Keratinase mutant, its encoding gene and expression vector and its application

Abstract

The present invention belongs to the field of genetic engineering technology, and more particularly relates to a keratinase mutant, its encoding gene, expression vector, and application thereof. The amino acid sequence of the keratinase mutant is shown in SEQ ID NO.2. The keratinase mutant provided by the present invention can effectively degrade keratin substrates, has good catalytic degradation activity on keratin, and has greatly improved enzyme activity compared to the wild-type protein. Moreover, the keratinase mutant can use keratin waste such as feathers as a direct substrate. In combination with the reducing agent DTT, it can basically decompose an entire feather within 24 hours, significantly improving the degradation efficiency of keratin-containing waste such as feathers, significantly reducing the production cost of keratinase and the degradation cost of keratin waste, and has broad market prospects and industrial application value in promoting the degradation of feather waste and the conversion, recycling, and utilization of keratin resources.

Description

Technical field

[0001] The present invention relates to the field of genetic engineering technology, and particularly to a keratinase mutant, its encoding gene and expression vector and its application. Background technology

[0002] Keratin is an insoluble protein that is widely found in hard structures such as animal feathers, hair, wool, nails, horns, hooves and scales. The crude protein content of this type of structure exceeds 80%, the total amount of amino acids is also higher than 70%, and the essential amino acid content is extremely high. It is a potential natural and high source of protein. However, the stable chemical structure of keratin makes it difficult to degrade, which hinders the resource utilization of these keratin wastes. For example, feathers are a by-product of poultry processing rich in keratin. Except for some waterfowl feathers used in high-value-added products such as down products and decorations, a large number of feathers are directly discarded or used as landfill or incinerated, causing pollution to the ecological environment and serious waste of protein resources.

[0003] At present, there have been research to degrade or partially degrade discarded feathers into mixtures containing oxidized keratin, amino acids and peptides as feed protein sources and soil improvers to promote animal and plant growth. In addition, feather keratin is also rich in umami amino acids such as glutamic acid and aspartic acid, which can be used to make new food additives and apply them to the food processing industry. The amino acids and derivatives obtained by keratin hydrolysis can also be used in pharmaceuticals, textiles, leather processing, composite materials and other industries. Therefore, studying the transformation of keratin resources and realizing the high-value recycling of waste resources is of great practical significance and has received more and more attention.

[0004] The traditional method of decomposing feathers mainly relies on physical and chemical methods, high temperature and high pressure, acid and alkali treatment, etc., but it has problems such as high energy consumption and serious nutrient loss. In contrast, enzymatic degradation of keratin has become a more promising solution with its low cost, mild treatment conditions, effective preservation of protein and amino acid resources in feathers, and green and environmentally friendly production process. Keratinase is a protease that specifically degrades keratin. It can be produced by bacteria, fungi, actinomycetes and other microorganisms. It has a wide range of substrate specificity and can effectively hydrolyze keratin from sources such as feathers, wool, keratin, human hair and nails at specific pH values and temperatures, producing high-value active peptides and free amino acids, and will not destroy the natural structure of amino acids. It is considered an ideal catalyst for the conversion of keratin waste.

[0005] The sources and distribution of keratinase-producing bacteria are very wide. However, the enzyme yield of wild-type keratinase-producing strains is low, and there are problems such as low catalytic efficiency of keratinase and long time to dikeratin hydrolysis, which greatly restricts the development and application of keratinase. Therefore, improving the activity of keratinase and helping to achieve high-value application of keratin waste such as feathers has good market application prospects and far-reaching social and economic benefits. Contents of invention

[0006] In response to the problems of low keratinase enzyme activity in the prior art, the present invention provides a new keratinase mutant and a nucleic acid molecule, expression vector and recombinant strain containing the gene encoding the keratin mutant, further providing the application of the keratin mutant in the treatment of keratin-containing substances.

[0007] The invention is specifically realized through the following technical solutions:

[0008] A first aspect of the present invention provides a keratinase mutant whose amino acid sequence is shown in SEQ ID NO.2.

[0009] A second aspect of the invention provides a nucleic acid molecule comprising a gene sequence encoding a keratinase mutant as described above.

[0010] Further, the gene sequence of the keratinase mutant is shown in SEQ ID NO.3.

[0011] Further, the nucleic acid molecule further comprises a gene sequence encoding a signal peptide, a promoter and / or a terminator, the keratinase mutant is located downstream of the signal peptide, the promoter is located upstream of the signal peptide, and the terminator is located downstream of the keratinase mutant.

[0012] Further, the signal peptide is a vsi signal peptide, and its gene sequence is shown in SEQ ID NO.4, the promoter is a kasOp* promoter, its gene sequence is shown in SEQ ID NO.5, the terminator is a T1 terminator, and its gene sequence is shown in SEQ ID NO.6.

[0013] A third aspect of the present invention provides an expression vector comprising a nucleic acid molecule as described above.

[0014] Further, the expression vector is Streptomyces integrated vector pSET152.

[0015] A fourth aspect of the present invention provides a recombinant strain, the recombinant strain comprising an expression vector as described above.

[0016] Further, the recombinant strain is Streptococcus acetacidum TK24.

[0017] The fifth aspect of the present invention provides the use of keratinase mutants as described above in the treatment of keratin-containing substances.

[0018] Further, the keratin-containing substance is selected from feathers, hairs or wools, and further, from avian feathers.

[0019] Further, the method of treating keratin-containing substances by the keratin-containing substances comprises: mixing the keratin-containing substances with the keratin-containing substances, adding a reducing agent, and performing a catalytic reaction at 50-60°C.

[0020] Further, the method for preparing the crude enzyme solution of the keratinase mutant includes: culturing the recombinant strain as described above, centrifuging the fermentation culture medium, filtering the supernatant, collecting the filtrate, and obtaining the crude enzyme solution containing the keratinase mutant.

[0021] Further, the reducing agent is dithiothreitol.

