A collagenase mutant, gene fragment, recombinant plasmid, recombinant expression system, collagen peptide, and their preparation method and uses.

Abstract

The present invention discloses a collagenase mutant, a gene fragment, a recombinant plasmid, a recombinant expression system, a collagen peptide, and a preparation method and use thereof, and belongs to the field of genetic engineering and fermentation engineering. The present invention obtains a collagenase mutant KT513 with high specific activity through recombinant expression in a Trichoderma reesei host, and its enzyme activity reaches 7423.18 U / mL. The optimum action pH of the enzyme is 3.0, and red snapper fish scale collagen can be enzymatically hydrolyzed within the range of pH 3.0-5.0, and has excellent enzymatic activity. After enzymatic hydrolysis of the collagenase mutant KT513 at 40 ° C for 4 h, the extraction rate of fish scale collagen reaches 80.09%, and the prepared collagen peptide has good antioxidant activity. When the added concentration is 10 mg / mL, the prepared collagen peptide has an in vitro scavenging activity of hydroxyl radicals of 99.26%, which is equivalent to ascorbic acid, and has broad application prospects in the preparation of antioxidant preparations.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and fermentation engineering, and specifically relates to a collagenase mutant, a gene fragment, a recombinant plasmid, a recombinant expression system, a collagen peptide, and a preparation method and use thereof. Background Art

[0002] As an important economic fish, red snapper produces a large amount of fish scales during processing, which not only wastes protein resources but also puts a certain amount of pressure on the environment. If it can be effectively utilized, the added value of red snapper processing can be greatly improved. The collagen in red snapper scales is type I collagen, which is rich in glycine, hydroxyproline, alanine, etc., among which the content of hydroxyproline is relatively high, and the amino acid sequence is different from collagen from other sources. This makes red snapper scale collagen unique in physical and chemical properties and biological activity, and is a high-quality raw material for the preparation of collagen peptides.

[0003] Acidic proteases are a type of protease that has catalytic activity under acidic conditions, with an optimal pH of 2.0-5.0. Under acidic conditions, the triple helix structure in fish scale collagen is loosened due to hydrogen bonds and salt ions being bound by hydrogen ions, and the solubility of the protein is usually improved, creating favorable conditions for enzymatic hydrolysis. At the same time, the amino acid residues on the surface of fish scale collagen molecules will be protonated in an acidic environment, and the charge properties will change, which will promote the binding of the enzyme to the substrate to a certain extent and improve the efficiency of enzymatic hydrolysis. Using acidic proteases to hydrolyze fish scale collagen can obtain more significant extraction efficiency and more active small molecular weight collagen peptides.

[0004] Therefore, it is of great value to develop acidic proteases with excellent enzymatic hydrolysis performance. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a collagenase mutant, a gene fragment, a recombinant plasmid, a recombinant expression system, a collagen peptide, and a preparation method and use thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides a collagenase mutant, wherein the amino acid sequence of the collagenase mutant has at least 99% sequence identity with the sequence shown in SEQ ID NO: 5.

[0008] Furthermore, the amino acid sequence of the enzyme collagenase mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identity with the sequence shown in SEQ ID NO: 5.

[0009] Furthermore, the enzyme collagenase mutant has an amino acid sequence as shown in SEQ ID NO: 5.

[0010] The present invention also provides a gene fragment encoding a collagenase mutant, the nucleotide sequence of the gene fragment is shown in SEQ ID NO: 7.

[0011] The present invention also provides a recombinant plasmid, which comprises the above gene fragment.

[0012] Furthermore, the plasmid is a plasmid obtained by inserting the above gene fragment into the pTR50 vector.

[0013] The present invention also provides a recombinant expression system, which contains a host bacterium and the recombinant plasmid.

[0014] Furthermore, the host bacteria of the recombinant expression system is Trichoderma reesei Trichoderma reesei KH7.

