CN117247923A - Keratinase mutant, engineering bacterium, preparation and application thereof - Google Patents
Keratinase mutant, engineering bacterium, preparation and application thereof Download PDFInfo
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- CN117247923A CN117247923A CN202311021723.5A CN202311021723A CN117247923A CN 117247923 A CN117247923 A CN 117247923A CN 202311021723 A CN202311021723 A CN 202311021723A CN 117247923 A CN117247923 A CN 117247923A
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- 102220471014 Dual specificity mitogen-activated protein kinase kinase 6_S207A_mutation Human genes 0.000 claims abstract description 77
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
- D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
- D06M2101/02—Natural fibres, other than mineral fibres
- D06M2101/10—Animal fibres
- D06M2101/12—Keratin fibres or silk
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M2200/00—Functionality of the treatment composition and/or properties imparted to the textile material
- D06M2200/45—Shrinking resistance, anti-felting properties
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Textile Engineering (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a keratinase mutant with reduced specific activity of casein enzyme obtained through site-directed mutagenesis so as to reduce the degradation effect of the casein enzyme activity in keratinase on casein components in wool, so that wool fibers have better performance. The mutant is obtained by mutating Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A or Q241A/S207A/S213A based on the wild type keratinase shown in SEQ ID NO. 1. The activity of the casein enzyme in the mutant is obviously reduced, which is beneficial to expanding the application of the mutant in the wool textile field.
Description
Technical field:
the invention belongs to the technical field of bioengineering, and particularly relates to a keratinase mutant with reduced specific activity of casein enzyme obtained through site-directed mutagenesis so as to reduce the degradation effect of the casein enzyme activity in keratinase on casein components in wool, so that wool fibers have better performance.
The background technology is as follows:
keratinase (keratanase) is a specific protease which, unlike conventional proteases, has a wide range of substrate specificities for a variety of insoluble, keratin-rich substrates, and can degrade keratin substrates including feathers, wool, nails, hair, and the like. Meanwhile, keratinase belongs to the class of proteases, and can degrade other common protein substrates, such as casein and the like.
Wool is an important raw material in textile industry, and wool cloth is a soft, warm and comfortable textile, and the structure of the wool is curled and is divided into a scale layer, a leather layer and a medulla layer. The flake layer mainly comprises keratin, so that wool has abrasion resistance and pollution resistance; the leather layer is a main component of wool fiber and is composed of proteins such as casein, so that the wool fiber has the advantages of good elasticity, good warmth retention property and the like.
Keratinase can degrade various soluble and insoluble proteins such as keratin, and gradually hydrolyze into polypeptides, oligopeptides and free amino acids. In industry, wool fibers are provided with scale layers arranged from roots to tips, the scale layers increase the gloss of the wool fabric, improve the pollution resistance and abrasion resistance of the wool fabric, and simultaneously bring serious felting defects to the wool fabric, so that the wool enzyme method anti-felting finishing effect is improved, and the scale layers of the wool are hydrolyzed by keratinase generally.
Bacillus is the main production strain of protease, and has the remarkable advantages of short fermentation period, rich materials and the like. In addition, bacillus has also made great progress in secretion and expression of foreign proteins, and has established effective bacillus expression system, and bacillus has the advantages of protein expression purification and secretion of active protein. Bacillus is now becoming increasingly widely used as a genetically engineered expression system.
Therefore, in the invention, the keratinase mutant with reduced specific activity of the casein is obtained by carrying out molecular modification on a keratinase gene derived from bacillus licheniformis (Bacillus licheniformis) and carrying out high-throughput screening by using a bacillus subtilis expression system.
The invention comprises the following steps:
as the keratinase has the activity of the caseinase, the caseins in wool can be degraded to different degrees in the application process, so that wool fibers are damaged to different degrees, and in order to reduce the specific activity of the caseinase, the keratinase with reduced specific activity of the caseinase is obtained, and the existing properties of the keratinase are required to be further improved.
The invention aims to obtain a keratinase mutant with reduced specific activity of casein, and the keratinase from bacillus licheniformis is a keratinase gene with relatively higher activity in the keratinase which is reported at present, so that the keratinase mutant takes the keratinase as a starting gene to modify the reduction of the activity of the casein. The invention constructs a recombinant expression vector pBSA43-bliker by using a keratinase gene (bliker) derived from bacillus licheniformis (Bacillus licheniformis) and a shuttle vector pBSA43, and expresses the recombinant expression vector pBSA43-bliker in bacillus subtilis WB 600; the key substrate binding region and the key acting amino acid site are determined through bioinformatics software analysis, the overlapping PCR technology is utilized to carry out site-directed mutagenesis on a bacillus licheniformis-derived keratinase gene (bliker), and the national standard method (Fu Lin Fenfa) is utilized to screen, so as to select keratinase mutants with reduced specific activity of the caseinase.