[0022] The advantages and positive effects of the present invention are:

[0023] The keratinase mutants provided by the present invention can effectively degrade keratin substrates, and dikeratin has good catalytic degradation activity, which greatly improves the enzyme activity of wild-type proteins. Moreover, this keratinase mutant can use keratin waste such as feathers as the direct substrate. With the combination of the reducing agent dithiothreitol (DTT), the whole feather can basically decompose the whole feather within 24 hours, significantly improve the degradation efficiency of keratin-containing waste such as feathers, significantly reduce the production cost of keratin and keratin waste, and has broad market prospects and industrial application value in promoting the degradation of feather waste and the conversion, recycling, and utilization of keratin resources. Attached description of the drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiment will be briefly introduced below. It is obvious that the accompanying drawings in the description below are only some embodiments of the invention. For those of ordinary skill in the art, other accompanying drawings can also be obtained based on these accompanying drawings without expending creative labor.

[0025] Figure 1 It is a map of the wild-type keratinase KT-ker expression vector of the embodiment of the present invention;

[0026] Figure 2 A map of the expression vector of KT1113 in the keratinase mutant KT1113 in the examples of the present invention;

[0027] Figure 3 Polyacrylamide gel electrophoresis diagram of the keratinase mutant KT1113 crude enzyme solution for example of the present invention;

[0028] Figure 4 This is the enzymatic effect diagram of the crude enzyme solution of keratinase mutant KT1113 in Examples of the present invention to feathers within 24 hours. Specific implementation methods

[0029] In order to make the object, technical scheme and advantages of the present invention clearer, the present invention will be further explained in detail below in conjunction with the examples. The equipment and reagents used in each example and test examples can be obtained commercially without any special description. The specific embodiments described herein are for the purpose of explaining the invention only and are not intended to limit the invention.

[0030] Various changes can be made to those skilled in the art according to the information contained in this application without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments and other descriptions are merely for the purpose of schematic illustration of specific aspects of the invention. Indeed, it is obvious that various changes to the embodiments of the invention can be made by those skilled in the art or related art. All are covered by the scope of the appended claims.

[0031] In order to better understand the invention rather than limit the scope of the invention, all numbers representing dosage, percentage, and other numerical values used in this application are to be understood in all cases as modified by the word "approximately". Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be varied according to the desired properties attempted to obtain. Each numerical parameter should be considered at least as obtained based on the reported valid digits and by conventional rounding methods.

[0032] In addition, it should be noted that unless otherwise defined, the scientific and technical terms used in the context of the present invention should have meanings commonly understood by those of ordinary skill in the art.

[0033] The terms "including", "comprising", "containing", "having" and other similar words are non-limiting, and other steps and other ingredients that do not affect the results can be added.

[0034] The term "and / or" shall be considered to be a specific disclosure of each of the two designated features or components with or without the other. For example, "A and / or B" will be considered to include the following situations: (i) A, (ii) B, and (iii) A and B.

[0035] The term "gene" is the entire nucleotide sequence required to produce a polypeptide chain or functional RNA. The gene may contain several operably linked nucleic acid fragments, such as a 5' non-coding region, a coding sequence, and a 3' non-coding region containing a polyadenylation site. Generally, non-coding regions have regulatory functions for gene expression, such as promoters and terminators.

[0036] The term "fusion gene" has the same meaning and refers to any gene that is not a natural gene, including regulatory and coding sequences that do not exist together in natural conditions. Thus, the fusion gene may include regulatory sequences and coding sequences derived from different organisms, or include regulatory sequences and coding sequences derived from the same organism but arranged in a different way than naturally occurring, such as the keratinase mutant expression cassette of the present invention.

[0037] The term "promoter" refers to a DNA sequence that controls the transcription of one or more nucleic acids or genes, usually upstream of the transcription direction of the gene transcription start site (ie, at the 5' end of the coding sequence). The promoter itself is not transcribed. It is structurally identified by the presence of the binding site of the DNA-dependent RNA polymerase and the presence of the transcription start site, and may also include any other DNA sequence, including but not limited to transcription factor binding sites, repressors and activating protein binding sites, as well as any other nucleotide sequences known to those skilled in the art that act directly or indirectly on the amount of transcription from the promoter.

[0038] The term "terminator" refers to the DNA sequence that provides a termination signal during the transcription process, which is located at the 3' end of the coding sequence, and acts at the RNA level through the transcribed terminator sequence to form a stem-loop structure, causing RNA synthesis to terminate.

[0039] The term "vector" refers to a self-replicating DNA molecule that transfers the gene of interest into a host cell, and is often in the form of a circular double-stranded DNA molecule. The vector containing an exogenous gene is a recombinant vector.

[0040] The term "expression vector" allows the gene of interest inserted into the vector to be expressed in the host cell, including regulatory elements, such as promoters and / or terminators, expressed in a designated host cell. The expression vector is introduced into the appropriate host cell to enable it to express the inserted gene of interest.

[0041] The term "introduction" or "transfer" refers to the transfer of the target gene into the host cell, resulting in stable genetic inheritance. The introduced nucleic acid molecule may be in the form of a plasmid retained in the host cell or may be integrated into the host cell genome. Nucleic acid molecules and / or vectors can be transferred into the host cell by "transfection", "transformation" or "transduction". The host cell containing the introduced nucleic acid molecule is referred to as a "transgenic" or "recombinant" or "transformed" organism or "engineered" organism. The introduction of vectors into host cells can be performed using conventional techniques well known to those skilled in the art.

[0042] Unless otherwise specified, similar words such as "nucleotide", "nucleic acid", "nucleic acid molecule" and "nucleic acid fragment" are interchangeable in the context of the present invention. The terms "gene", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid molecule" used in the present invention refer to the polyform of nucleotides of any length, which can be ribonucleotides or deoxyribonucleotides, and the term refers only to the primary structure of the molecule.

[0043] In order to make the above objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention will be explained in detail below.

[0044] Keratinase is a protease that is mainly produced by microorganisms such as fungi, actinomycetes, bacteria, etc., and can specifically degrade keratin. For example, keratin in feathers, wool, keratin, human hair, and nails can be degraded into active polypeptides and free amino acids. It has broad application prospects in industrial and environmental governance such as feed additives, soil improvement agents, food additives, leather softening, cosmetics, medicine, etc.

