was shaken 2-3 times and then performed in a water bath at 80° C.

TABLE-US-00003 TABLE 3 Reac- Reducing tion agent Whole cell extract Recombinant condi- DTT super- solublized protein tion No. (10 mM) natant cytosol membrane CDS CBP DSR 1 x x x x x x x 2 x ∘ x x x x x 3 ∘ ∘ x x x x x 4 x x ∘ x x x x 5 ∘ x ∘ x x x x 6 x x x ∘ x x x 7 ∘ x x ∘ x x x 8 x x ∘ x x x ∘ 9 ∘ x x x ∘ ∘ ∘ 10 x x x x ∘ ∘ ∘ 11 ∘ x ∘ x ∘ x x 12 ∘ x ∘ x x x ∘ 13 ∘ x ∘ x x ∘ x 14 ∘ x 0.05 mg x x x x 15 ∘ x  0.1 mg x x x x (∘: added enzyme, x: non-added enzyme)

[0118] As illustrated in FIG. 10, the decomposition forms of the chicken feather wastes was verified at a 24-hour unit, and finally, amino acid quantification was performed by a ninhydrin assay by using a culture medium for 72 h and the result was shown in FIG. 11. The ninhydrin assay was performed by adding the same amount of 10% trichloroacetic acid (TCA) solution to 50 μl of a culture broth recovered under each reaction condition and then vortexing, and a supernatant recovered through centrifugation at 13,000 rpm for 10 min was added with 15 μl of a 3% ninhydrin solution and 150 μl of an acetate cyanide buffer. The supernatant was boiled for 15 min and added with 660 μl of a pre-chilled isopropanol-water diluent, and then voltexing was performed and absorbance was measured at 570 nm by using an Ultraspec 8000 spectrophotometer (GE healthcare).

[0119] As the result of this experiment, the decomposition of the chicken feather wastes was verified in all of the whole cell extracts of the F. islandicum AW-1 strain having keratin decomposition ability, and the decomposition of the chicken feather wastes was confirmed in the reaction of adding both the whole cell extract and the recombinant protein. In addition, as a result of verifying a minimum amount of whole cell extract for the decomposition of 10 mg of chicken feather wastes (No. 14, 15), it was confirmed that a minimum of 0.05 mg of the whole cell extract was required.

Experimental Example 8: Decomposition Experiment for Chicken Feather Wastes Using Whole Cell Extract and Recombinant Proteins CDS and SufE of Colonies Cultured by Adding Glucose

[0120] As illustrated in FIG. 12, F. islandicum AW-1 cell having keratin decomposition ability was grinded in a culture broth added with 0.5% (v/v) glucose instead of the chicken feather wastes and then a whole cell extract was obtained and used in the decomposition experiment for the chicken feather wastes. The chicken feather wastes (10 mg) was added to a Hungate tube for the decomposition experiment of the chicken feather wastes by adding the whole cell extract and the recombinant protein SufE and a 50 mM HEPES buffer (pH 8.0) containing 50 mg of resazurin was dispensed by 5 ml. After replacing nitrogen gas for 10 min, a tube was sealed with a rubber stopper and an aluminum seal, and sterilized at 121° C. for 20 min, and then after cooling, 10 μl of Na.sub.2S was added to each sterilized tube. The whole cell extract, 0.5 mg of the recombinant protein CDS, and 2.5 mg of the recombinant protein SufE were used. Dithiothreitol (DTT), a reducing agent, was used to be a final 10 mM. The enzyme was added to each Hungate tube prepared in an anaerobic chamber under each condition (see Table 4), shaken several times, and then the reaction was started at 80° C. in a water bath.

TABLE-US-00004 TABLE 4 Recombinant Reaction Reducing agent Whole cell extract protein condition No. DTT (10 mM) whole cell extract CDS SufE 1 x x x x 2 ∘ x x x 3 ∘ ∘ x x 4 ∘ ∘ ∘ x 5 ∘ ∘ ∘ ∘ 6 ∘ ∘ x ∘ (∘: added enzyme, x: non-added enzyme)

[0121] As illustrated in FIG. 13, the decomposition pattern of the chicken feather wastes was verified by a 3-day unit and finally, quantification of amino acid was performed by a ninhydrin assay using the sampled culture broth. The result was shown in FIG. 14. The ninhydrin assay was performed by adding the same amount of 10% TCA solution to 50 μl of a culture solution recovered under each reaction condition and then vortexing, and 30 μl of a supernatant recovered through centrifugation at 13,000 rpm for 10 min was added with 150 μl of a 3% ninhydrin solution and 150 μl of an acetate cyanide buffer. The supernatant was boiled for 15 min after voltexing and added with 660 μl of a pre-chilled isopropanol-water diluent, and then voltexing was performed and absorbance was measured at 570 nm by using an Ultraspec 8000 spectrophotometer (GE healthcare).

[0122] As a result of this experiment, even in the whole cell extract of the F. islandicum AW-1 strain having keratin decomposition capability cultured in a medium added with glucose instead of the chicken feather, the chicken feather wastes decomposition was verified, and even in a reaction of adding both the whole cell extract and the recombinant protein, the decomposition of the chicken feather wastes was confirmed. In addition, in the case of using the whole cell extract derived from the glucose medium, compared with the case of using the whole cell extract derived from the chicken feather medium, it was demonstrated that the decomposition of the chicken feather wastes was slower and herein, it was demonstrated that when the recombinant proteins CDS and SufE were added, the chicken feather decomposition speed was faster.