Protease mutant, encoding gene and application thereof

Abstract

The present application relates to a kind of protease mutant and its coding gene and application, belong to the field of bioengineering technology.The protease mutant is obtained by the amino acid sequence shown in SEQ ID NO.1 by Y137C, E165D point mutation, it has more than 75% enzyme activity in the range of pH=1.5-4.5, temperature 40-70 ℃, still can keep more than 50% activity after 1h at 60 ℃, with the advantages of high enzyme activity, strong pH and temperature adaptability, good high-temperature resistance, it is advantageous to improve the flexibility of application of acid protease in different production processes, especially in higher temperature industrial production, acid protease can still realize efficient catalysis, show good market prospect and industrial application value in the fields such as brewing, feed processing, leather softening.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a protease mutant, its encoding gene, and its applications. Background Technology

[0002] Proteases are a class of hydrolytic enzymes that break down peptide bonds in proteins. They are widely found in animals, plants, and microorganisms. Based on their optimal pH range, proteases can be divided into three main categories: acidic proteases (pH 2.0-5.0), neutral proteases (pH 6.0-8.0), and alkaline proteases (pH 8.0-11.0). Acidic proteases are proteases that exhibit high catalytic activity in acidic environments, with most having an optimal pH between 2.5 and 4.5. These enzymes have wide applications in the food industry (such as soy sauce brewing, beer clarification, and cheese manufacturing), feed industry, leather processing, pharmaceutical preparations, and detergent additives. Particularly in the feed industry, acidic proteases can effectively compensate for protein digestion disorders caused by insufficient gastric acid secretion in young animals; in food processing, they can effectively hydrolyze proteins under low pH conditions, improving product flavor and texture. Although acidic proteases have important value in industrial applications, existing acidic proteases generally suffer from insufficient acid and alkali stability and limited heat stability. These defects make it difficult for them to be widely used in food and feed industries where high-temperature processes or acid and alkali treatments are common.

[0003] In the prior art, wild-type acidic proteases are modified using genetic engineering and other modification techniques, and then screened to obtain protease mutants with improved specific activity, resistance to cold and heat, or resistance to acids and alkalis. For example, Chinese patent CN120944859A mutated the wild-type acidic protease pap1 gene to obtain an acidic protease mutant with improved heat resistance. This mutant can maintain more than 55% of its enzyme activity after being kept at 55°C for 1 hour, but it is completely inactivated at 60°C. However, in food or feed processing, the high-temperature treatments involved usually reach 60°C or above. Therefore, the application of such acidic proteases is still limited, and their heat resistance needs to be further improved. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a protease mutant, its encoding gene, and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a protease mutant, which is obtained by point mutation of the amino acid sequence shown in SEQ ID NO.1 at Y137C and E165D; the amino acid sequence of the protease mutant is shown in SEQ ID NO.8.

[0006] This invention is based on wild-type acidic protease pap1. Through single-point or combined mutations at key sites in the amino acid sequence and subsequent screening, a heat-resistant acidic protease mutant, ACPⅡ23, is obtained. The optimal reaction pH for ACPⅡ23 is 2.0, the optimal reaction temperature is 60℃, and it exhibits over 75% enzyme activity within a pH range of 1.5-4.5 and over 75% enzyme activity within a temperature range of 40-70℃. After treatment at 60℃ for 1 hour, it retains over 50% of its activity. Compared to wild-type acidic protease pap1 and the known acidic protease mutant AQAP52, the acidic protease mutant ACPⅡ23 has a wider pH and temperature adaptability range and significantly enhanced heat resistance.

[0007] In a second aspect, the present invention provides the encoding gene of the protease mutant described in the first aspect.

[0008] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.9.

[0009] Thirdly, the present invention provides a recombinant vector comprising the coding gene described in the second aspect.

[0010] Furthermore, the vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, viral expression vectors, or shuttle vectors.

[0011] Preferably, the carrier is pUC57.

[0012] Fourthly, the present invention provides a recombinant strain containing the recombinant vector described in the third aspect.

[0013] Furthermore, the recombinant strain is selected from prokaryotic or eukaryotic cells.

[0014] Preferably, the recombinant strain is *Trichoderma reesei*, and more preferably *Trichoderma reesei* QM 9414.

