CN111172142A - Cephalosporin C acylase mutant with high thermal stability - Google Patents
Cephalosporin C acylase mutant with high thermal stability Download PDFInfo
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Abstract
The invention discloses a cephalosporin C acylase mutant with high thermal stability, which is constructed by a point mutation method, wherein the amino acid sequence of the cephalosporin C acylase mutant is selected from SEQ ID NOs: 3-7. Compared with the initial cephalosporin C acylase SEQ ID NO:1, the cephalosporin C acylase mutant has higher thermal stability and higher enzyme activity, and can be used for producing 7-aminocephalosporanic acid by a one-step enzyme method.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a cephalosporin C acylase mutant with high thermal stability constructed by a point mutation method and application thereof in producing 7-aminocephalosporanic acid (7-ACA) by a one-step enzyme method.
Background
The majority of cephalosporin antibiotics, which currently account for 40% of the global antibiotic market, are 7-ACA derivatives synthesized by 7-aminocephalosporanic acid (7-aminocephalosporanic acid, abbreviated as 7-ACA). 7-ACA is generally obtained by cleaving Cephalosporin C (CPC for short) chemically or biologically, and removing the side chain of the molecule. The biological enzyme method generally utilizes cephalosporin C acylase (CPC acylase) to catalyze CPC to remove a side chain to generate 7-ACA.
Referring to patent document PCT/CN2017/076688, through previous research, the inventor carries out genetic engineering transformation on CPC acylase from Pseudomonas sp.130, develops a series of CPC acylase mutants, and the mutants overcome the problem of product inhibition of CPC acylase from Pseudomonas sp.SE83 in the catalytic reaction process, and greatly improve the enzyme activity of CPC acylase from Pseudomonas sp.130 on cephalosporin C sodium salt. However, in the large-scale production of 7-ACA, the stability of CPC acylase mutants such as 130-ED2 and the like is not high, particularly the thermal stability is poor, the enzyme activity is obviously reduced and even inactivated after long-time reaction, enzyme needs to be added to maintain the reaction, so that the production cost of the 7-ACA is still high, and the industrial application is influenced.
Disclosure of Invention
In order to improve the thermal stability and the enzymatic activity of the CPC acylase, the invention utilizes the genetic engineering technology to transform and screen the prior CPC acylase and construct a CPC acylase mutant with high thermal stability and enzymatic activity, thereby promoting the realization of the industrialization of producing the 7-ACA by the enzymatic method.
A130-ED 2 mutant SEQ ID NO 1 in PCT/CN2017/076688 is continuously transformed by a random mutation technology, so that a CPC acylase mutant with high enzyme activity and high thermal stability, which takes CPC as a specific substrate, is obtained. Specifically, the present invention includes the following technical solutions.
A cephalosporin C acylase (CPC acylase) mutant, which is a mutant produced by changing one, two, three or four of the 12 th a, 199 th D, 306 th a and 494 th V of the amino acid sequence SEQ ID No. 1, wherein the changes include but are not limited to substitution, deletion, chemical modification by other amino acids; or a polypeptide having 85% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, more preferably 99% or more homology with the amino acid sequence.
Preferably, the above-mentioned changes are substitutions by other amino acids. For example, a is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or Z; d is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or Z; or V is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, Y or Z.
In one embodiment, the above changes are 12 th bit a replaced by N (i.e., a12N), 199 th bit D replaced by N (i.e., D199N), 306 th bit a replaced by T (i.e., a306T), 494 th bit V replaced by M (i.e., V494M), or a combination of two or more thereof.
Preferably, the amino acid sequence of the cephalosporin C acylase mutant is selected from the group consisting of:
3, which is a mutant of SEQ ID No. 1 with N substituted for D at position 199 and M substituted for V at position 494:
MEPTSTPQAPIAAYKPRSNEILWDDYGVPHIYGVDAPSAFYGYGWAQARSHGDNILRLYGEARGKGAEYWGPDYEQTTVWLLTNGVPERAQQWYAQQSPDFRANLDAFAAGINAYAQQNPDDISPDVRQVLPVSGADVVAHAHRLMNFLYVASPGRTLGEGDPPDLADQGSNSWAVAPGKTANGNALLLQNPHLSWTTNYFTYYEAHLVTPDFEVYGATQIGLPVIRVAFNQRMGITNTFNGMVGATNYRLTLQDGGYLYDGQVRPFERRQASYRLRQADGTTVDKPLEIRSSVHGPVFERADGTAVAVRVAGLDRPGMLEQTFDMITADSFDDYEAALARMQVPTLNIVYADREGTINYSFNGVAPKRAEGDIAFWQGLVPGDSSRYLWTETHPLDDLPRVTNPPGGFVQNSNDPPWTPTWPVTYTPKDFPSYLAPQTPHSLRAQQSVRLMSENDDLTLERFMALQLSHRAVMADRTLPDLIPAALIDPDPEMQAAARLLAAWDREFTSDSRAALLFEEWARLFAGQNFAGQAGFATPWSLDKPVSTPYGVRDPKAAVDQLRTAIANTKRKYGAIDRPFGDASRMILNDVNVPGAAGYGNLGSFRVFTWSDPDENGVRTPVHGETWVAMIEFSTPVRAYGLMSYGNSRQPGTTHYSDQIERVSRADFRELLLRREQVEAAVQERTPFNFKP(SEQ ID NO:3)；
4, which is a mutant of the V at position 494 of the amino acid sequence of SEQ ID NO. 1 replaced by M:
MEPTSTPQAPIAAYKPRSNEILWDDYGVPHIYGVDAPSAFYGYGWAQARSHGDNILRLYGEARGKGAEYWGPDYEQTTVWLLTNGVPERAQQWYAQQSPDFRANLDAFAAGINAYAQQNPDDISPDVRQVLPVSGADVVAHAHRLMNFLYVASPGRTLGEGDPPDLADQGSNSWAVAPGKTANGNALLLQNPHLSWTTDYFTYYEAHLVTPDFEVYGATQIGLPVIRVAFNQRMGITNTFNGMVGATNYRLTLQDGGYLYDGQVRPFERRQASYRLRQADGTTVDKPLEIRSSVHGPVFERADGTAVAVRVAGLDRPGMLEQTFDMITADSFDDYEAALARMQVPTLNIVYADREGTINYSFNGVAPKRAEGDIAFWQGLVPGDSSRYLWTETHPLDDLPRVTNPPGGFVQNSNDPPWTPTWPVTYTPKDFPSYLAPQTPHSLRAQQSVRLMSENDDLTLERFMALQLSHRAVMADRTLPDLIPAALIDPDPEMQAAARLLAAWDREFTSDSRAALLFEEWARLFAGQNFAGQAGFATPWSLDKPVSTPYGVRDPKAAVDQLRTAIANTKRKYGAIDRPFGDASRMILNDVNVPGAAGYGNLGSFRVFTWSDPDENGVRTPVHGETWVAMIEFSTPVRAYGLMSYGNSRQPGTTHYSDQIERVSRADFRELLLRREQVEAAVQERTPFNFKP(SEQ ID NO:4)；
5, which is a mutant of SEQ ID No. 1 with N substituted for a at position 12, N substituted for D at position 199, and M substituted for V at position 494:
MEPTSTPQAPINAYKPRSNEILWDDYGVPHIYGVDAPSAFYGYGWAQARSHGDNILRLYGEARGKGAEYWGPDYEQTTVWLLTNGVPERAQQWYAQQSPDFRANLDAFAAGINAYAQQNPDDISPDVRQVLPVSGADVVAHAHRLMNFLYVASPGRTLGEGDPPDLADQGSNSWAVAPGKTANGNALLLQNPHLSWTTNYFTYYEAHLVTPDFEVYGATQIGLPVIRVAFNQRMGITNTFNGMVGATNYRLTLQDGGYLYDGQVRPFERRQASYRLRQADGTTVDKPLEIRSSVHGPVFERADGTAVAVRVAGLDRPGMLEQTFDMITADSFDDYEAALARMQVPTLNIVYADREGTINYSFNGVAPKRAEGDIAFWQGLVPGDSSRYLWTETHPLDDLPRVTNPPGGFVQNSNDPPWTPTWPVTYTPKDFPSYLAPQTPHSLRAQQSVRLMSENDDLTLERFMALQLSHRAVMADRTLPDLIPAALIDPDPEMQAAARLLAAWDREFTSDSRAALLFEEWARLFAGQNFAGQAGFATPWSLDKPVSTPYGVRDPKAAVDQLRTAIANTKRKYGAIDRPFGDASRMILNDVNVPGAAGYGNLGSFRVFTWSDPDENGVRTPVHGETWVAMIEFSTPVRAYGLMSYGNSRQPGTTHYSDQIERVSRADFRELLLRREQVEAAVQERTPFNFKP(SEQ ID NO:5)；
6, which is a mutant of SEQ ID NO 1 with N at D199, T at A306, and M at V494:
MEPTSTPQAPIAAYKPRSNEILWDDYGVPHIYGVDAPSAFYGYGWAQARSHGDNILRLYGEARGKGAEYWGPDYEQTTVWLLTNGVPERAQQWYAQQSPDFRANLDAFAAGINAYAQQNPDDISPDVRQVLPVSGADVVAHAHRLMNFLYVASPGRTLGEGDPPDLADQGSNSWAVAPGKTANGNALLLQNPHLSWTTNYFTYYEAHLVTPDFEVYGATQIGLPVIRVAFNQRMGITNTFNGMVGATNYRLTLQDGGYLYDGQVRPFERRQASYRLRQADGTTVDKPLEIRSSVHGPVFERADGTTVAVRVAGLDRPGMLEQTFDMITADSFDDYEAALARMQVPTLNIVYADREGTINYSFNGVAPKRAEGDIAFWQGLVPGDSSRYLWTETHPLDDLPRVTNPPGGFVQNSNDPPWTPTWPVTYTPKDFPSYLAPQTPHSLRAQQSVRLMSENDDLTLERFMALQLSHRAVMADRTLPDLIPAALIDPDPEMQAAARLLAAWDREFTSDSRAALLFEEWARLFAGQNFAGQAGFATPWSLDKPVSTPYGVRDPKAAVDQLRTAIANTKRKYGAIDRPFGDASRMILNDVNVPGAAGYGNLGSFRVFTWSDPDENGVRTPVHGETWVAMIEFSTPVRAYGLMSYGNSRQPGTTHYSDQIERVSRADFRELLLRREQVEAAVQERTPFNFKP (SEQ ID NO: 6); or
7, which is a mutant of SEQ ID NO. 1 in which A at position 12 is replaced by N, D at position 199 is replaced by N, A at position 306 is replaced by T, and V at position 494 is replaced by M:
MEPTSTPQAPINAYKPRSNEILWDDYGVPHIYGVDAPSAFYGYGWAQARSHGDNILRLYGEARGKGAEYWGPDYEQTTVWLLTNGVPERAQQWYAQQSPDFRANLDAFAAGINAYAQQNPDDISPDVRQVLPVSGADVVAHAHRLMNFLYVASPGRTLGEGDPPDLADQGSNSWAVAPGKTANGNALLLQNPHLSWTTNYFTYYEAHLVTPDFEVYGATQIGLPVIRVAFNQRMGITNTFNGMVGATNYRLTLQDGGYLYDGQVRPFERRQASYRLRQADGTTVDKPLEIRSSVHGPVFERADGTTVAVRVAGLDRPGMLEQTFDMITADSFDDYEAALARMQVPTLNIVYADREGTINYSFNGVAPKRAEGDIAFWQGLVPGDSSRYLWTETHPLDDLPRVTNPPGGFVQNSNDPPWTPTWPVTYTPKDFPSYLAPQTPHSLRAQQSVRLMSENDDLTLERFMALQLSHRAVMADRTLPDLIPAALIDPDPEMQAAARLLAAWDREFTSDSRAALLFEEWARLFAGQNFAGQAGFATPWSLDKPVSTPYGVRDPKAAVDQLRTAIANTKRKYGAIDRPFGDASRMILNDVNVPGAAGYGNLGSFRVFTWSDPDENGVRTPVTGETWVAMIEFSTPVRAYGLMSYGNSRQPGTTHYSDQIERVSRADFRELLLRREQVEAAVQERTPFNFKP(SEQ ID NO:7)。
preferably, the cephalosporin C acylase mutant is SEQ ID NO 7.
