CN117757777A

CN117757777A - Keratinase mutant and application thereof

Info

Publication number: CN117757777A
Application number: CN202311790323.0A
Authority: CN
Inventors: 刘逸寒; 郑凯心; 李玉; 杨文华; 路福平
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2023-12-22
Filing date: 2023-12-22
Publication date: 2024-03-26

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a keratinase mutant with improved enzyme activity obtained through site-directed mutagenesis. The invention uses the overlapping PCR technology to carry out the site-directed mutagenesis of the keratinase gene from bacillus licheniformis (Bacillus licheniformis), screens to obtain the keratinase mutant with improved enzyme activity, and expresses the keratinase mutant in bacillus subtilis and bacillus amyloliquefaciens systems. The obtained keratinase mutant can be applied to the hydrolysis of keratin, in particular to the feed processing industry, the pharmaceutical industry, the tanning industry and the cosmetic industry, and solves the problem of high-efficiency hydrolysis of the keratin.

Description

Keratinase mutant and application thereof

Technical field:

the invention belongs to the technical field of bioengineering, and particularly relates to a keratinase mutant with improved enzyme activity obtained through site-directed mutagenesis.

The background technology is as follows:

keratin is a hard insoluble protein widely existing in nature, mainly existing in the structures of animal hair, scales, feathers, hooves, horns and the like, has stable chemical structure, and is insoluble in water and dilute acid and alkali. In agricultural production, millions of tons of keratin-rich waste or living byproducts are produced each year, and the protein content of the waste is high, so that the waste has recycling value. However, at present, the degradation of keratin adopts chemical or physical methods, the utilization rate of wastes cannot be ensured, acid-base hydrolysis also generates acidic and alkaline wastewater and steam, which affects the health of human bodies and pollutes the environment. Compared with the method, the method has the advantages of low cost for degrading the keratin by using an enzymatic method, mild treatment condition, less loss of protein nutritive value, high utilization rate of amino acid and great development space.

Keratinase is a very wide-substrate protease capable of degrading various soluble and insoluble proteins such as collagen, fibrin, bovine whey protein, hemoglobin, casein and the like, and most particularly, the keratinase can specifically degrade natural keratins, has specificity for keratins with higher sulfur content, can catalyze the denaturation and hydrolysis of the keratins into soluble amino acids and polypeptides, has mild action conditions and has obvious advantages compared with other traditional proteases. The discovery of keratinase enables people to find an ideal new way for changing keratin waste into valuable, but the keratinase needs to be modified to improve the activity of the keratinase so as to improve the degradation capability of the keratin.

The directed evolution of enzyme molecules is a rational design of proteins, and by artificially creating special evolution conditions, simulating a natural evolution mechanism, modifying genes of the enzyme molecules in vitro, and directionally screening to obtain certain enzymes with expected characteristics. A large number of enzyme molecules are successfully transformed by a site-directed mutagenesis technology, and the industrial enzyme with higher activity and better stability than the natural enzyme is obtained. At present, a certain progress is made in the subtilisin, and spatial resistance and electrostatic distribution of a substrate binding region in the subtilisin are closely related to the substrate specificity of the subtilisin, wherein the substrate binding regions of S1 and S4 play a main role, so that research on modification of the substrate binding regions of S1 and S4 is important.

The advantages of the bacillus subtilis are more remarkable when the bacillus subtilis secretes and expresses the keratinase, the bacillus subtilis also has a complete protein folding secretion mechanism, and the problem of codon preference is basically avoided when the keratinase gene derived from the bacillus is expressed, so that the keratinase derived from the bacillus can be over-expressed in a bacillus subtilis host.

Therefore, in the invention, the keratinase mutant with improved enzyme activity 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:

in order to improve the enzyme activity of keratinase and obtain a keratinase mutant with high activity, the existing properties of the keratinase mutant need to be further improved.

The invention aims to obtain a keratinase mutant with improved enzyme activity, which uses a keratinase gene derived from bacillus licheniformis (Bacillus licheniformis) as a starting gene for molecular modification. 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 the keratinase mutant with improved enzyme activity.

One of the technical schemes provided by the invention is a keratinase mutant, which is obtained by L136A mutation occurring on the basis of a wild keratinase zymogen region shown in SEQ ID NO. 1;

further, the keratinase mutant is an L136A mutant, and the amino acid sequence is shown as SEQ ID NO. 3;

furthermore, the coding gene blikerm1 of the L136A mutant has a nucleotide sequence shown in SEQ ID NO. 4.