[0015] The present invention also provides a method for preparing the above collagenase mutant, the method comprising the following steps:

[0016] (1) The orotate phosphoribosyltransferase gene as shown in SEQ ID NO: 1 pyr2 The sequence of the expression cassette, such as the sequence fragment containing the promoter, signal peptide, multiple cloning site and terminator as shown in SEQ ID NO: 2, and the sequence fragment containing the Escherichia coli replicon and ampicillin resistance gene as shown in SEQ ID NO: 3 were seamlessly cloned to obtain vector pTR50;

[0017] (2) The DNA fragment shown in SEQ ID NO: 7 was cloned into the vector pTR50 and transformed into Escherichia coli DH5α to obtain plasmid pT513;

[0018] (3) Transformation of plasmid pT513 into Trichoderma reesei Trichoderma reesei KH7, collect the colony spores, culture them, collect the fermentation supernatant enzyme liquid, and obtain the collagenase mutant.

[0019] The present invention also provides the use of the above-mentioned collagenase mutant, gene fragment, recombinant plasmid and recombinant expression system in the preparation of collagen peptides.

[0020] The present invention also provides a collagen peptide, which is a product obtained by using collagen as a raw material and enzymatically hydrolyzing the collagenase mutant.

[0021] Furthermore, the collagen is fish scale collagen.

[0022] Furthermore, the fish scale collagen is red snapper scale collagen.

[0023] The present invention also provides a method for preparing the above collagen peptide, which comprises the following steps: using collagen as a raw material, and enzymatically hydrolyzing it with the above collagenase mutant to obtain the collagen peptide.

[0024] Furthermore, the enzymatic hydrolysis temperature is 40° C., the enzymatic hydrolysis pH is 3.0, and the enzymatic hydrolysis time is 4 h.

[0025] The present invention also provides use of the above collagen peptide in preparing an antioxidant preparation.

[0026] The present invention has achieved the following beneficial effects:

[0027] The present invention obtains a collagenase mutant KT513 with high specific activity by recombinant expression in a Trichoderma reesei host, and its enzyme activity reaches 7423.18 U / mL. The optimal action pH of the enzyme is 3.0, and it can enzymatically hydrolyze red snapper scale collagen in the range of pH 3.0-5.0, and has excellent enzymatic activity. After enzymatic hydrolysis of the protease mutant KT513 at 40°C for 4 h, the extraction rate of fish scale collagen reaches 80.09%, and the prepared collagen peptide has good antioxidant activity. When the added concentration is 10 mg / mL, the in vitro scavenging activity of the prepared collagen peptide on hydroxyl free radicals reaches 99.26%, which is equivalent to ascorbic acid, and has broad application prospects in the preparation of antioxidant preparations.

[0028] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0029] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the SDS-PAGE image of collagenase KT501 and KT513.

[0031] Figure 2 is the relative enzymatic activity of collagenase KT501 and KT513 under acidic conditions.

[0032] Figure 3 pH stability of collagenase KT501 and KT513.

[0033] Figure 4 This is the enzymatic extraction rate of red snapper scale collagen by collagenase KT501 and KT513.

[0034] Figure 5 The in vitro scavenging activity of the enzymatic peptide solutions of collagenase KT501 and KT513 on hydroxyl free radicals. DETAILED DESCRIPTION

[0035] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0036] Example 1: Gene amplification and expression plasmid cloning

[0037] The cloning process of the basic plasmid pTR50 includes: chemical synthesis of the orotate phosphoribosyltransferase gene pyr2 The sequence of the expression cassette is shown in SEQ ID NO: 1; the sequence fragment containing the promoter, signal peptide, multiple cloning site and terminator is chemically synthesized as shown in SEQ ID NO: 2; the sequence fragment containing the Escherichia coli replicon and ampicillin resistance gene is chemically synthesized as shown in SEQ ID NO: 3; the above three DNA fragments were seamlessly cloned using the In-Fusion cloning kit of Takara Company, and the vector pTR50 was obtained by sequence determination.

[0038] The construction process of the expression plasmid pT501 includes: using the In-Fusion cloning kit of Takara Company, cloning the DNA fragment shown in SEQ ID NO: 6 into the vector pTR50 linearized by the restriction endonuclease NheI, transforming Escherichia coli DH5α, and obtaining the plasmid pT501 by sequence determination.

