CN116984353A - Application of Sp02200 enzyme in preparation of feather degradation preparation - Google Patents

Abstract

An application of Sp02200 enzyme in preparing feather degrading preparations relates to the technical field of bioengineering; the rSp02200 enzyme comprises an amino acid sequence shown in SEQ ID NO:1, the nucleotide sequence of the Sp02200 gene is shown as SEQ ID NO: 2. The Sp02200 enzyme has higher degradation rate on feathers, has stronger stability below 35 ℃ and stronger activity within the pH range of 4.0-11.0, so the Sp02200 enzyme can be applied to the preparation of feather degradation preparations and has huge development prospect.

Description

Application of Sp02200 enzyme in preparation of feather degradation preparation

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to application of Sp02200 enzyme in preparation of a feather degrading preparation.

Background

In recent years, with the rapid development of animal husbandry, millions of tons of feather waste are produced annually worldwide. Because of the unique natural structure of the feather, the feather is difficult to degrade in the natural environment and becomes waste which is difficult to dispose, and the feather also releases various pollutants (such as NO, H2S and the like) to become a pollution source, thus being a source for breeding pathogenic microorganisms and endangering the living environment of human beings.

Feathers mainly comprise keratin and are rich in various amino acids such as cysteine, arginine, threonine and the like and various minerals (such as K, ca, mg, fe, mn, zn, cu and the like); therefore, the hydrolysate of the feathers has potential to be applied to more industries such as animal feed, plant fertilizer, health food and the like. However, the feather keratin has a complex structure, and the secondary structure is rich in alpha-helix, beta-sheet and disulfide bonds, so that the feather keratin is not easy to hydrolyze by common proteases such as papain, trypsin and pepsin, and is difficult to be directly digested and absorbed by animals, and the application of the feather keratin as a dietary protein additive of animal feed, a plant fertilizer and the like is limited; therefore, the search for efficient degradation methods for feather waste has become a focus of attention. At present, physical, mechanical and chemical methods are often adopted to degrade feathers, but the efficiency is low, the byproducts are more, the environment is polluted, and the condition of large-scale industrial production is not satisfied.

The enzymatic degradation method has mild reaction conditions, is not destructive to the nutrition of feathers, does not generate byproducts, is environment-friendly, and is the most interesting environmental protection technical means. However, the existing feather degradation method based on the enzymatic degradation method is low in efficiency, and most enzymes are poor in stability, and the activity of the enzymes is greatly influenced by the factors such as the temperature, the pH value and the like of the environment, so that the industrial popularization is difficult.

Disclosure of Invention

In order to overcome the defects of the prior art, one of the purposes of the invention is to provide an application of Sp02200 enzyme in the preparation of feather degradation preparations, wherein the Sp02200 enzyme has high degradation rate and strong stability, and can keep high activity in a wider pH value range.

The second purpose of the invention is to provide an Sp02200 recombinant engineering bacterium which is used for efficiently expressing the Sp02200 enzyme.

The invention further aims to provide a construction method of Sp02200 recombinant engineering bacteria.

The fourth object of the invention is to provide a method for degrading feathers, which can realize efficient degradation of feathers, has mild reaction conditions, does not damage the nutrition of feathers, does not generate byproducts, and is environment-friendly.

One of the purposes of the invention is realized by adopting the following technical scheme:

an application of Sp02200 enzyme in preparing feather degrading preparation.

Further, the rSp02200 enzyme comprises an amino acid sequence shown in SEQ ID NO:1, a protein a represented by the formula 1,

or protein b derived from protein a by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by protein a and having the activity of protein a.

Further, the genes encoding the Sp02200 enzyme comprise Sp02200 genes, and the nucleotide sequence of the Sp02200 genes is shown as SEQ ID NO: 2.

Further, the Sp02200 enzyme is derived from a d.actinosclegusrm strain, the Sp02200 enzyme belonging to the serine protease of the S1C subfamily.