The technical route for achieving the purpose of the invention is summarized as follows:
site-directed mutagenesis of the keratinase gene (bliker) from Bacillus licheniformis (B.lichenifermis), screening with the Bacillus subtilis WB600 expression system to obtain the KER mutant S207A, S213A, T237A, Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q A/S207A/S213A, and the encoding genes blikerm1, blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8 and blikerm9 thereof, and the screened KER mutant with reduced specific activity of the casein enzyme is efficiently expressed in bacillus amyloliquefaciens, and the KER mutant with lower specific activity of the casein enzyme is obtained through fermentation, extraction and other technologies.
One of the technical schemes provided by the invention is a keratinase mutant, which is obtained by at least one of S207A, S213A, T237A, Q241A mutation occurring on the basis of a wild keratinase zymogen region shown in SEQ ID NO. 1;
further, the keratinase mutant is an S207A mutant, and the amino acid sequence is shown as SEQ ID NO. 3;
furthermore, the nucleotide sequence of the coding gene blikerm1 of the S207A mutant is shown as SEQ ID NO. 4;
further, the keratinase mutant is an S213A mutant, and the amino acid sequence is shown in SEQ ID NO. 5;
furthermore, the coding gene blikerm2 of the S213A mutant has a nucleotide sequence shown in SEQ ID NO. 6;
further, the keratinase mutant is a T237A mutant, and the amino acid sequence is shown as SEQ ID NO. 7;
furthermore, the nucleotide sequence of the coding gene blikerm3 of the T237A mutant is shown as SEQ ID NO. 8;
further, the keratinase mutant is a Q241A mutant, and the amino acid sequence is shown as SEQ ID NO. 9;
furthermore, the coding gene blikerm4 of the Q241A mutant has a nucleotide sequence shown in SEQ ID NO. 10;
further, the keratinase mutant is a Q241A/S207A mutant, and the amino acid sequence is shown as SEQ ID NO. 11;
furthermore, the coding gene blikerm5 of the Q241A/S207A mutant has a nucleotide sequence shown in SEQ ID NO. 12;
further, the keratinase mutant is a Q241A/S213A mutant, and the amino acid sequence is shown as SEQ ID NO. 13;
furthermore, the coding gene blikerm6 of the Q241A/S213A mutant has a nucleotide sequence shown in SEQ ID NO. 14.
Further, the keratinase mutant is a Q241A/T237A mutant, and the amino acid sequence is shown as SEQ ID NO. 15;
furthermore, the coding gene blikerm7 of the Q241A/T237A mutant has a nucleotide sequence shown in SEQ ID NO. 16;
further, the keratinase mutant is Q241A/S207A/T237A mutant, and the amino acid sequence is shown as SEQ ID NO. 17;
furthermore, the coding gene blikerm8 of the Q241A/S207A/T237A mutant has a nucleotide sequence shown in SEQ ID NO. 18;
further, the keratinase mutant is Q241A/S207A/S213A mutant, and the amino acid sequence is shown as SEQ ID NO. 19;
furthermore, the coding gene blikerm9 of the Q241A/S207A/S213A mutant has a nucleotide sequence shown in SEQ ID NO. 20.
The second technical scheme provided by the invention is a recombinant plasmid or recombinant strain containing the mutant coding gene;
further, the expression vector adopted by the recombinant plasmid is pBSA43;
further, the host cell adopted by the recombinant strain is bacillus subtilis or bacillus amyloliquefaciens;
further, the host cell is bacillus subtilis WB600 or bacillus amyloliquefaciens CGMCC No.11218;
preferably, the recombinant strain is obtained by connecting a mutant coding gene with an expression vector pBSA43 and then expressing the mutant coding gene in a host bacillus amyloliquefaciens CGMCC No.11218.
The third technical scheme provided by the invention is the application of the recombinant plasmid or recombinant strain, in particular the application in the production of the keratinase mutant in the first technical scheme.
The fourth technical scheme provided by the invention is the application of the keratinase mutant in the first technical scheme, particularly the application in hydrolyzing keratin or the application in wool anti-felting treatment.
The experimental scheme of the invention is as follows:
1. the obtaining of the KER mutant coding gene comprises the following steps:
(1) The wild KER coding gene bliker shown in SEQ ID No.2 is taken as a starting gene, an expression vector pBSA43-bliker is constructed, and site-directed mutagenesis is carried out.
(2) The mutated KER coding gene is transferred into bacillus subtilis WB600 after constructing recombinant plasmid, and the specific activity of the casein enzyme is measured by using a national standard method.
