Method for improving heat stability of alkaline protease, mutant thapt3-m6, m7, m8, m9 and application thereof

Abstract

This invention relates to the fields of genetic engineering and protein engineering, specifically to a method for improving the thermostability of alkaline proteases and mutants ThAPT3-M6, 7, 8, and 9, and their applications. This invention uses alkaline protease PA3 as the parent, and respectively... 18 Region-optimized mutants ThAPT3-M2, α8-helix region-optimized mutant ThAPT3-M4, and loop 19 The region-optimized mutant ThAPT3-M5 was subjected to two-region and three-region stacking, respectively, resulting in four mutants. The T mutants... m The values ​​increased by 7.2℃, 4.3℃, 5.7℃, and 7.5℃, with t at 55℃. 1 / 2 The levels were increased to 10.6 times, 6.9 times, 8.5 times, and 12.2 times that of the parent lines, respectively, at 60℃. 1 / 2 The specific activities of mutants M6, M7, M8, and M9 were increased to 21.2, 13.2, 17.6, and 39.7 times that of the parent, respectively. The specific activities of mutants M6, M7, M8, and M9 were increased by 23%, 25%, 20%, and 62% compared to the parent, respectively. Therefore, the alkaline protease mutants provided by this invention have better thermal stability and catalytic activity than the wild type, and have better prospects for industrial applications.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and protein engineering, specifically to a method and mutant for improving the thermostability of alkaline proteases. Th APT3-M6, 7, 8, 9 and Applications. Background Technology

[0002] Serine proteases are important peptide bond hydrolases, accounting for one-third of all known proteases. The subtilisin family, also known as the S8 family of serine proteases, is the second largest family of serine proteases. Therefore, they possess broad cleavage specificity and excellent organic solvent stability, making them widely used in various industrial sectors such as food, leather, medicine, and animal feed. Proteases hold a significant market share in enzyme preparations. However, proteases exhibit poor thermal stability; even the most widely used commercial protease in industry, Proteinase K, shows unsatisfactory stability at 60°C. Therefore, the stability of alkaline proteases at high temperatures is a major bottleneck limiting their further industrial applications. Summary of the Invention

[0003] The purpose of this invention is to provide... Torrubiella hemipterigena The alkaline protease PA3 derived from this strain is a mutant with improved thermostability and specific activity obtained from the parent. Th APT3-M6, Th APT3-M7, Th APT3-M8, Th APT3-M9.

[0004] Another object of the present invention is to provide the gene for the above-mentioned alkaline protease mutant.

[0005] Another object of the present invention is to provide a recombinant vector comprising the above-described alkaline protease mutant.

[0006] Another object of the present invention is to provide a recombinant strain comprising the above-described alkaline protease mutant.

[0007] Another object of the present invention is to provide a method for preparing an alkaline protease with improved thermal stability.

[0008] Another object of the present invention is to provide the application of the above-mentioned protease mutant.

[0009] This invention is based on Torrubiella hemipterigena The alkaline protease PA3 derived from the parent protein was mutated to obtain a thermostable mutant alkaline protease. The amino acid sequence of PA3 is shown in SEQ ID NO:1. (Underlined amino acid sequences indicate the sequence of the leader peptide.) SEQ ID NO:1: APSLARREEPAPLLEARGAQAIPGKFIVKLREGSPLAALQQAMSLLGGKADHVFQNVFSGFAASMNPA VIELMRNHPDVEYIEQDGKVNIN AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDYKTRGCPKGAVASMSLGGGKTQA VNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYLMGKDGAVAAGLCAKIAQTATRNVLRNIPAGTINALAFNGNPSG*.

