CN115975995A - Keratinase mutant without collagen hydrolytic activity - Google Patents

Abstract

The invention discloses a keratinase mutant without collagen hydrolytic activity, belonging to the technical field of genetic engineering and enzyme engineering. The invention provides a keratinase mutant without collagen hydrolysis activity, which is characterized in that methionine at 221 th position of keratinase with amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, and is named as KerZ1/M221I. The enzyme activity determination result shows that the keratinase mutant KerZ1/M221I retains the keratinase hydrolysis activity of 59235U/mL and has no collagen hydrolysis activity, so that the keratinase mutant can avoid the damage of collagenase during application and improve the quality of leather.

Description

Keratinase mutant without collagen hydrolytic activity

Technical Field

The invention relates to a keratinase mutant without collagen hydrolytic activity, belonging to the technical field of genetic engineering and enzyme engineering.

Background

Keratinase is a hydrolase which can catalyze the decomposition of keratin, and has wide application in the industries of detergents, cosmetics, pharmacy, leather making and the like. The wide application scene of the keratinase also reflects the characteristic of poor substrate specificity, not only has hydrolysis effect on the keratinase, but also has higher hydrolysis activity on other proteins, particularly collagen, which also limits the application of the keratinase in the aspects of cosmetics, leather making and the like, for example, in depilatory cream products, the collagen hydrolysis activity of the keratinase can cause skin damage, and the same problem also exists in leather depilatory applications. Thus, removal of the collagen activity of keratinase is of great importance to realize the commercial value of keratinase.

The prior stage of the subject group has constructed a keratinase recombinant strain which can be fermented on a 15L fermentation tank to obtain a keratinase product with keratinase hydrolysis activity of 426.60KU/mL (the keratinase is described in the biological transformation of keratan water to amino acids and active peptides based on cell free analysis literature), and the keratinase recombinant strain is the highest level of expression of the recombinant keratinase reported in the literature at present. However, the keratinase has higher collagen hydrolysis activity, and a keratinase mutant with completely removed collagen hydrolysis activity is screened by carrying out saturation mutation on the amino acid sites of parent enzymes.

Disclosure of Invention

The invention provides a keratinase mutant without collagen hydrolysis activity, which is characterized in that methionine at 221 th position of keratinase with amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, and is named as KerZ1/M221I.

In one embodiment of the invention, the nucleotide sequence encoding the parent enzyme keratinase is shown in SEQ ID NO. 2.

In one embodiment of the invention, the keratinase mutant KerZ1/M221I has an amino acid sequence shown in SEQ ID NO. 3.

In one embodiment of the invention, the nucleotide sequence encoding the keratinase mutant KerZ1/M221I is shown in SEQ ID NO. 4.

The invention also provides a gene for coding the keratinase mutant KerZ1/M221I.

The invention also provides a recombinant vector carrying the gene.

In one embodiment of the invention, pP43NMK is used as an expression vector.

In one embodiment of the present invention, the vector of the recombinant plasmid is a pP43NMK plasmid.

The invention also provides a recombinant cell expressing the keratinase mutant KerZ1/M221I, or carrying the gene or carrying the recombinant vector.

In one embodiment of the invention, the recombinant cell is a bacterial or fungal host cell.

In one embodiment of the invention, the host cell is Bacillus subtilis WB600.

The invention provides a recombinant Bacillus subtilis, which takes Bacillus subtilis WB600 as a host cell and pP43NMK plasmid as an expression vector to express a keratinase mutant KerZ1/M221I without collagen hydrolysis activity.

The invention also provides a method for removing collagen hydrolysis activity of keratinase, which is to mutate methionine at 221 th site of keratinase with amino acid sequence shown as SEQ ID NO.1 into isoleucine.

The invention also provides a product for degrading keratin, wherein the degraded keratin contains the keratin mutant KerZ1/M221I.

In one embodiment of the invention, the keratin-degrading product comprises: depilatory cream and leather depilatory.

The invention also provides a method for degrading keratin, which takes a substance containing keratin as a substrate and utilizes the keratinase mutant KerZ1/M221I or the recombinant cell or the recombinant bacillus subtilis to degrade the keratin.

In one embodiment of the invention, the keratin-containing material comprises fur, elastin, scales, fibers.

In one embodiment of the present invention, the recombinant cell is constructed by transferring a recombinant expression vector carrying a gene encoding the mutant enzyme into a host cell by electroporation or chemical transformation.

The invention provides the application of the keratinase mutant KerZ1/M221I, the gene, the expression vector or the host cell in animal husbandry, feed industry, leather industry, cosmetic industry and pharmaceutical industry.