The second aspect of the present invention is to provide a gene encoding the CPC acylase mutant described above.
When the cephalosporin C acylase mutant is SEQ ID NO. 7, the nucleotide sequence of the preferred coding gene is SEQ ID NO. 8:
atggaaccgacctccaccccgcaggctccgatcaacgcttacaaaccgcgttccaacgaaatcctgtgggacgactacggtgttccgcacatctacggtgttgacgctccgtccgctttctacggttacggttgggctcaggctcgttcccacggtgacaacatcctgcgtctgtacggtgaagctcgtggtaaaggtgctgaatactggggtccggactacgaacagaccaccgtttggctgctgaccaacggtgttccggaacgtgctcagcagtggtacgctcagcagtccccggacttccgtgctaacctggacgctttcgctgctggtatcaacgcttacgctcagcagaacccggacgacatctccccggacgttcgtcaggttctgccggtttccggtgctgacgttgttgctcacgctcaccgtctgatgaacttcctgtacgttgcttccccgggtcgtaccctgggtgaaggtgacccgccggacctggctgaccagggttccaactcctgggctgttgctccgggtaaaaccgctaacggtaacgctctgctgctgcagaacccgcacctgtcctggaccaccaactacttcacctactacgaagctcacctggttaccccggacttcgaagtttacggtgctacccagatcggtctgccggttatccgtgttgctttcaaccagcgtatgggtatcaccaacaccttcaacggtatggttggtgctaccaactaccgtctgaccctgcaggacggtggttacctgtacgacggtcaggttcgtccgttcgaacgtcgtcaggcttcctaccgtctgcgtcaggctgacggtaccaccgttgacaaaccgctggaaatccgttcctccgttcacggtccggttttcgaacgtgctgacggtaccactgttgctgttcgtgttgctggtctggaccgtccgggtatgctggaacagaccttcgacatgatcaccgctgactccttcgacgactacgaagctgctctggctcgtatgcaggttccgaccctgaacatcgtttacgctgaccgtgaaggtaccatcaactactccttcaacggtgttgctccgaaacgtgctgaaggtgacatcgctttctggcagggtctggttccgggtgactcctcccgttacctgtggaccgaaacccacccgctggacgacctgccgcgtgttaccaacccgccgggtggtttcgttcagaactccaacgacccgccgtggaccccgacctggccggttacctacaccccgaaagacttcccgtcctacctggctccgcagaccccgcactccctgcgtgctcagcagtccgttcgtctgatgtccgaaaacgacgacctgaccctggaacgtttcatggctctgcagctgtcccaccgtgctgttatggctgaccgtaccctgccggacctgatcccggctgctctgatcgacccggacccggaaatgcaggctgctgctcgtctgctggctgcttgggaccgtgaattcacctccgactcccgtgctgctctgctgttcgaagaatgggctcgtctgttcgctggtcagaacttcgctggtcaggctggtttcgctaccccgtggtccctggacaaaccggtttccaccccgtacggtgttcgtgacccgaaagctgctgttgaccagctgcgtaccgctatcgctaacaccaaacgtaaatacggtgctatcgaccgtccgttcggtgacgcttcccgtatgatcctgaacgacgttaacgttccgggtgctgctggttacggtaacctgggttccttccgtgttttcacctggtccgacccggacgaaaacggtgttcgtaccccggttaccggtgaaacctgggttgctatgatcgaattctccaccccggttcgtgcttacggtctgatgtcctacggtaactcccgtcagccgggtaccacccactactccgaccagatcgaacgtgtttcccgtgctgacttccgtgaactgctgctgcgtcgtgaacaggttgaagctgctgttcaggaacgtaccccgttcaacttcaaaccg(SEQ ID NO:8)。
the third aspect of the present invention is to provide a plasmid containing the above gene. The plasmid is a PET series vector, such as, but not limited to, PET24a (+).
The fourth aspect of the present invention is to provide a microorganism expressing the above-mentioned coding gene, which is transformed with the above-mentioned plasmid.
Preferably, the microorganism is selected from Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Acremonium acremonium, Penicillium chrysogenum, preferably Escherichia coli, Acremonium acremonium, more preferably Escherichia coli BL21(DE 3).
The fifth aspect of the present invention provides the use of the CPC acylase mutant or the microorganism described above for the production of 7-ACA.
The cephalosporin C acylase mutant or the microorganism can be used for producing 7-ACA, especially for producing 7-ACA by a one-step enzyme method.
In the production of 7-ACA, cephalosporin C (CPC) is used as a substrate raw material, and the cephalosporin C acylase mutant or microorganism is used as a catalyst to catalyze the reaction.
The 7-ACA can be produced by adopting conventional process conditions, for example, the concentration of the cephalosporin C can be selected from 1-3 wt%, preferably 2.5 wt%; the reaction temperature is selected from 10 to 37 ℃, preferably 12 to 35 ℃, more preferably 12 to 30 ℃, more preferably 14 to 25 ℃, and most preferably 15 +/-0.5 ℃.
In an alternative embodiment, when the reaction is catalyzed using the above cephalosporin C acylase mutant, the cephalosporin C acylase mutant is in the form of an immobilized enzyme, thereby further improving the stability and lifespan of the CPC acylase protein.
Compared with the initial enzyme SEQ ID NO. 1, the CPC acylase mutant SEQ ID NO. 3-7 of the invention has the advantages that the thermal stability and the enzyme activity are obviously improved, the enzyme activity is improved by 2.9 times to the maximum, and the thermal stability is improved by about 2.7 times to the maximum. When the immobilized enzyme is prepared by using the mutant SEQ ID NO. 7 pure enzyme and is used as a catalyst to carry out the one-step enzyme method for producing 7-ACA, the reaction is carried out for 40-60min at the temperature of 15 +/-0.5 ℃, the reaction is carried out for 50 batches continuously, and the conversion rate of CPC is over 97 percent.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.
For the sake of brevity, the amino acid abbreviations herein may be used in either the three or single letter English, as is well known to those skilled in the art, and are set forth in the following table:
TABLE 1 amino acids Chinese and English comparison and abbreviations
| Alanine | Alanine | A or Ala | Aliphatic group |
| Arginine | Arginine | R or Arg | Basic amino acids |
| Asparagine | Asparagine | N or Asn | Amides of amides |
| Aspartic acid | Aspartic acid | D or Asp | Acidic amino acids |
| Cysteine | Cysteine | C or Cys | Containing sulfur |
| Glutamine | Glutamine | Q or Gln | Amides of amides |
| Glutamic acid | Glutamic acid | E or Glu | Acidic amino acids |
| Glycine | Glycine | G or Gly | Aliphatic group |
| Histidine | Histidine | H or His | Basic amino acids |
| Isoleucine | Isoleucine | I or Ile | Aliphatic group |
| Leucine | Leucine | L or Leu | Aliphatic group |
| Lysine | Lysine | K or Lys | Basic amino acids |
| Methionine | Methionine | M or Met | Containing sulfur |
| Phenylalanine | Phenylalanine | F or Phe | Aromatic compounds |
| Proline | Proline | P or Pro | Imino acid |
| Serine | Serine | S or Ser | Hydroxy radicals |
| Threonine | Threonine | T or Thr | Hydroxy radicals |
| Tryptophan | Tryptophan | W or Trp | Aromatic compounds |
| Tyrosine | Tyrosine | Y or Tyr | Aromatic compounds |
| Valine | Valine | V or Val | Aliphatic group |
As a basic template for constructing a cephalosporin C acylase mutant, the cephalosporin C acylase mutant is CPC acylase 130-ED2 in PCT/CN2017/076688, namely SEQ ID NO: 1:
MEPTSTPQAPIAAYKPRSNEILWDDYGVPHIYGVDAPSAFYGYGWAQARSHGDNILRLYGEARGKGAEYWGPDYEQTTVWLLTNGVPERAQQWYAQQSPDFRANLDAFAAGINAYAQQNPDDISPDVRQVLPVSGADVVAHAHRLMNFLYVASPGRTLGEGDPPDLADQGSNSWAVAPGKTANGNALLLQNPHLSWTTDYFTYYEAHLVTPDFEVYGATQIGLPVIRVAFNQRMGITNTFNGMVGATNYRLTLQDGGYLYDGQVRPFERRQASYRLRQADGTTVDKPLEIRSSVHGPVFERADGTAVAVRVAGLDRPGMLEQTFDMITADSFDDYEAALARMQVPTLNIVYADREGTINYSFNGVAPKRAEGDIAFWQGLVPGDSSRYLWTETHPLDDLPRVTNPPGGFVQNSNDPPWTPTWPVTYTPKDFPSYLAPQTPHSLRAQQSVRLMSENDDLTLERFMALQLSHRAVMADRTLPDLIPAALIDPDPEVQAAARLLAAWDREFTSDSRAALLFEEWARLFAGQNFAGQAGFATPWSLDKPVSTPYGVRDPKAAVDQLRTAIANTKRKYGAIDRPFGDASRMILNDVNVPGAAGYGNLGSFRVFTWSDPDENGVRTPVTGETWVAMIEFSTPVRAYGLMSYGNSRQPGTTHYSDQIERVSRADFRELLLRREQVEAAVQERTPFNFKP(SEQ ID NO:1)。
the coding gene of SEQ ID NO. 1 is SEQ ID NO. 2:
atggaaccgacctccaccccgcaggctccgatcgccgcttacaaaccgcgttccaacgaaatcctgtgggacgactacggtgttccgcacatctacggtgttgacgctccgtccgctttctacggttacggttgggctcaggctcgttcccacggtgacaacatcctgcgtctgtacggtgaagctcgtggtaaaggtgctgaatactggggtccggactacgaacagaccaccgtttggctgctgaccaacggtgttccggaacgtgctcagcagtggtacgctcagcagtccccggacttccgtgctaacctggacgctttcgctgctggtatcaacgcttacgctcagcagaacccggacgacatctccccggacgttcgtcaggttctgccggtttccggtgctgacgttgttgctcacgctcaccgtctgatgaacttcctgtacgttgcttccccgggtcgtaccctgggtgaaggtgacccgccggacctggctgaccagggttccaactcctgggctgttgctccgggtaaaaccgctaacggtaacgctctgctgctgcagaacccgcacctgtcctggaccaccgactacttcacctactacgaagctcacctggttaccccggacttcgaagtttacggtgctacccagatcggtctgccggttatccgtgttgctttcaaccagcgtatgggtatcaccaacaccttcaacggtatggttggtgctaccaactaccgtctgaccctgcaggacggtggttacctgtacgacggtcaggttcgtccgttcgaacgtcgtcaggcttcctaccgtctgcgtcaggctgacggtaccaccgttgacaaaccgctggaaatccgttcctccgttcacggtccggttttcgaacgtgctgacggtaccgctgttgctgttcgtgttgctggtctggaccgtccgggtatgctggaacagaccttcgacatgatcaccgctgactccttcgacgactacgaagctgctctggctcgtatgcaggttccgaccctgaacatcgtttacgctgaccgtgaaggtaccatcaactactccttcaacggtgttgctccgaaacgtgctgaaggtgacatcgctttctggcagggtctggttccgggtgactcctcccgttacctgtggaccgaaacccacccgctggacgacctgccgcgtgttaccaacccgccgggtggtttcgttcagaactccaacgacccgccgtggaccccgacctggccggttacctacaccccgaaagacttcccgtcctacctggctccgcagaccccgcactccctgcgtgctcagcagtccgttcgtctgatgtccgaaaacgacgacctgaccctggaacgtttcatggctctgcagctgtcccaccgtgctgttatggctgaccgtaccctgccggacctgatcccggctgctctgatcgacccggacccggaagtgcaggctgctgctcgtctgctggctgcttgggaccgtgaattcacctccgactcccgtgctgctctgctgttcgaagaatgggctcgtctgttcgctggtcagaacttcgctggtcaggctggtttcgctaccccgtggtccctggacaaaccggtttccaccccgtacggtgttcgtgacccgaaagctgctgttgaccagctgcgtaccgctatcgctaacaccaaacgtaaatacggtgctatcgaccgtccgttcggtgacgcttcccgtatgatcctgaacgacgttaacgttccgggtgctgctggttacggtaacctgggttccttccgtgttttcacctggtccgacccggacgaaaacggtgttcgtaccccggttaccggtgaaacctgggttgctatgatcgaattctccaccccggttcgtgcttacggtctgatgtcctacggtaactcccgtcagccgggtaccacccactactccgaccagatcgaacgtgtttcccgtgctgacttccgtgaactgctgctgcgtcgtgaacaggttgaagctgctgttcaggaacgtaccccgttcaacttcaaaccg(SEQ ID NO:2)。
in order to obtain a CPC acylase mutant with high thermal stability and higher enzyme activity, the invention carries out point mutation on CPC acylase 130-ED2, namely the coding gene sequence SEQ ID NO. 2 of SEQ ID NO. 1. One or more amino acid site changes such as substituted mutant amino acid sequences are obtained through an error-prone PCR technology, 4 sites capable of improving the stability of CPC acylase or improving the enzyme activity are screened out, then a mutant with an amino acid sequence SEQ ID NO. 4 is obtained in a fixed-point combined mutation mode, finally a gene with the amino acid sequence SEQ ID NO. 4 mutant is used as a template, a second error-prone PCR technology is carried out, and the mutant with the amino acid sequence SEQ ID NO. 3-7 is obtained.