[0010] The alkaline protease mutant with improved thermal stability according to this application Th APT3-M6, wherein the amino acid sequence of the mutant is shown in SEQ ID NO:2 or SEQ ID NO:3, and the mutation site of the shown amino acid sequence is: loop. 18 The mutant M2, consisting of regional segment substitution and site-directed mutation (from M... 330 GKDGAVAAGLC 341 Replace with M 330 TLGRTTAANAC 341 Then, the mutant M4 (where amino acid at position 333 is mutated from G to N) and the two-point combination mutant M4 (where Ala at position 342 is mutated to Glu and Ala at position 345 is mutated to Gln) in the α8-helix region were superimposed. (Underlined amino acid sequences are the sequence corresponding to the leader peptide, and bold italicized amino acids are the mutant amino acids) SEQ ID NO:2 (containing the leader peptide sequence): APSLARREEPAPLLEARGAQAIPGKFIVKLREGSPLAALQQAMSLLGGKADHVFQNVFSGFAASMNPA VIELMRNHPDVEYIEQDGKVNIN AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDY KTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYL MTLNRTTAANACE KI QQTATRNVLRNIPAGTINALAFNGNPSG*.

[0011] SEQ ID NO:3 (excluding the leader peptide sequence): AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDY KTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYL MTLNRTTAANACE KI Q QTATRNVLRNIPAGTINALAFNGNPSG*.

[0012] The alkaline protease mutant with improved thermal stability according to this application Th APT3-M7, wherein the amino acid sequence of the mutant is shown in SEQ ID NO:4 or SEQ ID NO:5, and the mutation site of the shown amino acid sequence is: loop. 18 The mutant M2, consisting of regional segment substitution and site-directed mutation (from M... 330 GKDGAVAAGLC 341 Replace with M 330 TLGRTTAANAC 341 Then, the 333rd amino acid was mutated from G to N) and loop 19 The three-point combination mutant M5 (Arg at position 350 mutated to Leu, Ala at position 358 mutated to Glu, and Ser at position 371 mutated to Asn) is superimposed. (Underlined amino acid sequences are the corresponding amino acid sequences of the leader peptide, and bold italics are the mutant amino acids) SEQ ID NO:4 (containing the leader peptide sequence): APSLARREEPAPLLEARGAQAIPGKFIVKLREGSPLAALQQAMSLLGGKADHVFQNVFSGFAASMNPA VIELMRNHPDVEYIEQDGKVNIN AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDY KTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYL MTLNRTTAANAC AKIAQTAT L NVLRNIP E GTINALAFNGNP N G*.

[0013] SEQ ID NO:5 (excluding the leader peptide sequence): AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDY KTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYL MTLNRTTAANAC AKIAQTAT L NVLRNIP E GTINALAFNGNP N G *.

[0014] The alkaline protease mutant with improved thermal stability according to this application Th APT3-M8, wherein the amino acid sequence of the mutant is shown in SEQ ID NO:6 or SEQ ID NO:7, and the mutation sites of the shown amino acid sequence are: the double-point combination mutant M4 in the α8-helix region (Ala at position 342 is mutated to Glu, and Ala at position 345 is mutated to Gln) and loop. 19 The three-point combination mutant M5 (Arg at position 350 mutated to Leu, Ala at position 358 mutated to Glu, and Ser at position 371 mutated to Asn) is superimposed. (Underlined amino acid sequences are the corresponding amino acid sequences of the leader peptide, and bold italics are the mutant amino acids) SEQ ID NO:6 (containing the leader peptide sequence): APSLARREEPAPLLEARGAQAIPGKFIVKLREGSPLAALQQAMSLLGGKADHVFQNVFSGFAASMNPA VIELMRNHPDVEYIEQDGKVNIN AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDYKTRGCP KGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYLMGKDGAVAAGLC E KI Q QTAT L NVLRNIP E GTINALAFNGNP N G*.

[0015] SEQ ID NO:7 (excluding the leader peptide sequence): AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDYKTRGCP KGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYLMGKDGAVAAGLC E KI Q QTAT L NVLRNIP E GTINALAFNGNP N G *.