The second purpose of the invention is realized by adopting the following technical scheme:

an Sp02200 recombinant engineering bacterium, the Sp02200 recombinant engineering bacterium being used for expressing an Sp02200 enzyme in the application of the Sp02200 enzyme in the preparation of a feather degradation preparation.

The third purpose of the invention is realized by adopting the following technical scheme:

the construction method of the Sp02200 recombinant engineering bacteria comprises the following steps:

s101, cloning a gene fragment encoding Sp02200 enzyme;

s102, connecting the gene segment to an expression vector to construct a recombinant plasmid;

s103, electrically transforming the recombinant plasmid into host bacteria to obtain the Sp02200 recombinant engineering bacteria.

Further, in step S101, the specific operations include: taking a DNA genome of a D.acteosclegusRM strain as a template, carrying out PCR reaction by adopting a P1 primer and a P2 primer, collecting a PCR product, cloning the PCR product onto a pEASY-Blunt vector to construct a pEASY-Blunt/Sp02200 plasmid, converting the pEASY-Blunt/Sp02200 plasmid into escherichia coli DH5 alpha, screening positive clone, and sequencing to obtain a gene fragment for encoding Sp02200 enzyme;

wherein the P1 primer is 5'-CGGAATTCGCCCCCACCAGTCC-3'; the P2 primer is 5'-CCGCTCGAGATTGCTCAGCTTG-3'.

Further, in step S102, the expression vector is a pICH vector;

in step S103, the host bacterium is pichia pastoris GS115 bacterium.

The fourth purpose of the invention is realized by adopting the following technical scheme:

a method for degrading feathers, comprising the steps of:

s201, carrying out induced expression on Sp02200 recombinant engineering bacteria to obtain Sp02200 enzyme solution;

s202, mixing the feather to be treated with the Sp02200 enzyme liquid, and culturing and fermenting to finish the degradation of the feather.

Further, the Sp02200 enzyme content in the Sp02200 enzyme liquid is 370-400 U.ml ^-1 The mass fraction of the feathers in the mixed solution of the feathers and the Sp02200 enzyme is 0.1-4%.

Compared with the prior art, the invention has the beneficial effects that:

the Sp02200 enzyme has higher degradation rate on feathers, has stronger stability below 35 ℃ and stronger activity within the pH range of 4.0-11.0, so the Sp02200 enzyme can be applied to the preparation of feather degradation preparations and has huge development prospect.

The construction method of the Sp02200 recombinant engineering bacteria realizes the transformation of the Sp02200 recombinant engineering bacteria, so that the recombinant bacteria have higher activity and can efficiently secrete and express the Sp02200 enzyme.

Drawings

FIG. 1 is a diagram showing the genomic electrophoresis of RM strain in example 1 of the present invention; wherein M is DNA marker; RM strain genome.

FIG. 2 is an electrophoretogram of sp02200 gene in example 1 of the present invention; wherein M is DNA marker;1:sp02200PCR product.

FIG. 3 is a 3D block diagram of homology modeling of the Sp02200 gene in example 1 of the present invention.

FIG. 4 is a schematic diagram of a transparent hydrolytic circle produced by recombinant Pichia pastoris in MM milk medium in example 2 according to the present invention.

FIG. 5 is an electrophoretogram of the genomic PCR product of recombinant Pichia pastoris in example 2 of the present invention; wherein M is a DNA standard; 1, PCR products amplified by the primers P1 and P2; 2:5'AOX sequencing primer and 3' AOX sequence primer amplified PCR product.

FIG. 6 is a graph showing rSp02200 activity and wet weight of cells of recombinant Pichia pastoris in accordance with example 2 of the present invention, expressed by induction in a triangular flask.

FIG. 7 is a SDS-PAGE analysis of purified rSp02200 of example 2 of the present invention; wherein M is a protein molecular weight marker; salting out; sephadexG75 chromatography.

FIG. 8 is a schematic representation of the effect of temperature on rSp02200 enzyme activity in example 2 of the present invention.