Wherein, SEQ ID NO. 1 is the common sequence of the amino acid sequences SEQ ID NO. 3-7, the amino acid sequences are all mutants obtained by replacing 1, 2, 3 or 4 amino acids on the basis of SEQ ID NO. 1, and the amino acid sequences of the mutants keep more than 99 percent of homology.
In the present invention, the terms "CPC acylase mutant", "mutant CPC acylase" and "mutant enzyme" are used in the same sense, and all refer to mutants formed by amino acid changes based on the original cephalosporin C acylase 130-ED 2.
In the present invention, the terms "initial enzyme" and "initial enzyme" mean the same meaning, and refer to CPC acylase 130-ED2(SEQ ID NO: 1).
The CPC acylase mutant of the present invention has the number of amino acids of only 692 and a definite structure, so that a person skilled in the art can easily obtain the encoding genes thereof, expression cassettes and plasmids containing the genes, and transformants containing the plasmids.
For optimal expression of the proteins SEQ ID NO 3-7 in different microorganisms, codon optimization can be performed for specific microorganisms such as E.coli or Cephalosporium acremonium. Codon optimization is one technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms often show a special preference for one of several codons encoding the same amino acid due to mutation tendencies and natural selection. For example, in rapidly growing microorganisms such as E.coli, the optimized codons reflect the composition of their respective pools of genomic tRNA's. Thus, in a fast growing microorganism, the low frequency codons of an amino acid can be replaced with codons for the same amino acid but with a high frequency. Thus, expression of optimized DNA sequences is improved in fast growing microorganisms. The gene sequence SEQ ID NO 8 provided herein is a codon-optimized nucleotide sequence for the CPC acylase mutant SEQ ID NO 7, but the CPC acylase mutant SEQ ID NO 7 expression gene of the present invention is not limited thereto.
These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction means well known to those skilled in the art.
The above-mentioned transformant host may be any microorganism suitable for expressing CPC acylase, including bacteria and fungi. Preferably the microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Cephalosporium acremonium or Penicillium chrysogenum, preferably Escherichia coli or Cephalosporium acremonium, more preferably Escherichia coli BL21(DE 3).
When used as a biocatalyst for the production of 7-ACA, the CPC acylase of the present invention may be in the form of an enzyme or in the form of a bacterium. The enzyme forms comprise free enzyme and immobilized enzyme, including purified enzyme, crude enzyme, fermentation liquor, enzyme immobilized by a carrier and the like; the form of the thallus comprises a viable thallus and a dead thallus.
The CPC acylase mutant of the present invention is also well known to those skilled in the art in terms of isolation and purification, including immobilized enzyme preparation techniques. For example, the enzyme immobilization may be carried out by a conventional carrier immobilization method such as adsorption, entrapment, carrier coupling, bifunctional or multifunctional crosslinking reagent crosslinking, or may be carried out by a carrier-free immobilization method such as crosslinking of lytic enzymes, crosslinking of enzyme crystals, crosslinking of enzyme polymers, etc., but is not limited thereto. So as to fix the CPC acylase mutant and keep the enzyme activity to the maximum extent, thereby further improving the stability and the service life of enzyme protein.
Examples
The whole gene synthesis, primer synthesis and sequencing in the examples were carried out by Suzhou Jinzhi corporation.
The molecular biological experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, culture medium preparation, and the like, and are mainly performed with reference to "molecular cloning experimental manual" (third edition), sambrook, d.w. rasel (american), translation of huang peitang et al, scientific press, beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary. PCR amplification experiments were performed according to the reaction conditions or instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.
PCR amplification experiments were performed according to the reaction conditions or instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.
LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.2. (20 g/L agar powder was additionally added to LB solid medium.)
TB culture medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K2HPO4.3H2O、2.31g/LKH2PO45g/L of glycerol, and the pH value is 7.0-7.5. (20 g/L agar powder was additionally added to TB solid medium.)
EXAMPLE 1 construction of initial CPC acylase 130-ED2 expression Strain
The total gene synthesis was carried out according to the CPC acylase 130-ED2 encoding gene (SEQ ID NO:13 as such) published in PCT/CN2017/076688, herein sequence SEQ ID NO: 2. Restriction endonuclease sites NdeI and XhoI are designed at two ends of the gene, and are subcloned to corresponding sites of a vector pET24a (Novagen) to obtain a recombinant plasmid pET24a-130ED2, and a host escherichia coli BL21(DE3) is transformed to obtain a recombinant escherichia coli expressing initial CPC acylase, wherein the number of the recombinant escherichia coli is still 130-ED 2.
Example 2 construction of random mutation Point library by error-prone PCR method and screening
2.1 construction of random mutation Point library by error-prone PCR method
2 is used as a template, and an error-prone PCR technology is applied to construct a random mutant library.
Forward primers C-F: 5' -CATATGGAACCGACCTCCACCCCGCAG-3’，
Reverse primer C-R: 5' -CTCGAGCGGTTTGAAGTTGAACGGGGTACGTTC-3’。
The 50 μ L error-prone PCR reaction system included: 50ng plasmid template pET24a-13ED2, 30pmol of a pair of primers C-F and C-R, 1 XTaq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl2，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl22.5 units of Taq enzyme (Fermentas). The PCR reaction conditions are as follows: 5min at 95 ℃; 94 ℃ for 30s, 55 ℃ for 30s and 72 DEG C
2 min/kbp; 30 cycles; 10min at 72 ℃. Gel 2.0kb random mutant fragment was recovered as a large primer, and Megaprimer PCR was performed using KOD-plus DNA polymerase: 5min at 94 ℃; 10s at 98 ℃, 30s at 60 ℃, 2min/kbp at 68 ℃ and 25 cycles; 10min at 68 ℃. DpnI digestion of the plasmid template and electroporation of E.coli BL21(DE3) gave more than 104Random mutant pools of individual clones.
2.2 high throughput screening of mutant pools
Transformants in the mutant pool were selected and inoculated into a 96-well deep-well plate containing 700. mu.L of LB medium containing 100. mu.g/mL kanamycin and 0.1mM IPTG, cultured at 37 ℃ for 6 hours, then cooled to 25 ℃ and cultured overnight. Centrifuging at 5000rpm for 10min, discarding supernatant, freezing at-70 deg.C for 1h, and thawing at room temperature for 30 min. Adding 200 μ L of 0.1M potassium phosphate buffer (pH8.0) containing 1mg/mL lysozyme, resuspending the thallus, incubating at 37 deg.C for 1h, centrifuging at 4 deg.C and 5000rpm for 20min, collecting 20 μ L of supernatant, heating at 40 deg.C for 10min, and determining CPC activity.
2.3 high throughput enzyme Activity assay
Substrate 1 reaction solution: a 0.1M potassium phosphate salt buffer (pH8.0) containing 2% by weight of CPC sodium salt,
substrate 2 reaction solution: 0.1M potassium phosphate salt buffer (pH8.0) containing 2 wt% GL-7ACA sodium salt,
terminating the reaction solution: 0.05M NaOH, 20% v/v glacial acetic acid,
color developing agent: 0.5 wt% PDAB (p-dimethylaminobenzaldehyde ) in methanol was contained.
Definition of enzyme activity: the amount of enzyme required to catalyze the substrate to produce 1 micromole (. mu.mol) of 7-ACA per minute at 37 ℃ is defined as 1 unit (U).
mu.L of the supernatant obtained in step 2.2 was added to 20. mu.L of the substrate reaction solution, reacted at 37 ℃ for 3 hours, 200. mu.L of the reaction-terminating solution was added, and the mixture was centrifuged at 5000rpm for 10 min. And (3) taking 200 mu L of centrifugal supernatant, adding 40 mu L of color developing agent, reacting at room temperature for 10min, and detecting the absorbance at 415 nm.
2.4 Heat treatment of enzymes
And (3) taking the supernatant obtained in the step 2.2, and preserving the heat for 48 hours in a constant-temperature water bath at 70 ℃. The enzyme activity was then determined.
The results of screening the random mutation library show that at least two mutation points of D199N and V494M can obviously improve the thermal stability of the enzyme. The results are shown in table 2.
TABLE 2 results of relative activity test of crude enzyme solution fermented by random mutant bacteria at 37 deg.C
Note: all relative specific activities refer to the percentage of enzyme activity relative to the starting strain 130-ED2 in the absence of heat treatment.
Example 3 construction and screening of random mutation Point library by second round error-prone PCR method
3.1 construction of random mutation Point library by error-prone PCR method
Referring to the method of example 2, plasmids of CPC acylase mutant strain 130-V1 strain obtained in example 2 were extracted as a template, and an error-prone PCR technique was applied to construct a random mutant library. The error-prone PCR system was the same as in step 2.1 of example 2, and error-prone PCR was continued using forward primer C-F and reverse primer C-R.
3.2 high throughput screening, Heat treatment and Activity assays of mutant pools
The method is the same as step 2.2, step 2.3 and step 2.4 of example 2.
As a result of screening the random mutation library, two strains 130-V3 and 130-V4 which have significantly improved substrate activity relative to CPC sodium salt but have no improved thermal stability were selected, and sequencing results showed that A12N mutation occurred in 130-V3 and A306T mutation occurred in 130-V4, and the results are shown in Table 3.
TABLE 3 results of relative activity test of crude enzyme solution fermented by random mutant bacteria at 37 deg.C
Note: all relative specific activities refer to the percentage of enzyme activity relative to the starting strain 130-V1 under non-heat-treated conditions.
EXAMPLE 4 construction of site-directed combination mutant CPC acylase Strain
Based on the 2 sites A12N and A306T screened in example 3, mutation point primers were designed, and a site-directed combinatorial library was constructed using pET24a-130-V3 plasmid as a template.
4.1 construction of directed combinatorial mutant strains by site-directed mutagenesis
Circular PCR was performed using pET24a-130-V3 plasmid as a template and 306-F and 306-R primer pairs to construct a site-directed combinatorial library. The primer sequence is 306-F: 5'-CGTGCTGACGGTACCACTGTTGCTGTTCGTGTTG-3', respectively;
306-R：5’-CAACACGAACAGCAACAGTGGTACCGTCAGCACG-3’。
the third PCR was Megaprimer PCR using KOD-plus DNA polymerase with fragment P as the big primer.
A50. mu.L circular PCR reaction system included: 100ng of plasmid template, 30pmol 306-F, 30pmol 306-R, 1xKODplus buffer, 0.2mM dNTP, 1.5mM MgSO45 units of KOD-plus DNA polymerase.
