Use of a thioredoxin in promoting enzymatic digestion of keratin

Abstract

The application belongs to the technical field of genetic engineering. The application provides application of a thiol-oxidoreductase in promoting keratin enzymolysis and a method for promoting keratin enzymolysis. The thiol-oxidoreductase is applied to a keratin enzymolysis system, and the enzymolysis effect is obviously improved. Through testing, the enzymolysis effect of the thiol-oxidoreductase combined with a protease on keratin is not only obviously higher than the enzymolysis effect of a single protease, but also is better than the hydrolysis of keratin by a chemical reducing agent with the same concentration. The application retains the advantages of enzyme method "green chemistry" and is friendly to the environment.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to the application of a sulfur-oxygen-reduction protein. Background Technology

[0002] Keratin is a widely distributed protein material, and a large amount of keratin waste is generated annually during light industrial production. This waste not only wastes protein resources but also causes serious environmental problems (Peng Z, Zhang J, Du G, et al. Keratin waste recycling based on microbial degradation: mechanisms and prospects[J].ACS sustainable chemistry & engineering, 2019, 7(11): 9727-9736.). Numerous studies have been conducted on the applications of keratin, with keratin decomposition at its core. Furthermore, keratin decomposition is also involved in many light industrial production processes, such as leather depilation. The main methods for keratin decomposition include physical, chemical, and enzymatic methods. Physical methods mainly include hot water hydrolysis and steam explosion; chemical methods mainly use chemical reagents, such as acids, alkalis, and ionic liquids, to hydrolyze keratin; enzymatic methods mainly rely on various proteases to hydrolyze keratin. The ability of microorganisms to degrade keratin is also based on the various proteases produced by microorganisms (Eslahi N, Dadashian F, Nejad NH. An investigation on keratin extraction from wool and feather waste by enzymatic hydrolysis[J]. Preparative Biochemistry and Biotechnology, 2013, 43(7):624-648.).

[0003] Currently used keratin decomposition methods all have certain problems. Physical methods generally require high temperature and high pressure treatment, which places high demands on equipment and consumes a lot of energy. At the same time, due to the limitations of the principle and equipment, this method is not suitable for the hair removal production process in leather making. Chemical methods are lower in cost, simple to operate, and have low equipment requirements, but the large-scale use of various chemicals will generate a lot of polluted wastewater, and the toxic chemicals may harm human health. Compared with traditional chemical methods, enzymatic methods are milder and safer and greener, but traditional enzymatic processes use a single protease to hydrolyze keratin, which is less effective and results in a lower degree of hydrolysis.

[0004] Therefore, the enzymatic hydrolysis of keratin using the more mild and environmentally friendly protease method requires further improvement in its hydrolysis efficiency. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, the present invention aims to provide an application of a thioreduction protein in promoting the enzymatic hydrolysis of keratin, wherein the amino acid sequence of the thioreduction protein is shown in SEQ ID NO.1.

[0006] Furthermore, the gene sequence encoding the sulfur oxide reductase protein is shown in SEQ ID NO.2.

[0007] Furthermore, the sulfur-oxygen-reducing protein is a reduced sulfur-oxygen-reducing protein.

[0008] The present invention also aims to provide a method for promoting the enzymatic hydrolysis of keratin, wherein a thioreduction protein encoded by an amino acid sequence as shown in SEQ ID NO.1 or a gene sequence as shown in SEQ ID NO.2 is added to a reaction system containing at least one protease and keratin as a substrate for reaction.

[0009] Further, the sulfur-oxygen-reducing protein or the protein encoded by the encoding gene is subjected to reduction treatment.

[0010] Furthermore, the reducing agent used in the reduction treatment is a thiol reducing agent.

