分离的多肽、核酸及其应用

Isolated polypeptides, nucleic acids and uses thereof

文档序号：3289 发布日期：2021-09-17 浏览：83次中文

1. An isolated polypeptide having an amino acid mutation at position 310 as compared to wild-type carnosine synthase, SEQ ID NO: 1.

2. The polypeptide of claim 1, wherein the polypeptide has p.gly310ala or p.gly310ser as compared to wild-type carnosine synthetase SEQ ID No. 1.

3. The polypeptide of claim 2, wherein the polypeptide has an amino acid sequence as set forth in SEQ ID NO 2 or 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO 2 or 3.

4. An isolated nucleic acid having at least one of the following combinations of mutations compared to the wild-type carnosine synthase gene of SEQ ID No. 4:

c.929G > C and c.930C > T; and

c.928G > Cc.929G > C and c.930C > T.

5. The nucleic acid according to claim 4, wherein the nucleic acid has a nucleotide sequence as set forth in any one of SEQ ID Nos. 5 to 7 or a nucleotide sequence having at least 95% homology with the nucleotide sequence set forth in any one of SEQ ID Nos. 5 to 7.

6. A construct comprising the nucleic acid of any one of claims 3 to 4.

7. A recombinant cell obtained by transforming a recipient cell with the construct of claim 5.

8. The recombinant cell of claim 7, wherein the recombinant cell is E.coli.

9. A method of increasing the activity of a carnosine synthase, comprising mutating the amino acid 310 of the carnosine synthase;

preferably, the Gly at position 310 of the carnosine synthase is mutated to Ala or Ser.

10. Use of the polypeptide of claims 1 to 3, the nucleic acid of any one of claims 4 to 5, the construct of claim 6, the recombinant cell of claims 7 to 8 for the preparation of L-carnosine.

11. A method of producing carnosine, comprising:

catalytically reacting the polypeptide of claim 2 with β -alanine and/or a β -alanine derivative to obtain the carnosine.

12. The method according to claim 11, wherein the polypeptide is expressed by the recombinant cell according to any one of claims 7 to 8;

optionally, the method comprises subjecting the recombinant cell to a fermentation treatment in the presence of the beta-alanine and/or beta-alanine derivative, so as to obtain the carnosine;

preferably, the beta-alanine derivative is beta-alanine methyl ester and/or beta-alanine methyl ester salt;

more preferably, the beta-alanine methyl ester salt is beta-alanine methyl ester hydrochloride.

13. The method according to claim 12, wherein the fermentation treatment is carried out in a fermentation medium comprising histidine;

preferably, the molar ratio of the histidine to the beta-alanine and/or the beta-alanine derivative is (1-12): 5, preferably 1: 1;

optionally, the fermentation treatment is carried out for 3-6 hours at a temperature of 25-35 ℃, preferably 30 ℃;

optionally, the initial pH of the fermentation treatment is 6-8, preferably 7.

14. A method of producing carnosine, comprising:

carrying out fermentation treatment on the escherichia coli in a fermentation medium for 3-6 hours, wherein the fermentation medium comprises L-histidine and beta-alanine methyl ester hydrochloride, the temperature of the fermentation treatment is 25-35 ℃, and the initial pH of the fermentation treatment is 6-8;

wherein the molar ratio of the L-histidine to the beta-alanine methyl ester hydrochloride is (1-12): 5, preferably 1: 1.

Background

L-carnosine (β -alanyl-L-histidine) is a dipeptide consisting of β -alanine and histidine, with higher concentrations in the skeletal muscle and central nervous system of vertebrates (boldyv, 2013). L-carnosine has many important physiological functions in vivo, including antioxidant, anti-glycation, and intercellular buffering and free radical elimination. Therefore, L-carnosine has attracted much attention as a bioactive compound and is widely used in the fields of medicines, cosmetics, food additives and the like (Yin et al.2019). The research on the production of L-carnosine by chemical synthesis has been widely reported and is the main method for industrial production at present. The chemical synthesis method has complex process, can generate a plurality of toxic substances, has harsh reaction conditions and large energy consumption, and does not meet the requirement of green chemistry.

Therefore, it is necessary to develop a new method for substitution.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.