The PCR reaction conditions are as follows: 94 ℃ for 2 min; 10s at 98 ℃, 30s at 57 ℃, 2min/kbp at 68 ℃ and 25 cycles; 10min at 68 ℃. Digesting a plasmid template by DpnI, chemically transforming Escherichia coli E.coli BL21(DE3), randomly picking, cloning, sequencing and identifying to obtain the combined mutant strain 130-V34.
4.2 high throughput screening, Heat treatment and Activity assays of mutant pools
The procedure was the same as in example 2, step 2.2, step 2.3 and step 2.4, and the results are shown in Table 4.
TABLE 4 results of relative activity test of crude enzyme solution fermented by combination mutant bacteria at 37 deg.C
Note: all relative specific activities refer to the percentage of enzyme activity relative to the starting strain 130-V1 under non-heat-treated conditions.
Example 5 fermentation of Strain, enzyme extraction and determination of pure enzyme specific Activity
5.1 Shake flask fermentation
A single colony of 130-V34 was picked, inoculated into 5mL of LB liquid medium containing 50. mu.g/mL kanamycin sulfate, and cultured at 37 ℃ overnight at 250 rpm. Inoculating 2mL of overnight culture into 200mL of TB medium, and shake-culturing at 37 deg.C and 250rpm for 2-3h to OD600About 0.6-0.8, 0.1mM IPTG was added and incubated overnight at 28 ℃ and 200 rpm. Centrifuging at 4 deg.C and 10000rpm for 10min, and collecting thallus.
5.2 extraction of the enzyme
The cells were resuspended in 50mL of an equilibration buffer (50mM potassium phosphate buffer, 200mM NaCl, pH8.0), then disrupted by sonication, and the disrupted cells were centrifuged at 12000rpm at 4 ℃ for 20min to collect the supernatant. The supernatant was applied to an affinity column containing 10mL of Ni-NAT matrix at a rate of 1mL/min, and the column was then washed with an equilibration buffer containing 30mM imidazole to elute impurities. Finally, the target protein is removed by washing with an equilibrium buffer containing 500mM imidazole, and the peak eluent is collected.
Desalting the eluent by an ultrafiltration tube with the molecular weight cutoff of 10kDa to obtain pure enzyme.
5.3 determination of specific Activity of pure enzyme
The solution used for this step was the same as the reagent used in step 2.3 of example 2.
mu.L of the desalted solution obtained in step 5.2 was added to 20. mu.L of the substrate reaction solution, reacted at 37 ℃ for 5 minutes, 200. mu.L of the terminating reaction solution was added, and then centrifuged at 5000rpm for 10 minutes. Taking 200 mu L of centrifugal supernatant, adding 40 mu L of color developing agent, reacting at room temperature for 10min, detecting the absorbance at 415nm, and comparing and quantifying with a 7-ACA quantitative standard curve.
Meanwhile, the Protein concentration of the pure enzyme is measured by adopting a BCA Protein Assay Kit of Thermo Scientific company, so that the specific activity of the pure enzyme is obtained.
TABLE 5 comparison of enzyme activities at 37 deg.C
Note: all relative specific activities refer to the percentage of enzyme activity relative to the initial enzyme 130-ED2 under non-heat treated conditions.
As can be seen from Table 5, compared with the initial CPC acylase SEQ ID NO:1, the cephalosporin C acylase mutant of the present invention has improved thermal stability of SEQ ID NO:7 by about 2.7 times and improved enzyme activity by 2.9 times.
Example 6 immobilization of CPC acylase mutant
1.50g of pure enzyme was dissolved in water to a protein concentration of 18 to 20mg/ml, and then about 5.4g of potassium dihydrogenphosphate and 16.5g of dipotassium hydrogenphosphate solid were added to this solution to correct the pH of the enzyme solution at 8.0. + -. 0.2 using dipotassium hydrogenphosphate solid, followed by addition of 10g of LX-1000EP epoxy carrier for immobilization by covalent bonding under the conditions: after being fixed at 25 ℃ and 150rpm for 48 hours and then allowed to stand for 24 hours, the supernatant was removed, washed 3 times with 3-fold volume of pure water, and then drained for use.
EXAMPLE 77 production of ACA
Weighing 2.8g of CPC sodium salt, adding water to dissolve, cooling to 15 ℃, adjusting the pH to 8.2, adding 3000U of the immobilized enzyme prepared in example 6, and stirring for reaction. Controlling the temperature to be 15 +/-0.5 ℃ and the pH value to be 8.0 +/-0.2 in the reaction process, reacting for 40-80min, detecting a reaction sample, performing suction filtration to separate the immobilized enzyme when the content of CPC sodium salt in the sample is lower than 2 wt%, directly adding a substrate to the immobilized enzyme after suction filtration to perform reaction of the next batch, and detecting the reaction condition of 50 batches of the immobilized enzyme continuously used.
The HPLC detection method of the sample comprises the following steps: accurately measuring 100 mu L of reacted sample, using water to fix the volume to 10mL, and carrying out HPLC analysis under the analysis conditions: c18200 mm × 4.6 column, wavelength 262nm, mobile phase 0.02M sodium acetate ph5.5 acetonitrile (═ 93:7), temperature 25 ℃.
The results show that 50 batches of continuous reaction are carried out at the temperature of 15 +/-0.5 ℃ and the pH value of 8.0 +/-0.2, the reaction time is 40-60min, and the conversion rate of CPC sodium salt is over 97 percent.
In conclusion, compared with the initial CPC acylase SEQ ID NO:1, the CPC acylase mutant SEQ ID NOs:3-7 constructed by the invention has improved enzyme activity and thermal stability, wherein the enzyme activity of the mutant SEQ ID NO:7 is improved by 2.9 times at most, and the thermal stability is improved by about 2.7 times. When the immobilized mutase SEQ ID NO 7 is used for catalyzing CPC sodium salt to react to produce 7-ACA, the reaction is carried out for 40-60min at the temperature of 15 +/-0.5 ℃, 50 batches of reactions are continuously carried out, the CPC conversion rate exceeds 97 percent, and a good effect is obtained.
Sequence listing
<110> Shanghai Toudan Cheng Biotechnology Limited responsibility company
<120> cephalosporin C acylase mutant with high heat stability
<130>SHPI2010062
<160>8
<170>SIPOSequenceListing 1.0
<210>1
<211>692
<212>PRT
<213> Artificial sequence ()
<400>1
Met Glu Pro Thr Ser Thr Pro Gln Ala Pro Ile Ala Ala Tyr Lys Pro
1 5 10 15
Arg Ser Asn Glu Ile Leu Trp Asp Asp Tyr Gly Val Pro His Ile Tyr
20 25 30
Gly Val Asp Ala Pro Ser Ala Phe Tyr Gly Tyr Gly Trp Ala Gln Ala
35 40 45
Arg Ser His Gly Asp Asn Ile Leu Arg Leu Tyr Gly Glu Ala Arg Gly
50 55 60
Lys Gly Ala Glu Tyr Trp Gly Pro Asp Tyr Glu Gln Thr Thr Val Trp
65 70 75 80
Leu Leu Thr Asn Gly Val Pro Glu Arg Ala Gln Gln Trp Tyr Ala Gln
85 90 95
Gln Ser Pro Asp Phe Arg Ala Asn Leu Asp Ala Phe Ala Ala Gly Ile
100105 110
Asn Ala Tyr Ala Gln Gln Asn Pro Asp Asp Ile Ser Pro Asp Val Arg
115 120 125
Gln Val Leu Pro Val Ser Gly Ala Asp Val Val Ala His Ala His Arg
130 135 140
Leu Met Asn Phe Leu Tyr Val Ala Ser Pro Gly Arg Thr Leu Gly Glu
145 150 155 160
Gly Asp Pro Pro Asp Leu Ala Asp Gln Gly Ser Asn Ser Trp Ala Val
165 170 175
Ala Pro Gly Lys Thr Ala Asn Gly Asn Ala Leu Leu Leu Gln Asn Pro
180 185 190
His Leu Ser Trp Thr Thr Asp Tyr Phe Thr Tyr Tyr Glu Ala His Leu
195 200 205
Val Thr Pro Asp Phe Glu Val Tyr Gly Ala Thr Gln Ile Gly Leu Pro
210 215 220
Val Ile Arg Val Ala Phe Asn Gln Arg Met Gly Ile Thr Asn Thr Phe
225 230 235 240
Asn Gly Met Val Gly Ala Thr Asn Tyr Arg Leu Thr Leu Gln Asp Gly
245 250 255
Gly Tyr Leu Tyr Asp Gly Gln Val Arg Pro Phe Glu Arg Arg Gln Ala
260 265270
Ser Tyr Arg Leu Arg Gln Ala Asp Gly Thr Thr Val Asp Lys Pro Leu
275 280 285
Glu Ile Arg Ser Ser Val His Gly Pro Val Phe Glu Arg Ala Asp Gly
290 295 300
Thr Ala Val Ala Val Arg Val Ala Gly Leu Asp Arg Pro Gly Met Leu
305 310 315 320
Glu Gln Thr Phe Asp Met Ile Thr Ala Asp Ser Phe Asp Asp Tyr Glu
325 330 335
Ala Ala Leu Ala Arg Met Gln Val Pro Thr Leu Asn Ile Val Tyr Ala