The invention also claims the application of the keratinase mutant KerZ1/M221I, the gene, the expression vector or the host cell in degrading skin, hair, casein, elastin, hair, nails, scales, fibers and hair keratin.

Advantageous effects

The keratinase mutant KerZ1/M221I provided by the invention has no collagen hydrolysis activity, so that the keratinase mutant can avoid damage of collagenase in application. For example, in the leather-making industry, enzymatic unhairing is used, since the keratinase is serine protease and has no substrate specificity, collagen on the surface of leather is easily hydrolyzed indiscriminately in the application process to cause leather damage, and the quality of the leather is reduced, and the keratinase mutant KerZ1/M221I has no collagen hydrolysis activity, so that the leather is not damaged, and therefore, the keratinase mutant KerZ1/M221I without collagen activity provided by the invention has more application value and potential.

Drawings

FIG. 1 is a comparison of the keratinase and collagen hydrolysis activities of different mutants with control bacteria.

FIG. 2 is a SDS-PAGE gel electrophoresis of the Bacillus subtilis fermentation supernatants of the keratinase parent and the mutant KerZ 1/M221I; where M represents the protein molecular weight standard, the different lanes represent the different keratinases, and the arrow indicates the position of the protein band of interest.

Detailed Description

Escherichia coli JM109 referred to in the following examples was purchased from North Nay organisms; the pP43NMK plasmids referred to in the examples below were purchased from the Feng Hui organism; bacillus subtilis WB600 referred to in the following examples is described in patent application publication No. CN 102492645A.

The media involved in the following examples are as follows:

LB liquid medium: yeast powder 5 g.L ¹ Tryptone 10 g. L ¹ 、NaCl 10g·L ¹ 。

LB solid medium: yeast powder 5 g.L ¹ Tryptone 10 g. L ¹ 、NaCl 10g·L ¹ 20 g.L agar powder ¹ 。

B, bacillus subtilis fermentation culture medium: peptone 20 g.L ¹ Yeast powder 10 g.L ¹ 20 g.L of sucrose ¹ 、KH ₂ PO ₄ 3g·L ¹ 、Na ₂ HPO ₄ 6g·L ¹ 、MgSO ₄ 0.3g·L ¹ 。

The detection methods referred to in the following examples are as follows:

keratin hydrolysis activity assay method: taking 50 mu L of fermentation supernatant which is diluted properly, adding 150 mu L of Gly/NaOH solution with the concentration of 50mM as buffer solution and 100 mu L of water-soluble keratin with the concentration of 2.5% (purchased from Chiloeei (Shanghai) chemical industry development limited company, product code: K0043) as substrate, mixing uniformly, and reacting for 20min at 60 ℃; the reaction was stopped by adding 200. Mu.L of 4% (w/v) trichloroacetic acid (TCA) and centrifuged at 8000r/min at room temperature for 3min. The supernatant was taken to 200. Mu.L, and 1mL of 4% (w/v) Na was added ₂ CO ₃ Mixing with 200 μ L Folin phenol reagent, mixing, developing at 50 deg.C for 10min, and using 0.5cm stoneMeasuring the light absorption value of the clear liquid in an English cuvette at 660 nm; the experiment group is 3 parallels, blank control is that reaction terminator TCA is added before adding substrate, and other operations are the same.

Definition of enzyme activity unit: OD at 60 ℃ and pH 10 ₆₆₀ The enzyme quantity required is 0.001 per liter, which is one enzyme activity unit (U).

The collagen hydrolysis activity determination method comprises the following steps: 1mg of gelatin, 0.5mL of Tris-HCl as a buffer solution, 0.1mL of a suitably diluted fermentation supernatant were taken, reacted at 60 ℃ for 30min, and 0.5mL of 10% trichloroacetic acid was added to terminate the reaction. The ninhydrin color development method is used for determining the amount of amino acid released by reaction, and enzyme activity is calculated by contrasting with a glycine standard curve; the experiment group is 3 parallels, blank control is that reaction terminator TCA is added before adding substrate, and other operations are the same.

The unit of enzyme activity is defined as: the amount of enzyme that hydrolyzes collagen per minute at 60 ℃ and pH 7.5 to produce 1. Mu.g glycine per minute is 1 enzyme activity unit (U).

Example 1: construction of keratinase mutants

1. Construction of mutants

(1) The construction of the pP43NMK-ker recombinant vector is described in patent US20210079371A 1.