[0045] Compared with traditional physical and chemical methods to decompose keratin, keratin is used to degrade keratin, which has the advantages of mild reaction conditions, can effectively retain protein, peptide, and amino acid resources in waste such as feathers, and is more green and environmentally friendly. It is of great significance to realize the efficient utilization and high-value application of keratin resources. Therefore, keratinase is considered an ideal catalyst for keratin waste conversion and has received a lot of attention in recent years. However, the catalytic efficiency of keratinases reported is not high, and the degradation time of feathers and other substrates is long, so it is not suitable for large-scale industrial applications. Therefore, comprehensive and systematic screening, researching and developing new keratinases to improve their enzyme catalytic activity and using efficient keratinases to achieve effective feather utilization has become an important research direction in the field of feather waste resource utilization.

[0046] As an important evolutionary method, site-directed mutation technology has been successfully applied to the transformation of a variety of enzymes, including improving enzyme activity and stability. This application uses the wild-type keratinase KT-ker (amino acid sequence shown in SEQ ID NO.1) derived from Streptomyces as the basis, and constructs its mutant library through error-prone PCR. Then, by fusing expression elements such as signal peptides derived from Streptomyces coelicolor, kasOp* promoter and T1 terminator with each keratinase mutant gene, Streptomyces is used to fusion Streptomyces (Streptomyces coelicolor) with the genes of each keratinase mutant, and uses Streptomyces (Streptomyces) to Streptomyces (Streptomyces) to fusion with the genes of each keratinase mutant. Lividans)TK24, as a host bacteria, expressed a fusion gene. By comparing the degradation ability of the fermentation broth supernatant of Streptocytic recombinant strain fermentation broth, a mutant KT1113 (V99A / E116N / I117G / Y160S) with significantly improved enzyme activity was obtained. Compared with wild-type KT-ker, the enzyme activity was increased by 28%.

[0047] The mutant KT1113 has amino acid substitution at four sites 99, 116, 117 and 160 of wild-type keratinase KT-ker. The 99th amino acid mutates from V to A (V99A), the 116th amino acid mutates from E to N (E116N), the 117th amino acid mutates from I to G (I117G), and the 160th amino acid mutates from Y to S (Y160S).

[0048] Based on this, the embodiment of the present invention provides a keratinase mutant whose amino acid sequence is as follows:

[0049] ETADATRASVAELARVSDAVLDADVPGTAWYTDAESGKLVVTADATVSAAELAQLKKAAGDKAGAVEIKRTPGTFNKLIAGGEAIYAAGGGRSSLGFN A RSSSGATYALTAGHCT NG ASTWYTNSGQTSLAGTRAGTSFPGNDYGLIRHSNASAADGRV S LYNGSYRDITGAGNAYVGQTVQRSGSTTGLHSGRVTGLNATVNYGGGDIVSGLIQTNVCAEPGDSGGALFAGSTALGLTSGGSGNCRTGGTTFFQPVTEALSAYGVSII (see SEQ ID NO.2), underlined as the mutation site.

[0050] The keratinase mutant provided by the present invention can effectively degrade keratin substrates and have good catalytic degradation activity on keratin. The keratin mutant gene is expressed through the Streptomyces expression system. After testing, it was found that the enzyme activity of the recombinant bacteria fermentation supernatant was 2127.58 U / mL, and the enzyme activity of the wild-type fermentation supernatant under the same culture system and culture conditions was 1662.17 U / mL. The enzyme catalytic activity of the mutant was increased by 28% compared to the wild-type protein. Moreover, the keratinase mutants of the present invention can use keratin waste such as feathers as direct substrate, and with the combination of the reducing agent DTT, the recombinant bacteria fermentation supernatant as crude enzyme solution, can basically decompose the whole feathers within 24 hours, significantly improve the degradation efficiency of keratin-containing waste such as feathers. Compared with the traditional engineering strains that use carbohydrate carbon sources and organic nitrogen sources as culture medium components, it can significantly reduce the production cost of keratinase and the degradation cost of keratin waste, and can be better applicable to the industrial field. It has broad market prospects and industrial application value in promoting the degradation of feather waste and the conversion, recycling, and utilization of keratin resources.

[0051] Another embodiment of the present invention provides a nucleic acid molecule, the nucleic acid molecule comprises a gene sequence encoding a keratinase mutant (V99A / E116N / I117G / Y160S) as described above.

[0052] The advantages of the nucleic acid molecule over the prior art are the same as those of the keratinase mutants as described above compared to the prior art, and will not be described here.

[0053] Nucleic acid molecules include DNA molecules (eg genomic DNA or cDNA) and / or RNA molecules (eg mRNA), which may be single-stranded or double-stranded.

[0054] The sequence of nucleic acid molecules can be derived based on the keratinase mutant amino acid (AA) sequence through conventional means such as codon coding rules. The full-length sequence of nucleic acid molecules or fragments thereof can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0055] Exemplary, the gene (DNA) sequence encoding the keratinase mutant as described above is shown in SEQ ID NO.3.

[0056] Those skilled in the art will understand that due to the degeneracy of the genetic code, gene sequences different from those described above can also encode keratinase mutants of the present invention, and therefore, the sequences of the above examples should not be used as a limitation on the scope of protection of the present invention.

[0057] Optionally, the nucleic acid molecule also includes a gene sequence encoding expression elements such as signal peptide, promoter and / or terminator. The keratinase mutant is located downstream of the signal peptide, the promoter is located upstream of the signal peptide, and the terminator is located downstream of the keratinase mutant. That is, when all the above-mentioned expression elements are included, the gene connection sequence is from upstream to downstream: promoter-signal peptide-keratinase mutant-terminator. Signal peptides are used to guide keratinase to achieve secretion expression in host bacteria, making it easier to separate and purify. The promoter is used to initiate the transcription process of keratinase, and the terminator is used to terminate the transcription of keratinase to form a complete keratinase expression cassette. Signal peptides, promoters and terminators are adaptively selected according to the type of expression vector and host cell; and when keratin is actually expressed, each expression element can also be selected according to actual needs. If secretion expression is not required or the selected vector has a signal peptide, there is no need to add additional signal peptide genes upstream of the keratinase mutant gene.

[0058] Another embodiment of the present invention provides an expression vector comprising a nucleic acid molecule as described above.