[0039] The construction process of the expression plasmid pT513 includes: using the In-Fusion cloning kit of Takara Company, cloning the DNA fragment shown in SEQ ID NO: 7 into the vector pTR50 linearized with the restriction endonuclease NheI, transforming Escherichia coli DH5α, and obtaining the plasmid pT513 by sequencing.

[0040] The DNA fragment shown in SEQ ID NO: 7 can be obtained by chemical synthesis and PCR amplification.

[0041] Example 2: Heterologous expression of collagenase KT501 and KT513 and preparation of fermentation supernatant enzyme solution

[0042] Method for heterologous expression of collagenase KT501 and KT513 and preparation of fermentation supernatant enzyme solution: The expression plasmids pT501 and pT513 provided in the above embodiment are respectively transferred into the uracil auxotrophic host Trichoderma reesei Trichoderma reesei Protoplasts of KH7; positive colonies were screened on protoplast transformation and regeneration medium (bottom culture medium: malt extract 30 g / L, sorbitol 1 mol / L, agar powder 1.5%; top culture medium: malt extract 30 g / L, sorbitol 1 mol / L, agarose 0.7%), and cultured at 30°C for 4-5 days; possible positive colonies grown on the transformation plate were picked onto a solid plate of MM basal medium without uridine, cultured at 30°C for 2-3 days, and rescreened. pyr2 The gene expression cassette is back-complemented into the positive clones integrated into the genome; the colonies that grow normally on the rescreening plate are inoculated onto a PDA solid plate without uridine added, and cultured at 30°C for 7-8 days until the green spores cover the plate.

[0043] Screening of recombinant strains by shake flask fermentation: spores of each colony were collected from the PDA plate and diluted to 10 6 spores / mL, 500 µL of spore suspension was transferred to 20 mL of seed medium (glucose 2%, yeast powder 1%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.02%, pH 6.0), and cultured at 30℃ and 220 r / min for 48 h. 2 mL of seed solution was transferred to 50 mL of fermentation medium (glucose 2%, tryptone 2%, potassium dihydrogen phosphate 0.1%, potassium dihydrogen phosphate 0.1%, anhydrous calcium chloride 0.01%, magnesium sulfate 0.005%, 1 mL of trace elements; trace elements: manganese sulfate 0.16%, ferrous sulfate 0.2%, zinc sulfate 0.14%, cobalt chloride 0.2%), and cultured at 30℃ and 220 r / min for 96 h. The fermentation supernatant enzyme solution was collected, and the fermentation supernatant enzyme solution containing the target protease KT501 and the fermentation supernatant enzyme solution containing the target protease KT513 were obtained respectively. The molecular weight of protease KT501 and KT513 were analyzed by SDS-PAGE, and the results were as follows: Figure 1 shown.

[0044] The amino acid sequence of the target protease KT513 is shown in SEQ ID NO: 5.

[0045] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0046] Experimental Example 1: Determination of enzyme activity of the mutant enzyme of the present invention under acidic conditions

[0047] Prepare 0.1 mol / L Britton-Robinson buffers of pH 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, and dilute the fermentation supernatant enzyme solution containing protease KT501 and the fermentation supernatant enzyme solution containing protease KT513 obtained in Example 2 to 10-15 U / mL with the buffer solutions of the above pH, respectively. Determine the enzyme activity of protease KT501 and KT513 on the substrate casein at 40°C and the corresponding pH conditions. The highest enzyme activity is defined as 100%, and the corresponding pH is the optimal pH of the enzyme on casein, and the relative enzyme activity under other pH conditions is calculated.

[0048] Referring to GBT 23527-2009 Protease Preparation, the activity of acidic protease was determined by spectrophotometry. Casein was used as substrate, and the reaction was carried out in a water bath at 40°C for 10 min. The reaction was terminated with trichloroacetic acid (TCA). After filtering the precipitate, the reaction was carried out with Folin reagent for 20 min, and the absorbance at 680 nm was measured by spectrophotometer.