[0016] The alkaline protease mutant with improved thermal stability according to this application Th APT3-M9, wherein the amino acid sequence of the mutant is shown in SEQ ID NO:8 or SEQ ID NO:9, and the mutation site of the shown amino acid sequence is: loop. 18 The mutant M2, consisting of regional segment substitution and site-directed mutation (from M... 330 GKDGAVAAGLC341 Replace with M 330 TLGRTTAANAC 341 Subsequently, the two-point combination mutant M4 (mutating Ala at position 333 from G to N), the α8-helix region (mutating Ala at position 342 to Glu and Ala at position 345 to Gln), and the loop were mutated. 19 The three-point combination mutant M5 (mutation of Arg at position 350 to Leu, Ala at position 358 to Glu, and Ser at position 371 to Asn) was superimposed.

[0017] SEQ ID NO: 8 (containing the leader peptide sequence): APSLARREEPAPLLEARGAQAIPGKFIVKLREGSPLAALQQAMSLLGGKADHVFQNVFSGFAASMNPA VIELMRNHPDVEYIEQDGKVNIN AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDY KTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYL MTLNRTTAANACE KI Q QTAT L NVLRNIP E GTINALAFNGNP N G*.

[0018] SEQ ID NO: 9 (excluding the leader peptide sequence): AYTTQTGAPWGLGRISHRAKGSTSYTYDTSAGEGTCVYVIDTGVEDTHPEFEGRAKLIKTYYGNRDGHGHGTHCSGTIGSKTYGVAKKTKIYGVKVLDDNGSGTFSNIIAGVDFVANDY KTRGCPKGAVASMSLGGGKTQAVNDAVARLQRAGVFVAVAAGNDNTDAANTSPASEPSVCTVGASDKDDVRSTFSNYGSVVDIFAPGTAILSTWIGGRTNTISGTSMATPHIAGLAAYL MTLNRTTAANACE KI Q QTAT L NVLRNIP E GTINALAFNGNPN G *.

[0019] According to a specific embodiment of the present invention, the gene sequence of alkaline protease PA3 is shown in SEQ ID NO:10.

[0020] SEQ ID NO: 10: GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTGCTCAAGCTATCCC AGGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCTGCATTGCAACAAGCTATGTCCTTGCTTGGTGGT AAGGCTGACCACGTTTTCCAGAACGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGA GAAACCATCCAGACGTCGAGTACATTGAGCAGGACGGTAAGGTTAACATCAAC .

[0021] This invention provides a gene encoding the above-mentioned protease mutant.

[0022] According to a specific embodiment of the present invention, an alkaline protease mutant Th The nucleotide sequence of the APT3-M6 encoding gene is shown in SEQ ID NO:11 or SEQ ID NO:12.

[0023] SEQ ID NO: 11 (comprising the leader peptide sequence): GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTGCTCAAGCTATCCC AGGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCTGCATTGCAACAAGCTATGTCCTTGCTTGGTGGT AAGGCTGACCACGTTTTCCAGAACGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGA GAAACCATCCAGACGTCGAGTACATTGAGCAGGACGGTAAGGTTAACATCAAC GCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTG ATGACTTTGAACAGAACTACTGCTGCCAACGCATGTGAA AAGATT CAA CAGACTGCCACCAGAAACGTCCTGAGAAATATTCCAGCTGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCATCTGGTTAA.

[0024] SEQ ID NO: 12 (without the leader peptide sequence): GCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTG ATGACTTTGAACAGAACTACTGCTGCCAACGCATGTGA A AAGATT CAA CAGACTGCCACCAGAAACGTCCTGAGAAATATTCCAGCTGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCATCTGGTTAA.