(3) The KER mutant with reduced specific activity of the casein enzyme relative to wild type keratinase is obtained through screening, the KER mutant coding genes blikerm1, blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8 and blikerm9 are obtained through sequencing, and plasmids pBSA43-blikerm1, pBSA43-blikerm2, pBSA43-blikerm3, pBSA43-blikerm4, pBSA43-blikerm5, pBSA43-blikerm6, pBSA43-blikerm7, pBSA43-blikerm8 and pBSA43-blikerm9 containing the KER mutant coding genes with reduced specific activity of the casein enzyme are stored.
Fermenting and culturing the keratinase mutant with the reduced specific activity of the screened caseinase, and purifying to obtain the KER protein.
2. The bacillus amyloliquefaciens recombinant strain containing the KER mutant encoding gene and the process for preparing keratinase with reduced specific activity of the caseinase by using the bacillus amyloliquefaciens recombinant strain comprise the following steps:
(1) The KER mutant encoding gene blikerm1-9 is connected with the bacillus amyloliquefaciens expression plasmid pBSA43 to obtain a new recombinant plasmid pBSA43-blikerm1-9;
(2) Transferring the recombinant plasmid pBSA43-blikerm1-9 into bacillus amyloliquefaciens CGMCC No.11218, screening with resistance to kanamycin (Kan), enzyme cutting to verify to obtain recombinant strain, and culturing and fermenting the recombinant strain to obtain keratinase.
The following definitions are employed in the present invention:
1. nomenclature of amino acids and DNA nucleic acid sequences
Using the accepted IUPAC nomenclature for amino acid residues, in single letter or three letter codes. The DNA nucleic acid sequence uses accepted IUPAC nomenclature.
2. Identification of keratinase mutants
"amino acid substituted at the original amino acid position" is used to denote the mutated amino acid in the KER mutant. As Ser207Ala, it is indicated that the amino acid at position 207 is replaced by Ser of the wild-type KER with Ala, the numbering of the position corresponding to the amino acid sequence numbering of the proenzyme region of the wild-type KER in SEQ ID NO. 1.
In the present invention, the lower case italics bliker represents the gene encoding the wild type keratinase KER, the lower case italics blikerm1 represents the gene encoding the mutant S207A, the lower case italics blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8, blikerm9 represent the gene encoding the mutant S213A, T237A, Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A/S213A, respectively, and the specific information is as follows.
The beneficial effects are that:
1. the invention uses site-directed mutagenesis technology to mutate the wild type of KER to obtain mutants S207A, S213A, T237A, Q241A, Q A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A/S213A with the specific activity of the casein enzyme reduced relative to the wild type at 60 ℃, the specific activity of the casein enzyme of the wild type KER is 949.55U/mg in a bacillus subtilis expression system, and the specific activity of the casein enzyme of the mutants is 827.48U/mg, 861.69U/mg, 826.70U/mg, 812.76U/mg, 744.11U/mg, 751.97U/mg, 760.97U/mg, 674.88U/mg, 700.41U/mg respectively. The specific activities of the keratinase of the wild-type KER and the mutants are 750.14U/mg, 703.35U/mg, 775.52U/mg, 752.30U/mg, 772.12U/mg, 796.19U/mg, 789.57U/mg, 776.19U/mg, 742.36U/mg and 665.38U/mg respectively.
2. Wild-type KER and KER mutants S207A, S213A, T237A, Q241, A, Q A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A/S213A fermented caseinase activity values in the Bacillus amyloliquefaciens expression system were 9685.43U/mL, 8026.53U/mL, 8272.23U/mL, 8184.39U/mL, 7221.25U/mL, 7515.51U/mL, 7369.36U/mL, 7001.04U/mL, 6141.43U/mL, 6583.82U/mL.
3. The invention realizes the efficient expression and preparation of the KER mutant with improved enzyme activity by using the bacillus amyloliquefaciens expression system.
Description of the drawings:
FIG. 1 is a PCR amplification electrophoretogram of wild-type procaspase gene
Wherein: m is DNA Marker,1 is keratinase zymogen gene bliker;
FIG. 2 is a diagram showing the cleavage assay of pBSA43-bliker plasmid, wherein: m is DNA Marker,1 is pBSA43-bliker, and the two enzyme digestion patterns of BamHI and SmaI are adopted.
The specific embodiment is as follows:
the technical contents of the present invention will be further described with reference to examples, but the present invention is not limited to these examples, and the scope of the present invention is not limited to the following examples.
The culture medium used in the embodiment of the invention is as follows:
LB medium (g/L): yeast extract 5.0, tryptone 10.0, naCl 10.0, the balance being water;
2% agar was added to the solid medium.