[0049] Enzyme activity is defined as the amount of enzyme required to hydrolyze casein to produce 1 µg of tyrosine per minute under specific conditions, which is 1 unit of enzyme activity. At 40°C and pH 3.0, the highest expressed enzyme activity of enzyme KT501 was 4231.63 U / mL, and the highest expressed enzyme activity of mutant enzyme KT513 was 7423.18 U / mL, which was 1.75 times higher.

[0050] The results are as follows Figure 2 As shown in the figure, both enzymes KT501 and KT513 have casein degradation activity in the pH range of 2.0-7.0, and the optimal pH is 3.0, indicating that both enzymes are acidic proteases. Among them, in the pH range of 3.0-5.0, KT501 maintains a relative enzyme activity of more than 65.77%; the mutant enzyme KT513 can maintain a relative enzyme activity of more than 70.21%, which is significantly higher than that of KT501. The results of pH curve measurement show that the mutant enzyme KT513 has better applicability under acidic conditions.

[0051] The above results show that compared with the enzyme KT501, the enzyme activity of the mutant enzyme KT513 of the present invention is significantly improved and has better applicability under acidic conditions.

[0052] Experimental Example 2: pH stability of the mutant enzyme of the present invention

[0053] The fermentation supernatant enzyme solution containing protease KT501 and the fermentation supernatant enzyme solution containing protease KT513 obtained in Example 2 were diluted to 10-15 U / mL with 0.1 mol / L Britton-Robinson buffer at pH 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, and allowed to stand for 2 h. The residual enzyme activity under each pH condition was measured at 40°C, and the enzyme activity of the original fermentation supernatant enzyme solution without acid treatment was defined as 100%.

[0054] The results are as follows Figure 3 As shown, after being treated with a buffer solution of pH 3.0-6.0 for 2 h, the mutant enzyme KT513 showed higher stability than the original enzyme KT501. The specific data are as follows: after incubation for 2 h under pH 3.0 conditions, KT501 can maintain 60.46% of residual enzyme activity, while the relative enzyme activity of the mutant enzyme KT513 reached 70.19%, and the stability was improved by 16.09%; after being treated for 2 h under pH 4.0-6.0 conditions, the original enzyme KT501 had 70.13%-81.50% of residual activity; the relative enzyme activity of the mutant enzyme KT513 was between 76.17%-88.06%, which further confirmed that within the pH 3.0-6.0 range, the stability of the mutant enzyme of the present invention was also significantly improved.

[0055] Experimental Example 3: Collagenase hydrolysis performance of the mutant enzyme of the present invention

[0056] Rinse the collected red snapper scales with running water to remove impurities such as mud, mucus and blood attached to the surface. Then soak the scales in 12 times the volume of 5% (w / v) citric acid solution and stir at 4°C for decalcification. The scales will gradually become transparent and soft. When the decalcification solution (i.e. the solution after the fish scales react with 5% (w / v) citric acid solution) is mixed with the ammonium oxalate solution, there is no white precipitate, indicating that decalcification is basically completed. Take the fish scales out of the citric acid solution and rinse them repeatedly with running water to remove the residual citric acid solution until the rinse water is neutral, and dry the fish scales.

[0057] Add water equivalent to 4 times the volume of fish scales, stir well, and adjust the pH value of the mixed solution to 7.0-8.0. Add lipase at a ratio of 0.2‰ of the weight of the fish skin, and stir the reaction at 30°C for 2 h to complete the defatting of the fish scales. After the defatting reaction is completed, the temperature of the reaction solution is raised to 100°C and boiled for 20 min to inactivate the lipase. The reaction solution is cooled to 45-50°C, and the initial pH of the reaction solution is adjusted to 5.0 with acetic acid solution. 1‰ (w / w) of the fermentation supernatant enzyme solution containing protease KT501 and the fermentation supernatant enzyme solution containing protease KT513 obtained in Example 2 are added respectively, and the reaction is stirred at 40°C to obtain an aqueous solution of red snapper fish scale collagen peptide hydrolyzed by acidic proteases KT501 and KT513. It was tested that the pH of the reaction solution was maintained in the range of 3.0-5.0 during the enzymolysis process. The extraction rate of collagen in the enzymatic hydrolyzate (also called reaction solution) was determined at 1 h, 2 h, 3 h, 4 h, and 5 h of reaction, respectively. The extraction rate of collagen was calculated as follows:

[0058] Referring to GB 5009.5-2016 "National Food Safety Standard for Determination of Protein in Food", the total nitrogen content in red snapper scale collagen was determined by Kjeldahl method. The mass concentration of L-hydroxyproline standard was taken as the abscissa, and the absorbance at 560 nm was taken as the ordinate to draw the standard curve. The regression equation was y=0.4332x+0.0003 (R 2 =0.99995), and the hydroxyproline content in the enzymatic hydrolysis of fish scale collagen was determined and calculated. The extraction rate of collagen was expressed by hydroxyproline concentration using the following formula.

[0059] Extraction rate (%) = m1 × dilution factor × 11.1 / m0 × 100

[0060] Wherein, m1 is the hydroxyproline content, g; 11.1 is the coefficient of converting hydroxyproline to collagen; m0 is the total amount of red snapper scale protein.

[0061] Depend on Figure 4 It can be seen that the extraction rate of collagen increases with the extension of enzymatic hydrolysis time. After 2 h of enzymatic hydrolysis with enzyme KT501, the extraction rate of collagen was 60.46%; after 3 h, the extraction rate increased slowly and reached 70.56% after 4 h. After 2 h of enzymatic hydrolysis with enzyme KT513, the extraction rate of collagen was 67.21%, which was 111.16% of the control group enzyme KT501; after 4 h of enzymatic hydrolysis with enzyme KT513, the extraction rate of collagen reached 80.90%, which was 114.65% of the control group enzyme KT501.

[0062] The above results show that compared with the enzyme KT501, the mutant enzyme KT513 of the present invention has significantly improved enzymatic hydrolysis ability on collagen and significantly improved collagen extraction rate.

[0063] Experimental Example 4: Molecular weight determination of peptide solution obtained after enzymatic hydrolysis with the mutant enzyme of the present invention

[0064] The molecular weight distribution of peptides in the aqueous solution of red snapper scale collagen peptide prepared by enzymatic hydrolysis of proteases KT501 and KT513 for 4 h in Experimental Example 3 was detected by HPLC. Five standards were selected, including: cytochrome C (Mr=12500Da), aprotinin (Mr=6500 Da), bacitracin (Mr=1450 Da), tetrapeptide GGYR (Mr=451 Da) and tripeptide GGG (Mr=189 Da). The TSK gel G2000 SWXL chromatographic column (specification 7.8×300mm) produced by Tosoh Company of Japan was used, with a 45% acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid as the mobile phase, a flow rate of 0.5 mL / min, and the determination was carried out at a wavelength of 220nm. The standard curve was drawn based on the peak elution time and molecular weight of each standard. The two peptide solutions prepared in the above examples were diluted to 10 mg / mL, filtered through a 0.22 µm membrane, and 20 µL was taken for measurement to calculate the molecular weight of the peptide segments and the proportion of each component in the peptide solution prepared after enzymatic hydrolysis with proteases KT501 and KT513.

[0065] Table 1 Molecular weight of peptides in aqueous solution of red snapper scale collagen peptide

[0066]

[0067] The results are shown in Table 1. After 4 h of enzymatic hydrolysis, the red snapper scale collagen peptide aqueous solution prepared by the original enzyme KT501 hydrolysis was mainly composed of peptides with a molecular weight of less than 3000 Da, accounting for 90.11%, of which the content of peptides with a molecular weight of less than 1000 Da was 68.88%. In the red snapper scale collagen peptide aqueous solution prepared by the mutant enzyme KT513 hydrolysis, the proportion of peptides with a molecular weight of less than 3000 Da reached 92.03%, of which the content of peptides with a molecular weight of less than 1000 Da increased to 77.42%, especially the proportion of peptides with a molecular weight of less than 500 Da reached 33.85%, which was 1.8 times that of the original enzyme KT501. The above experimental results show that the mutant enzyme KT513 can prepare a high content of small molecular weight peptides, which is more conducive to maintaining the better physiological activity of collagen peptides.