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 L136A mutant.

The fourth technical scheme provided by the invention is the application of the L136A mutant, particularly the application in hydrolyzing keratin, and more particularly the application in feed processing industry, pharmaceutical industry, tanning industry and cosmetic industry.

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 coding gene is transferred into bacillus subtilis WB600 after constructing recombinant plasmid, and the activity of keratinase is measured by using national standard method.

(3) The KER mutant coding gene blikerm1 with the increased activity relative to the wild type keratinase is obtained through screening, and the plasmid pBSA43-blikerm1 containing the KER mutant coding gene with the increased activity of keratinase is stored.

Fermenting and culturing the keratinase mutant with the keratinase activity improved, and purifying to obtain the KER mutant protein.

2. The bacillus amyloliquefaciens recombinant strain containing the KER mutant coding gene and the process for preparing the keratinase mutant by using the bacillus amyloliquefaciens recombinant strain comprise the following steps:

(1) The KER mutant encoding gene blikerm1 and the bacillus amyloliquefaciens expression plasmid pBSA43 are connected to obtain a new recombinant plasmid pBSA43-blikerm1;

(2) Transferring the recombinant plasmid pBSA43-blikerm1 into bacillus amyloliquefaciens CGMCC No.11218, screening with resistance of kananamycin (Kan), performing enzyme digestion verification to obtain recombinant strain, and culturing and fermenting the recombinant strain to obtain the keratinase mutant.

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 Leu136Ala, it is indicated that the amino acid at position 136 is replaced by Leu of the wild-type KER with Ala, the numbering of the position corresponding to the amino acid sequence numbering of the wild-type KER zymogen region in SEQ ID NO. 1.

In the present invention, the lower case italic bliker represents the gene encoding the wild type keratinase KER, and the lower case italic bliker 1 represents the gene encoding the mutant L136A, 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 the KER to obtain mutant L136A with the activity of keratinase improved relative to that of the wild type at 60 ℃, the specific activities of the keratinase of the wild type KER and the mutant L136A are 780.5U/mg and 1069.4U/mg respectively in a bacillus subtilis expression system,

2. 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 L136A mutant is shown in SEQ ID NO. 3: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVADTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

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) ATCC14580 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 improved keratinase Activity

1. Site-directed mutagenesis was performed based on overlap PCR technique to construct novel keratinase and the mutation primers were designed as follows:

in the first step of overlapping PCR reaction system, P1 is used as an upstream primer, 136-R is used as a downstream primer, and plasmid pBSA43-bliker is used as a template to perform PCR1 reaction to obtain an upstream fragment; PCR1 reaction was performed using P2 as the upstream primer, 136-F as the downstream primer, and the plasmid pBSA43-bliker as the template to obtain a downstream fragment.

The reaction system for amplifying the upstream fragment is as follows:

P1	2μL
		136-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
		136-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 L136A.

4. The keratinase site-directed mutant gene bliker L136A 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 L136A.

And then the recombinant plasmid pBSA43-bliker L136A is transformed into bacillus subtilis WB600 to obtain recombinant strain WB600/pBSA43-bliker L136A. 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 activity of keratinase:

keratinase hydrolyzes keratin at a temperature and pH (the temperature of the invention is 60 ℃ and the pH is 10, as not specifically described) to produce amino acids containing phenolic groups, which are reduced with Fu Lin Fen reagent to produce tungsten blue, and the absorbance of the solution is measured at 680nm using an ultraviolet spectrophotometer. The specific activity of the keratinase can be calculated by directly proportional to the absorbance. The measurement method is as follows:

adding 1mL enzyme solution into blank group, and preserving heat at 60 DEG C2min, adding 2mL of trichloroacetic acid, reacting at 60deg.C for 10min, adding 1mL of 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 keratin (10 g/L) solution, reacting for 10min at 60 ℃, adding 2mL trichloroacetic acid, taking out and 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-blikerL136A is proposed to be sent to Jin Weizhi company for sequencing, and after the correct mutation site is determined, the coding gene of the keratinase mutant with the 136 th amino acid Leu mutated into Ala is named as blikerm1.