[0011] Animal hair itself has a complex, multi-layered nested structure. The keratin molecules that make up hair have a large number of covalent bonds and a small number of ionic and hydrogen bonds to maintain their dense molecular structure. This complex structure hinders the action of protease molecules on peptide bonds, resulting in low efficiency of keratinase hydrolysis using proteases alone. The main function of thioreduction proteins in cells is to maintain the redox environment and act on the covalent forces between cysteine ​​residues inside proteins to promote proper protein folding and avoid intracellular protein aggregation and inactivation (Nakamura H, Nakamura K, Yodoi J. Redox regulation of cellular activation[J]. Annual review of immunology, 1997, 15(1). 351-369.). This invention studies the sequence, function and mechanism of action of thioreduction proteins, establishes a heterologous expression and purification method, obtains high-purity thioreduction proteins, and applies them to assist in keratinase hydrolysis.

[0012] The beneficial effects of this invention are as follows:

[0013] Compared with the prior art, the present invention applies the above-mentioned sulfur-oxygen-reduction protein to the keratin hydrolysis system, which significantly improves the hydrolysis effect. After testing, the hydrolysis effect of sulfur-oxygen-reduction protein combined with protease on keratin is not only significantly higher than that of using a single protease, but also better than the hydrolysis of keratin by chemical reducing agents of the same concentration. The present invention retains the advantages of "green chemistry" of enzymatic methods and is environmentally friendly. Attached Figure Description

[0014] Figure 1 Electrophoresis diagram of the Thioredoxin target gene (M is marker);

[0015] Figure 2 Electrophoresis image of recombinant plasmid pET-17b containing the Thioredoxin target gene;

[0016] Figure 3 Image of SDS-PAGE gel electrophoresis analysis of sulfur oxide reductase protein (15% gel);

[0017] Figure 4 A magnified view of the SDS-PAGE gel electrophoresis analysis of thioreductase protein (15% gel);

[0018] Figure 5 In the bovine insulin reduction experiment at a wavelength of 650 nm, A was used in the Trx group and the DTT group. 650 Changes;

[0019] Figure 6 Qualitative observation diagram of hair structure changes before and after hydrolysis of sulfur-oxygen-reducing protein / chemical reducing agent based on cow hair;

[0020] Figure 7 For the alkaline protease hydrolysis water pretreatment group, GSH, DTT, BME, and Trx experimental groups, ΔA 280 The relative value;

[0021] Figure 8 ΔA for each group after keratinase hydrolysis 280 The relative value;

[0022] Figure 9 ΔA for each group after trypsin hydrolysis 280 The relative value;

[0023] Figure 10 A qualitative observation diagram of hair structure changes before and after hydrolysis of sulfur-oxygen-reducing proteins / chemical reducing agents based on wool;

[0024] Figure 11 For the alkaline protease hydrolysis water pretreatment group, GSH, DTT, BME, and Trx experimental groups, ΔA 280 The relative value;

[0025] Figure 12 ΔA for each group after keratinase hydrolysis 280 The relative value;

[0026] Figure 13 ΔA for each group after trypsin hydrolysis 280 The relative value. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Thioreduction proteins are widely distributed in various organisms and participate in most intracellular redox and electron transport processes. Their role in maintaining covalent bonds between cysteine ​​residues in protein molecules also stems from their ability to maintain the intracellular redox environment.

[0029] Based on this, embodiments of the present invention select the nucleotide sequence of thioreduction protein from the oxygen-tolerant anaerobic microorganism Clostridium perfringens as the target gene, construct it in an Escherichia coli heterologous expression system for fermentation expression and purification, use the obtained high-purity thioreduction protein to pretreat cow hair and wool, and combine it with a variety of commercially available proteases to enzymatically hydrolyze the hair.