Fermentation medium (g/L): corn flour 64, bean cake flour 40, amylase 2.7, na 2 HPO 4 4，KH 2 PO 4 0.3, the balance being water; preserving heat at 90 ℃ for 30min and sterilizing at 121 ℃ for 20min.
In the present invention, the zymogen region sequence of the wild type keratinase KER is shown in SEQ ID NO. 1: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.
In the present invention, the zymogen region sequence of the keratinase S207A mutant is shown in SEQ ID NO. 3: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGAYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.
In the present invention, the zymogen region sequence of the keratinase S213A mutant is shown in SEQ ID NO. 5: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVAGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.
In the present invention, the zymogen region sequence of the keratinase T237A mutant is shown in SEQ ID NO. 7: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSAAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.
In the present invention, the zymogen region sequence of the keratinase Q241A mutant is shown in SEQ ID NO. 9: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKAAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.
The invention will be further illustrated by the following examples.
EXAMPLE 1 obtaining wild-type keratinase Gene
1. Genomic DNA of Bacillus licheniformis (Bacillus licheniformis ATCC 14580) was extracted using a kit (OMEGA: bacterial DNA Kit) as follows:
(1) Strains were inoculated onto LB solid plates with an inoculating loop and incubated overnight at 37 ℃.
(2) Single colonies were picked from plates for culturing the cells and inoculated into a liquid test tube medium, and shake-cultured at 37℃and 220r/min overnight.
(3) 3mL-5mL of the bacterial liquid is placed in a sterilized EP tube, and the bacterial liquid is centrifuged for 2min at 12000r/min, and the supernatant is discarded.
(4) 200 mu L of sterile water is added into an EP tube to resuspend the thalli, 50 mu L of lysozyme is added, and the mixture is blown and sucked uniformly, and the temperature is kept at 37 ℃ for 20min.
(5) 100. Mu.L of BTL buffer and 20. Mu.L of proteinase K are added into an EP tube, mixed by vortex oscillation, kept at 55 ℃ for 40min, and mixed by oscillation every 20min.
(6) Add 5. Mu.L RNase and mix upside down several times and leave at room temperature for 10min.
(7) Centrifuge 12000r/min for 2min, remove undigested fractions, transfer supernatant to fresh EP tube, add 220. Mu.L BDL buffer, water bath at 65℃for 15min.
(8) 220 mu L absolute ethyl alcohol is added, and the mixture is blown and sucked uniformly.
(9) Transferring the liquid in the EP pipe into a recovery column, standing for 1min, centrifuging for 1min at 12000r/min, pouring the filtrate into the recovery column again, repeating for two times, and pouring out the waste liquid.
(10) Add 500. Mu.L HBC buffer, centrifuge for 1min at 12000r/min, discard the filtrate.
(11) 700 mu L DNA wash buffer was added, left stand for 1min, centrifuged at 12000r/min for 1min, and the filtrate was discarded.
(12) 500 mu L DNA wash buffer was added, left stand for 1min, centrifuged at 12000r/min for 1min, and the filtrate was discarded.
(13) 12000r/min was allowed to air-space for 2min, the waste tube was discarded, and the recovery column was placed in a new EP tube.
(14) And (5) placing in a metal bath at 55 ℃ for drying for 10min.
(15) 50. Mu.L of 55℃sterile water was added, left to stand at room temperature for 5min, centrifuged at 12000r/min for 2min, and the recovery column was discarded, and the liquid in the EP tube was the genome.
2. The genome of the extracted bacillus licheniformis is taken as a template, a pair of primers are designed at the upstream and downstream of an ORF frame, and restriction enzyme sites BamHI and SmaI are respectively introduced, and the amplification primers of the keratinase gene bliker are as follows:
the upstream primer P1:
5’-CGCGGATCC ATGATGAGGAAAAAGAGTTTTTGGCT-3’
downstream primer P2:
5’-TCCCCCGGGTTAGTGATGATGATGATGATGTTGAGCGGCACCTTCGA-3’
p1 and P2 are used as an upstream primer and a downstream primer, and the bacillus licheniformis genome is used as a template for amplification.
The reaction system for amplification is as follows:
| upstream primer P1 | 2.0μL |
| Downstream primer P2 | 2.0μL |
| DNA template | 2.0μL |
| PrimerStar Max enzyme | 25μL |
| ddH 2 O | 19μL |
The amplification procedure was: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 57℃for 20s, extension at 72℃for 6s, 30 cycles; extending at 72℃for 10min. The PCR amplified product was subjected to 0.8% agarose gel electrophoresis to obtain 1140bp band (FIG. 1), and the PCR product was recovered by using a small amount of DNA recovery kit to obtain the wild-type procaryotic keratinase gene bliker (SEQ ID NO. 2) of the present invention. The bliker and pBSA43 plasmids are respectively subjected to double enzyme digestion by restriction enzymes BamHI and SmaI, the bliker recovered by the gel digestion is connected with a vector pBSA43 to obtain a recombinant plasmid pBSA43-bliker, enzyme digestion verification is shown in figure 2, and the recombinant plasmid pBSA43-bliker is transformed into escherichia coli JM109 and bacillus subtilis WB600 to obtain bacillus subtilis recombinant strain WB600/pBSA43-bliker.