[0068] Experimental Example 5: In vitro hydroxyl radical scavenging activity of the peptide solution obtained after enzymatic hydrolysis by the mutant enzyme of the present invention

[0069] Hydroxyl free radicals (•OH) can cause a series of oxidative stress-related diseases. Studies have shown that collagen peptides have antioxidant activity, especially the scavenging effect on hydroxyl free radicals. The Fenton reaction was used to detect the scavenging effect of collagen peptides in the aqueous solution of red snapper scale collagen peptides prepared by enzymatic hydrolysis of proteases KT501 and KT513 for 4 h in Experimental Example 3 on hydroxyl free radicals.

[0070] Prepare the red snapper scale collagen peptide aqueous solution (also known as enzymatic peptide solution) with a concentration of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 mg / mL. Add 1 mL of enzymatic peptide solution, 1 mL of 9 mmol / L salicylic acid-ethanol solution, 1 mL of 9 mmol / L ferrous sulfate solution, and 1 mL of 8.8 mmol / L H2O2 solution to the test tube to start the reaction. Incubate in a 37℃ constant temperature water bath for 30 min, and measure the absorbance of the mixed solution at 510 nm. Under the same conditions, an equal volume of distilled water was used to replace the 8.8 mmol / L H2O2 solution as the background group; an equal volume of distilled water was used to replace the sample as the blank control group, and an equal volume of ascorbic acid was used to replace the sample as the positive control group.

[0071] The free radical scavenging rate was calculated using the following formula:

[0072] Clearance rate (%) = [1 - (A1-A2) / A0] × 100

[0073] Where A1 is the absorbance of the sample, A2 is the absorbance of the background group, and A0 is the absorbance of the blank control group.

[0074] Depend on Figure 5 It can be seen that the scavenging ability of the enzymatic peptide solution with a concentration of 1-10 mg / mL for hydroxyl radicals (•OH) increases with the increase of the solution concentration. The peptide solution prepared after enzymatic hydrolysis with the original enzyme KT501 has a maximum scavenging activity of 86.31% when the addition amount is 10 mg / mL. The peptide solution prepared after enzymatic hydrolysis with the mutant enzyme KT513 has a scavenging activity of 99.26% when the addition amount is 10 mg / mL, which is equivalent to the scavenging ability of ascorbic acid. The above experimental results show that compared with the peptide solution prepared after enzymatic hydrolysis with the original enzyme KT501, the ability of the peptide solution prepared after enzymatic hydrolysis with the mutant enzyme KT513 to scavenging hydroxyl radicals has been significantly improved.

[0075] In summary, the present invention obtains a collagenase mutant KT513 with high specific activity through recombinant expression in a Trichoderma reesei host, and its enzyme activity reaches 7423.18 U / mL. The optimal action pH of the enzyme is 3.0, and it can enzymatically hydrolyze red snapper scale collagen in the range of pH 3.0-5.0, and has excellent enzymatic activity. After enzymatic hydrolysis of the protease mutant KT513 at 40°C for 4 h, the extraction rate of fish scale collagen reached 80.09%, and the prepared collagen peptide had good antioxidant activity. At an added concentration of 10 mg / mL, the in vitro scavenging activity of the prepared collagen peptide for hydroxyl free radicals reached 99.26%, which is equivalent to ascorbic acid, and has broad application prospects in the preparation of antioxidant preparations.