[0030] Example 1: Construction of recombinant sulfur-oxygen reduction protein particles

[0031] (1) The target gene of thioredoxin (gene sequence shown in SEQ ID NO.1) was analyzed by PCR. The electrophoresis diagram of the thioredoxin target gene is shown below. Figure 1 As shown, M: standard molecular weight marker; (1)(2)(3): negative control; (4)(5)(6): target gene band) fragments were amplified in large quantities, with EcoRI and NotI restriction enzyme sites at both ends. The obtained sulfur oxide reductase target gene and vector plasmid were double-digested with EcoRI and NotI restriction enzymes, respectively. The restriction enzyme reaction was carried out at 30℃ for 30 min, and the system was prepared as follows:

[0032] Thioredoxin target gene / pET-17b 32μL FastDigestEcoRI restriction endonuclease 2μL FastDigestNotI restriction endonuclease 2μL 10× enzyme digestion buffer 4μL Total volume of the reaction system 40μL

[0033] (2) The target gene and the vector were ligated using T4 DNA ligase to obtain a recombinant plasmid containing the target gene. The ligation reaction was carried out at 22℃ for 30 min, and the system was prepared as follows:

[0034] pET-17b after double enzyme digestion 2μL Thioredoxin target gene after double enzyme digestion 6μL T4 DNA ligase 1μL 10× Ligation Buffer 1μL Total volume of the reaction system 10μL

[0035] (3) The recombinant plasmid was transformed into the E. coli DH5α cloning host.

[0036] The recombinant plasmid was transformed into competent E. coli cells using the heat shock method, as follows:

[0037] 1. Take out the competent E. coli DH5α cells stored in the -80℃ freezer and immediately place them in an ice bath for 5 minutes to allow the bacterial block to thaw;

[0038] 2. In a sterile laminar flow hood, add 10 μL of recombinant plasmid to 100 μL of thawed competent cells, and gently tap the centrifuge tube to mix, avoiding pipetting. After mixing, incubate on ice for 25 min.

[0039] 3. The mixed competent cells were subjected to heat shock in a 42℃ water bath for 45 seconds. After the heat shock, the competent cells were quickly placed back into an ice bath and left to stand for 2 minutes.

[0040] 4. Add 700 μL of antibiotic-free LB liquid medium to competent cells and incubate at 37°C and 250 rpm for 1 h.

[0041] 5. Centrifuge the cultured bacterial cells at 5000 rpm for 1 min, discard most of the supernatant, and retain only 100 μL. Gently pipette the retained supernatant to resuspend the bacterial cells, mix well, and spread it onto LB solid medium containing 100 μg / mL Amp. Invert the plate and incubate overnight at 37°C. 6. Pick single colonies growing on the LB solid plate, inoculate them into 1 mL of LB liquid medium containing 100 μg / mL Amp, and label them. Incubate at 37°C and 250 rpm. Sequencing is performed after the bacterial culture becomes turbid.

[0042] (3) Extract plasmids from recombinant strains with positive sequencing results. The steps are as follows: inoculate the plasmid-amplified strain into 10 mL LB liquid medium (containing 100 μg / mL Amp), and culture overnight at 37°C and 250 rpm until OD600 exceeds 0.8. Extract plasmids using a plasmid DNA mini-extraction kit.

[0043] Example 2

[0044] A heterologous expression system for sulfoxide-reduction protein in E. coli was constructed by transforming the extracted recombinant plasmid into competent E. coli BL21 (AI) cells, as follows:

[0045] (1) Take out the competent cells E.coli BL21 (AI) stored in the -80℃ freezer and immediately place them in an ice bath for 5 minutes to allow the bacterial block to thaw;

[0046] (2) In a sterile laminar flow hood, add 10 μL of recombinant plasmid to 100 μL of thawed competent cells and gently tap the centrifuge tube to mix, avoiding pipetting. After mixing, incubate on ice for 25 min.

[0047] (3) The mixed competent cells were subjected to heat shock in a 42℃ water bath for 45 seconds. After the heat shock, the competent cells were quickly placed back into an ice bath and left to stand for 2 minutes.

[0048] (4) Add 700 μL of antibiotic-free LB liquid medium to competent cells and culture at 37°C and 250 rpm for 1 h.

[0049] (5) Centrifuge the cultured cells at 5000 rpm for 1 min, discard most of the supernatant, keep only 100 μL, gently pipette the remaining supernatant to resuspend the cells, mix well and spread it onto LB solid medium containing 100 μg / mL Amp, invert and place in a 37°C incubator for overnight culture.