EXAMPLE 2 construction of a library of keratinase mutants screening of keratinase mutants with reduced specific Casein Activity
1. Site-directed mutagenesis is performed based on the overlapping PCR technology, a novel keratinase is constructed, and mutation primers are respectively designed aiming at different mutation sites as follows:
in the first step of overlapping PCR reaction system, P1 is used as an upstream primer, 207-R, 213-R, 237-R and 241-R are respectively used as downstream primers, and plasmid pBSA43-bliker is used as a template to carry out PCR1 reaction to respectively obtain upstream fragments; PCR1 reaction was performed using P2 as the upstream primer, 207-F, 213-F, 237-F, 241-F as the downstream primer, and plasmid pBSA43-bliker as the template, respectively, to obtain downstream fragments. Taking the S207A mutation as an example:
the reaction system for amplifying the upstream fragment is as follows:
| P1 | 2μL |
| 207-R | 2μL |
| plasmid pBSA43-bliker | 2μL |
| PrimerStar Max enzyme | 25μL |
| ddH 2 O | 19μL |
The reaction system for amplifying the downstream fragment is as follows:
| P2 | 2μL |
| 207-F | 2μL |
| plasmid pBSA43-bliker | 2μL |
| PrimerStar Max enzyme | 25μL |
| ddH 2 O | 19μL |
The amplification procedure was: pre-denaturation at 98 ℃ for 30min; denaturation at 98℃for 10s, annealing at 57℃for 20s, extension at 72℃for 6s for 30 cycles; extending at 72℃for 10min.
2. And (3) cutting the gel, recovering the upstream fragment and the downstream fragment, and then carrying out PCR2, wherein the reaction system is as follows:
| upstream fragment | 2.0μL |
| Downstream fragment | 2.0μL |
| PrimerStar Max enzyme | 25μL |
| ddH 2 O | 17μL |
The amplification procedure was: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 57℃for 20s, extension at 72℃for 6s, 5 cycles; extending at 72℃for 10min.
3. After the end of PCR2, 2. Mu.L of each of the primers P1 and P2 was added to the system, and the PCR 3 amplification procedure was performed as follows: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 10s, annealing at 57℃for 20s, extension at 72℃for 6s, 30 cycles; extending at 72℃for 10min. The PCR amplified product was subjected to 0.8% agarose gel electrophoresis, and the PCR product was recovered using a small amount of DNA recovery kit to obtain the keratinase site-directed mutant gene bliker S207A. The corresponding primers were replaced in the same manner to obtain other keratinase site-directed mutant genes bliker S213A, blikerT237A, blikerQ241A.
4. The keratinase site-directed mutant gene bliker S207A, blikerS213A, blikerT237A, blikerQ241A is connected with an expression vector pBSA43, then is transformed into Escherichia coli JM109, and plasmids thereof are extracted to obtain recombinant plasmids pBSA43-bliker S207A, pBSA43-bliker S213A, pBSA43-bliker T237A, pBSA43-bliker Q241A.
Then the recombinant plasmid pBSA43-bliker S207A, pBSA-bliker S213A, pBSA-bliker T237A, pBSA-bliker Q241A is transformed into bacillus subtilis WB600 to obtain recombinant strain WB600/pBSA43-bliker S207A, WB600/pBSA43-bliker S213A, WB600/pBSA43-bliker T237A, WB/pBSA 43-bliker Q241A. The transformants transformed with the subtilis were activated on a new partitioned streaked Kan plate, cultivated upside down at 37℃for 12 hours, and then the mutant strains were screened, as follows:
(1) Under aseptic conditions, mutant single colonies and wild type single colonies (i.e., WB600/pBSA 43-bliker) were picked and inoculated into 5mL liquid LB tubes containing Kan resistance, and cultured overnight at 37℃with shaking at 220 r/min.
(2) 1mL of the bacterial liquid in the test tube is sucked, and the bacterial liquid is added into 50mL of liquid LB culture medium containing Kan resistance, and the liquid is subjected to shaking culture at 37 ℃ for 48h at 220 r/min.
(3) After the cultivation, the flask was taken out, and the bacterial concentration of the bacterial liquid was measured at OD 600.