(2) Construction of mutants

The keratinase with an amino acid sequence of SEQ ID NO.2 is used as a parent enzyme, and 8 amino acid sites (threonine 33, glycine 62, tyrosine 208, proline 209, leucine 216, aspartic acid 217, threonine 219 and methionine 221) of the keratinase are selected for carrying out saturation mutation transformation.

According to the sequence of keratinase (the nucleotide sequence is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1), mutation primers (shown as table 1) are respectively designed, and site-directed saturation mutation is carried out on the keratinase gene on the pP43NMK plasmid.

TABLE 1 primers

The PCR reaction systems are as follows: primeSTAR Max Premix (2X) 25. Mu.L, 10. Mu.M forward primer 1. Mu.L, 10. Mu.M reverse primer 1. Mu.L, template DNA 1. Mu.L, double distilled water was added to 50. Mu.L;

the PCR product amplification conditions were all: pre-denaturation at 98 ℃ for 3min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 2min, and 30 cycles; finally keeping the temperature at 72 ℃ for 10min;

detecting the PCR amplification product by using 1% agarose gel electrophoresis, after confirming that the size of the amplification product is correct, adding 0.5 mu L of methylated template digestive enzyme (DpnI) into 10 mu L of the amplification product, uniformly mixing, reacting for 1.5h at 37 ℃, converting the amplification product treated by the DpnI into escherichia coli JM109 competence, coating the conversion product on an LB solid culture medium containing ampicillin resistance, and culturing for 8-10 h at 37 ℃. Transferring single colonies on the LB solid to a bacteria shaking tube by using an LB liquid culture medium, culturing at 37 ℃ and 220rpm for 2-3 h, and extracting plasmids to obtain recombinant plasmids containing different mutant genes.

Example 2: construction of recombinant plasmid and expression of keratinase

The method comprises the following specific steps:

(1) Respectively transforming the recombinant plasmids obtained in the example 1 into Bacillus subtilis WB600 competence, coating the transformation products on an LB solid culture medium containing kanamycin resistance, culturing for 10-12 h at 37 ℃, respectively preparing recombinant strains, inoculating single bacterial colonies growing on a plate culture medium into a 96 deep-hole plate of an LB liquid culture medium containing kanamycin resistance, culturing for 10-12 h at 220rpm at 37 ℃, and respectively preparing seed solutions;

(2) Transferring the seed liquid obtained in the step (1) to a 96-deep-well plate containing a bacillus subtilis fermentation culture medium according to the proportion of 5%, and culturing for 24h at 37 ℃ and 220rpm to obtain fermentation liquor containing each mutant enzyme.

(3) Respectively centrifuging the fermentation liquor at 4 ℃ and 5000rpm for 10min to obtain fermentation supernatant and crude enzyme liquid of each mutant enzyme.

(4) The results of SDS-PAGE gel electrophoresis of the crude enzyme solutions of the respective mutants obtained in step (3) are shown in FIG. 2 (showing only the effects of the advantageous mutants of the present application).

The results show that: the recombinant strain WB600/pP43NMK-KerZ1/M221I can produce keratinase with the same molecular weight and concentration as those of the original strain.

(5) The results of examining the crude enzyme solutions of the respective mutants for the keratin hydrolyzing activity and the collagen hydrolyzing activity are shown in table 2 and fig. 1:

table 2: keratinolytic activity and collagenolytic activity in crude enzyme solutions of respective mutants

The results show that: the mutant KerZ1/M221I completely eliminates the collagen hydrolysis activity under the condition of retaining the keratin hydrolysis activity of 59235U/mL, and the characteristic of the mutant is favorable for the application of the mutant in the aspects of depilatory cream and enzyme method leather making.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A keratinase mutant without collagen hydrolysis activity, characterized in that the mutant is obtained by mutating methionine 221 of keratinase with amino acid sequence as shown in SEQ ID NO.1 into isoleucine.

2. A gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector according to claim 3, wherein pP43NMK is the expression vector.

5. A recombinant cell expressing the mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3 or 4.

6. The recombinant cell of claim 5, wherein the recombinant cell is a bacterial or fungal host cell.

7. A method for removing collagen hydrolysis activity of keratinase, which comprises mutating methionine at 221-position of keratinase having an amino acid sequence as shown in SEQ ID NO.1 to isoleucine.

8. A keratin-degrading product comprising the keratin mutant of claim 1 in an epilatory product.

9. A method for degrading keratin, comprising adding the mutant according to claim 1 or the recombinant cell according to claim 5 or 6 to a reaction system containing keratin to degrade keratin.

10. The method of claim 7, wherein the keratin-containing material comprises fur, elastin, scales, fibers.

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