FIG. 9 is a schematic representation of the effect of temperature on the stability of rSp02200 enzyme in example 2 of the present invention.

FIG. 10 is a schematic representation of the effect of pH on rSp02200 enzyme activity in example 2 of the present invention.

FIG. 11 is a graph showing the effect of rSp02200 enzyme on chicken feather degradation in example 3 of the present invention; wherein, A is a control group; rSp02200 and 02200 enzymatic hydrolysis.

FIG. 12 is an SEM image of feathers after enzymatic hydrolysis of rSp02200 enzyme in example 3 of the present invention; a is a control group; B. c, D the enzymatic hydrolysis of rSp 02200.02200 on different parts.

Detailed Description

The present invention will be further described with reference to the following specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below.

The materials used in the invention can be obtained from the market except for the other descriptions; wherein the D.actinosclerus RM strain is provided by the Shao college of English east organism and agricultural college of microbiology laboratory deposit; pichia pastoris GS115 (his-, mut+) strain, pPIC9K vector and Trizol reagent were purchased from Invitrogen (California, USA), pEASY-Blunt plasmid was purchased from Beijing full gold Biotechnology Co., LTD (Beijing, china); coli DH 5. Alpha. And enzymes for manipulation of DNA or RNA were purchased from Takara (Beijing, china); the primers used in the present invention were from SangonBiotech (Shanghai) Inc. (China Co., ltd.); the culture media for Pichia pastoris GS115 strain were all configured according to the Pichia expression kit handbook of Invitrogen (Calif., U.S.A.).

Example 1

screening and cloning of sp02200 Gene

1.1 Whole genome sequencing of RM strains

RM strain was inoculated in LB medium, cultured with shaking at 180rpm for 4d, and centrifuged at 12000rpm for 3min to collect the cells. Extracting genome DNA thereof, sending the genome DNA to Guangzhou sequencing department of biological engineering (Shanghai) stock, carrying out high-throughput sequencing and sequence assembly on genome of RM strain by using an Illumina Hiseq 2500 second-generation sequencing platform, submitting sequences obtained by sequencing to a GenBank database, and annotating the sequences according to PGAP of NCBI.

Genome sequencing results of RM strains show that the genome has the full length of 4013254bp and the GC content of 67.81 percent; in the sequence splicing, the size of N50contig is 21827bp, the longest contig is 79609bp, the average length is 12088.92bp, and the number of contigs is 332. Prokaryotic Genome Annotation Pipeline (PGAP) using NCBI showed that the RM genome contained 4132 genes, of which 3931 could encode the amino acid sequence. The entire genomic sequence has been submitted to the GenBank database for access No. qycq00000000.1. And (3) combining the morphological structure of the RM strain, the 16SrDNA sequence comparison and the physiological and biochemical test result to determine that the RM strain is the D.actinoscleus strain.

1.2 transcriptome sequencing of RM strains

The RM strain is inoculated in chicken feather fermentation medium, cultured for 2d at 28 ℃ and 160rpm, then centrifuged to collect thalli, washed 3 times with sterile water, centrifuged for 2min at 12000rpm to collect thalli, and sent to Shanghai Paenosen Biotechnology Co., ltd for sequencing by utilizing an Illumina platform, wherein the sequencing strategy is PE150.

According to the result of transcriptome sequencing analysis, performing statistical analysis on the gene expression quantity of the encodable amino acid sequence by using FPKM, wherein the RM strain expression base factor is 3844 (FPKM > 1) accounting for 97.79% of the total number of genes; the number of genes that were not expressed or were expressed was 87 (FPKM < 1), accounting for 2.81% of the total number of genes. The present invention selects sp02200 (fpkm= 1731.5) gene with one of high expression levels as the next study object, which contains 444 genes of extracellular expression proteins of signal peptide, 26 genes of extracellular expression proteins are annotated by NR, swissprot, KEGG database, and 4 genes of high expression level protease (FPKM > 1000) are annotated as proteases.