(4) The bacterial liquid is received into a 50mL centrifuge tube, put into a centrifuge, centrifuged for 10min at 8000r/min, and the supernatant is taken as enzyme liquid for enzyme activity measurement.
5. The national standard method (Fu Lin Fenfa) is used for measuring the specific activities of the caseinase and the keratinase:
keratinase hydrolyzes casein or keratin at a certain temperature and pH (the temperature of the invention is 60 ℃ and the pH is 10, as not specifically described), generates amino acid containing phenolic groups, reduces the amino acid with Fu Lin Fen reagent to generate tungsten blue, and measures the absorbance of the solution at 680nm by using an ultraviolet spectrophotometer. The specific activities of the caseinase and the keratinase can be calculated in proportion to the absorbance. The measurement method is as follows:
adding 1mL of enzyme solution into the blank group, preserving heat for 2min at 60 ℃,adding 2mL of trichloroacetic acid, reacting at 60deg.C for 10min, adding 1mL of casein or keratin (10 g/L) solution, taking out, standing for 10min, centrifuging for 2min at 12000r/min, collecting 0.5mL of supernatant, adding 2.5mL of Na 2 CO 3 Fu Lin Fen reagent 0.5mL was added and developed at 60℃for 20min, and the absorbance of the solution was measured with a UV spectrophotometer at 680nm using a 10mm cuvette.
Adding 1mL enzyme solution into sample group, keeping the temperature at 60 ℃ for 2min, adding 1mL casein or keratin (10 g/L) solution, reacting for 10min at 60 ℃, adding 2mL trichloroacetic acid, taking out, standing for 10min, centrifuging for 2min 12000r/min, taking 0.5mL supernatant, adding 2.5mL Na 2 CO 3 Fu Lin Fen reagent 0.5mL was added and developed at 60℃for 20min, and the absorbance of the solution was measured with a UV spectrophotometer at 680nm using a 10mm cuvette.
Subtracting the OD value of the blank group from the OD value measured by the sample group to obtain delta OD, and substituting the delta OD into the following formula to calculate the corresponding enzyme activity:
(N is the dilution of the sample)
Meanwhile, the mutant strain plasmid pBSA43-bliker S207A, pBSA-bliker S213A, pBSA-bliker T237A, pBSA-bliker Q241A was proposed to be sent to Jin Weizhi company for sequencing, after each mutation site was determined to be correct, the gene of the keratinase mutant in which the 207 th amino acid Ser was mutated to Ala was named bliker m1, the gene of the keratinase mutant in which the 213 th amino acid Ser was mutated to Ala was named bliker m2, the gene of the keratinase mutant in which the 237 th amino acid Thr was mutated to Ala was named bliker m3, and the gene of the keratinase mutant in which the 241 th amino acid Gln was mutated to Ala was named bliker m4.
Screening to obtain mutant Q241A with lowest specific activity of casein enzyme at 60 ℃ and lower than that of wild type. Further, the mutant Q241A and S207A, S213A, T A are combined and mutated to obtain mutant Q241A/S207A, Q241A/S213A, Q A/T237A, Q A/S207A/T237A, Q241A/S207A/S213A and encoding genes blikerm5, blikerm6, blikerm7, blikerm8 and blikerm9. The specific combinatorial mutation process is as follows:
based on the coding gene of mutant Q241A, a combined mutation primer is designed, P1 is used as an upstream primer, Q241A/S207A-R, Q241A/S213A-R, Q A/T237A-R are respectively used as a downstream primer, P2 is used as an upstream primer, Q241A/S207A-F, Q241A/S213A-F, Q241A/T237A-F are respectively used as a downstream primer, and a plasmid pBSA43-bliker Q241A is used as a template for carrying out PCR1 amplification reaction to respectively obtain an upstream fragment and a downstream fragment. And (3) cutting the gel, recovering the upstream and downstream fragments, then carrying out PCR2, adding primers P1 and P2 into a system after the PCR2 is finished, and carrying out PCR 3 to obtain Q241A/S207A, Q A/S213A, Q241A/T237A coding genes blikerm5, blikerm6 and blikerm7, wherein the steps 1, 2 and 3 are implemented by specific systems and condition references.
Similarly, based on mutant Q241A/S207A, a combined mutation primer Q241A/S207A/T237A-F, Q241A/S207A/T237A-R is designed for amplification to obtain a gene blikerm8 encoding the Q241A/S207A/T237; the combined mutation primer Q241A/S207A/S213A-F, Q A/S207A/S213A-R is designed to amplify to obtain the coding gene blikerm9 of Q241A/S207A/S213A.