[0015] Furthermore, the method for preparing the protease mutant includes the following steps: transforming the host strain with the recombinant vector described in the third aspect to obtain a recombinant strain; culturing the recombinant strain in liquid to obtain a fermentation broth; and centrifuging and filtering the fermentation broth to obtain a filtrate, thereby obtaining a crude enzyme solution containing the protease mutant.

[0016] Preferably, the host cell is *Trichoderma reesei*, and more preferably *Trichoderma reesei* QM 9414.

[0017] Furthermore, the liquid culture is carried out at 27-30℃ and 150-250 r / min for 5-7 days.

[0018] Furthermore, the preparation method of protease mutants also includes purification steps, including but not limited to ammonium sulfate precipitation, ion exchange chromatography, gel filtration chromatography, etc.

[0019] Fifthly, the present invention provides the application of the protease mutant described in the first aspect in food processing, feed processing or leather processing.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a protease mutant, obtained by point mutation of the amino acid sequence shown in SEQ ID NO.1 at Y137C and E165D. The protease mutant is an acidic protease mutant, exhibiting over 75% enzyme activity within a pH range of 1.5-4.5 and a temperature range of 40-70℃. After treatment at 60℃ for 1 hour, it retains over 50% activity, demonstrating advantages such as high enzyme activity, strong pH and temperature adaptability, and good heat resistance. This improves the flexibility of acidic protease applications in different production processes, especially enabling efficient catalysis in high-temperature industrial production. It shows promising market prospects and industrial application value in fields such as brewing, feed processing, and leather softening. Attached Figure Description

[0021] Figure 1 The vector map of the recombinant expression vector pUCⅡ23; Figure 2 The relative enzyme activities of various acidic proteases at different pH values; Figure 3 The relative enzyme activities of various acidic proteases at different temperatures; Figure 4 Results on the heat stability of various acidic proteases. Detailed Implementation

[0022] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0023] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all materials, reagents, and biological materials used are commercially available.

[0024] Unless otherwise specified, the experimental methods in this invention are conventional methods. For molecular biology experimental methods not specifically described, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al., or follow the instructions of commercial reagent kits.

[0025] The strains or vectors used in this invention: Escherichia coli DH5α and Trichoderma reesei QM 9414 are commercially available; the expression vector pUC57 is commercially available.

[0026] Example 1: Screening of acidic protease mutants This invention utilizes Swiss Model (https: / / swissmodel.expasy.org / ) to perform homology modeling of the wild-type acidic protease pap1 (amino acid sequence shown in SEQ ID NO.1). After quality verification of the model, the enzyme-substrate molecule docking and analysis were performed using AutoDock software to screen out key sites affecting enzyme-substrate interaction, including 10 amino acid sites such as tyrosine at position 137 (Y137) and glutamic acid at position 165 (E165). Then, based on the wild-type acidic protease pap1 gene, and combining the key sites and mutation types designed at each site obtained from the aforementioned analysis, multiple single-site and combined mutation protease mutants were chemically synthesized. These protease mutants were expressed in *Trichoderma reesei*. The protease mutant genes contained the signal peptide of the pap1 gene (amino acid sequence MQTFGAFLVSFLAASGLAAA (as shown in SEQ ID NO.2)) as the secretory expression signal peptide. The chb1 promoter (nucleotide sequence as shown in SEQ ID NO.3) and chb1 terminator (nucleotide sequence as shown in SEQ ID NO.4) were fused with each acidic protease mutant gene. Subsequently, these were ligated with the *E. coli* ori replicon (nucleotide sequence as shown in SEQ ID NO.5), the ampicillin (Amp) gene (nucleotide sequence as shown in SEQ ID NO.6), and the pyr2 gene expression cassette (nucleotide sequence as shown in SEQ ID NO.7) to form a recombinant expression vector. Some primer sequences involved in constructing the recombinant expression vector are shown in Table 1. The obtained recombinant expression vector was transformed into *Trichoderma reesei* QM After inducing the secretory expression of the target gene in the pyr2 gene-deficient strain 9414, the temperature resistance of the acidic protease in the fermentation supernatant was measured, and the acidic protease mutant ACPⅡ23 with the best temperature resistance was finally obtained.