[0025] According to a specific embodiment of the present invention, the alkaline protease mutant Th The nucleotide sequence of the APT3-M7 encoding gene is as shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0026] SEQ ID NO: 13 (comprising the leader peptide sequence): GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTGCTCAAGCTATCCC AGGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCTGCATTGCAACAAGCTATGTCCTTGCTTGGTGGT AAGGCTGACCACGTTTTCCAGAACGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGA GAAACCATCCAGACGTCGAGTACATTGAGCAGGACGGTAAGGTTAACATCAAC GCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTG ATGACTTTGAACAGAACTACTGCTGCCAACGCAT GTGCTAAGATTGCTCAGACTGCCACC TTG AACGTCCTGA GAA ATATTCCAGAAGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCA AAC GGTTAA

[0027] SEQ ID NO: 14 (without leader peptide sequence): GCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTG ATGACTTTGAACAGAACTACTGCTGCCAACGCAT GTGCTAAGATTGCTCAGACTGCCACC TTG AACGTCCTGA GAA ATATTCCAGAAGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCA AAC GGTTAA.

[0028] According to a specific embodiment of the present invention, the alkaline protease mutant Th The nucleotide sequence of the APT3-M8 encoding gene is as shown in SEQ ID NO:15 or SEQ ID NO:16.

[0029] SEQ ID NO: 15 (comprising leader peptide sequence): GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTGCTCAAGCTATCCC AGGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCTGCATTGCAACAAGCTATGTCCTTGCTTGGTGGT AAGGCTGACCACGTTTTCCAGAACGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGA GAAACCATCCAGACGTCGAGTACATTGAGCAGGACGGTAAGGTTAACATCAAC GCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTGATGGGTAAAGATGGTGCAGTTGCTGCAGGTTTGTGT GAA AAGATT CAA CAGACTGCCACC TTG AACGTCCTGAGAAATATTCCA GAA GGTACTATCAACGCCCTGGCCTTTAACGGTAATCCA AAC GGTTAA.

[0030] SEQ ID NO: 16 (excluding the leader peptide sequence): GCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTGATGGGTAAAGATGGTGCAGTTGCTGCAGGTTTGTGT GA A AAGATT CAA CAGACTGCCACC TTG AACGTCCTGAGAAATATTCCA GAA GGTACTATCAACGCCCTGGCCTTTAACGGTAATCCA AAC GGTTAA.

[0031] According to the specific embodiment of the present invention, the alkaline protease mutant Th The nucleotide sequence of the APT3-M9 encoding gene is shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0032] SEQ ID NO: 17 (including the leader peptide sequence): GCTCCATCCTTGGCTAGAAGAGAAGAACCAGCTCCTTTGTTGGAAGCTAGAGGTGCTCAAGCTATCCC AGGTAAGTTCATCGTCAAGTTGAGAGAGGGTTCTCCATTGGCTGCATTGCAACAAGCTATGTCCTTGCTTGGTGGT AAGGCTGACCACGTTTTCCAGAACGTTTTCTCTGGTTTCGCCGCCTCTATGAACCCAGCTGTTATTGAGTTGATGA GAAACCATCCAGACGTCGAGTACATTGAGCAGGACGGTAAGGTTAACATCAAC GCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTG ATGACTTTGAACAGAACTACTGCTGCCAACGCATGTGAA AAGATT CAA CAGACTGCCACC TTG AACGTCCTGA GAAATATTCCAGAAGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCA AAC GGTTAA.

[0033] SEQ ID NO: 18 (without leader peptide sequence): GCCTACACTACTCAGACTGGTGCTCCATGGGGTTTGGGTAGAATTTCTCATAGAGCTAAGGGTTCCACCTCCTACACTTACGATACTTCCGCTGGTGAGGGTACTTGTGTTTACGTTATCGACACTGGTGTCGAGGACACTCACCCAGAATTTGAGGGTAGAGCCAAGCTGATCAAGACCTACTACGGTAACAGAGATGGTCACGGTCATGGTACTCACTGTTCCGGTACTATTGGTTCCAAGACTTACGGTGTCGCCAAAAAGACCAAAATCTACGGTGTCAAGGTCCTGGACGATAACGGTTCTGGTACTTTCTCCAACATTATCGCCGGTGTTGACTTCGTTGCCAACGACTACAAGACTAGAGGTTGTCCAAAGGGTGCTGTTGCCTCTATGTCTCTTGGTGGTGGAAAGACTCAAGCTGTTAACGACGCTGTTGCTAGATTGCAACGTGCCGGTGTTTTTGTTGCTGTTGCTGCTGGTAACGACAACACTGATGCTGCTAATACTTCTCCAGCTTCTGAGCCATCCGTCTGTACTGTTGGTGCTTCTGATAAGGACGACGTCAGATCCACCTTCTCTAACTACGGTTCCGTTGTTGACATCTTCGCTCCAGGTACTGCTATCTTGTCCACTTGGATTGGTGGTAGGACTAACACCATCTCCGGTACTTCTATGGCTACTCCACACATTGCTGGTTTGGCTGCTTACCTG ATGACTTTGAACAGAACTACTGCTGCCAACGCATGTGA A AAGATT CAA CAGACTGCCACC TTG AACGTCCTGA GAA ATATTCCAGAAGGTACTATCAACGCCCTGGCCTTTAACGGTAATCCA AACGGTTAA.