340 345 350
Asp Arg Glu Gly Thr Ile Asn Tyr Ser Phe Asn Gly Val Ala Pro Lys
355 360 365
Arg Ala Glu Gly Asp Ile Ala Phe Trp Gln Gly Leu Val Pro Gly Asp
370 375 380
Ser Ser Arg Tyr Leu Trp Thr Glu Thr His Pro Leu Asp Asp Leu Pro
385 390 395 400
Arg Val Thr Asn Pro Pro Gly Gly Phe Val Gln Asn Ser Asn Asp Pro
405 410 415
Pro Trp Thr Pro Thr Trp Pro Val Thr Tyr Thr Pro Lys Asp Phe Pro
420 425430
Ser Tyr Leu Ala Pro Gln Thr Pro His Ser Leu Arg Ala Gln Gln Ser
435 440 445
Val Arg Leu Met Ser Glu Asn Asp Asp Leu Thr Leu Glu Arg Phe Met
450 455 460
Ala Leu Gln Leu Ser His Arg Ala Val Met Ala Asp Arg Thr Leu Pro
465 470 475 480
Asp Leu Ile Pro Ala Ala Leu Ile Asp Pro Asp Pro Glu Val Gln Ala
485 490 495
Ala Ala Arg Leu Leu Ala Ala Trp Asp Arg Glu Phe Thr Ser Asp Ser
500 505 510
Arg Ala Ala Leu Leu Phe Glu Glu Trp Ala Arg Leu Phe Ala Gly Gln
515 520 525
Asn Phe Ala Gly Gln Ala Gly Phe Ala Thr Pro Trp Ser Leu Asp Lys
530 535 540
Pro Val Ser Thr Pro Tyr Gly Val Arg Asp Pro Lys Ala Ala Val Asp
545 550 555 560
Gln Leu Arg Thr Ala Ile Ala Asn Thr Lys Arg Lys Tyr Gly Ala Ile
565 570 575
Asp Arg Pro Phe Gly Asp Ala Ser Arg Met Ile Leu Asn Asp Val Asn
580 585 590
Val Pro Gly Ala Ala Gly Tyr Gly Asn Leu Gly Ser Phe Arg Val Phe
595 600 605
Thr Trp Ser Asp Pro Asp Glu Asn Gly Val Arg Thr Pro Val Thr Gly
610 615 620
Glu Thr Trp Val Ala Met Ile Glu Phe Ser Thr Pro Val Arg Ala Tyr
625 630 635 640
Gly Leu Met Ser Tyr Gly Asn Ser Arg Gln Pro Gly Thr Thr His Tyr
645 650 655
Ser Asp Gln Ile Glu Arg Val Ser Arg Ala Asp Phe Arg Glu Leu Leu
660 665 670
Leu Arg Arg Glu Gln Val Glu Ala Ala Val Gln Glu Arg Thr Pro Phe
675 680 685
Asn Phe Lys Pro
690
<210>2
<211>2076
<212>DNA
<213> Artificial sequence ()
<400>2
atggaaccga cctccacccc gcaggctccg atcgccgctt acaaaccgcg ttccaacgaa 60
atcctgtggg acgactacgg tgttccgcac atctacggtg ttgacgctcc gtccgctttc 120
tacggttacg gttgggctca ggctcgttcc cacggtgaca acatcctgcg tctgtacggt 180
gaagctcgtg gtaaaggtgc tgaatactgg ggtccggactacgaacagac caccgtttgg 240
ctgctgacca acggtgttcc ggaacgtgct cagcagtggt acgctcagca gtccccggac 300
ttccgtgcta acctggacgc tttcgctgct ggtatcaacg cttacgctca gcagaacccg 360
gacgacatct ccccggacgt tcgtcaggtt ctgccggttt ccggtgctga cgttgttgct 420
cacgctcacc gtctgatgaa cttcctgtac gttgcttccc cgggtcgtac cctgggtgaa 480
ggtgacccgc cggacctggc tgaccagggt tccaactcct gggctgttgc tccgggtaaa 540
accgctaacg gtaacgctct gctgctgcag aacccgcacc tgtcctggac caccgactac 600
ttcacctact acgaagctca cctggttacc ccggacttcg aagtttacgg tgctacccag 660
atcggtctgc cggttatccg tgttgctttc aaccagcgta tgggtatcac caacaccttc 720
aacggtatgg ttggtgctac caactaccgt ctgaccctgc aggacggtgg ttacctgtac 780
gacggtcagg ttcgtccgtt cgaacgtcgt caggcttcct accgtctgcg tcaggctgac 840
ggtaccaccg ttgacaaacc gctggaaatc cgttcctccg ttcacggtcc ggttttcgaa 900
cgtgctgacg gtaccgctgt tgctgttcgt gttgctggtc tggaccgtcc gggtatgctg 960
gaacagacct tcgacatgat caccgctgac tccttcgacg actacgaagc tgctctggct 1020
cgtatgcagg ttccgaccct gaacatcgtt tacgctgacc gtgaaggtac catcaactac 1080
tccttcaacg gtgttgctcc gaaacgtgct gaaggtgaca tcgctttctg gcagggtctg 1140
gttccgggtg actcctcccg ttacctgtgg accgaaaccc acccgctgga cgacctgccg 1200
cgtgttacca acccgccggg tggtttcgtt cagaactcca acgacccgcc gtggaccccg 1260
acctggccgg ttacctacac cccgaaagac ttcccgtcct acctggctcc gcagaccccg 1320
cactccctgc gtgctcagca gtccgttcgt ctgatgtccg aaaacgacga cctgaccctg 1380
gaacgtttca tggctctgca gctgtcccac cgtgctgtta tggctgaccg taccctgccg 1440
gacctgatcc cggctgctct gatcgacccg gacccggaag tgcaggctgc tgctcgtctg 1500
ctggctgctt gggaccgtga attcacctcc gactcccgtg ctgctctgct gttcgaagaa 1560
tgggctcgtc tgttcgctgg tcagaacttc gctggtcagg ctggtttcgc taccccgtgg 1620
tccctggaca aaccggtttc caccccgtac ggtgttcgtg acccgaaagc tgctgttgac 1680
cagctgcgta ccgctatcgc taacaccaaa cgtaaatacg gtgctatcga ccgtccgttc 1740
ggtgacgctt cccgtatgat cctgaacgac gttaacgttc cgggtgctgc tggttacggt 1800
aacctgggtt ccttccgtgt tttcacctgg tccgacccgg acgaaaacgg tgttcgtacc 1860
ccggttaccg gtgaaacctg ggttgctatg atcgaattct ccaccccggt tcgtgcttac 1920
ggtctgatgt cctacggtaa ctcccgtcag ccgggtacca cccactactc cgaccagatc 1980
gaacgtgttt cccgtgctga cttccgtgaa ctgctgctgc gtcgtgaaca ggttgaagct 2040
gctgttcagg aacgtacccc gttcaacttc aaaccg 2076
<210>3
<211>692
<212>PRT
<213> Artificial sequence ()
<400>3
Met Glu Pro Thr Ser Thr Pro Gln Ala Pro Ile Ala Ala Tyr Lys Pro
1 5 10 15
Arg Ser Asn Glu Ile Leu Trp Asp Asp Tyr Gly Val Pro His IleTyr
20 25 30
Gly Val Asp Ala Pro Ser Ala Phe Tyr Gly Tyr Gly Trp Ala Gln Ala
35 40 45
Arg Ser His Gly Asp Asn Ile Leu Arg Leu Tyr Gly Glu Ala Arg Gly
50 55 60
Lys Gly Ala Glu Tyr Trp Gly Pro Asp Tyr Glu Gln Thr Thr Val Trp
65 70 75 80
Leu Leu Thr Asn Gly Val Pro Glu Arg Ala Gln Gln Trp Tyr Ala Gln
85 90 95
Gln Ser Pro Asp Phe Arg Ala Asn Leu Asp Ala Phe Ala Ala Gly Ile
100 105 110
Asn Ala Tyr Ala Gln Gln Asn Pro Asp Asp Ile Ser Pro Asp Val Arg
115 120 125
Gln Val Leu Pro Val Ser Gly Ala Asp Val Val Ala His Ala His Arg
130 135 140
Leu Met Asn Phe Leu Tyr Val Ala Ser Pro Gly Arg Thr Leu Gly Glu
145 150 155 160
Gly Asp Pro Pro Asp Leu Ala Asp Gln Gly Ser Asn Ser Trp Ala Val
165 170 175
Ala Pro Gly Lys Thr Ala Asn Gly Asn Ala Leu Leu Leu Gln Asn Pro
180 185 190
His Leu Ser Trp Thr Thr Asn Tyr Phe Thr Tyr Tyr Glu Ala His Leu
195 200 205
Val Thr Pro Asp Phe Glu Val Tyr Gly Ala Thr Gln Ile Gly Leu Pro
210 215 220
Val Ile Arg Val Ala Phe Asn Gln Arg Met Gly Ile Thr Asn Thr Phe
225 230 235 240
Asn Gly Met Val Gly Ala Thr Asn Tyr Arg Leu Thr Leu Gln Asp Gly
245 250 255
Gly Tyr Leu Tyr Asp Gly Gln Val Arg Pro Phe Glu Arg Arg Gln Ala
260 265 270
Ser Tyr Arg Leu Arg Gln Ala Asp Gly Thr Thr Val Asp Lys Pro Leu
275 280 285
Glu Ile Arg Ser Ser Val His Gly Pro Val Phe Glu Arg Ala Asp Gly
290 295 300
Thr Ala Val Ala Val Arg Val Ala Gly Leu Asp Arg Pro Gly Met Leu
305 310 315 320
Glu Gln Thr Phe Asp Met Ile Thr Ala Asp Ser Phe Asp Asp Tyr Glu
325 330 335
Ala Ala Leu Ala Arg Met Gln Val Pro Thr Leu Asn Ile Val Tyr Ala
340 345 350
Asp Arg Glu Gly Thr Ile Asn Tyr Ser Phe Asn Gly Val Ala Pro Lys
355 360 365
Arg Ala Glu Gly Asp Ile Ala Phe Trp Gln Gly Leu Val Pro Gly Asp
370 375 380
Ser Ser Arg Tyr Leu Trp Thr Glu Thr His Pro Leu Asp Asp Leu Pro
385 390 395 400
Arg Val Thr Asn Pro Pro Gly Gly Phe Val Gln Asn Ser Asn Asp Pro
405 410 415
Pro Trp Thr Pro Thr Trp Pro Val Thr Tyr Thr Pro Lys Asp Phe Pro
420 425 430
Ser Tyr Leu Ala Pro Gln Thr Pro His Ser Leu Arg Ala Gln Gln Ser
435 440 445
Val Arg Leu Met Ser Glu Asn Asp Asp Leu Thr Leu Glu Arg Phe Met
450 455 460
Ala Leu Gln Leu Ser His Arg Ala Val Met Ala Asp Arg Thr Leu Pro
465 470 475 480
Asp Leu Ile Pro Ala Ala Leu Ile Asp Pro Asp Pro Glu Met Gln Ala
485 490 495
Ala Ala Arg Leu Leu Ala Ala Trp Asp Arg Glu Phe Thr Ser Asp Ser
500 505 510
Arg Ala Ala Leu Leu Phe Glu Glu Trp Ala Arg Leu Phe Ala Gly Gln
515 520 525
Asn Phe Ala Gly Gln Ala Gly Phe Ala Thr Pro Trp Ser Leu Asp Lys
530 535 540
Pro Val Ser Thr Pro Tyr Gly Val Arg Asp Pro Lys Ala Ala Val Asp
545 550 555 560
Gln Leu Arg Thr Ala Ile Ala Asn Thr Lys Arg Lys Tyr Gly Ala Ile
565 570 575
Asp Arg Pro Phe Gly Asp Ala Ser Arg Met Ile Leu Asn Asp Val Asn
580 585 590
Val Pro Gly Ala Ala Gly Tyr Gly Asn Leu Gly Ser Phe Arg Val Phe
595 600 605
Thr Trp Ser Asp Pro Asp Glu Asn Gly Val Arg Thr Pro Val His Gly
610 615 620
Glu Thr Trp Val Ala Met Ile Glu Phe Ser Thr Pro Val Arg Ala Tyr
625 630 635 640
Gly Leu Met Ser Tyr Gly Asn Ser Arg Gln Pro Gly Thr Thr His Tyr
645 650 655
Ser Asp Gln Ile Glu Arg Val Ser Arg Ala Asp Phe Arg Glu Leu Leu
660 665 670
Leu Arg Arg Glu Gln Val Glu Ala Ala Val Gln Glu Arg Thr Pro Phe
675 680 685
Asn Phe Lys Pro
690
<210>4
<211>692
<212>PRT
<213> Artificial sequence ()
<400>4
Met Glu Pro Thr Ser Thr Pro Gln Ala Pro Ile Ala Ala Tyr Lys Pro
1 5 10 15
Arg Ser Asn Glu Ile Leu Trp Asp Asp Tyr Gly Val Pro His Ile Tyr
20 25 30
Gly Val Asp Ala Pro Ser Ala Phe Tyr Gly Tyr Gly Trp Ala Gln Ala
35 40 45
Arg Ser His Gly Asp Asn Ile Leu Arg Leu Tyr Gly Glu Ala Arg Gly
50 55 60
Lys Gly Ala Glu Tyr Trp Gly Pro Asp Tyr Glu Gln Thr Thr Val Trp