[0050] (6) Pick single colonies growing on LB solid plates, inoculate them into 1 mL of LB liquid medium containing 100 μg / mL LAmp, label them, and incubate at 37℃ and 250 rpm. After the bacterial culture becomes turbid, sequence the culture and retain the recombinant plasmid strains that show positive results. The recombinant plasmid pET-17b containing the Thioredoxin target gene is shown below. Figure 2 As shown.

[0051] Example 3: Expression and purification of recombinant thioreductase protein

[0052] The expression and purification of recombinant thioredoxin are performed as follows:

[0053] (1) Expression of recombinant thioreductase protein

[0054] 1. Bacterial cell activation

[0055] The *E. coli* BL21(AI)-pET-17b-Thioredoxin strain, preserved at -80℃, was removed and placed on an ice bath. After thawing, the cells were streaked onto LB agar plates containing 100 μg / mL Amp under aseptic conditions. The plates were then incubated upside down at 37℃ for 12 hours. Well-grown expression strains were picked from the plates and inoculated into 10 mL of LB liquid medium containing 100 μg / mL Amp. The medium was incubated overnight at 37℃ and 250 rpm until turbidity was reached, serving as the activation seed culture.

[0056] 2. Induction of expression and harvesting

[0057] Take 8 mL of the above seed culture and transfer it to 400 mL of LB liquid medium containing 100 μg / mL Amp under aseptic conditions. Incubate at 37°C and 250 rpm until the bacterial culture reaches OD500. 600 The value reached 0.5-0.6. Add 2 mL of sterile 400 mg / mL L-arabinose solution and induce culture at 37℃ and 250 rpm for 3 h. After fermentation, collect the bacteria by pouring the bacterial solution into a 500 mL centrifuge cup and centrifuging at 4℃ and 5000×g for 5 min.

[0058] 3. Bacterial cell disruption

[0059] The collected bacterial cells were resuspended in protein purification equilibration buffer, and the cells were disrupted using an ultrasonic disruptor. The cells were then centrifuged at 18000 x g for 40 min at 4°C to remove insoluble impurities, and the supernatant was retained.

[0060] (2) Purification of recombinant thioreduction protein

[0061] Purification was performed using the AKTAPure protein purifier and an affinity chromatography nickel column. The purification steps are as follows:

[0062] Take 1L of ultrapure water, filter it using a 0.22μm filter membrane, and degas it by sonication for 30 minutes;

[0063] 2. System flushing: Flush the AKTA system pipeline with filtered ultrapure water for 60 min. The system flow rate is set to 5 mL / min and the system pressure to 0.5 MPa.

[0064] 3. Install the HisTrapHP 5mL affinity chromatography nickel column: Unscrew the cable connecting the injection valve and the detector. When liquid flows out of the outlet, unscrew the nut on the top of the column and connect it to the outlet of the injection valve. Unscrew the nut at the bottom of the column and wait for liquid to drip out. Once the dripping liquid fills the connection port on the detector, screw the bottom of the column into the connection port.

[0065] Avoid introducing air bubbles during the process;

[0066] 4. Column washing: Rinse the chromatography column with filtered ultrapure water. The system settings are: column washing for 60 min, flow rate 3 mL / min, and column pressure 0.5 MPa.

[0067] 5. Column equilibration: The system was set to equilibrate the column with equilibration fluid for 120 min, flow rate 3 mL / min, and column pressure 0.5 MPa;

[0068] 6. Loading: Add 80 mL of filtered cell lysis supernatant to a 150 mL Superloop loading loop, remove air bubbles, connect to the AKTA purification system, flow rate 3 mL / min, column pressure 0.5 MPa;