[0027] The amino acid sequence of the acidic protease mutant ACPⅡ23 was determined to be as shown in SEQ ID NO. 8: LPTEGQKTASVEVQYNKNYVPHGPTALFKAKRKYGAPISDNLKSLVAARQAKQALAKRQTGSAPNHPSDSADSEYITSVSIGTPAQVLPLDFDTGSSDLWVFSSETPKSSATGHAIYTPSKSSTSKKVSGASWSISCGDGSSSSGDVYTDKVTIGGFSVNTQGVDSATRVSTEFVQDTVISGLVGLAFDSGNQVRPHPQKTWFSNAASSLAEPLFTADLRHGQNGSYNFGYIDTSVAKGPVAYTPVDNSQGFWEFTASGYSVGGKLNRNSIDGIADGTTLLLLDDNVVDAYYANVQSAQYDNQQEGVVFDCDEDLPSFSFGVGSSTITIPGDLNLTPPLEEGSSTCFGGLQSSSGIGINIFGDVALKAALVVFDLGNERLGWAQQ;

[0028] Table 1. Partial primer sequences involved in constructing the expression vector. sequence name sequence F1 (5'→3') (SEQ ID NO.10) gtcaaccgcggactgcgcaccatgcagacctttggagctttc R1 (5'→3') (SEQ ID NO.11) caggctttcgccacggagctttacttctgagcccagcccag F2 (5'→3') (SEQ ID NO.12) gttattgtctcatggcggccgctctagagttgtgaagtcgg R2 (5'→3') (SEQ ID NO.13) gaaagctccaaaggtctgcatggtgcgcagtccgcggttgac F3 (5'→3') (SEQ ID NO.14) ctgggctgggctcagaagtaaagctccgtggcgaaagcctg R3 (5'→3') (SEQ ID NO.15) cgatccagatgtgcttctggctacgggttatgaacgggatg F4 (5'→3') (SEQ ID NO.16) catcccgttcataacccgtagccagaagcacatctggatcg R4 (5'→3') (SEQ ID NO.17) ctatggaaaaacgccagcaacgtagctagttacgcttgtttatttacg F5 (5'→3') (SEQ ID NO.18) cgtaaataaacaagcgtaactagctacgttgctggcgtttttccatag R5 (5'→3') (SEQ ID NO.19) ccgacttcacaactctagagcggccgccatgagacaataac F6 (5'→3') (SEQ ID NO.20) cactgtacgctttgacttccatgtg R6 (5'→3') (SEQ ID NO.21) gcagttgtacggaggctagctag Example 2 Preparation of acidic protease mutants (1) The chemically synthesized acidic protease mutant ACPⅡ23 gene carries a signal peptide sequence. Using the synthesized target gene as a template, the target gene fragment was amplified by PCR using F1 and R1 primer pairs. Using F2 and R2 primer pairs, F3 and R3 primer pairs, and F4 and R4 primer pairs, and using Trichoderma reesei QM 9414 genomic DNA as a template, the gene fragments of the chb1 promoter, chb1 terminator, and pyr2 expression cassette were amplified by PCR. Using F5 and R5 primer pairs, and using the pUC57 vector as a template, the fusion fragments of the Escherichia coli ori replicon and Amp resistance gene were amplified by PCR. The amplified target gene fragments, chb1 promoter, chb1 terminator, pyr2 expression cassette, ori replicon, and Amp resistance gene fragment were assembled by Gibson to obtain the recombinant expression vector pUCⅡ23. The vector map is shown below. Figure 1 As shown, the recombinant expression vector pUCⅡ23 was transformed into Escherichia coli DH5α, and the plasmid was extracted from the positive clone. The correct expression vector pUCⅡ23 was obtained by sequence determination.

[0029] (2) Construction of recombinant acid protease strains Using the pyr2 gene-deficient strain of Trichoderma reesei QM 9414 as the host strain, the recombinant expression vector pUCⅡ23 was transformed into the pyr2 gene-deficient strain of Trichoderma reesei QM 9414 via polyethylene glycol (PEG)-mediated protoplast transformation to obtain recombinant strain Ⅱ23 expressing the acidic protease mutant ACPⅡ23. The method for constructing the pyr2 gene-deficient strain of Trichoderma reesei QM 9414 is disclosed in the prior art CN120944859A, and includes the following steps: Trichoderma reesei QM 9414 was inoculated onto PDA plates and activated by incubation at 30°C for 6 days. Sterile water was then added to the activated QM 9414 plates to prepare a spore suspension. The spore suspension was then diluted 10... 3 10 4 10 5 The culture was spread onto PDA+U+5'-FOA plates and incubated at 30°C for 4 days. Single colonies with good growth were picked, and colony PCR was performed using F6 and R6 primer pairs, followed by sequencing verification to obtain the pyr2 gene-deficient strain.