65 70 75 80
Leu Leu Thr Asn Gly Val Pro Glu Arg Ala Gln Gln Trp Tyr Ala Gln
85 90 95
Gln Ser Pro Asp Phe Arg Ala Asn Leu Asp Ala Phe Ala Ala Gly Ile
100 105 110
Asn Ala Tyr Ala Gln Gln Asn Pro Asp Asp Ile Ser Pro Asp Val Arg
115 120 125
Gln Val Leu Pro Val Ser Gly Ala Asp Val Val Ala His Ala His Arg
130 135 140
Leu Met Asn Phe Leu Tyr Val Ala Ser Pro Gly Arg Thr Leu Gly Glu
145 150 155 160
Gly Asp Pro Pro Asp Leu Ala Asp Gln Gly Ser Asn Ser Trp Ala Val
165 170 175
Ala Pro Gly Lys Thr Ala Asn Gly Asn Ala Leu Leu Leu Gln Asn Pro
180 185 190
His Leu Ser Trp Thr Thr Asp Tyr Phe Thr Tyr Tyr Glu Ala His Leu
195 200 205
Val Thr Pro Asp Phe Glu Val Tyr Gly Ala Thr Gln Ile Gly Leu Pro
210 215 220
Val Ile Arg Val Ala Phe Asn Gln Arg Met Gly Ile Thr Asn Thr Phe
225 230 235 240
Asn Gly Met Val Gly Ala Thr Asn Tyr Arg Leu Thr Leu Gln Asp Gly
245 250 255
Gly Tyr Leu Tyr Asp Gly Gln Val Arg Pro Phe Glu Arg Arg Gln Ala
260 265 270
Ser Tyr Arg Leu Arg Gln Ala Asp Gly Thr Thr Val Asp Lys Pro Leu
275 280 285
Glu Ile Arg Ser Ser Val His Gly Pro Val Phe Glu Arg Ala Asp Gly
290 295 300
Thr Ala Val Ala Val Arg Val Ala Gly Leu Asp Arg Pro Gly Met Leu
305 310 315 320
Glu Gln Thr Phe Asp Met Ile Thr Ala Asp Ser Phe Asp Asp Tyr Glu
325 330 335
Ala Ala Leu Ala Arg Met Gln Val Pro Thr Leu Asn Ile Val Tyr Ala
340 345 350
Asp Arg Glu Gly Thr Ile Asn Tyr Ser Phe Asn Gly Val Ala Pro Lys
355 360 365
Arg Ala Glu Gly Asp Ile Ala Phe Trp Gln Gly Leu Val Pro Gly Asp
370 375 380
Ser Ser Arg Tyr Leu Trp Thr Glu Thr His Pro Leu Asp Asp Leu Pro
385 390 395 400
Arg Val Thr Asn Pro Pro Gly Gly Phe Val Gln Asn Ser Asn Asp Pro
405 410 415
Pro Trp Thr Pro Thr Trp Pro Val Thr Tyr Thr Pro Lys Asp Phe Pro
420425 430
Ser Tyr Leu Ala Pro Gln Thr Pro His Ser Leu Arg Ala Gln Gln Ser
435 440 445
Val Arg Leu Met Ser Glu Asn Asp Asp Leu Thr Leu Glu Arg Phe Met
450 455 460
Ala Leu Gln Leu Ser His Arg Ala Val Met Ala Asp Arg Thr Leu Pro
465 470 475 480
Asp Leu Ile Pro Ala Ala Leu Ile Asp Pro Asp Pro Glu Met Gln Ala
485 490 495
Ala Ala Arg Leu Leu Ala Ala Trp Asp Arg Glu Phe Thr Ser Asp Ser
500 505 510
Arg Ala Ala Leu Leu Phe Glu Glu Trp Ala Arg Leu Phe Ala Gly Gln
515 520 525
Asn Phe Ala Gly Gln Ala Gly Phe Ala Thr Pro Trp Ser Leu Asp Lys
530 535 540
Pro Val Ser Thr Pro Tyr Gly Val Arg Asp Pro Lys Ala Ala Val Asp
545 550 555 560
Gln Leu Arg Thr Ala Ile Ala Asn Thr Lys Arg Lys Tyr Gly Ala Ile
565 570 575
Asp Arg Pro Phe Gly Asp Ala Ser Arg Met Ile Leu Asn Asp Val Asn
580585 590
Val Pro Gly Ala Ala Gly Tyr Gly Asn Leu Gly Ser Phe Arg Val Phe
595 600 605
Thr Trp Ser Asp Pro Asp Glu Asn Gly Val Arg Thr Pro Val His Gly
610 615 620
Glu Thr Trp Val Ala Met Ile Glu Phe Ser Thr Pro Val Arg Ala Tyr
625 630 635 640
Gly Leu Met Ser Tyr Gly Asn Ser Arg Gln Pro Gly Thr Thr His Tyr
645 650 655
Ser Asp Gln Ile Glu Arg Val Ser Arg Ala Asp Phe Arg Glu Leu Leu
660 665 670
Leu Arg Arg Glu Gln Val Glu Ala Ala Val Gln Glu Arg Thr Pro Phe
675 680 685
Asn Phe Lys Pro
690
<210>5
<211>692
<212>PRT
<213> Artificial sequence ()
<400>5
Met Glu Pro Thr Ser Thr Pro Gln Ala Pro Ile Asn Ala Tyr Lys Pro
1 5 10 15
Arg Ser Asn Glu Ile Leu Trp Asp Asp Tyr Gly Val Pro His Ile Tyr
20 2530
Gly Val Asp Ala Pro Ser Ala Phe Tyr Gly Tyr Gly Trp Ala Gln Ala
35 40 45
Arg Ser His Gly Asp Asn Ile Leu Arg Leu Tyr Gly Glu Ala Arg Gly
50 55 60
Lys Gly Ala Glu Tyr Trp Gly Pro Asp Tyr Glu Gln Thr Thr Val Trp
65 70 75 80
Leu Leu Thr Asn Gly Val Pro Glu Arg Ala Gln Gln Trp Tyr Ala Gln
85 90 95
Gln Ser Pro Asp Phe Arg Ala Asn Leu Asp Ala Phe Ala Ala Gly Ile
100 105 110
Asn Ala Tyr Ala Gln Gln Asn Pro Asp Asp Ile Ser Pro Asp Val Arg
115 120 125
Gln Val Leu Pro Val Ser Gly Ala Asp Val Val Ala His Ala His Arg
130 135 140
Leu Met Asn Phe Leu Tyr Val Ala Ser Pro Gly Arg Thr Leu Gly Glu
145 150 155 160
Gly Asp Pro Pro Asp Leu Ala Asp Gln Gly Ser Asn Ser Trp Ala Val
165 170 175
Ala Pro Gly Lys Thr Ala Asn Gly Asn Ala Leu Leu Leu Gln Asn Pro
180 185 190
His Leu Ser Trp Thr Thr Asn Tyr Phe Thr Tyr Tyr Glu Ala His Leu
195 200 205
Val Thr Pro Asp Phe Glu Val Tyr Gly Ala Thr Gln Ile Gly Leu Pro
210 215 220
Val Ile Arg Val Ala Phe Asn Gln Arg Met Gly Ile Thr Asn Thr Phe
225 230 235 240
Asn Gly Met Val Gly Ala Thr Asn Tyr Arg Leu Thr Leu Gln Asp Gly
245 250 255
Gly Tyr Leu Tyr Asp Gly Gln Val Arg Pro Phe Glu Arg Arg Gln Ala
260 265 270
Ser Tyr Arg Leu Arg Gln Ala Asp Gly Thr Thr Val Asp Lys Pro Leu
275 280 285
Glu Ile Arg Ser Ser Val His Gly Pro Val Phe Glu Arg Ala Asp Gly
290 295 300
Thr Ala Val Ala Val Arg Val Ala Gly Leu Asp Arg Pro Gly Met Leu
305 310 315 320
Glu Gln Thr Phe Asp Met Ile Thr Ala Asp Ser Phe Asp Asp Tyr Glu
325 330 335
Ala Ala Leu Ala Arg Met Gln Val Pro Thr Leu Asn Ile Val Tyr Ala
340 345 350
Asp Arg Glu Gly Thr Ile Asn Tyr Ser Phe Asn Gly Val Ala Pro Lys
355 360 365
Arg Ala Glu Gly Asp Ile Ala Phe Trp Gln Gly Leu Val Pro Gly Asp
370 375 380
Ser Ser Arg Tyr Leu Trp Thr Glu Thr His Pro Leu Asp Asp Leu Pro
385 390 395 400
Arg Val Thr Asn Pro Pro Gly Gly Phe Val Gln Asn Ser Asn Asp Pro
405 410 415
Pro Trp Thr Pro Thr Trp Pro Val Thr Tyr Thr Pro Lys Asp Phe Pro
420 425 430
Ser Tyr Leu Ala Pro Gln Thr Pro His Ser Leu Arg Ala Gln Gln Ser
435 440 445
Val Arg Leu Met Ser Glu Asn Asp Asp Leu Thr Leu Glu Arg Phe Met
450 455 460
Ala Leu Gln Leu Ser His Arg Ala Val Met Ala Asp Arg Thr Leu Pro
465 470 475 480
Asp Leu Ile Pro Ala Ala Leu Ile Asp Pro Asp Pro Glu Met Gln Ala
485 490 495
Ala Ala Arg Leu Leu Ala Ala Trp Asp Arg Glu Phe Thr Ser Asp Ser
500 505 510
Arg Ala Ala Leu Leu Phe Glu Glu Trp Ala Arg Leu Phe Ala Gly Gln
515 520 525
Asn Phe Ala Gly Gln Ala Gly Phe Ala Thr Pro Trp Ser Leu Asp Lys
530 535 540
Pro Val Ser Thr Pro Tyr Gly Val Arg Asp Pro Lys Ala Ala Val Asp
545 550 555 560
Gln Leu Arg Thr Ala Ile Ala Asn Thr Lys Arg Lys Tyr Gly Ala Ile
565 570 575
Asp Arg Pro Phe Gly Asp Ala Ser Arg Met Ile Leu Asn Asp Val Asn
580 585 590
Val Pro Gly Ala Ala Gly Tyr Gly Asn Leu Gly Ser Phe Arg Val Phe
595 600 605
Thr Trp Ser Asp Pro Asp Glu Asn Gly Val Arg Thr Pro Val His Gly
610 615 620
Glu Thr Trp Val Ala Met Ile Glu Phe Ser Thr Pro Val Arg Ala Tyr
625 630 635 640
Gly Leu Met Ser Tyr Gly Asn Ser Arg Gln Pro Gly Thr Thr His Tyr
645 650 655
Ser Asp Gln Ile Glu Arg Val Ser Arg Ala Asp Phe Arg Glu Leu Leu
660 665 670
Leu Arg Arg Glu Gln Val Glu Ala Ala Val Gln Glu Arg Thr Pro Phe
675 680 685
Asn Phe Lys Pro
690
<210>6
<211>692
<212>PRT
<213> Artificial sequence ()
<400>6
Met Glu Pro Thr Ser Thr Pro Gln Ala Pro Ile Ala Ala Tyr Lys Pro
1 5 10 15
Arg Ser Asn Glu Ile Leu Trp Asp Asp Tyr Gly Val Pro His Ile Tyr
20 25 30
Gly Val Asp Ala Pro Ser Ala Phe Tyr Gly Tyr Gly Trp Ala Gln Ala
35 40 45
Arg Ser His Gly Asp Asn Ile Leu Arg Leu Tyr Gly Glu Ala Arg Gly
50 55 60
Lys Gly Ala Glu Tyr Trp Gly Pro Asp Tyr Glu Gln Thr Thr Val Trp
65 70 75 80
Leu Leu Thr Asn Gly Val Pro Glu Arg Ala Gln Gln Trp Tyr Ala Gln
85 90 95
Gln Ser Pro Asp Phe Arg Ala Asn Leu Asp Ala Phe Ala Ala Gly Ile
100 105 110
Asn Ala Tyr Ala Gln Gln Asn Pro Asp Asp Ile Ser Pro Asp Val Arg
115 120 125
Gln Val Leu Pro Val Ser Gly Ala Asp Val Val Ala His Ala His Arg
130 135 140
Leu Met Asn Phe Leu Tyr Val Ala Ser Pro Gly Arg Thr Leu Gly Glu
145 150 155 160
Gly Asp Pro Pro Asp Leu Ala Asp Gln Gly Ser Asn Ser Trp Ala Val
165 170 175
Ala Pro Gly Lys Thr Ala Asn Gly Asn Ala Leu Leu Leu Gln Asn Pro
180 185 190
His Leu Ser Trp Thr Thr Asn Tyr Phe Thr Tyr Tyr Glu Ala His Leu
195 200 205
Val Thr Pro Asp Phe Glu Val Tyr Gly Ala Thr Gln Ile Gly Leu Pro
210 215 220
Val Ile Arg Val Ala Phe Asn Gln Arg Met Gly Ile Thr Asn Thr Phe
225 230 235 240
Asn Gly Met Val Gly Ala Thr Asn Tyr Arg Leu Thr Leu Gln Asp Gly
245 250 255
Gly Tyr Leu Tyr Asp Gly Gln Val Arg Pro Phe Glu Arg Arg Gln Ala
260 265 270
Ser Tyr Arg Leu Arg Gln Ala Asp Gly Thr Thr Val Asp Lys Pro Leu
275 280 285
Glu Ile Arg Ser Ser Val His Gly Pro Val Phe Glu Arg Ala Asp Gly
290 295 300
Thr Thr Val Ala Val Arg Val Ala Gly Leu Asp Arg Pro Gly Met Leu
305 310 315 320