[0034] This invention provides a recombinant vector pPICZαA- containing the above-mentioned alkaline protease mutant encoding gene. Th APT3-m6, 7, 8, 9.

[0035] The present invention also provides a recombinant strain GS115- containing the above-mentioned alkaline protease mutant encoding gene. Th APT3-M6, 7, 8, 9.

[0036] The present invention also provides a method for preparing a protease with improved thermal stability, the method comprising the following steps: Recombinant strains were obtained by transforming Pichia pastoris GS115 host cells with a recombinant vector containing the parental gene encoding alkaline protease. Recombinant strains were cultured to induce alkaline protease expression; The recombinant expressed alkaline protease was recovered and purified.

[0037] The beneficial effects of this invention are: This invention uses alkaline protease PA3 as a parent, and separately... 18 Region-optimized mutants Th APT3-M2 and α8-helix region optimized mutants Th APT3-M4 and loop 19 Region-optimized mutants Th APT3-M5 was used for two-region and three-region overlay respectively, producing... Th Four mutants, APT3-M6 (M2+M4), M7 (M2+M5), M8 (M4+M5), and M9 (M2+M4+M5), were identified. Enzymatic characterization revealed that these four mutants... T m The values ​​increased by 7.2℃, 4.3℃, 5.7℃, and 7.5℃ at 55℃. t 1 / 2 The levels were increased to 10.6 times, 6.9 times, 8.5 times, and 12.2 times that of the parent lines, respectively, at 60℃. t 1 / 2 The specific activities of the mutants M6, M7, M8, and M9 were increased to 21.2, 13.2, 17.6, and 39.7 times that of the parent, respectively. Furthermore, the specific activities of the mutants M6, M7, M8, and M9 were increased by 23%, 25%, 20%, and 62% compared to the parent, respectively. Therefore, the alkaline protease mutants provided by this invention exhibit better thermal stability and catalytic activity than the wild type, and have better prospects for industrial applications. Attached Figure Description

[0038] Figure 1 Display alkaline protease PA3 and mutants.Th APT3-M6, 7, 8, and 9 Coomassie protein staining gel images; Figure 2 Display alkaline protease PA3 and mutants. Th APT3-M6, 7, 8, and 9 glycoprotein staining gel images; Figure 3 Displays alkaline protease PA3 and a two-region combination mutant. Th Specific activity of APT3-M6, 7, and 8 at different temperatures; Figure 4 Displays alkaline protease PA3 and a two-region combination mutant. Th Thermal stability of APT3-M6, 7, and 8 at 55 and 60°C; Figure 5 Displaying alkaline protease PA3 and three-point combination mutants. Th The optimal temperature for APT3-M9; Figure 6 Displaying alkaline protease PA3 and three-point combination mutants. Th Thermal stability of APT3-M9. Detailed Implementation

[0039] Experimental materials and reagents: Strains and vectors: expression host is Pichia pastoris GS115, the expression plasmid vector is pPICZαA.

[0040] Enzymes and other biochemical reagents: Restriction endonucleases and other reagents can be purchased from general biochemical reagent companies.