In a first aspect of the invention, the invention features an isolated polypeptide. According to an embodiment of the present invention, the polypeptide has an amino acid mutation at position 310 as compared to wild-type carnosine synthetase SEQ ID NO: 1. The inventor finds that mutation on the 310 th amino acid of the wild type carnosine synthetase can change the activity of the carnosine synthetase, and the mutation has different types of amino acids and different influences on the enzyme activity, for example, some types of amino acids can greatly reduce the enzyme activity, some types of amino acids can make the enzyme lose the activity of catalyzing carnosine synthesis, and some types of amino acids can greatly improve the enzyme activity, so that the conversion rate of synthesizing carnosine is improved.

According to an embodiment of the invention, the isolated polypeptide further has the following additional technical features:

according to an embodiment of the invention, the polypeptide has p.gly310ala or p.gly310ser as compared to wild type carnosine synthetase SEQ ID NO: 1. The inventor finds that when the amino acid at position 310 of the wild type carnosine synthetase is mutated from glycine to alanine, the activity of the carnosine synthetase can be greatly improved, and the enzyme activity can be improved by 26%; the activity of the wild-type carnosine synthetase can also be improved when the 310 th amino acid is mutated from glycine to alanine to serine.

According to an embodiment of the invention, the polypeptide has the amino acid sequence shown in SEQ ID NO 2 or 3 or an amino acid sequence with at least 95% identity compared to the amino acid sequence shown in SEQ ID NO 2 or 3.

LIISSVSAAEPIRARDLGIPFDGQPGSLNAITDVAGVEVGQVTLIDGEGALLVGSGPVRTGVTVIHPRGRNSTDPVFAGWFALNASGEMTGTTWLEERGMVDGPIAITNTHSVGVVRDAAVAWMVEQGWPADWHAPVVAETYDGGLNDINGFHVTREHALEAMAKARTGVVEEGVVGGGTGMVCNGFKGGIGTSSRVFDALGRSFTVGILVQCNYNWDGEQDLRIGGKNMSGLLPVGKHCFIYRDVPRHVNWYPYCDDSSANDELDKPTRDGSIIIIVATDAPLLPHQLRRLAKRPALGLGRLGGISSDSSGDIFLAFSTASPGLINENEESTISMFPNNGLSVVFEAAVQATEEAIVNAMVAAETVVGASGLQVEEMPEDQLRAIFLD (SEQ ID NO:3) in a second aspect of the invention, the invention features an isolated nucleic acid. According to an embodiment of the invention, the isolated nucleic acid has at least one of the following combinations of mutations compared to the wild-type carnosine synthase gene SEQ ID NO: 4: c.929G > C and c.930C > T; and c.928G > Cc.929G > C and c.930C > T.

According to an embodiment of the invention, the isolated nucleic acid described above has the following additional technical features:

according to an embodiment of the invention, the nucleic acid has a nucleotide sequence as set forth in any one of SEQ ID NOs 5 to 7 or a nucleotide sequence having at least 95% homology with the nucleotide sequence as set forth in any one of SEQ ID NOs 5 to 7.

CTGATCATCAGCAGCGTTAGCGCGGCGGAACCGATCCGTGCGCGTGATCTGGGTATTCCGTTCGACGGTCAGCCGGGTTCTCTGAATGCTATTACTGATGTTGCAGGTGTTGAAGTGGGTCAAGTTACCCTGATTGATGGTGAAGGTGCACTGTTAGTGGGTTCCGGCCCGGTTCGTACTGGCGTTACCGTTATCCACCCGCGTGGCCGTAACTCCACTGACCCGGTTTTTGCTGGTTGGTTTGCTCTTAACGCTTCTGGTGAAATGACCGGTACCACTTGGCTGGAAGAACGTGGTATGGTTGACGGTCCGATTGCTATCACCAACACCCACTCTGTGGGCGTTGTTCGTGATGCGGCGGTTGCGTGGATGGTTGAACAGGGTTGGCCGGCGGATTGGCACGCGCCGGTTGTTGCCGAAACCTATGACGGTGGTCTGAACGACATCAACGGCTTCCACGTTACCCGCGAACACGCGCTGGAAGCGATGGCGAAAGCGCGTACCGGCGTTGTTGAAGAAGGCGTTGTTGGTGGTGGTACCGGTATGGTTTGCAACGGCTTCAAAGGCGGTATCGGCACTTCTAGCCGTGTTTTTGACGCACTGGGCCGTAGCTTCACCGTAGGTATCCTGGTTCAGTGCAACTATAACTGGGATGGTGAACAGGACCTGCGTATCGGCGGCAAAAACATGAGCGGTCTGCTGCCGGTTGGCAAACATTGCTTTATCTACCGTGACGTGCCGCGTCACGTAAACTGGTACCCGTACTGCGATGATAGCTCCGCGAACGATGAACTGGATAAACCGACCCGTGACGGTTCCATCATCATCATCGTGGCGACCGATGCGCCGCTGCTGCCGCACCAGCTGCGCCGCCTGGCGAAACGTCCGGCTCTGGGTCTGGGTCGTCTGGGCGGCATCTCTTCCGATGGCTCTGGCGACATCTTCCTGGCGTTCTCTACCGCGTCGCCGGGCCTGATTAACGAAAACGAAGAATCCACCATTTCCATGTTCCCGAACAACGGCCTGTCTGTTGTTTTCGAAGCGGCGGTGCAGGCGACCGAAGAAGCGATCGTTAACGCGATGGTTGCGGCGGAAACCGTTGTGGGTGCGAGCGGTCTGCAGGTTGAAGAAATGCCGGAAGATCAGCTGCGTGCTATCTTCCTGGAT(SEQ ID NO:4)