Glu Gln Thr Phe Asp Met Ile Thr Ala Asp Ser Phe Asp Asp Tyr Glu
325 330 335
Ala Ala Leu Ala Arg Met Gln Val Pro Thr Leu Asn Ile Val Tyr Ala
340 345 350
Asp Arg Glu Gly Thr Ile Asn Tyr Ser Phe Asn Gly Val Ala Pro Lys
355 360 365
Arg Ala Glu Gly Asp Ile Ala Phe Trp Gln Gly Leu Val Pro Gly Asp
370 375 380
Ser Ser Arg Tyr Leu Trp Thr Glu Thr His Pro Leu Asp Asp Leu Pro
385 390 395 400
Arg Val Thr Asn Pro Pro Gly Gly Phe Val Gln Asn Ser Asn Asp Pro
405 410 415
Pro Trp Thr Pro Thr Trp Pro Val Thr Tyr Thr Pro Lys Asp Phe Pro
420 425 430
Ser Tyr Leu Ala Pro Gln Thr Pro His Ser Leu Arg Ala Gln Gln Ser
435 440 445
Val Arg Leu Met Ser Glu Asn Asp Asp Leu Thr Leu Glu Arg Phe Met
450 455 460
Ala Leu Gln Leu Ser His Arg Ala Val Met Ala Asp Arg Thr Leu Pro
465 470 475 480
Asp Leu Ile Pro Ala Ala Leu Ile Asp Pro Asp Pro Glu Met Gln Ala
485 490 495
Ala Ala Arg Leu Leu Ala Ala Trp Asp Arg Glu Phe Thr Ser Asp Ser
500 505 510
Arg Ala Ala Leu Leu Phe Glu Glu Trp Ala Arg Leu Phe Ala Gly Gln
515 520 525
Asn Phe Ala Gly Gln Ala Gly Phe Ala Thr Pro Trp Ser Leu Asp Lys
530 535 540
Pro Val Ser Thr Pro Tyr Gly Val Arg Asp Pro Lys Ala Ala Val Asp
545 550 555 560
Gln Leu Arg Thr Ala Ile Ala Asn Thr Lys Arg Lys Tyr Gly Ala Ile
565 570 575
Asp Arg Pro Phe Gly Asp Ala Ser Arg Met Ile Leu Asn Asp Val Asn
580 585 590
Val Pro Gly Ala Ala Gly Tyr Gly Asn Leu Gly Ser Phe Arg Val Phe
595 600 605
Thr Trp Ser Asp Pro Asp Glu Asn Gly Val Arg Thr Pro Val His Gly
610 615 620
Glu Thr Trp Val Ala Met Ile Glu Phe Ser Thr Pro Val Arg Ala Tyr
625 630 635 640
Gly Leu Met Ser Tyr Gly Asn Ser Arg Gln Pro Gly Thr Thr His Tyr
645 650 655
Ser Asp Gln Ile Glu Arg Val Ser Arg Ala Asp Phe Arg Glu Leu Leu
660 665 670
Leu Arg Arg Glu Gln Val Glu Ala Ala Val Gln Glu Arg Thr Pro Phe
675 680 685
Asn Phe Lys Pro
690
<210>7
<211>692
<212>PRT
<213> Artificial sequence ()
<400>7
Met Glu Pro Thr Ser Thr Pro Gln Ala Pro Ile Asn Ala Tyr Lys Pro
1 5 10 15
Arg Ser Asn Glu Ile Leu Trp Asp Asp Tyr Gly Val Pro His Ile Tyr
20 25 30
Gly Val Asp Ala Pro Ser Ala Phe TyrGly Tyr Gly Trp Ala Gln Ala
35 40 45
Arg Ser His Gly Asp Asn Ile Leu Arg Leu Tyr Gly Glu Ala Arg Gly
50 55 60
Lys Gly Ala Glu Tyr Trp Gly Pro Asp Tyr Glu Gln Thr Thr Val Trp
65 70 75 80
Leu Leu Thr Asn Gly Val Pro Glu Arg Ala Gln Gln Trp Tyr Ala Gln
85 90 95
Gln Ser Pro Asp Phe Arg Ala Asn Leu Asp Ala Phe Ala Ala Gly Ile
100 105 110
Asn Ala Tyr Ala Gln Gln Asn Pro Asp Asp Ile Ser Pro Asp Val Arg
115 120 125
Gln Val Leu Pro Val Ser Gly Ala Asp Val Val Ala His Ala His Arg
130 135 140
Leu Met Asn Phe Leu Tyr Val Ala Ser Pro Gly Arg Thr Leu Gly Glu
145 150 155 160
Gly Asp Pro Pro Asp Leu Ala Asp Gln Gly Ser Asn Ser Trp Ala Val
165 170 175
Ala Pro Gly Lys Thr Ala Asn Gly Asn Ala Leu Leu Leu Gln Asn Pro
180 185 190
His Leu Ser Trp Thr Thr Asn Tyr Phe Thr Tyr TyrGlu Ala His Leu
195 200 205
Val Thr Pro Asp Phe Glu Val Tyr Gly Ala Thr Gln Ile Gly Leu Pro
210 215 220
Val Ile Arg Val Ala Phe Asn Gln Arg Met Gly Ile Thr Asn Thr Phe
225 230 235 240
Asn Gly Met Val Gly Ala Thr Asn Tyr Arg Leu Thr Leu Gln Asp Gly
245 250 255
Gly Tyr Leu Tyr Asp Gly Gln Val Arg Pro Phe Glu Arg Arg Gln Ala
260 265 270
Ser Tyr Arg Leu Arg Gln Ala Asp Gly Thr Thr Val Asp Lys Pro Leu
275 280 285
Glu Ile Arg Ser Ser Val His Gly Pro Val Phe Glu Arg Ala Asp Gly
290 295 300
Thr Thr Val Ala Val Arg Val Ala Gly Leu Asp Arg Pro Gly Met Leu
305 310 315 320
Glu Gln Thr Phe Asp Met Ile Thr Ala Asp Ser Phe Asp Asp Tyr Glu
325 330 335
Ala Ala Leu Ala Arg Met Gln Val Pro Thr Leu Asn Ile Val Tyr Ala
340 345 350
Asp Arg Glu Gly Thr Ile Asn Tyr Ser Phe Asn Gly Val AlaPro Lys
355 360 365
Arg Ala Glu Gly Asp Ile Ala Phe Trp Gln Gly Leu Val Pro Gly Asp
370 375 380
Ser Ser Arg Tyr Leu Trp Thr Glu Thr His Pro Leu Asp Asp Leu Pro
385 390 395 400
Arg Val Thr Asn Pro Pro Gly Gly Phe Val Gln Asn Ser Asn Asp Pro
405 410 415
Pro Trp Thr Pro Thr Trp Pro Val Thr Tyr Thr Pro Lys Asp Phe Pro
420 425 430
Ser Tyr Leu Ala Pro Gln Thr Pro His Ser Leu Arg Ala Gln Gln Ser
435 440 445
Val Arg Leu Met Ser Glu Asn Asp Asp Leu Thr Leu Glu Arg Phe Met
450 455 460
Ala Leu Gln Leu Ser His Arg Ala Val Met Ala Asp Arg Thr Leu Pro
465 470 475 480
Asp Leu Ile Pro Ala Ala Leu Ile Asp Pro Asp Pro Glu Met Gln Ala
485 490 495
Ala Ala Arg Leu Leu Ala Ala Trp Asp Arg Glu Phe Thr Ser Asp Ser
500 505 510
Arg Ala Ala Leu Leu Phe Glu Glu Trp Ala Arg Leu Phe Ala Gly Gln
515 520 525
Asn Phe Ala Gly Gln Ala Gly Phe Ala Thr Pro Trp Ser Leu Asp Lys
530 535 540
Pro Val Ser Thr Pro Tyr Gly Val Arg Asp Pro Lys Ala Ala Val Asp
545 550 555 560
Gln Leu Arg Thr Ala Ile Ala Asn Thr Lys Arg Lys Tyr Gly Ala Ile
565 570 575
Asp Arg Pro Phe Gly Asp Ala Ser Arg Met Ile Leu Asn Asp Val Asn
580 585 590
Val Pro Gly Ala Ala Gly Tyr Gly Asn Leu Gly Ser Phe Arg Val Phe
595 600 605
Thr Trp Ser Asp Pro Asp Glu Asn Gly Val Arg Thr Pro Val Thr Gly
610 615 620
Glu Thr Trp Val Ala Met Ile Glu Phe Ser Thr Pro Val Arg Ala Tyr
625 630 635 640
Gly Leu Met Ser Tyr Gly Asn Ser Arg Gln Pro Gly Thr Thr His Tyr
645 650 655
Ser Asp Gln Ile Glu Arg Val Ser Arg Ala Asp Phe Arg Glu Leu Leu
660 665 670
Leu Arg Arg Glu Gln Val Glu Ala Ala Val Gln Glu Arg Thr Pro Phe
675 680 685
Asn Phe Lys Pro
690
<210>8
<211>2076
<212>DNA
<213> Artificial sequence ()
<400>8
atggaaccga cctccacccc gcaggctccg atcaacgctt acaaaccgcg ttccaacgaa 60
atcctgtggg acgactacgg tgttccgcac atctacggtg ttgacgctcc gtccgctttc 120
tacggttacg gttgggctca ggctcgttcc cacggtgaca acatcctgcg tctgtacggt 180
gaagctcgtg gtaaaggtgc tgaatactgg ggtccggact acgaacagac caccgtttgg 240
ctgctgacca acggtgttcc ggaacgtgct cagcagtggt acgctcagca gtccccggac 300
ttccgtgcta acctggacgc tttcgctgct ggtatcaacg cttacgctca gcagaacccg 360
gacgacatct ccccggacgt tcgtcaggtt ctgccggttt ccggtgctga cgttgttgct 420
cacgctcacc gtctgatgaa cttcctgtac gttgcttccc cgggtcgtac cctgggtgaa 480
ggtgacccgc cggacctggc tgaccagggt tccaactcct gggctgttgc tccgggtaaa 540
accgctaacg gtaacgctct gctgctgcag aacccgcacc tgtcctggac caccaactac 600
ttcacctact acgaagctca cctggttacc ccggacttcg aagtttacgg tgctacccag 660
atcggtctgc cggttatccg tgttgctttc aaccagcgta tgggtatcac caacaccttc 720
aacggtatgg ttggtgctac caactaccgt ctgaccctgc aggacggtgg ttacctgtac 780
gacggtcagg ttcgtccgtt cgaacgtcgt caggcttcct accgtctgcg tcaggctgac 840
ggtaccaccg ttgacaaacc gctggaaatc cgttcctccg ttcacggtcc ggttttcgaa 900
cgtgctgacg gtaccactgt tgctgttcgt gttgctggtc tggaccgtcc gggtatgctg 960
gaacagacct tcgacatgat caccgctgac tccttcgacg actacgaagc tgctctggct 1020
cgtatgcagg ttccgaccct gaacatcgtt tacgctgacc gtgaaggtac catcaactac 1080
tccttcaacg gtgttgctcc gaaacgtgct gaaggtgaca tcgctttctg gcagggtctg 1140
gttccgggtg actcctcccg ttacctgtgg accgaaaccc acccgctgga cgacctgccg 1200
cgtgttacca acccgccggg tggtttcgtt cagaactcca acgacccgcc gtggaccccg 1260
acctggccgg ttacctacac cccgaaagac ttcccgtcct acctggctcc gcagaccccg 1320
cactccctgc gtgctcagca gtccgttcgt ctgatgtccg aaaacgacga cctgaccctg 1380
gaacgtttca tggctctgca gctgtcccac cgtgctgtta tggctgaccg taccctgccg 1440
gacctgatcc cggctgctct gatcgacccg gacccggaaa tgcaggctgc tgctcgtctg 1500
ctggctgctt gggaccgtga attcacctcc gactcccgtg ctgctctgct gttcgaagaa 1560
tgggctcgtc tgttcgctgg tcagaacttc gctggtcagg ctggtttcgc taccccgtgg 1620
tccctggaca aaccggtttc caccccgtac ggtgttcgtg acccgaaagc tgctgttgac 1680
cagctgcgta ccgctatcgc taacaccaaa cgtaaatacg gtgctatcga ccgtccgttc 1740
ggtgacgctt cccgtatgat cctgaacgac gttaacgttc cgggtgctgc tggttacggt 1800
aacctgggtt ccttccgtgt tttcacctgg tccgacccgg acgaaaacgg tgttcgtacc 1860
ccggttaccg gtgaaacctg ggttgctatg atcgaattct ccaccccggt tcgtgcttac 1920
ggtctgatgt cctacggtaa ctcccgtcag ccgggtacca cccactactc cgaccagatc 1980
gaacgtgttt cccgtgctga cttccgtgaa ctgctgctgc gtcgtgaaca ggttgaagct 2040
gctgttcagg aacgtacccc gttcaacttc aaaccg 2076
Claims (10)
1. A cephalosporin C acylase mutant is a mutant generated by changing one, two, three or four of 12 th A, 199 th D, 306 th A and 494 th V of an amino acid sequence SEQ ID NO 1, wherein the change refers to the substitution, deletion or chemical modification by other amino acids.