[0030] Preparation of PDA+U+5'-FOA plates: Take 200 g of peeled fresh potato pieces, add 1000 mL of water, boil for 20 min, filter with gauze, add 20 g / L glucose, 2 g / L uracil, 1 g / L 5-FOA and 20 g / L agar, mix well and autoclave for later use.

[0031] (3) Preparation of crude enzyme solution of acidic protease The obtained recombinant strain II23 was inoculated on PDA plates and cultured for 7 days. A bacterial block of about 1 cm × 1 cm was cut and inoculated into an appropriate amount of liquid fermentation medium. The culture was cultured at 27℃ and 200 rpm for 7 days. The culture was centrifuged at 12000 r / min for 20 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain the crude enzyme solution of acidic protease mutant ACPⅡ23. The liquid fermentation medium formula is as follows: 10 g / L glucose, 20 g / L lactose, 20 g / L corn steep liquor, 5 g / L ammonium sulfate, 1 g / L magnesium sulfate, 10 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, and 1 mL trace elements, diluted to 1.0 L with deionized water. The trace element composition is 5.0 g / L FeSO4·7H2O, 1.6 g / L MnSO4·H2O, 1.4 g / L ZnSO4·7H2O, and 2.0 g / L CoCl2·2H2O. After mixing, the mixture is autoclaved and then used for further processing. The crude enzyme solutions of wild-type acidic protease pap1, acidic protease mutant ACPⅠ04 (mutation site: Y137C), and acidic protease mutant ACPⅡ18 (mutation site: Y137C+E165N) were prepared according to the above methods. In addition, the acidic protease mutant AQAP52 was prepared according to the content disclosed in patent CN120944859A.

[0032] Determination of the enzymatic properties of protease mutants in test cases 1. Referring to GB / T 23527.1-2023 Quality Requirements for Enzyme Preparations Part 1: Protease Preparations, the acidic protease activities of wild-type acidic protease pap1, acidic protease mutant ACPⅡ23, acidic protease mutant ACPⅠ04, and acidic protease mutant ACPⅡ18 were determined. The specific determination method is as follows: (1) Sample group: Mix 1 mL of appropriately diluted crude enzyme solution with 1 mL of casein substrate, react for 10 min at pH 3.0 and 40℃, and then add 2 mL of trichloroacetic acid (TCA) solution to terminate the reaction; Blank group: Mix 1 mL of crude enzyme solution with 2 mL of TCA, react for 10 min at pH 3.0 and 40℃, and then add 1 mL of casein substrate and mix evenly.

[0033] (2) Take the sample group and blank group after the reaction and filter them respectively. Take 1 mL of filtrate, add 5 mL of sodium carbonate solution and 1 mL of Folin reagent, incubate in a water bath at 40℃ for 20 min, and then measure the OD. 680 Absorbance. A standard curve was prepared using L-tyrosine, and the enzyme activity of the crude enzyme solution was calculated.

[0034] (3) Catalytic efficiency analysis and comparison: The enzyme activity of wild-type acidic protease was taken as 100%, and the relative enzyme activity of acidic protease mutant was calculated. The relative enzyme activity (%) = crude enzyme activity of acidic protease mutant / crude enzyme activity of wild-type acidic protease * 100%. The results are shown in Table 2.

[0035] Table 2. Results of enzymatic property determination of acidic protease mutant enzymes Group Relative enzyme activity (%) Mutation method Acidic protease mutant ACPⅡ23 138 Y137C+E165D Acidic protease mutant ACPⅠ04 115 Y137C Acidic protease mutant ACPⅡ18 103 Y137C+E165Q Wild-type acidic protease pap1 100 / 2. Determination of the optimal pH for protease mutants Appropriately diluted crude enzyme solutions of wild-type acidic protease pap1, acidic protease mutant ACPⅡ23, and acidic protease mutant AQAP52 were subjected to enzymatic hydrolysis reactions under different pH conditions (1.0-6.0). The optimal reaction pH was determined at a reaction temperature of 40℃. The relative enzyme activities of each acidic protease at different pH values ​​were calculated, with the maximum enzyme activity at the time of measurement taken as 100%. The results are shown below. Figure 2 As shown.