[0069] The protein was eluted using the following system elution conditions: Solution A: Nickel column equilibration buffer (500 mM NaCl, 20 mM Tris-HCl, 30 mM imidazole); Solution B: Nickel column elution buffer (500 mM NaCl, 20 mM Tris-HCl, 30 mM imidazole). A linear ratio was set for protein elution at a flow rate of 3 mL / min and a column pressure of 0.5 MPa. Ten column volumes were eluted, and 2 mL of eluent was collected per tube. The collected enzymes were verified by SDS-PAGE gel electrophoresis, and the results are shown below. Figure 3 , Figure 4 As shown, the SDS-PAGE gel electrophoresis analysis (15% gel) of thioreductin was performed. M: standard molecular weight marker. (1) Fermentation supernatant; (2) Cell fragment resuspension; (3) Cell lysate; (4) Purified product of the target protein. The amino acid sequence of thioreductin is shown in SEQ ID NO.2.

[0070] (3) Concentration and concentration determination of recombinant thioreductase protein

[0071] The target protein was subjected to imidazole replacement using AmiconUllra (microfiltration) tubes with a molecular weight of 3 kDa. Replacement was performed with protein replacement buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.5), and centrifuged at 4800 x g at 4°C for at least three times, 50 minutes each time. The concentration of the purified target protein was determined using a BCA protein quantification kit.

[0072] (4) Assay of recombinant thioreductase activity

[0073] Accurately weigh 0.0184 g of bovine insulin, dissolve it in phosphate buffer (pH 6.5) and bring the volume to 2 mL to prepare a 1.6 mM bovine insulin preparation solution. Store at 4°C for later use.

[0074] Prepare a 2 mM EDTA solution using pH 6.5 phosphate buffer. Mix 4657 μL of the EDTA solution with 825 μL of a 1.6 mM bovine insulin preparation solution to prepare a substrate solution suitable for 10 samples.

[0075] Take an appropriate amount of concentrated enzyme solution stored at 4℃, add DTT solution to the final DTT concentration of 10mM, pretreat at 25℃ for 30min, and replace the buffer with pH 6.5 phosphate buffer using an ultrafiltration tube at 4℃ to remove DTT from the solution. Determine the protein concentration after replacement.

[0076] Trx experimental group: Take 100 μL of the pretreated concentrated enzyme solution of thioredoxin, add pH 6.5 phosphate buffer to dilute to 0.25 mM according to the initial concentration, and prepare according to the following systems:

[0077]

[0078] DTT group: Replace the diluted enzyme sample in the Trx experimental group with the same volume of 10mM DTT solution.

[0079] After the above systems were mixed thoroughly, 200 μL of each was added to a 96-well microplate. The microplate reader was set to kinetic measurement, the temperature was 25℃, the reaction time was 40 min, the kinetic interval was 1 min, and continuous shaking was performed. The measurement wavelength was 650 nm. The results of the thioreduction protein Trx group and the DTT group in the bovine insulin reduction experiment at a wavelength of 650 nm were obtained. 650 Changes, such as Figure 5 As shown.

[0080] After the measurement, absorbance values ​​per minute were plotted and the trend was analyzed based on ΔA. 650 The disulfide reductase activity of thioreductin to bovine insulin substrate was calculated. Enzyme activity was defined as: in the bovine insulin reduction experiment at 25°C, 1 U of thioreductin produced 1.0 ΔA within 1 min. 650 .

[0081] The specific vitality calculation method is as follows:

[0082] Specific activity (U / mg) = ΔA 650 (m×t)

[0083] Where m is the mass of the sulfur-oxidizing protein in the reaction system (unit: milligrams, mg), and t is ΔA. 650 The obtained reaction time (min).

[0084] Example 4: Experimental analysis of the promoting effect of recombinant thioreductase protein on keratinase hydrolysis.

[0085] Take fresh cow hair and sheep wool, accurately weigh them, and dispense them into small centrifuge tubes, 0.05g of cow hair per tube. Divide each type of hair into 15 tubes for 3 parallel experiments. Prepare and operate according to the following groupings:

[0086] Water pretreatment experimental group: Take three small centrifuge tubes containing cow hair / wool (label them respectively, corresponding to alkaline protease, keratinase, and trypsin), and add 1 mL of ultrapure water to each tube;

[0087] GSH experimental group: Take three small centrifuge tubes containing cow hair / wool (label them respectively, corresponding to alkaline protease, keratinase, and trypsin), and add 1 mL of 10 mM GSH solution to each tube.