6. The activities of the caseins and keratins of the mutants were measured in the same manner as in steps 4 and 5 of example 2, and the specific activities of the caseins and keratins of the wild-type KER and the mutants at 60℃were finally calculated, as shown in the following table.
| Keratinase | Specific activity of casein enzyme (U/mg) | Specific activity of keratinase (U/mg) |
| WT | 949.55 | 750.14 |
| S207A | 827.48 | 703.35 |
| S213A | 861.69 | 775.52 |
| T237A | 826.70 | 752.30 |
| Q241A | 812.76 | 772.12 |
| Q241A/S207A | 744.11 | 796.19 |
| Q241A/S213A | 751.97 | 789.57 |
| Q241A/T237A | 760.97 | 776.19 |
| Q241A/S207A/T237A | 674.88 | 742.36 |
| Q241A/S207A/S213A | 700.41 | 665.38 |
EXAMPLE 3 expression and preparation of keratinase mutants in recombinant strains of Bacillus amyloliquefaciens
The KER wild-type encoding gene bliker, and mutant S207A, S213A, T237A, Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/S237A, Q241A/S207A/S213A encoding genes bliker 1-9 are respectively connected with a bacillus amyloliquefaciens expression plasmid pBSA43 to obtain new recombinant plasmids pBSA43-bliker, pBSA43-bliker 1, pBSA43-bliker m2, pBSA43-bliker m3 … … pBSA43-bliker m9;
the recombinant plasmid pBSA43-blikerm1 … is respectively transferred into bacillus amyloliquefaciens CGMCC No.11218, and is subjected to resistance screening of kananamycin (Kan), and enzyme digestion verification to obtain wild recombinant strains CGMCC No.11218/pBSA43-bliker, mutant recombinant bacteria CGMCC No.11218/pBSA43-bliker m1, CGMCC No.11218/pBSA43-bliker m2, CGMCC No.11218/pBSA43-bliker m3 … … CGMCC No.11218/pBSA43-bliker m9.
The Bacillus amyloliquefaciens mutant recombinant strain CGMCC No.11218/pBSA43-blikerm1 … and the wild-type recombinant strain CGMCC No.11218/pBSA43-bliker were inoculated into 5mL of fermentation medium (containing kanamycin, 50. Mu.g/mL) and cultured overnight at 37℃at 220r/min, and transferred into 50mL of fresh fermentation medium (containing kanamycin, 50. Mu.g/mL) according to an inoculum size of 2%, and further cultured at 37℃at 220r/min for 48 hours.
Fermentation medium (g/L): corn flour 64, bean cake flour 40, amylase 2.7, na 2 HPO 4 4，KH 2 PO 4 0.3, the balance being water; preserving heat at 90 ℃ for 30min and sterilizing at 121 ℃ for 20min.
The supernatant was centrifuged to determine the enzyme activity, and the casein enzyme activity obtained by fermentation with Bacillus amyloliquefaciens was determined by using the national standard method in example 2. The wild-type KER in Bacillus amyloliquefaciens had a caseinate activity of 9685.43U/mL, and the mutant S207A, S213A, T237A, Q241A, Q A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A/S213A had a caseinate activity of 8026.53U/mL, 8272.23U/mL, 8184.39U/mL, 7221.25U/mL, 7515.51U/mL, 7369.36U/mL, 7001.04U/mL, 6141.43U/mL, 6583.82U/mL.
Preparation of protease pure enzyme powder: the supernatant obtained by centrifuging the prepared fermentation broth is salted out by ammonium sulfate with 25 percent of saturation to remove the impurity protein, and then the saturation is increased to 65 percent to precipitate the target protein. Dissolving, dialyzing for desalting, dissolving the active component obtained after salting out and desalting by using 0.02mol/L Tris-HCl (pH 7.0) buffer solution, loading the solution to a cellulose ion exchange chromatographic column, eluting unadsorbed protein by using the same buffer solution, performing gradient elution by using 0.02mol/L Tris-HCl (pH 7.0) buffer solution containing NaCl with different concentrations (0-1 mol/L), and collecting target protein. The active components obtained by ion exchange are firstly balanced by 0.02mol/L Tris-HCl (pH 7.0) buffer solution containing 0.15mol/L NaCl, are loaded to a sephadex g25 gel chromatographic column and are eluted by the same buffer solution at the speed of 0.5mL/min, so as to obtain purified enzyme solution, and are frozen and dried to obtain the pure keratinase enzyme powder. The prepared keratinase mutant enzyme powder can be applied to the fields of tanning, food, feed and the like.