The mutant L136A with higher keratinase activity than the wild type at 60 ℃ is obtained through enzyme activity measurement. The enzyme activities are shown in the following table:

keratinase	Enzyme activity (U/mL)	Specific activity (U/mg)
			WT	1404.9	780.5
L136A	2031.7	1069.4

EXAMPLE 3 expression and preparation of keratinase mutants in recombinant strains of Bacillus amyloliquefaciens

The KER wild-type encoding gene bliker and the mutant L136A encoding gene bliker m1 are respectively connected with a bacillus amyloliquefaciens expression plasmid pBSA43 to obtain new recombinant plasmids pBSA43-bliker and pBSA43-bliker m1;

the recombinant plasmids pBSA43-bliker and pBSA43-bliker 1 are respectively transferred into bacillus amyloliquefaciens CGMCC No.11218, and wild recombinant strains CGMCC No.11218/pBSA43-bliker and mutant recombinant strains CGMCC No.11218/pBSA43-bliker 1 are obtained through resistance screening of kanamycin (Kan) and enzyme digestion verification.

Bacillus amyloliquefaciens recombinant strain CGMCC No.11218/pBSA43-blikerm1 and wild recombinant strain CGMCC No.11218/pBSA43-bliker are inoculated into 5mL of fermentation medium (containing kanamycin and 50 mug/mL) and cultured overnight at 37 ℃ and 220r/min, and transferred into 50mL of fresh fermentation medium (containing kanamycin and 50 mug/mL) according to the inoculum size of 2%, and the culture is continued at 37 ℃ and 220r/min for 48 hours.

The supernatant was centrifuged to determine the enzyme activity, and the keratinase activity obtained by fermentation with Bacillus amyloliquefaciens was determined by the national standard method in example 2. The keratinase activity of the wild-type KER in Bacillus amyloliquefaciens is 3512.3U/mL, and the enzyme activity of the mutant L136A is 5025.9U/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 dialyzing 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, and then carrying out gradient elution by using 0.02mol/L Tris-HCl (pH 7.0) buffer solution containing NaCl with different concentrations (0-1 mol/L), thereby 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 for wool hydrolysis

Wool is a natural protein fiber, and has been widely used in textile industry due to its excellent properties such as warmth retention, softness, easy dyeing, etc. However, due to the existence of the flake layer of the wool fiber structure, the fabric tends to be felted and deformed after washing, and itchy feeling and the like are generated in the wearing process, and in order to overcome the defect, the flake is required to be removed by using keratinase in the processing process, so that the performance of the wool fabric is improved.

The wool tops are cleaned and dried at 60 ℃, wool (1 g) with the same quality is weighed and placed in 50mL Gly-NaOH buffer solution, 0.1g of wild keratinase and mutant L136A are added respectively, and the two are reacted with the wool tops which are not treated by the enzyme in a water bath shaking table at 40 ℃ for 6 hours. Then the enzyme is deactivated by heat preservation for 10min at 85 ℃, finally the enzyme is washed by water and dried at 60 ℃. The ultraviolet absorption spectrum of the hydrolysis residual liquid after the wool is treated by enzyme is tested by an ultraviolet-visible spectrophotometer, and the effect on wool fibers is analyzed according to the absorbance at the wavelength of 280 nm.

The keratinase successfully breaks disulfide bonds in the wool fiber flake layer and hydrolyzes the cleaved protein peptide fragments further into amino acids which are dissolved in the solution, so that the absorbance is increased to some extent. The result shows that the absorbance of the hydrolysis liquid of untreated wool at 280nm is 0.14, the absorbance of the hydrolysis liquid of wild type keratinase treated wool is 0.33, and the absorbance of the hydrolysis liquid of mutant L136A treated wool is 0.58, because the absorbance of the solution of the mutant L136A treated wool tops is obviously increased relative to the absorbance of the solution of the wild type keratinase treated wool tops, namely the content of amino acid in the solution is increased, the mutant L136A is more effective in degrading the flake layer of wool, the hydrolysis efficiency of wool flakes is improved, and the performance of wool products is improved.

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

1. The keratinase mutant is obtained by carrying out L136A mutation on the basis of wild keratinase shown in SEQ ID NO.1, and the amino acid sequence of the keratinase mutant is shown in SEQ ID NO. 3.

2. A gene encoding the keratinase mutant of claim 1.

3. A recombinant plasmid or recombinant strain comprising the mutant encoding gene of claim 2.

4. Use of the recombinant plasmid or recombinant strain of claim 3 for the production of the keratinase mutant of claim 1.

5. Use of a keratinase mutant of claim 1.

6. The use according to claim 5, in the hydrolysis of keratin.

7. The use according to claim 6, in the feed processing industry, the pharmaceutical industry, the tanning industry or the cosmetic industry.