[0088] DTT experimental group: Take three small centrifuge tubes containing cow hair / wool (label them respectively, corresponding to alkaline protease, keratinase, and trypsin), and add 1 mL of 10 mM DTT solution to each tube.

[0089] BME experimental group: Take three small centrifuge tubes containing cow hair / wool (label them respectively, corresponding to alkaline protease, keratinase, and trypsin), and add 1 mL of 10 mM BME solution to each tube.

[0090] Trx experimental group: Take three small centrifuge tubes containing cow hair / wool (corresponding to alkaline protease, keratinase, and trypsin, respectively), and add 1 mL of 10 mM reduced thioreductin solution to each tube (pretreated with DTT and the buffer replaced; heterologously expressed thioreductin is in its oxidized form and needs to be reduced with DTT before use. Reduced thioreductin was used in this experiment).

[0091] All experimental groups were immersed for 24 hours at 25℃ and 600 rpm.

[0092] After 24 hours of pretreatment, the centrifuge tubes containing hair were removed from the shaking reactor. All liquid was aspirated from the tubes using a pipette, and 1 mL of ultrapure water was added. The tubes were repeatedly aspirated and re-aspirated to wash away any remaining pretreatment liquid from the hair. After five washes, 1 mL each of alkaline protease, keratinase, and trypsinase solutions were added to their respective labeled centrifuge tubes. The hair was then processed according to the instructions for the proteases used, under the following conditions:

[0093] Alkaline protease group: treated at 50℃ and 600 rpm for 24 h;

[0094] Keratinome: treated at 50℃ with shaking at 600 rpm for 24 h;

[0095] Trypsinase group: treated at 37℃ and 600 rpm for 24 h.

[0096] After adding the enzyme solution, the initial morphology of the hair before hydrolysis was recorded by taking photos, and then it was placed in a shaking reactor for processing.

[0097] Take 3 mL of each of the three protease solutions and add them to 9 empty centrifuge tubes to form 3 parallel blank control groups. After labeling, shake under the corresponding conditions for 24 h.

[0098] Assessment of the degree of hair hydrolysis

[0099] Qualitative assessment of the degree of hydrolysis was conducted by observing the morphology of the remaining hair and comparing it with that before the reaction; quantitative determination of the degree of hydrolysis was based on the change in absorbance of the supernatant at 280 nm before and after the reaction.

[0100] After 24 hours of hydrolysis, all centrifuge tubes were removed from the shaking reactor, and the hair morphology was photographed. 500 μL of the supernatant from the blank control group (without added hair) and each experimental group was taken and labeled into blank centrifuge tubes, and centrifuged at 4℃ and 15000×g for 10 min. A certain amount of the centrifuged supernatant was diluted 20 times with ultrapure water and used as the test sample.

[0101] Take 200 μL of the diluted supernatant into a 96-well microplate, measure the absorbance at 280 nm using a microplate reader, and subtract the value of the blank control. Using the water pretreatment group value as 100%, calculate the relative values ​​of each experimental group to the water pretreatment group value.

[0102] Experimental results for promoting the hydrolysis of reduced sulfur-oxidation proteins / chemical reducing agents using cow hair as a substrate are as follows: Figure 6 , 7 As shown in Figures 8 and 9, among which Figure 6 To qualitatively observe the changes in hair structure before and after hydrolysis (in the pretreatment agent composition column, + indicates the presence of the corresponding component in the pretreatment solution, and - indicates the absence of the corresponding component), a certain amount of the soaking solution was taken, diluted 20 times, and ΔA was measured and calculated. 280 , Figure 7 , 8 9 represent the ΔA values ​​after hydrolysis by alkaline protease (labeled A), keratinase (labeled K), and trypsin (labeled T), respectively. 280 With water pretreatment group ΔA 280 The relative values; the error values ​​of the relative value data are calculated based on three sets of parallel experiments, and the error bars are marked on the graph.