[0059] Optionally, the expression vector includes a prokaryotic expression vector, an eukaryotic expression vector, or a viral expression vector (eg, lentivirus, adenovirus). Correspondingly, the host cells transformed or transfected with expression vectors can be prokaryotic cells and eukaryotic cells. The type of expression vector is selected. For example, when it is a prokaryotic expression vector, the host cell uses prokaryotic cells. Examples of commonly used prokaryotic cells include E. coli, Bacillus, Corynebacterium, Zystrophimosis, Streptomyces, etc.; when it is an eukaryotic expression vector, the host cell uses eukaryotic cells, and the examples of commonly used eukaryotic cells include S. cerevisiae, Bacillus yeast, filamentous fungi, etc.

[0060] Typical vectors include plasmids (such as pUC series, pET series, pWB series, pGEX series, pDXW series, pBR322, pEZ15A, pMA5, pPICZα, PIC9K, pSET152), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmecetes and microchromosomes. Plasmids are the most commonly used vectors, so in the context of the present invention, plasmids and vectors are used interchangeably if not specified.

[0061] As an important protein expression host, Streptomyces has the advantages of mature industrial culture, convenient genetic operation, high safety, strong metabolic activity, high protein secretion efficiency, rich transcription signals, large yield of exogenous proteins and no endotoxin production, which makes it have extensive application prospects and great development potential in the field of biotechnology. In a typical embodiment of the present invention, a Streptomyces expression system is adopted, and the expression vector used is Streptomyces integrated vector pSET152, the host (or receptor) cell is Streptomyces TK24, the signal peptide is Streptomyces vsi signal peptide, the promoter is Streptomyces kasOp* promoter, and the terminator is Streptomyces T1 terminator. Through the above-mentioned expression elements and keratinase mutant genes, the expression vector is constructed, and the recombinant Streptocytica transformed expression vector is obtained. After conventional fermentation and culture, the efficient secretion and expression of keratin can be achieved. The proportion of target enzymes in the extracellular fermentation broth is higher than 99%. Therefore, the fermentation supernatant of the recombinant strain can be used as a source of high-quality keratinase enzymes for hydrolyzing keratin, which is conducive to improving the production efficiency of keratin and reducing production costs, and is conducive to reducing the conversion and utilization costs of keratin waste such as feathers, and meeting the market's demand for green and efficient products.

[0062] The gene (DNA) sequence of the above-mentioned vsi signal peptide is shown in SEQ ID NO.4, the gene (DNA) sequence of the kasOp* promoter is shown in SEQ ID NO.5, and the gene (DNA) sequence of the T1 terminator is shown in SEQ ID NO.6; the fusion gene (DNA) sequence formed with the keratinase mutant through the aforementioned expression elements is shown in SEQ ID NO.7.

[0063] Another embodiment of the present invention provides a recombinant strain containing a nucleic acid molecule as described above or an expression vector as described above.

[0064] Optionally, the starting strain of the recombinant strain is Streptocytica, which may be specifically Streptocytica TK24.

[0065] Transformation or transfection of nucleic acid molecules or expression vectors into host cells can be done by various methods known in the art, including: CaCl 2 Transformation method, calcium phosphate-DNA coprecipitation, electroporation, gene gun bombardment, microinjection, junction transfer, liposome mediated transfection, liposome fusion, liposome transfection and protoplast fusion, etc.

[0066] The embodiments of the present invention also provide a method for preparing keratinase mutants, including the following steps:

[0067] Cultivate the recombinant strain as described above, centrifuge the fermentation culture medium, filter the supernatant, and collect the filtrate to obtain a crude enzyme solution containing keratinase mutants.

[0068] The embodiments of the present invention further provide the application of the keratinase mutant as described above in the treatment of keratin-containing substances, and provide related treatment methods; the keratin-containing substances include but are not limited to: feathers, hair, wool, etc., and are preferably bird feathers.

[0069] The method of treating keratin-containing substances by keratin-containing substances includes: mixing the keratin-containing substances or their crude enzyme solution with the keratin-containing substances and adding a reducing agent. The reducing agent can be dithiothreitol (DTT), and performing a catalytic reaction at 50-60°C.

[0070] Optionally, the concentration of reducing agent in the reaction solution is usually 1-5 mM.

[0071] The present invention will be further explained in the following in connection with specific examples. The experimental methods that do not specify specific conditions in the following examples are usually subject to conventional conditions, such as those described in the "Guidelines for Molecular Cloning Experiments (Fourth Edition)" published by Cold Spring Harbor Laboratory, or generally according to the conditions recommended by the manufacturer.

[0072] In the following examples, the main biological materials include:

[0073] 1) Strain: E. coli DH5α, commercially available; Streptomyces lividans, S. lividans TK24 is derived from Streptomyces group of John Innes Centre, Norwich, NR4 7UH, England.

[0074] 2) Expression vector: Streptomyces integrated vector pSET152. For vector information, see GenBank accessionnumber: AJ414670.1. This vector includes expression elements such as int, phiC31 attP site, Factor Xa site, oriT-RP4, TraJ, aac(3)IV, ori, and screening antibiotics: aporamycin and naphthyroidic acid.

[0075] 3) Enzymes and kits in vector construction: DNA ligase (T4 DNA Ligase, Catalog No.: C301-01), PCR reagent (2× KeyPo Master Mix (Dye Plus), Catalog No.: PK511-01), plasmid extraction kit (FastpurePlasmid Mini Kit-BOX 2, Catalog No.: DC201-01), gel purification and recovery kit (FastPure Gel DNAExtraction Mini Kit, Catalog No.: DC301-01), ClonExpress Ultra One StepCloning Kit V3, item number: C117-01) and others were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0076] 4) Culture medium: (a) Escherichia coli medium (LB medium): yeast extract 5g / L, peptone 10g / L and NaCl 5g / L, pH 7.0; (b) LB aporamycin resistance medium: Add 50μg / mL aporamycin to LB medium, pH 7.0; (c) LB kanamycin resistance medium: Add 50μg / mL kanamycin to LB medium, pH 7.0; (d) Conjugation and transfer medium (Modified-ISP4 medium): soluble starch 10g / L, K 2 HPO 4 1.0g / L, NaCl 1.0g / L, (NH4) 2 SO 4 2.0g / L, CaCO 3 2.0g / L, yeast extract 0.5g / L, peptone 1g / L, inorganic salt solution 1mL / L and agar 20g / L, inorganic salt solution includes: FeSO 4 1g / L,MnCl 2 1g / L,ZnSO 4 1g / L, pH 7.2-7.4, add MgCl before using the conjugation transfer medium 2 The final concentration was 30 mM; (e) Protein secretion expression medium (TSB medium): trypsin soy broth 30g / L, pH 7.2-7.4.