1.3 Gene expression analysis and screening of protease Gene

The raw data obtained from the sequencing was filtered to remove band-ends and low quality Reads, yielding high quality clearreads. After a reference genome index was established by Bowtie2, filtered Reads were aligned and analyzed with genomic sequence (accessno. qycq00000000.1) using Bowtie 2. In order to explore the gene expression condition in the feather degradation process, HTSeq0.6.1p2 software is adopted to statistically compare the ReadCount value of each gene as a reference value of the original expression quantity of the gene; in order to compare the comparability of different gene expression levels, FPKM (Fragmentsperkilobaseofexonpermillionfragmentsmapped) is adopted to analyze and compare the gene expression quantity, when the FPKM is more than 1, the gene is expressed, and the larger the FPKM value is, the higher the gene expression quantity is; therefore, a protease gene having high extracellular secretion and high expression level was selected based on a gene annotated as peptase (orprotease, proteinase) containing a signal peptide and having a high FPKM value, and an sp02200 gene was obtained.

1.4 cloning of sp02200 Gene

As known from the analysis of the sp02200 gene sequence by using SignalP-5.0Server online software, the 1-33 amino acid residue sequence is a signal peptide sequence, and a primer P1 and a primer P2 for cloning the sp02200 gene are designed, and the PCR product does not contain the signal peptide sequence;

p1 primer: 5'-CGGAATTCGCCCCCACCAGTCC-3';

p2 primer: 5'-CCGCTCGAGATTGCTCAGCTTG-3'.

And carrying out PCR reaction by using the whole genome DNA as a template and adopting P1 and P2 primers. The reaction procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 30s; annealing at 63 ℃ for 30s; extending at 72 ℃ for 2min;32 cycles, constant temperature of 72℃for 5min. The PCR product was purified and recovered, cloned into pEASY-Blunt vector to construct pEASY-Blunt/sp02200 plasmid, transformed into E.coli DH 5. Alpha. And screened for positive clone sequencing confirmation.

As shown in FIGS. 1 and 2, PCR amplification was performed using the P1 and P2 primers with the RM strain genome as a template, the size of the PCR product was consistent with the expected 1071bp size, the PCR product was ligated to the cloning vector pEASY-Blunt, and after transformation to Escherichia coli DH5α, sequencing was performed, and the sequencing result was consistent with the genomic sequence (accession No. QYCQ01000028 REGION: 9232..10404), which indicated that the sp02200 gene was successfully cloned.

The Sp02200 gene codes for 357 amino acid residues in total, and the sequence analysis of the Sp02200 gene by using GenBank database Conserved Domain Search Service shows that the Sp02200 gene is serine proteinase (COG 0265: degQ) of an S1C subfamily, and specifically, the rSp02200 enzyme comprises an amino acid sequence shown in SEQ ID NO:1, the nucleotide sequence of the Sp02200 gene is shown as SEQ ID NO:2 is shown in the figure; however, the sequence has low sequence homology with the family members, the highest homology being 33.56% for DegQ of Escherichia coli (PDB code:3STJ.1. A), 27.61% for DegQ of Legionella fallonii (PDB code:3Pv4.1.A.1. A), and 26.94% for DegQ of Legionella pneumophila (PDB code:4YO1.1. A).

According to the scoring and evaluation results of homology modeling, a 3D structure of rSp02200 is subjected to homology modeling by taking DegQ (PDB code:3STJ.1. A) of Escherichia coli as a template, as shown in FIG. 3, wherein the column shape represents an alpha helix; the bands represent beta sheets; the line shape represents curl; the catalytically active residues S154 (upper), H50 (middle), D80 (lower) are represented by the CPK model.