[0041] Culture medium: (1) Low-salt LB (LLB) medium for Escherichia coli (1% peptone, 0.5% yeast extract, 0.5% NaCl, natural pH); (2) Pichia pastoris culture medium YPD (1% yeast extract, 2% peptone, 2% glucose, natural pH); (3) BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB, 0.00004% biotin, natural pH). (4) BMMY medium (1% yeast extract, 2% peptone, 0.5% methanol, 1.34% YNB, 0.00004% biotin, natural pH). Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0042] Example 1: Alkaline protease mutant recombinant vector pPICZαA- Th Preparation of APT3-m6, 7, 8, and 9 The parental alkaline protease PA3 (pre-mutation) sequence fragment (with signal peptide removed) was cloned into the expression vector pPIC-ZαA, and the recombinant vector was named pPICZαA- Th APT3 - wt; with recombinant vector pPICZαA- Th APT3 - Using wt as a template, the vector is amplified by primers carrying mutation sites to obtain recombinant vectors carrying the sequences of each mutant.

[0043] Table 1. Alkaline protease mutants Th Specific primers for APT3-m6, 7, 8, and 9 .

[0044] Example 2 Construction of alkaline protease mutant expression strain

[0045] (1) Expression vector electrotransformation of expression host Use restriction endonucleases Dra The linearized expression vector was electroporated into the expression host GS115 competent cells. After incubation with sorbitol for 30 min, the cells were plated on YPD plates containing bleomycin resistance (100 μL / 100 mL) and cultured at 30 °C for 2 days.

[0046] (2) Screening of transformants with high protease activity Using a sterilized toothpick, pick a single colony from a YPD plate containing transformants and inoculate it onto a milk double-layer screening solid plate. Incubate overnight at 30°C. Once a visible milk hydrolysis clear zone appears on the milk plate, select the transformant with the largest hydrolysis zone and inoculate it into 30 mL of YPD medium.

[0047] Example 3 Expression and purification of recombinant protease mutants

[0048] (1) Protease mutant Th Shaking-flask level expression of APT3-M6, 7, 8, and 9 Transformants with high enzyme activity were inoculated at a 1% inoculum rate into 300 mL of BMGY medium. After incubation at 30°C and 200 rpm for 48 h with shaking, the culture medium was centrifuged at 4500 rpm for 5 min, and the supernatant was discarded. The culture was resuspended in 200 mL of BMGY medium and incubated at 30°C and 200 rpm for another 48 h with shaking. During the incubation period, 0.5% methanol was added to the medium every 24 h. After the incubation period, the supernatant fermentation broth was collected by centrifugation at 12000 rpm for 10 min for purification.

[0049] (2) Protease mutant Th APT3-M6, 7, 8, 9 purification The collected fermentation broth was concentrated to 15 mL using a 5 kDa membrane bag. Salt ions were removed by overnight dialyzing in 20 mM citrate-phosphate buffer (pH 5.5) using a 3 kDa dialysis bag. Purification was performed by cation exchange chromatography using 20 mM citrate-phosphate buffer (pH 5.5) solutions A and B. Enzyme activity was detected and analyzed by SDS-PAGE electrophoresis of the 1 M NaCl linear elution buffer, and staining was performed using Coomassie Brilliant Blue G250. Figure 1 The mutants are the same size as the parent proteins. The mutation sites in mutants M6, M7, and M9 all involve a mutation of G to N at position 333, successfully introducing glycosylation modification. This mutation result was verified using the Pierce™ Glycoprotein Staining Kit for glycoprotein staining. Figure 2 ).