[0077] 1. Screening of keratinase mutants

[0078] Based on wild keratinase KT-ker, the gene is amplified by error-prone PCR to construct a keratinase mutant library. Then, the vsi signal peptide derived from Streptococcus azure (amino acid sequence: MRRTLKAVGAAAAAATCVLAATAGTAQA), the strong promoter kasOp* and terminator T1 are fused with each keratinase mutant gene, and the fusion gene is recombinant with the integrated expression vector pSET152, and the recombinant expression vector for keratinase gene secretion was successfully constructed. The method of indirect transfer of E. coli-Streptomyces sp. was introduced into Streptococcus azure TK24 for efficient secretion and expression. The keratinase activity of the fermentation supernatant was then measured, and the keratinase combination mutant KT1113 with increased enzyme activity was screened. Compared with the wild-type keratinase KT-ker, it contains the following amino acid replacement V99A / E116N / I117G / Y160S.

[0079] The amino acid sequence of wild-type keratinase KT-ker is as follows:

[0080] ETADATRASVAELARVSDAVLDADVPGTAWYTDAESGKLVVTADATVSAAELAQLKKAAGDKAGAVEIKRTPGTFNKLIAGGEAIYAAGGGRSSLGFN V RSSSGATYALTAGHCT EI ASTWYTNSGQTSLAGTRAGTSFPGNDYGLIRHSNASAADGRV Y LYNGSYRDITGAGNAYVGQTVQRSGSTTGLHSGRVTGLNATVNYGGGDIVSGLIQTNVCAEPGDSGGALFAGSTALGLTSGGSGNCRTGGTTFFQPVTEALSAYGVSII (see SEQ ID NO.1), italicized as the site to be mutated.

[0081] The amino acid sequence of the keratinase mutant KT1113 is as follows:

[0082] ETADATRASVAELARVSDAVLDADVPGTAWYTDAESGKLVVTADATVSAAELAQLKKAAGDKAGAVEIKRTPGTFNKLIAGGEAIYAAGGGRSSLGFN A RSSSGATYALTAGHCT NG ASTWYTNSGQTSLAGTRAGTSFPGNDYGLIRHSNASAADGRV S LYNGSYRDITGAGNAYVGQTVQRSGSTTGLHSGRVTGLNATVNYGGGDIVSGLIQTNVCAEPGDSGGALFAGSTALGLTSGGSGNCRTGGTTFFQPVTEALSAYGVSII (see SEQ ID NO.2), underlined as the mutation site.

[0083] The gene sequences used to construct keratinase expression vectors are shown in Table 1.

[0084] Table 1 Genes and primer sequences used in Examples of the present invention

[0085] Sequence name DNA sequence information KT1113 mutant GAGACCGCCGACGCCACCCGCGCCTCCGTCGCCGAGCTGGCCCGCGTCTCCGACGCCGTCCTGGACGCCGACGTCCCGGGCACCGCCTGGTACACCGACGCCGAGTCCGGCAAGCTGGTCGTCACCGCCGACGCCACCGTCTCCGCCGCCGAGCTGGCCCAGCTGAAGAAGGCCGCCGGCGACAAGGCCGGCGCCGTCGAGATCAAGCGCACCCCGGGCACCTTCAACAAGCTGATCGCCGGCGGCGAGGCCATCTACGCCGCCGGCGGCGGCCGCTCCTCCCTGGGCTTCAACGCGCGCTCCTCCTCCGGCGCCACCTACGCCCTGACCGCCGGCCACTGCACCAACGGCGCCTCCACCTGGTACACCAACTCCGGCCAGACCTCCCTGGCCGGCACCCGCGCCGGCACCTCCTTCCCGGGCAACGACTACGGCCTGATCCGCCACTCCAACGCCTCCGCCGCCGACGGCCGCGTCTCGCTGTACAACGGCTCCTACCGCGACATCACCGGCGCCGGCAACGCCTACGTCGGCCAGACCGTCCAGCGCTCCGGCTCCACCACCGGCCTGCACTCCGGCCGCGTCACCGGCCTGAACGCCACCGTCAACTACGGCGGCGGCGACATCGTCTCCGGCCTGATCCAGACCAACGTCTGCGCCGAGCCGGGCGACTCCGGCGGCGCCCTGTTCGCCGGCTCCACCGCCCTGGGCCTGACCTCCGGCGGCTCCGGCAACTGCCGCACCGGCGGCACCACCTTCTTCCAGCCGGTCACCGAGGCCCTGTCCGCCTACGGCGTCTCCATCATCTGA (See SEQ ID NO.3) vsi signal peptide ATGCGTCGCACCCTCAAGGCCGTGGGAGCAGCCGCGGCGGCGGCCACCTGCGTCCTCGCCGCGACGGCAGGCACCGCGCAGGCC (See SEQ ID NO.4) kasOp* promoter TGTTCACATTCGAACGGTCTCTGCTTTGACAACATGCTGTGCGGTGTTGTAAAGTCGTGGCCAGGAGAATACGACAGCGTGCAGGACTGGGGGAGTT (See SEQ ID NO.5) T1 terminator AGCAGTGCTGAAGGCCGTCAAGGGGCGTTCGTGGTGGGAAGA (See SEQ ID NO.6) KT1113 Expression Cassette TGTTCACATTCGAACGGTCTCTGCTTTGACAACATGCTGTGCGGTGTTGTAAAGTCGTGGCCAGGAGAATACGACAGCGTGCAGGACTGGGGGAGTTATGCGTCGCACCCTCAAGGCCGTGGGAGCAGCCGCGGCGGCGGCCACCTGCGTCCTCGCCGCGACGGCAGGCACCGCGCAGGCCGAGACCGCCGACGCCACCCGCGCCTCCGTCGCCGAGCTGGCCCGCGTCTCCGACGCCGTCCTGGACGCCGACGTCCCGGGCACCGCCTGGTACACCGACGCCGAGTCCGGCAAGCTGGTCGTCACCGCCGACGCCACCGTCTCCGCCGCCGAGCTGGCCCAGCTGAAGAAGGCCGCCGGCGACAAGGCCGGCGCCGTCGAGATCAAGCGCACCCCGGGCACCTTCAACAAGCTGATCGCCGGCGGCGAGGCCATCTACGCCGCCGGCGGCGGCCGCTCCTCCCTGGGCTTCAACGCGCGCTCCTCCTCCGGCGCCACCTACGCCCTGACCGCCGGCCACTGCACCAACGGCGCCTCCACCTGGTACACCAACTCCGGCCAGACCTCCCTGGCCGGCACCCGCGCCGGCACCTCCTTCCCGGGCAACGACTACGGCCTGATCCGCCACTCCAACGCCTCCGCCGCCGACGGCCGCGTCTCGCTGTACAACGGCTCCTACCGCGACATCACCGGCGCCGGCAACGCCTACGTCGGCCAGACCGTCCAGCGCTCCGGCTCCACCACCGGCCTGCACTCCGGCCGCGTCACCGGCCTGAACGCCACCGTCAACTACGGCGGCGGCGACATCGTCTCCGGCCTGATCCAGACCAACGTCTGCGCCGAGCCGGGCGACTCCGGCGGCGCCCTGTTCGCCGGCTCCACCGCCCTGGGCCTGACCTCCGGCGGCTCCGGCAACTGCCGCACCGGCGGCACCACCTTCTTCCAGCCGGTCACCGAGGCCCTGTCCGCCTACGGCGTCTCCATCATCTGAAGCAGTGCTGAAGGCCGTCAAGGGGCGTTCGTGGTGGGAAGA (See SEQ ID NO.7) Vector-F CCATCATCTGAAGCAGTGCTGAAGGCCGTCAAG (See SEQ ID NO.8) Vector-R GTGCGACGCATAACTCCCCCAGTCCTGCACGCTG (See SEQ ID NO.9) KT-keratin-F GGGGGAGTTATGCGTCGCACCCTCAAGGCCG (See SEQ ID NO.10) KT-keratin-R CAGCACTGCTTCAGATGATGGAGACGCCGTAG (See SEQ ID NO.11) error-prone-F CTGGGGGAGTTATGCGTCGCAC (See SEQ IDNO.12) error-prone-R CAGCACTGCTTCAGATGATGG (See SEQ IDNO.13)