From this 3D structure, rSp02200 can be seen to contain 6 alpha helices, 1 310 helices, 17 beta-sheets, and multiple random coil compositions, (for ease of description, the first amino acid residue E number in rSp022003-D structure is defined as 1, and so on), 1-204 are the catalytic domains of serine proteases, where H50, D80, and S154 are the conserved catalytic triplet active residues; 205-322 are PDZ domains, which are mainly auxiliary domains that bind to substrates and control protease function. The main function of DegQ-type proteases is to cleave protein substrates in misfolded cells. The rSp02200 has larger difference with DegQ enzyme in the aspects of amino acid sequence composition, secondary structure, loop ring and the like; therefore, sp02200 is a novel protease, and has a value of intensive research on its structure and function.

Example 2

Construction and expression of Sp02200 recombinant engineering bacteria

2.1 construction of recombinant sp02200 expression vectors

PCR was performed using the pEASY-Blunt/sp02200 plasmid obtained in example 1 as a template, and the P1 primer and the P2 primer, and the procedure and system for PCR reaction were the same as those in example 1.

The PCR product and pICH expression vector recovered by purification (pPIC 9K vector is obtained by replacing NotI restriction site with XhoI restriction site and then modifying the pICH expression vector) are respectively subjected to EcoRI and XhoI double digestion, then are connected by T4DNA ligase to construct recombinant expression vector pICH/sp02200, are transformed into escherichia coli DH5 alpha, and are picked up for single colony sequencing confirmation.

2.2, transformation and screening of Sp02200 recombinant Pichia pastoris

After StuI linearization of pICH/sp02200 expression vector, the vector was transformed into Pichia pastoris GS115 strain by electric shock with a gene pulse Xcell electroporation system (Bio-Rad, hercules, calif., USA), spread on MD plates for 2-4d, single colonies were picked up to MM milk medium (1.34% YNB,1.0% methanol, 1.5% skim milk, 1.5% agar, 100ml20mM phosphate buffer solution), cultured on plates for 2-4d, curd appeared around the colonies, continued culture became transparent hydrolysis circles (FIG. 4), while the recombinant transformants obtained by the empty expression vector pICH were electrically transferred into Pichia pastoris GS115 strain as a control, and transparent hydrolysis circles were not produced around colonies of the recombinant Pichia pastoris GS115 strain. Selecting a transformant with the largest hydrolysis ring diameter for carrying out PCR identification of a yeast genome, wherein the size of a PCR product is consistent with that of an expected product, and the P1 and P2 primers amplify sp02200 genes; the AOX primers amplified 2 PCR products, respectively the AOX1 gene of pichia pastoris and the expression vector pICH/sp02200 sequence containing the sp02200 gene (fig. 5), indicating that the sp02200 gene has been integrated into the pichia pastoris genome and successfully expressed.

2.3, rSp02200 inducible expression

Inoculating recombinant Pichia pastoris GS115 strain into a 250mL triangular flask containing 50mL of MGY culture medium (pH=6.0), shaking and culturing at 28 ℃ for 1d, centrifuging at 12000rpm for 2min to collect thalli, transferring (the initial inoculum size light absorption value OD600 is about 5.0-6.0) to a 500mL triangular flask containing 150mL of the MGM culture medium for culturing, adding 1.0% (volume fraction) methanol every 24h for induction expression, and centrifuging at 12000rpm for 10min to collect fermentation supernatant after the induction is finished to obtain crude enzyme liquid.

As shown in FIG. 6, the protease activity of the fermentation broth was 25.4U/mL at maximum when the recombinant Pichia pastoris was induced to express in a flask for 120 hours, and the wet weight of the recombinant Pichia pastoris was 29g/L at maximum when the recombinant Pichia pastoris was induced to express in a 500mL flask, and then remained substantially constant, as found by the analysis of rSp02200 activity and the wet weight of the thallus.