[0036] 3. Determination of the optimal temperature for protease mutants Wild-type acidic protease pap1, acidic protease mutant ACPⅡ23, and acidic protease mutant AQAP52 were appropriately diluted crude enzyme solutions and subjected to enzymatic hydrolysis reactions at different temperatures (30-70℃). The optimal reaction temperature was determined at pH 3.0. The relative enzyme activities of each acidic protease at different temperatures were calculated, with the maximum enzyme activity at the time of measurement taken as 100%. The results are shown below. Figure 3 As shown.

[0037] 3. Stability determination of protease mutants The thermostability of wild-type acidic protease pap1, acidic protease mutant ACPⅡ23, and acidic protease mutant AQAP52 was determined using the following methods: Crude enzyme solutions of wild-type acidic protease pap1, acidic protease mutant ACPⅡ23, and acidic protease mutant AQAP52, diluted appropriately, were used as test samples. These solutions were incubated in water baths at 40℃, 50℃, 60℃, and 70℃ for 1 hour, respectively. Enzyme activity was then measured at pH 3.0 and 40℃. The residual enzyme activity was calculated with the enzyme activity before incubation as 100%. The results are as follows: Figure 4 As shown.

[0038] Results Analysis: As shown in Table 2, the enzyme activities of acidic protease mutants ACPⅡ23, ACPⅠ04, and ACPⅡ18 were increased by 38%, 15%, and 3% respectively compared to wild-type acidic protease. This indicates that the enzyme activity of acidic protease mutants containing dual mutations of Y137C and E165D was significantly increased, while the enzyme activity of acidic protease mutants containing only a single mutation of Y137C was increased to a certain extent. The enzyme activity of acidic protease mutants containing dual mutations of Y137C and E165Q was comparable to that of wild-type acidic protease pap1. This suggests that the mutation sites and the types of mutated amino acids have a significant impact on the activity of enzyme mutants.

[0039] Depend on Figure 2 , 3 The results show that the optimal reaction pH for the acidic protease mutant ACPⅡ23 is 2.0, the optimal reaction temperature is 60℃, and it exhibits over 75% enzyme activity within the pH range of 1.5-4.5 and over 75% enzyme activity within the temperature range of 40-70℃. The optimal reaction pH for both the wild-type acidic protease pap1 and the acidic protease mutant AQAP52 is 2.5, and the optimal reaction temperature is 55℃. The acidic protease mutant ACPⅡ23 provided by this invention has a wider pH and temperature adaptability range. Figure 4 The results show that the acidic protease mutant ACPⅡ23 provided by the present invention still retains more than 50% of its activity after treatment at 60℃ for 1 hour, while the wild-type acidic protease pap1 and the acidic protease mutant AQAP52 completely lose their enzyme activity after treatment at 60℃ for 1 hour. This indicates that the heat resistance of the acidic protease mutant ACPⅡ23 provided by the present invention has been significantly improved, and it can be used more widely in high-temperature environments.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A protease mutant, characterized in that, The protease mutant was obtained by point mutation of the amino acid sequence shown in SEQ ID NO.1 at Y137C and E165D; the amino acid sequence of the protease mutant is shown in SEQ ID NO.

8.

2. The encoding gene of the protease mutant according to claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

9.

4. A recombinant vector, characterized in that, The recombinant vector comprises the coding gene as described in claim 2 or 3.

5. The recombinant vector as described in claim 4, characterized in that, The carrier is pUC57.

6. A recombinant bacterial strain, characterized in that, Contains the recombinant vector as described in claim 4 or 5.

7. The recombinant strain according to claim 6, characterized in that, The recombinant strain is Trichoderma reesei.

8. The method for preparing the protease mutant according to claim 1, characterized in that, The process includes the following steps: transforming the host strain with the recombinant vector described in claim 4 to obtain a recombinant strain; culturing the recombinant strain in liquid to obtain a fermentation broth; and centrifuging and filtering the fermentation broth to obtain a filtrate, which is a crude enzyme solution containing a protease mutant.

9. The preparation method according to claim 8, characterized in that, The liquid culture was carried out at 27-30℃ and 150-250 r / min for 5-7 days.

10. The use of the protease mutant of claim 1 in food processing, feed processing or leather processing.

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