[0086] 1.1 Construction of wild-type keratinase expression vector

[0087] The basic vector pSET152-kasOp*-T1 was constructed: The fusion fragment kasOp*-T1 of the promoter kasOp* (sequence see SEQ ID NO.5) and terminator T1 (sequence see SEQ ID NO.6) was synthesized by Qingke Biotechnology Co., Ltd., and the restriction endonuclease BamHI cleavage site was added before the promoter kasOp* and the restriction endonuclease EcoRV cleavage site was added after the terminator T1. The kasOp*-T1 fragment and vector pSET152 were cleaved with restriction enzymes BamHI and EcoRV respectively. After purification and recovery, the kasOp*-T1 fragment was linked to the expression vector pSET152, and Escherichia coli DH5α was transformed. After the sequence determination was correct, the basic expression vector pSET152-kasOp*-T1 was obtained.

[0088] The keratinase (KT-ker) gene derived from Streptomyces was optimized according to Streptomyces codon preference, and secretion signal peptide vsi was added at its N-terminus. The optimized nucleotide sequence was synthesized by Qingke Biotechnology Co., Ltd. The keratinase gene attached to the N-terminal signal peptide vsi is named SSvsi-KT-ker.

[0089] Using high-fidelity DNA polymerase, PCR amplification was used to obtain the linearized pSET152-kasOp*-T1 vector by using a high-fidelity DNA polymerase by using Vector-F (sequence see SEQ ID NO.8) and Vector-R (sequence see SEQ ID NO.9) primer pairs, and then the SSvsi-KT-keratin-F (sequence see SEQ ID NO.10) and KT-keratin-R (sequence see SEQ ID NO.11) primer pairs were amplified to obtain the SSvsi-KT-keratin gene. Purify the PCR product using a gel purification kit. The linearized pSET152-kasOp*-T1 and SSvsi-KT-ker gene fragments were linked through a one-step cloning kit. Then the ligation product was transformed into E. coli DH5α, and the transformed strain was cultured with aporamycin-resistant LB medium. The positive clones were screened for sequencing. The recombinant expression plasmid pSET152-kasOp*-SSvsi-KT-ker-T1 of the keratinase gene KT-ker with a completely correct sequence. The vector map is shown in the vector map of the keratinase gene KT-ker. Figure 1 ,KT-ker represents the wild-type keratinase gene, SSvsi signal represents the vsi signal peptide gene, kasOp* represents the promoter kasOp* gene, and T1 terminator represents the T1 terminator gene.

[0090] 1.2 Construction of keratinase mutant library

[0091] Random mutations were introduced through error-prone PCR (error-prone PCR), and protein engineering and mutant screening were carried out on keratinase. The primers of random PCR mutations were designed to be error-prone-F (sequence see SEQ ID NO.12) and error-prone-R (sequence see SEQ ID NO.13) and PCR amplification was performed using the aforementioned error-prone PCR primer using the random mutation PCR kit (purchased from Bomais, product name GeneMorph II RandomMutagenesis Kit). The kit can generate equal amounts of ATGC mutation chance during the amplification process. After the amplification was completed, the amplified products were recovered, linked to the linearized pSET152-kasOp*-T1 vector, converted into E. coli DH5α, coated on aporamycin-resistant LB plate, and cultured at 37°C inverted.