2.4, rSp, 02200 purification

Dialyzing the crude enzyme solution in 20mM phosphate buffer solution with pH of 7.5 for overnight to reduce the viscosity of the crude enzyme solution, and concentrating by adopting a 50.0kDa and 10.0kDa tangential flow membrane filtration separation system; solid ammonium sulfate was added to the concentrate to 85.0% saturation overnight for salting out, centrifuging, collecting precipitate, dissolving in 20mM phosphate buffer (pH 7.5), dialyzing to remove salt ions, filtering with 0.15um filter membrane, and concentrating by removing impurities with 100.0kDa, 50.0kDa ultrafiltration centrifuge tube (Merck Millipore, german). Then, the mixture was subjected to SephadexG-75 gel chromatography (GEHealthcare) to conduct chromatography on rSp02200, elution was conducted with 20mM phosphate buffer (pH 7.5) at a flow rate of 30.0mL/h, and the collected eluate was concentrated with an ultrafiltration centrifuge tube to obtain purified rSp02200, and purity was identified by SDS-PAGE.

The specific activity of rSp02200 obtained by purification was 766.4U/mg, and it was identified by SDS-PAGE electrophoresis, and as a result, as shown in FIG. 7, a single protein band was visible at 35.0kDa, the size of which substantially matches the molecular weight deduced from the amino acid sequence of rSp02200, and as a result, it was found that the single protein band was rSp02200, and as a result, it was revealed that rSp02200 of this example had been purified to electrophoretic purity.

2.5, rSp02200 determination of enzymatic Properties

The purified rSp02200 was subjected to determination of optimum temperature, optimum pH, temperature stability, pH stability and influence of metal ions and chemical reagents on its properties.

As shown in fig. 8-9, rSp02200 had an optimum reaction temperature of 37 ℃; has strong stability below 30 ℃, gradually reduces when the temperature exceeds 35 ℃, and almost loses all activity after 10min of heat preservation when the temperature is higher than 50 ℃.

As shown in FIG. 10, rSp02200 has an optimal reaction pH of 7.5-8.0, and can maintain stability of over 70% in a wide pH range of 4.0-11.0, which indicates that rSp02200 has strong pH stability.

The effect of metal ions and inhibitors on rSp02200 activity is shown in table 1.

TABLE 1

As can be seen from table 1, the effect of different metal ions and inhibitors on rSp02200 activity was different. Fe (Fe) ³⁺ 、Fe ²⁺ 、Mn ²⁺ 、Cu ²⁺ Has inhibitory effect on rSp02200 activity, wherein Fe ³⁺ The inhibition effect is most remarkable (the inhibition rate reaches 24.29 percent), and the Zn is the most remarkable ²⁺ 、Mg ²⁺ 、K ⁺ 、Ni ²⁺ Has an accelerating effect on rSp02200 activity, and therefore, the above-mentioned ion can be used for the activity accelerator of rSp02200 of the present invention. While Ca ²⁺ The effect on the activity of the Pepstatin is not obvious, rSp02200 can be obviously inhibited by PMFS, pepstatin has no effect on the enzyme activity, and EDTA has a certain inhibiting effect on rSp 02200. While the surfactant SDS and the surfactant TrionX-100 have remarkable inhibitory effect on rSp02200, and rSp02200 is completely deactivated when the concentration of SDS is 0.5%.

Example 3

Method for degrading feather by rSp02200

Naturally air-drying the cleaned chicken feather, placing the chicken feather into a test tube, sterilizing for 30min at 121 ℃, cooling, adding 5ml of rSp02200 (386.88 U.ml < -1 >) solution containing 5mM DTT into the chicken feather, performing enzymolysis at 37 ℃ for 2d, filtering to obtain undegraded residual chicken feather, naturally air-drying, and observing the enzymolysis condition of the residual chicken feather by adopting a desk-top scanning electron microscope TM3030 (HITACHI, japan).

As shown in fig. 11, after the chicken feather degradation product obtained by the enzymolysis of rSp02200 is filtered and dried, it can be seen that after the chicken feather is degraded by rSp02200, the larger feather shaft is broken, and the feather small branches are degraded into broken floccules (fig. 10. B).