EXAMPLE 4 use of keratinase
1. Determination of amino nitrogen content in keratin enzymatic hydrolysis casein hydrolysate
5g of casein is weighed into a beaker, dissolved with NaOH solution, and after the casein is completely dissolved, the casein is fixed to 100mL. Taking 5mL of casein solution into two beakers respectively, placing the beakers into a constant-temperature water bath kettle with the temperature of 50 ℃ for preheating, then adding 0.1g of wild type keratinase and mutant Q241A/S207A/T237A into the two beakers respectively, carrying out enzymolysis for 150min at the temperature of 50 ℃, continuously stirring in the enzymolysis process, heating the enzymolysis solution at the temperature of 95 ℃ for 10min for inactivating enzyme after the reaction is finished, cooling to the room temperature, and centrifuging for 15min at 10000 r/min. The amino nitrogen content was determined by a double indicator formaldehyde titration method to determine the residual casein content of the hydrolysate. The results showed that the casein content remaining after the casein treatment with the wild-type keratinase was 0.96g and the casein content remaining after the casein treatment with the keratinase mutant Q241A/S207A/T237A was 2.28g. Compared with wild type keratinase, the casein content remained after casein treatment by the mutant Q241A/S207A/T237A is improved by 137.50%, which proves that the casein hydrolysis degree of the mutant Q241A/S207A/T237A is lower and the casein component is more remained.
2. Analysis of mechanical properties of wool
The wool (2 g) was weighed in equal amounts and placed in 10mL Gly-NaOH buffer, and 0.1g of wild type keratinase and mutant Q241A/S207A/T237A were added, respectively, and reacted in a 50℃water bath shaker for 1h. After the reaction time, the wool was dried to a constant mass in an oven at 105 ℃, conditioned for 24 hours in a standard environment at a relative humidity of 65% and a temperature of 20 ℃, cut into test specimens with a length of 250mm, and tested for elongation at break using an INSTRON5590 universal material tester. Test parameter setting: and the test fabric is subjected to constant-speed equal-elongation stretching, the clamping length of a test sample is 200mm, and the stretching speed is 200mm/min. And testing 30 groups of fabrics in each group, and taking the average value of the elongation at break after eliminating abnormal data in the testing process.
The results showed that the elongation at break of the wild type keratinase treated wool sample was 12.86% and that of the mutant Q241A/S207A/T237A treated wool sample was 22.74% with an increase in elongation at break of 76.83%. As the wool sample treated by the mutant Q241A/S207A/T237A retains more casein components, the elasticity of the wool fiber is increased under the same elongation condition, so that the elongation at break is increased, and the wool fabric has better mechanical property.
Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims (10)
1. A mutant of a keratinase, characterized in that the mutant is obtained by mutating Q241A, Q241A/S207A, Q a/S213A, Q241A/T237A, Q241A/S207A/T237A or Q241A/S207A/S213A on the basis of the wild type keratinase shown in SEQ ID No. 1.
2. A keratinase mutant as defined in claim 2 wherein,
the amino acid sequence of the Q241A mutant is shown as SEQ ID NO. 9;
the amino acid sequence of the Q241A/S207A mutant is shown as SEQ ID NO. 11;
the amino acid sequence of the Q241A/S213A mutant is shown as SEQ ID NO. 13;
the amino acid sequence of the Q241A/T237A mutant is shown as SEQ ID NO. 15;
the amino acid sequence of the Q241A/S207A/T237A mutant is shown as SEQ ID NO. 17;
the amino acid sequence of the Q241A/S207A/S213A mutant is shown as SEQ ID NO. 19.
3. A gene encoding the keratinase mutant of claim 1.
4. The coding gene of claim 3, wherein the gene is,
the coding gene of the Q241A mutant has a nucleotide sequence shown as SEQ ID NO. 10;
the coding gene of the Q241A/S207A mutant has a nucleotide sequence shown in SEQ ID NO. 12;
the coding gene of the Q241A/S213A mutant has a nucleotide sequence shown in SEQ ID NO. 14;
the coding gene of the Q241A/T237A mutant has a nucleotide sequence shown as SEQ ID NO. 16;
the coding gene of the Q241A/S207A/T237A mutant has a nucleotide sequence shown as SEQ ID NO. 18;
the coding gene of the Q241A/S207A/S213A mutant has a nucleotide sequence shown in SEQ ID NO. 20.
5. A recombinant plasmid or recombinant strain comprising the coding gene of claim 3.
6. The recombinant plasmid or recombinant strain according to claim 5, wherein the expression vector used is pBSA43, the host cell is Bacillus subtilis WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No.11218.
7. Use of the recombinant plasmid or recombinant strain of claim 5 for the production of the keratinase mutant of claim 1.
8. Use of a keratinase mutant of claim 1.
9. The use according to claim 8, in the hydrolysis of keratin.
10. The use according to claim 8, in a wool anti-felting treatment.
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