[0092] 1.3 Construction of recombinant strains of keratinase mutant library

[0093] The keratinase mutant library expression vector was transferred into the host cell Streptocytica by engaging transfer. The specific operations are as follows:

[0094] (1) Pick the transformants grown on the aporamycin-resistant LB plate and monoclonalize them into a 96-well plate. Add 50 μL of LB culture medium containing aporamycin to each well, culture at 37℃ and 220 rpm for about 6 hours, mix the bacteria in the well plate, transfer to 50 mL of LB culture medium containing aporamycin to expand the culture, culture at 37℃ and 220 rpm for about 3 hours, to OD 600 Centrifuge the bacteria for 4 min at 0.5-0.6,5000 rpm, wash them once with LB medium, and finally suspend them with 2 mL of LB medium to obtain the donor DH5α recombinant bacteria suspension.

[0095] (2) Inoculate the three parental ligation and transfer auxiliary strain ET12567 / pUB307 of E. coli-Streptomyces, take 500 μL of overnight culture bacterial solution and transfer it to 50 mL of LB medium containing kanamycin and chloramphenicol. Cultivate for about 37℃ and 220 rpm for about 3 hours to OD 600 Centrifuge the bacteria for 4 min at 0.5-0.6,5000 rpm, clean it once with LB medium, and suspend it with 2 mL of LB medium to obtain the suspension of auxiliary E. coli ET12567 / pUB307.

[0096] (3) Take 2 mL of spore suspension of Streptomyces S. lividans TK24, centrifuge at 10,000 rpm for 2 min, discard the glycerin, wash it once with TSB medium, and suspend the bacteria with 10 mL of TSB medium; heat shock in a 50℃ water bath for 10 min, shake and recover for 3 h at 30℃, then wash it once with TSB, and suspend the bacteria with 1 mL of TSB to obtain the receptor Streptomyces TK24 suspension.

[0097] (4) Take 200 μL of donor DH5α recombinant bacteria suspension, 200 μL of assisted E. coli ET12567 / pUB307 suspension and 100 μL of acceptor Streptomyces TK24 suspension, mix well, and apply the mixture to the conjugation transfer medium (add the final concentration of 30 mM MgCl before pouring the plate 2 ) Cultivate on a plate at 28℃ for 16-20 h. Then cover it with aporamycin (50 mg / mL of mother liquor) and nalidic acid (25 mg / mL of mother liquor), add 25 μL of aporamycin and 50 μL of nalidic acid to 1 mL of sterile water, mix evenly, cover the entire plate, that is, the plate contains a final concentration of 50 μg / mL of aporamycin and 50 μg / mL of nalidic acid; blow the plate in the ultra-clean table for about 1 h until completely dry. Cultivate at 28°C for 5-7 days, and the grown conjugates were picked on aporamycin and nalidicone antibiotic selection plates, and colonies were separated.

[0098] 1.4 Screening of keratinase mutants

[0099] Pick single colonies from the selection plates of the above steps and add 200 μL of TSB culture medium to each well, and culture at 28°C and 220 rpm for about 96 hours. The supernatant of the fermentation broth was taken for keratinase activity determination.

[0100] The determination of enzyme activity of keratinase refers to the national recommended standard "Spectrophotometry for the determination of acidic and neutral protease activity of feed additives, standard number: GB / T 28715-2012" and the recommended standard of the Ministry of Agriculture, "Spectrophotometry for the determination of keratinase activity, standard number: NY / T 4362-2023". The determination principle is: keratinase hydrolyzes keratin at a certain temperature and pH condition (if not specified, the temperature of the present invention is 60℃ and the pH is 10), and produces amino acids containing phenol groups, which are reduced by the forrin phenol reagent to produce tungsten blue. The solution absorption value is measured at 680 nm with an ultraviolet spectrophotometer. The enzyme activity is proportional to the absorbance, so the specific vitality of keratin can be calculated. The measurement method is as follows:

[0101] Blank group: Take out 20 μL of fermentation supernatant to a new 96-well plate, keep incubate at 60 ℃ for 2 min, add 40 μL of trichloroacetic acid, react at 60 ℃ for 10 min, add 20 μL of feather fragment solution containing 10 g / L, take out and let stand for 10 min, centrifuge at 12000r / min for 2 min, take 50 μL of supernatant and add 50 μL Na 2 CO 3 , 10 μL of forrin phenol reagent was added, and the color was developed at 60°C for 20 min. The solution absorbance value was measured at 680 nm using an ultraviolet spectrophotometer. Trichloroacetic acid is used to inactivate the enzyme, and the blank group is used to deduct background values.

[0102] Sample group: Take out 20 μL of the fermentation broth supernatant to a new 96-well plate, keep incubate for 2 min at 60℃, add 20 μL of the feather fragment solution, react for 10 min at 60℃, add 40 μL of trichloroacetic acid, take out and let stand for 10 min, 12000 r / min for 2 min, take 50 μL of supernatant and add 50 μL of Na2CO3, add 10 μL of forrin phenol reagent, develop color for 20 min at 60℃, and determine the absorbance value of the solution at 680 nm using an ultraviolet spectrophotometer.

[0103] By comparing the OD value between samples of different fermentation supernatants, the activity of secreting keratin expression in the supernatants of recombinant strains containing different mutant enzymes was determined. The recombinant strain of Streptocytica with significantly improved keratinase enzyme activity was extracted, genomic DNA was extracted for PCR, and the mutated gene fragments were amplified and the fragments were sequenced. The mutant KT1113 (V99A / E116N / I117G / Y160S) was screened for significantly improving keratinase activity without affecting its original enzymatic properties.

[0104] 2. Analysis of enzyme activity of wild-type keratinase and keratinase mutants

[0105] The mutant gene fragment obtained by the above amplification was linked to the linearized pSET152-kasOp*-T1 vector and converted into E. coli DH5α. To ensure accuracy, several clones were sequenced and the plasmid was purified from the E. coli clone with correct sequencing results, and the mutant expression vector pSET152-kasOp*-SSvis-KT1113-T1 was obtained. The vector map of the Figure 2 , KT1113 represents the keratinase mutant gene, SSvsi signal represents the vsi signal peptide gene, kasOp* represents the promoter kasOp* gene, and T1 terminator represents the T1 terminator gene.