CTGATCATCAGCAGCGTTAGCGCGGCGGAACCGATCCGTGCGCGTGATCTGGGTATTCCGTTCGACGGTCAGCCGGGTTCTCTGAATGCTATTACTGATGTTGCAGGTGTTGAAGTGGGTCAAGTTACCCTGATTGATGGTGAAGGTGCACTGTTAGTGGGTTCCGGCCCGGTTCGTACTGGCGTTACCGTTATCCACCCGCGTGGCCGTAACTCCACTGACCCGGTTTTTGCTGGTTGGTTTGCTCTTAACGCTTCTGGTGAAATGACCGGTACCACTTGGCTGGAAGAACGTGGTATGGTTGACGGTCCGATTGCTATCACCAACACCCACTCTGTGGGCGTTGTTCGTGATGCGGCGGTTGCGTGGATGGTTGAACAGGGTTGGCCGGCGGATTGGCACGCGCCGGTTGTTGCCGAAACCTATGACGGTGGTCTGAACGACATCAACGGCTTCCACGTTACCCGCGAACACGCGCTGGAAGCGATGGCGAAAGCGCGTACCGGCGTTGTTGAAGAAGGCGTTGTTGGTGGTGGTACCGGTATGGTTTGCAACGGCTTCAAAGGCGGTATCGGCACTTCTAGCCGTGTTTTTGACGCACTGGGCCGTAGCTTCACCGTAGGTATCCTGGTTCAGTGCAACTATAACTGGGATGGTGAACAGGACCTGCGTATCGGCGGCAAAAACATGAGCGGTCTGCTGCCGGTTGGCAAACATTGCTTTATCTACCGTGACGTGCCGCGTCACGTAAACTGGTACCCGTACTGCGATGATAGCTCCGCGAACGATGAACTGGATAAACCGACCCGTGACGGTTCCATCATCATCATCGTGGCGACCGATGCGCCGCTGCTGCCGCACCAGCTGCGCCGCCTGGCGAAACGTCCGGCTCTGGGTCTGGGTCGTCTGGGCGGCATCTCTTCCGATGGCTCTGCTGACATCTTCCTGGCGTTCTCTACCGCGTCGCCGGGCCTGATTAACGAAAACGAAGAATCCACCATTTCCATGTTCCCGAACAACGGCCTGTCTGTTGTTTTCGAAGCGGCGGTGCAGGCGACCGAAGAAGCGATCGTTAACGCGATGGTTGCGGCGGAAACCGTTGTGGGTGCGAGCGGTCTGCAGGTTGAAGAAATGCCGGAAGATCAGCTGCGTGCTATCTTCCTGGAT(SEQ ID NO:5)