[0050] Example 4 Enzymatic property analysis of alkaline protease PA3 and its two-region superimposed mutants M6, M7 and M8

[0051] The enzymatic properties of the protease of this invention were determined using the Folin-Ciocalteu method. Casein was used as the substrate. 500 μL of substrate (1% w / w) and 500 μL of enzyme solution of appropriate dilution were reacted at different temperatures for 20 min. The reaction was terminated by adding 1 mL of 0.4 M trichloroacetic acid solution. The reaction system was transferred to a 2 mL EP tube, centrifuged at 12000 rpm for 3 min, and 500 μL of the supernatant was added to 2.5 mL of 0.4 M sodium carbonate solution, followed by 500 μL of Folin-Ciocalteu reagent. The mixture was incubated at 40 °C for 30 min. After cooling, 250 μL of the incubation system was read, and the absorbance at 680 nm was recorded. The protease activity unit is defined as the amount of enzyme required to break down casein to produce 1 µmol of tyrosine per minute under certain conditions.

[0052] Comparison of specific activity between parental lines and double-region superimposed mutants at different temperatures In a borax-NaOH buffer system, the specific activities of the purified enzyme solution were determined at 55℃, 60℃, and 65℃ for both the parent PA3 and the mutant. Figure 3 As shown, compared with the wild type, the optimum temperature for the double-region superposition mutants was 65℃, which was 5℃ higher than the optimum temperature of the parents. In addition, the specific activity of the three double-region superposition mutants was increased to some extent compared with the parents.

[0053] Comparison of thermal stability of parental lines and double-region superposition mutants at 55 and 60 °C The purified parental and mutant enzyme solutions were diluted to 100 ng / ml, and 200 μL was placed in 1.5 mL EP tubes. The tubes were incubated at 55℃ and 60℃ for 2 min, 5 min, 10 min, 20 min, 30 min, and 60 min, respectively, and the residual activity was measured. The enzyme solution without heat treatment was used as a control, and its residual relative activity was calculated. Results are as follows: Figure 5 As shown, the mutant exhibits significantly improved thermal stability compared to the parent. After incubation at 55℃ for 1 h, it retains 40% of its residual activity, while the parent is essentially inactivated after 1 h of treatment. After treatment at 60℃ for 10 min, it retains 40% of its residual activity, which is also significantly better than the parent.

[0054] half life t 1 / 2 Thermostat is one of the commonly used characterization parameters for enzyme thermostability; a higher value indicates better enzyme thermostability. It refers to the time required for the initial activity to decrease by 50% at a given temperature, and is calculated using the following formula: in, k d The deactivation rate constant can be obtained using the following formula: In the formula, A t A0 refers to the residual activity, A0 is the initial activity, and t refers to the treatment time at a given treatment temperature.

[0055] Melting temperature T m This refers to the temperature at which a protein unfolds to 50% of its original volume. Differential scanning calorimetry (DSC) is commonly used to determine the temperature at which a protein unfolds to 50% of its original volume. T m .

[0056] The results are as follows Figure 4As shown, the thermostability of the dual-region superposition mutants was superior to that of the parents. M6, M7, M8, and M9 exhibited significantly improved thermostability at 55℃; the parents were essentially inactivated after 1 h of treatment, while the mutants retained over 80% of their activity. The mutants also showed a significant improvement in thermostability at 60℃. The Tm value and t1 / 2 of the mutants were also significantly higher than those of the parents, as shown in Table 2. T m The values ​​increased by 7.2℃, 4.3℃, 5.7℃, and 7.5℃ respectively at 55℃. t 1 / 2 The levels were increased to 10.6 times, 6.9 times, 8.5 times, and 12.2 times that of the parent lines, respectively, at 60℃. t 1 / 2 The levels were increased to 21.2 times, 13.2 times, 17.6 times, and 39.7 times that of the parent lines, respectively.

[0057] Table 2 Parents and Mutants Th APT3-M6, M7 and M8 T m Value and t 1 / 2 parameter .

[0058] Enzymatic Properties Analysis of APT3-M9

[0059] (1) Determination of optimal temperature In a borax-NaOH buffer system at pH 9.5, PA3 and the three-region superimposed mutant were subjected to different temperatures (30℃, 40℃, 50℃, 55℃, 60℃, 65℃, 70℃ and 80℃). Th The optimal temperature for APT3-M9 enzyme activity was determined by measuring its activity. The activity corresponding to the optimal temperature was defined as 100%, and the relative activities at other temperatures were calculated. For example... Figure 5 As shown, the optimal temperature for the parent PA3 is 60℃, while the optimal temperature for the mutant M9 is 5℃ higher than that for the parent.