[0106] The expression vector pSET152-kasOp*-SSvis-KT1113-T1 was transferred into the receptor cell Streptocytica TK24 through binding and transfer, and a mutant recombinant strain was obtained, named TK24::pSET152-kasOp*-SSvsi-KT1113-T1. Pick a single colony of the mutant recombinant strain and put it on a 96-well plate. Add 200 μL of TSB culture medium to each well, culture at 28℃ and 220 rpm for 96 hours, centrifuge the fermentation broth at 12,000 rpm for 15 minutes, and then filter the supernatant on a positive pressure filter with a 0.22 μm membrane to obtain the keratinase fermentation supernatant, and use a crude enzyme solution to determine the mutant enzyme activity. S. lividans TK24 (TK24:: pSET152-kasOp*-SSvsi-KT-ker-T1) transformed with pSET152-kasOp*-SSvsi-KT-ker-T1 was used as the control strain.

[0107] The results of the enzyme activity determination of mutant and wild-type keratinase crude enzyme liquid are shown in Table 2. It can be seen that the enzyme activity of the four-site mutant KT1113 of keratinase is 2127.58 U / mL, and the enzyme activity of wild-type keratinase is 1662.17 U / mL; the enzyme activity of keratinase KT1113 is 28% higher than that of wild-type keratinase KT-ker, and the enzyme activity of the keratinase mutant KT1113 after protein engineering has been significantly improved.

[0108] Table 2 Enzyme activity of mutant and wild-type keratinase crude enzyme solution (test conditions 60°C, pH 10)

[0109] Keratinase Genotype / mutation site Enzyme activity (U / mL) KT-ker Wild type 1662.17 KT1113 V99A / E116N / I117G / Y160S 2127.58

[0110] In addition, polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the mutant crude enzyme solution to verify the size and expression of the keratinase mutant KT1113 secreted. See the results. Figure 3 . It can be seen that the fermentation supernatant has a clear keratinase band at 25 kDa, which is consistent with its theoretical size of 26.7 kDa. At the same time, the keratinase concentration in the supernatant is high, its content is 1.45 g / L, and its purity exceeds 99%.

[0111] 3. Establishment and evaluation of feather degradation technology of keratinase mutants

[0112] Take 10 μL of TK24::pSET152-kasOp*-SSvsi-KT1113-T1 spore suspension, inoculate it into a PA bottle containing 5 mL TSB medium and 25 μg / mL aporamycin, and culture at 28°C for 2 days to prepare seed liquid. Transfer 2 mL of seed solution to 50 mL of TSB medium, culture at 28°C for 4 days, centrifuge, collect the supernatant, which is the fermentation supernatant of recombinant keratinase, and then filter the supernatant on a positive pressure filter with a 0.22 μm membrane to obtain the crude keratinase enzyme solution.

[0113] To the crude keratinase enzyme solution, DTT with a final concentration of 2 mM was added, and then the complete feather was added. The feather degradation was tested at 50 °C. The results were shown in Figure 4 , Among them, the picture on the left is the actual image of the feathers when they were put into the keratinase KT1113 enzyme solution for 0 h, and the picture on the right is the actual image of the keratinase KT1113 enzyme solution and the feathers for 24 h. It can be seen from the figure that the keratinase mutants show good feather degradation effects, and most feathers can degrade within 24 hours. It is shown that the recombinant keratinase KT1113 of the present invention and the reducing agent DTT can effectively degrade feathers.

[0114] The crude protein content in feathers accounts for about 80%, which is a potentially excellent protein resource. The products of feather waste degraded by keratinase are rich in amino acids (such as cystine, glycine, proline) and polypeptide nutrients. It has been proven to be able to partially replace relatively expensive protein feeds such as fish meal and soybean meal, and can also improve environmental pollution problems caused by feather waste. Therefore, the development of highly active keratinase has important practical significance and research value. The four-site mutant KT1113 of Streptomyces keratinase provided by the present invention can directly degrade feathers through mild reactions under the presence of DTT. Compared with the traditional keratin-producing bacteria that use carbohydrate carbon sources and organic nitrogen sources for fermentation and hydrolysis, it can significantly reduce the conversion cost of keratin, solve the problem of efficient and low-cost hydrolysis of feathers, and has the advantages of good degradation effect and green environmental protection.

[0115] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A keratinase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that The invention comprises a gene sequence encoding the keratinase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein The gene sequence of the keratinase mutant is shown in SEQ ID NO.

3.

4. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule also includes a gene sequence encoding a signal peptide, a promoter and a terminator. The keratinase mutant is located downstream of the signal peptide, the promoter is located upstream of the signal peptide, and the terminator is located downstream of the keratinase mutant.

5. The nucleic acid molecule according to claim 4, wherein The signal peptide is a vsi signal peptide, the gene sequence of the vsi signal peptide is shown in SEQ ID NO.4, the promoter is a kasOp* promoter, the gene sequence of the kasOp* promoter is shown in SEQ ID NO.5, the terminator is a T1 terminator, and the gene sequence of the T1 terminator is shown in SEQ ID NO.

6.

6. An expression vector or recombinant strain, characterized in that: The recombinant strain comprises the expression vector, and the expression vector comprises the nucleic acid molecule according to any one of claims 3 to 5.

7. The expression vector or recombinant strain according to claim 6, characterized in that The expression vector is the Streptomyces integration vector pSET152, and the recombinant strain is Streptomyces lividans TK24.

8. Use of the keratinase mutant according to claim 1 in treating keratin-containing materials.

9. Use of the keratinase mutant according to claim 8 in treating keratin-containing materials, characterized in that: The keratin-containing material is selected from feathers or hair.

10. Use of the keratinase mutant according to claim 9 in treating keratin-containing materials, characterized in that: The hair is wool.

11. Use of the keratinase mutant according to claim 9 in treating keratin-containing materials, characterized in that: The method for treating keratin-containing materials with the keratinase mutant comprises: The keratinase mutant or its crude enzyme solution is mixed with keratin-containing material, a reducing agent is added, and a catalytic reaction is carried out at 50-60° C. wherein the reducing agent is dithiothreitol.

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