[0103] according to Figure 6 , 7 Results 8 and 9 showed that pretreatment of the bovine hair substrate with reduced thioreduction protein / chemical reducing agent intensified its protease hydrolysis; for each group, ΔA 280Relative values ​​show that, for bovine hair substrates, at a concentration of 10 mM, the promoting hydrolysis effect of reduced thioreductol protein on bovine hair substrates is comparable to that of other chemical reducing agents at the same thiol concentration. In the experimental group that combined keratin (labeled K) with hair hydrolysis, reduced thioreductol protein showed a certain degree of advantage, with its ΔA... 280 The relative value with the blank control group reached 192.105%, which was higher than the three experimental groups GSH (158.851%), DTT (124.922%) and BME (130.665%).

[0104] Figure 10 , 11 12 and 13 are the experimental results using wool as the substrate, among which Figure 10 To conduct a qualitative observation of the changes in hair structure before and after hydrolysis, Figure 11 , 12 1 and 13 represent the ΔA values ​​after hydrolysis by alkaline protease (labeled A), keratinase (labeled K), and trypsin (labeled T), respectively. 280 With water pretreatment group ΔA 280 The relative values ​​are calculated based on the error values ​​from three sets of parallel experiments, and the error bars are marked on the graph.

[0105] according to Figure 10 As shown, after 24 hours of protease hydrolysis, the wool substrates in each group exhibited varying degrees of color change. In the experimental group treated with a chemical reducing agent / reduced sulfur oxide-reducing protein, a small number of segmented hairs appeared within the initially clump-like wool substrate, and precipitation occurred. In the three groups of experiments divided according to the protease used, a certain degree of color deepening of the soaking solution was observed after 24 hours of hydrolysis. A certain amount of each soaking solution was diluted 20 times and ΔA was measured and calculated. 280 ,according to Figure 11 , 12 The results in group 13 showed that pretreatment of wool substrate with reduced thioreductol protein / chemical reducing agent enhanced the degree of protease hydrolysis, thus demonstrating the feasibility of the reduced thioreductol protein-induced hydrolysis method for wool substrates; for each group ΔA 280 Relative values ​​show that, for wool substrates at a concentration of 10 mM, reduced sulfur-oxidation protein exhibits a certain advantage in promoting the hydrolysis of wool substrates compared to other chemical reducing agents at the same concentration. In the experimental group that hydrolyzed hair in conjunction with basic protein (labeled A), the ΔA of the reduced sulfur-oxidation protein experimental group was significantly higher. 280The relative value compared to the blank control group reached 417.844%, which was higher than that of the three experimental groups: GSH (319.765%), DTT (278.235%), and BME (317.342%). Tests showed that the enzymatic hydrolysis effect of the heterologously expressed thioreductase combined with protease on hair was at least 150% higher than that achieved with a single protease, exceeding 400% of the effect. This pro-hydrolysis effect was superior to that of chemical reducing agents commonly used in chemical methods. Therefore, the thioreductase combined with protease in this invention significantly improves the enzymatic hydrolysis effect on keratin, increasing the efficiency of enzymatic hydrolysis of keratin and demonstrating superiority over chemical reducing agents of the same concentration in the hydrolysis of keratin.

[0106] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.

Claims

1. The application of a sulfur-oxygen-reducing protein in promoting the enzymatic hydrolysis of keratin, characterized in that, The gene sequence encoding the sulfur oxide reductase protein is shown in SEQ ID NO.2, and the sulfur oxide reductase protein is a reduced sulfur oxide reductase protein.

2. A method for promoting the enzymatic hydrolysis of keratin, characterized in that, The protein encoded by the gene according to claim 1 is added to a reaction system containing at least one protease and keratin as a substrate to reduce the protein encoded by the gene.

3. A method for promoting the enzymatic hydrolysis of keratin according to claim 2, characterized in that, The reducing agent used in the reduction treatment is a thiol reducing agent.

Images