2. The cephalosporin C acylase mutant of claim 1 wherein the alteration is a substitution with another amino acid.
3. The cephalosporin C acylase mutant of claim 1 wherein the alteration is a substitution of A at position 12 to N, D at position 199 to N, A at position 306 to T, V at position 494 to M, or a combination of two or more thereof.
4. The cephalosporin C acylase mutant of claim 1 wherein the amino acid sequence is:
3, which is a mutant of SEQ ID NO 1 in which D at position 199 is replaced by N and V at position 494 is replaced by M;
4, which is a mutant of the 494 th V of the SEQ ID NO. 1 replaced by M;
5, which is a mutant of SEQ ID NO 1 in which A at position 12 is replaced by N, D at position 199 is replaced by N, and V at position 494 is replaced by M;
6, which is a mutant of SEQ ID NO 1 wherein D at position 199 is replaced by N, A at position 306 is replaced by T, and V at position 494 is replaced by M; or
7, which is a mutant of SEQ ID NO. 1 wherein A at position 12 is replaced by N, D at position 199 is replaced by N, A at position 306 is replaced by T, and V at position 494 is replaced by M.
5. The cephalosporin C acylase mutant of claim 1, wherein the amino acid sequence is SEQ ID NO 7.
6. A gene encoding the cephalosporin C acylase mutant of claim 4 or 5.
7. The gene encoding the cephalosporin C acylase mutant of claim 5, SEQ ID NO 7, having the sequence of SEQ ID NO 8.
8. A microorganism expressing the gene of claim 6 or 7.
9. The microorganism of claim 8, wherein the microorganism is selected from the group consisting of bacillus subtilis, pichia pastoris, saccharomyces cerevisiae, escherichia coli, cephalosporium acremonium, penicillium chrysogenum.
10. Use of a cephalosporin C acylase mutant according to any of claims 1 to 5 or a microorganism according to claim 8 for the production of 7-aminocephalosporanic acid.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129305A (en) * | 2019-05-28 | 2019-08-16 | 河北凯恩利生物技术有限公司 | A kind of Cephalosporin C acylase mutant being used to prepare 7-ACA |
| CN112662655A (en) * | 2020-12-29 | 2021-04-16 | 山东金城柯瑞化学有限公司 | Cephalosporin C acylase mutant and preparation method and application thereof |
| CN115851687A (en) * | 2021-12-24 | 2023-03-28 | 艾美科健株式会社 | Polypeptide with cephalosporin C acyltransferase activity and application thereof |
Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5457032A (en) * | 1990-04-18 | 1995-10-10 | Gist-Brocades Nv | Mutated β-lactam acylase genes |
| US6033823A (en) * | 1994-08-12 | 2000-03-07 | Van Der Laan; Jan M. | Mutated penicillin G acylase genes |
| EP1553175A1 (en) * | 2004-01-12 | 2005-07-13 | Antibioticos S.p.A. | Cephalosporin C acylase mutants |
| US20070207519A1 (en) * | 2003-08-11 | 2007-09-06 | Sandoz Gmbh | Cephalosporin C Acylase Mutant and Method for Preparing 7-ACA Using Same |
| CN102321603A (en) * | 2011-09-30 | 2012-01-18 | 清华大学 | Cephalosporin acylase mutant and encoding gene and application thereof |
| CN102925423A (en) * | 2012-11-16 | 2013-02-13 | 清华大学 | Mutated cephalosporin C acylase |
| CN102978192A (en) * | 2012-12-25 | 2013-03-20 | 湖南福来格生物技术有限公司 | Mutant cephalosporin C acylase, method for preparing same and method for converting 7-aminocephalosporin acid (ACA) |
| CN103060298A (en) * | 2012-12-31 | 2013-04-24 | 安徽丰原基因工程技术有限公司 | Cephalosporin C acylase mutant as well as coding gene and application thereof |
| KR20140094150A (en) * | 2013-01-21 | 2014-07-30 | 아미코젠주식회사 | A mutant enzyme for production of cephalosporin antibiotics |
| CN105543201A (en) * | 2016-02-23 | 2016-05-04 | 上海星维生物技术有限公司 | Cephalosporin c acylase mutant |
| CN106676090A (en) * | 2016-11-09 | 2017-05-17 | 武汉瀚海新酶生物科技有限公司 | Cephalosporin C acylase mutant with higher heat stability and construction method thereof |
| CN107099523A (en) * | 2017-06-15 | 2017-08-29 | 清华大学 | Cefradine synthase mutant and its encoding gene |
| WO2018001034A1 (en) * | 2016-07-01 | 2018-01-04 | 百瑞全球有限公司 | Cephalosporin hydroxylase mutant, encoding dna of mutant, method for using mutant and application thereof |
| CN108220276A (en) * | 2017-10-30 | 2018-06-29 | 南京朗恩生物科技有限公司 | A kind of Cephalosporin C acylase mutant and its application in 7-amino-cephalosporanic acid production |
| WO2018165881A1 (en) * | 2017-03-15 | 2018-09-20 | 上海星维生物技术有限公司 | Cephalosporin c acylase mutants and applications thereof |
| CN109913436A (en) * | 2017-12-12 | 2019-06-21 | 石药集团圣雪葡萄糖有限责任公司 | Cephalosporin C acylase mutant and preparation method thereof containing one or several point mutation |
| CN110129305A (en) * | 2019-05-28 | 2019-08-16 | 河北凯恩利生物技术有限公司 | A kind of Cephalosporin C acylase mutant being used to prepare 7-ACA |
| CN112342233A (en) * | 2020-11-10 | 2021-02-09 | 上海陶宇晟生物技术有限责任公司 | Polynucleotide for increasing c-di-AMP production when bacteria express DacA |
-
2020
- 2020-02-14 CN CN202010092450.3A patent/CN111172142B/en active Active
Patent Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5457032A (en) * | 1990-04-18 | 1995-10-10 | Gist-Brocades Nv | Mutated β-lactam acylase genes |
| US6033823A (en) * | 1994-08-12 | 2000-03-07 | Van Der Laan; Jan M. | Mutated penicillin G acylase genes |
| US20070207519A1 (en) * | 2003-08-11 | 2007-09-06 | Sandoz Gmbh | Cephalosporin C Acylase Mutant and Method for Preparing 7-ACA Using Same |
| EP1553175A1 (en) * | 2004-01-12 | 2005-07-13 | Antibioticos S.p.A. | Cephalosporin C acylase mutants |
| CN102321603A (en) * | 2011-09-30 | 2012-01-18 | 清华大学 | Cephalosporin acylase mutant and encoding gene and application thereof |
| CN102925423A (en) * | 2012-11-16 | 2013-02-13 | 清华大学 | Mutated cephalosporin C acylase |
| CN102978192A (en) * | 2012-12-25 | 2013-03-20 | 湖南福来格生物技术有限公司 | Mutant cephalosporin C acylase, method for preparing same and method for converting 7-aminocephalosporin acid (ACA) |
| CN103060298A (en) * | 2012-12-31 | 2013-04-24 | 安徽丰原基因工程技术有限公司 | Cephalosporin C acylase mutant as well as coding gene and application thereof |
| KR20140094150A (en) * | 2013-01-21 | 2014-07-30 | 아미코젠주식회사 | A mutant enzyme for production of cephalosporin antibiotics |
| CN105543201A (en) * | 2016-02-23 | 2016-05-04 | 上海星维生物技术有限公司 | Cephalosporin c acylase mutant |
| WO2018001034A1 (en) * | 2016-07-01 | 2018-01-04 | 百瑞全球有限公司 | Cephalosporin hydroxylase mutant, encoding dna of mutant, method for using mutant and application thereof |
| US20190330673A1 (en) * | 2016-07-01 | 2019-10-31 | Yuk Sun Lui | Deacetoxycephalosporin C Hydroxylase Mutants, DNA Encoding the Mutants, Method for Utilizing the Mutants and Application Thereof |
| CN106676090A (en) * | 2016-11-09 | 2017-05-17 | 武汉瀚海新酶生物科技有限公司 | Cephalosporin C acylase mutant with higher heat stability and construction method thereof |
| WO2018165881A1 (en) * | 2017-03-15 | 2018-09-20 | 上海星维生物技术有限公司 | Cephalosporin c acylase mutants and applications thereof |
| CN109072215A (en) * | 2017-03-15 | 2018-12-21 | 上海星维生物技术有限公司 | A kind of Cephalosporin C acylase mutant and its application |
| CN107099523A (en) * | 2017-06-15 | 2017-08-29 | 清华大学 | Cefradine synthase mutant and its encoding gene |
| CN108220276A (en) * | 2017-10-30 | 2018-06-29 | 南京朗恩生物科技有限公司 | A kind of Cephalosporin C acylase mutant and its application in 7-amino-cephalosporanic acid production |
| CN109913436A (en) * | 2017-12-12 | 2019-06-21 | 石药集团圣雪葡萄糖有限责任公司 | Cephalosporin C acylase mutant and preparation method thereof containing one or several point mutation |
| CN110129305A (en) * | 2019-05-28 | 2019-08-16 | 河北凯恩利生物技术有限公司 | A kind of Cephalosporin C acylase mutant being used to prepare 7-ACA |
| CN112342233A (en) * | 2020-11-10 | 2021-02-09 | 上海陶宇晟生物技术有限责任公司 | Polynucleotide for increasing c-di-AMP production when bacteria express DacA |
Non-Patent Citations (12)
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129305A (en) * | 2019-05-28 | 2019-08-16 | 河北凯恩利生物技术有限公司 | A kind of Cephalosporin C acylase mutant being used to prepare 7-ACA |
| CN110129305B (en) * | 2019-05-28 | 2022-10-28 | 河北凯恩利生物技术有限公司 | Cephalosporin C acylase mutant for preparing 7-ACA |
| CN112662655A (en) * | 2020-12-29 | 2021-04-16 | 山东金城柯瑞化学有限公司 | Cephalosporin C acylase mutant and preparation method and application thereof |
| CN115851687A (en) * | 2021-12-24 | 2023-03-28 | 艾美科健株式会社 | Polypeptide with cephalosporin C acyltransferase activity and application thereof |
| JP2023095798A (en) * | 2021-12-24 | 2023-07-06 | アミコージェン・インコーポレイテッド | Polypeptide having cephalosporin C acylase activity and use thereof |
| EP4202044A3 (en) * | 2021-12-24 | 2023-08-23 | Amicogen, Inc. | Polypeptide having cephalosporin c acylase activity and use thereof |
| US12024729B2 (en) | 2021-12-24 | 2024-07-02 | Amicogen, Inc. | Polypeptide having cephalosporin C acylase activity and use thereof |
| JP7527674B2 (en) | 2021-12-24 | 2024-08-05 | アミコージェン・インコーポレイテッド | Polypeptides having cephalosporin C acylase activity and uses thereof |
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