As shown in fig. 12, the chicken feather control not digested with rSp02200 was observed by a scanning electron microscope TM3030, and it can be seen that the feather branches of the chicken feather are smooth in surface and complete in structure (fig. 11. A); and the feather small-branch fluff after being subjected to rSp02200 enzymolysis is shriveled and softened (fig. 11. B), part of the down Mao Tuola (fig. 11. C), the surface of the feather small-branch is wrinkled, rough and broken and shed (fig. 11. D).

Therefore, in the degradation process of rSp02200 on chicken feather, long and thick feather shafts or long and thick feather branches of the chicken feather are broken into fine feather shafts and feather branches through enzymolysis, and the finer feather branches and the feather branches become amino acids or small peptides to be dissolved in a solution after enzymolysis, so that efficient degradation of the feather is realized.

The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.

Claims (10)

1. An application of Sp02200 enzyme in preparing feather degrading preparation.

2. The use of Sp02200 enzyme according to claim 1 for the preparation of a feather degrading formulation, characterized in that: the rSp02200 enzyme comprises an amino acid sequence shown in SEQ ID NO:1, a protein a represented by the formula 1,

or protein b derived from protein a by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by protein a and having the activity of protein a.

3. The use of Sp02200 enzyme according to claim 2, for the preparation of a feather degrading formulation, characterized in that: the gene encoding the Sp02200 enzyme comprises an Sp02200 gene, and the nucleotide sequence of the Sp02200 gene is shown as SEQ ID NO: 2.

4. The use of Sp02200 enzyme according to claim 1 for the preparation of a feather degrading formulation, characterized in that: the Sp02200 enzyme is derived from a D.acteosclegusRM strain, and the Sp02200 enzyme belongs to serine proteinase of S1C subfamily.

5. An Sp02200 recombinant engineering bacterium, wherein the Sp02200 recombinant engineering bacterium is used for expressing the Sp02200 enzyme in the application of the Sp02200 enzyme in the preparation of a feather degradation preparation according to any one of claims 1 to 4.

6. A method for constructing the Sp02200 recombinant engineering bacterium according to claim 5, which comprises the following steps:

s101, cloning a gene fragment encoding Sp02200 enzyme;

s102, connecting the gene segment to an expression vector to construct a recombinant plasmid;

s103, electrically transforming the recombinant plasmid into host bacteria to obtain the Sp02200 recombinant engineering bacteria.

7. The method of constructing an Sp02200 recombinant engineering bacterium according to claim 6, wherein in the step S101, the specific operations include: taking a DNA genome of a D.acteosclegusRM strain as a template, carrying out PCR reaction by adopting a P1 primer and a P2 primer, collecting a PCR product, cloning the PCR product onto a pEASY-Blunt vector to construct a pEASY-Blunt/Sp02200 plasmid, converting the pEASY-Blunt/Sp02200 plasmid into escherichia coli DH5 alpha, screening positive clone, and sequencing to obtain a gene fragment for encoding Sp02200 enzyme;

wherein the P1 primer is 5'-CGGAATTCGCCCCCACCAGTCC-3'; the P2 primer is 5'-CCGCTCGAGATTGCTCAGCTTG-3'.

8. The method of claim 6, wherein in step S102, the expression vector is a pICH vector;

in step S103, the host bacterium is pichia pastoris GS115 bacterium.

9. The feather degradation method is characterized by comprising the following steps of:

s201, carrying out induced expression on the Sp02200 recombinant engineering bacteria in claim 5 to obtain Sp02200 enzyme solution;

s202, mixing the feather to be treated with the Sp02200 enzyme liquid, and culturing and fermenting to finish the degradation of the feather.

10. The method for degrading feathers according to claim 9, wherein the Sp02200 enzyme solution has a Sp02200 enzyme content of 370-400 U.ml ^-1 The mass fraction of the feathers in the mixed solution of the feathers and the Sp02200 enzyme is 0.1-4%.