[0060] (2) Thermal stability Thermal stability results are as follows Figure 6 As shown, the thermostability of the mutant after the three regions were superimposed was significantly better than that of the parent. After treatment at 55℃ for 1 h, the activity increased from the initial 5% to more than 80%, and after treatment at 60℃ for 1 h, 60% of the residual enzyme activity was still retained. t 1 / 2 and T m The analysis results are shown in Table 3. T m The temperature was 7.5°C higher than the wild type, at 55°C. t1 / 2 Increased to 12.2 times that of the parent, at 60℃ t 1 / 2 It is 39.7 times that of the parent.

[0061] Table 3 Parents and Mutants Th APT3-M9 T m Value and t 1 / 2 parameter .

[0062] (3) Determination of kinetic constants Casein was prepared at concentrations of 0.5, 0.8, 1.0, 1.3, 1.5, 2.0, 2.5, 5.0, 8.0, and 10.0 mg / mL as substrates. The purified enzyme solutions were appropriately diluted, and reacted with different concentrations of substrate at the optimal temperature and pH for 10 min, respectively. Enzyme activity was then measured. The Michaelis-Menten equation constants were fitted using GraphPad Prism version 5.01 software, and the enzyme activity was calculated. K m and V max The results are shown in Table 4. The mutant... Th APT3-M9 exhibits a 62% higher specific activity compared to its parent, resulting in higher catalytic activity and efficiency.

[0063] Table 4 Parents and Mutants Th APT3-M9 dynamic parameters .

[0064] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for improving the thermal stability and specific activity of alkaline protease, characterized in that, The method includes the step of mutating the alkaline protease parent PA3, whose amino acid sequence is shown in SEQ ID NO:1, as follows: By M 330 GKDGAVAAGLC 341 Replace with M 330 TLGRTTAANAC 341 Then, the amino acid at position 333 was mutated from G to N, position 342 (Ala) to Glu, and position 345 (Ala) to Gln; or By M 330 GKDGAVAAGLC 341 Replace with M 330 TLGRTTAANAC 341 Then, the amino acid at position 333 was mutated from G to N, position 350 from Arg to Leu, position 358 from Ala to Glu, and position 371 from Ser to Asn; or By M 330 GKDGAVAAGLC 341 Replace with M 330 TLGRTTAANAC 341 Then, the amino acid at position 333 was mutated from G to N, position 342 from Ala to Glu, position 345 from Ala to Gln, position 350 from Arg to Leu, position 358 from Ala to Glu, and position 371 from Ser to Asn.

2. A method for improving the thermal stability and specific activity of alkaline protease, characterized in that, The method includes the step of mutating the alkaline protease parent PA3, whose amino acid sequence is shown in SEQ ID NO:1, as follows: The Ala at position 342 is mutated to Glu, the Ala at position 345 is mutated to Gln, the Arg at position 350 is mutated to Leu, the Ala at position 358 is mutated to Glu, and the Ser at position 371 is mutated to Asn.

3. An alkaline protease mutant, characterized in that, The amino acid sequences of the alkaline protease mutants are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

4. An alkaline protease gene, characterized in that, The alkaline protease gene encodes the alkaline protease mutant of claim 3.

5. The alkaline protease gene according to claim 4, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:

18.

6. A recombinant expression vector comprising the alkaline protease gene of claim 4.

7. A recombinant strain comprising the alkaline protease gene of claim 4.

8. A method for preparing alkaline protease, characterized in that, Includes the following steps: Transform the host cell with the recombinant expression vector of claim 6; Inducing host cell expression; Alkaline protease was obtained by isolation and purification.

9. The application of the alkaline protease mutant of claim 3 to hydrolyze casein.

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