[0076] Sequence Listing:

[0077] SEQ ID NO: 1

[0078]

[0079] SEQ ID NO: 2

[0080]

[0081] SEQ ID NO: 3

[0082]

[0083] SEQ ID NO: 4

[0084] msdmekpwkegeearavlqgharaqepqavdkgpvagdermavtvvlrrqradalaahverqaaiapharehlkreafaashgaslddfaelrrfadahglaldranvaagtavlsgpvdainrafgvelrhfdhpdgsyrsylgevtvpasiapmieavlgldtrpvarphfrmqrraeggfearsqaaaptaytpldvaqayqfpegldgqgqciaiielgggydeaslaqyfaslgvpapqvvsvsvdgasnqptgdpsgpdgeveldievag alapgakiavyfapntdagfldaittaihdptlkpsvvsiswggpedswpsaaiaamnrafldaaalgvtvlaaagdsgstdgeqdglyhvdfpaaspyvlacggtrlvasggriaqetvwndgpdggatgggvsrifplpawqehanvppsanpgassgrgvpdlagnadpatgyevvidgeatviggtsavaplfaalvarinqklgkavgylnptlyqlpadvfhditegnndianraqiyqagpgwdpctglgspigvrllqallpsasqpqp

[0085] SEQ ID NO: 5

[0086] msdmekpwkegeearavlqgharaqepqavdkgpvagdermavtvvlrrqradalaahverqaaiapharehlkreafaashgaslddfaelrrfadahglaldranvaagtavlsgpvdainrafgvelrhfdhpdgsyrsylgevtvpasiapmieavlgldtrpvarphfrmqrraeggfearsqaaaptaytpldvaqayqfpegldgqgqciaiielgggydeaslaqyfaslgvpapqvvsvsvdgasnqptgdpegpdghvtldievagalapgakiavyfapdttagfldaittaihdptlkpsvvsiswggpedswpsaaiaamnrafldaaalgvtvlaaagnqgstsgeqdglyhvdfpaaspyvlacggtrlvasggriaqetvwndgpdggatgggvsrifplpawqehanvppsanpgassgrgvpdlagnadpatgyevvidgeatviggtsavaplfaalvarinqklgkavgylnptlyqlpadvfhditegnndianraqiyqagpgwdpctglgspigvrllqallpsasqpqp

[0087] SEQ ID NO: 6

[0088]

[0089] SEQ ID NO: 7

[0090]

Claims

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

5.

2. A gene fragment encoding a collagenase mutant, characterized in that: The nucleotide sequence of the gene fragment is shown in SEQ ID NO:

7.

3. A recombinant plasmid, characterized in that: The plasmid comprises the gene fragment according to claim 2.

4. A recombinant expression system, characterized in that: The recombinant expression system contains a host bacterium and the recombinant plasmid according to claim 3.

5. A method for preparing the collagenase mutant according to claim 1, characterized in that: The method comprises the following steps: (1) seamlessly cloning the sequence containing the orotate phosphoribosyltransferase gene pyr2 expression cassette as shown in SEQ ID NO:1, the sequence fragment containing the promoter, signal peptide, multiple cloning site and terminator as shown in SEQ ID NO:2, and the sequence fragment containing the Escherichia coli replicon and ampicillin resistance gene as shown in SEQ ID NO:3 to obtain vector pTR50; (2) The DNA fragment shown in SEQ ID NO:7 was cloned into vector pTR50 and transformed into Escherichia coli DH5α to obtain plasmid pT513; (3) The plasmid pT513 was transformed into Trichoderma reesei KH7, the colony spores were collected, cultured, and the fermentation supernatant enzyme liquid was collected to obtain the collagenase mutant.

6. Use of the collagenase mutant according to claim 1, the gene fragment according to claim 2, the recombinant plasmid according to claim 3, and the recombinant expression system according to claim 4 in the preparation of collagen peptides.

7. A collagen peptide, characterized in that: The collagen peptide is a product obtained by using collagen as a raw material and enzymatically hydrolyzing it with the collagenase mutant according to claim 1.

8. A method for preparing the collagen peptide according to claim 7, characterized in that: The method comprises the following steps: using collagen as a raw material, enzymatically hydrolyzing it with the collagenase mutant according to claim 1 to obtain collagen peptides.

9. Use of the collagen peptide according to claim 7 in the preparation of antioxidant preparations.

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