CTGATCATCAGCAGCGTTAGCGCGGCGGAACCGATCCGTGCGCGTGATCTGGGTATTCCGTTCGACGGTCAGCCGGGTTCTCTGAATGCTATTACTGATGTTGCAGGTGTTGAAGTGGGTCAAGTTACCCTGATTGATGGTGAAGGTGCACTGTTAGTGGGTTCCGGCCCGGTTCGTACTGGCGTTACCGTTATCCACCCGCGTGGCCGTAACTCCACTGACCCGGTTTTTGCTGGTTGGTTTGCTCTTAACGCTTCTGGTGAAATGACCGGTACCACTTGGCTGGAAGAACGTGGTATGGTTGACGGTCCGATTGCTATCACCAACACCCACTCTGTGGGCGTTGTTCGTGATGCGGCGGTTGCGTGGATGGTTGAACAGGGTTGGCCGGCGGATTGGCACGCGCCGGTTGTTGCCGAAACCTATGACGGTGGTCTGAACGACATCAACGGCTTCCACGTTACCCGCGAACACGCGCTGGAAGCGATGGCGAAAGCGCGTACCGGCGTTGTTGAAGAAGGCGTTGTTGGTGGTGGTACCGGTATGGTTTGCAACGGCTTCAAAGGCGGTATCGGCACTTCTAGCCGTGTTTTTGACGCACTGGGCCGTAGCTTCACCGTAGGTATCCTGGTTCAGTGCAACTATAACTGGGATGGTGAACAGGACCTGCGTATCGGCGGCAAAAACATGAGCGGTCTGCTGCCGGTTGGCAAACATTGCTTTATCTACCGTGACGTGCCGCGTCACGTAAACTGGTACCCGTACTGCGATGATAGCTCCGCGAACGATGAACTGGATAAACCGACCCGTGACGGTTCCATCATCATCATCGTGGCGACCGATGCGCCGCTGCTGCCGCACCAGCTGCGCCGCCTGGCGAAACGTCCGGCTCTGGGTCTGGGTCGTCTGGGCGGCATCTCTTCCGATTCTTCTGGCGACATCTTCCTGGCGTTCTCTACCGCGTCGCCGGGCCTGATTAACGAAAACGAAGAATCCACCATTTCCATGTTCCCGAACAACGGCCTGTCTGTTGTTTTCGAAGCGGCGGTGCAGGCGACCGAAGAAGCGATCGTTAACGCGATGGTTGCGGCGGAAACCGTTGTGGGTGCGAGCGGTCTGCAGGTTGAAGAAATGCCGGAAGATCAGCTGCGTGCTATCTTCCTGGAT(SEQ ID NO:6)

CTGATCATCAGCAGCGTTAGCGCGGCGGAACCGATCCGTGCGCGTGATCTGGGTATTCCGTTCGACGGTCAGCCGGGTTCTCTGAATGCTATTACTGATGTTGCAGGTGTTGAAGTGGGTCAAGTTACCCTGATTGATGGTGAAGGTGCACTGTTAGTGGGTTCCGGCCCGGTTCGTACTGGCGTTACCGTTATCCACCCGCGTGGCCGTAACTCCACTGACCCGGTTTTTGCTGGTTGGTTTGCTCTTAACGCTTCTGGTGAAATGACCGGTACCACTTGGCTGGAAGAACGTGGTATGGTTGACGGTCCGATTGCTATCACCAACACCCACTCTGTGGGCGTTGTTCGTGATGCGGCGGTTGCGTGGATGGTTGAACAGGGTTGGCCGGCGGATTGGCACGCGCCGGTTGTTGCCGAAACCTATGACGGTGGTCTGAACGACATCAACGGCTTCCACGTTACCCGCGAACACGCGCTGGAAGCGATGGCGAAAGCGCGTACCGGCGTTGTTGAAGAAGGCGTTGTTGGTGGTGGTACCGGTATGGTTTGCAACGGCTTCAAAGGCGGTATCGGCACTTCTAGCCGTGTTTTTGACGCACTGGGCCGTAGCTTCACCGTAGGTATCCTGGTTCAGTGCAACTATAACTGGGATGGTGAACAGGACCTGCGTATCGGCGGCAAAAACATGAGCGGTCTGCTGCCGGTTGGCAAACATTGCTTTATCTACCGTGACGTGCCGCGTCACGTAAACTGGTACCCGTACTGCGATGATAGCTCCGCGAACGATGAACTGGATAAACCGACCCGTGACGGTTCCATCATCATCATCGTGGCGACCGATGCGCCGCTGCTGCCGCACCAGCTGCGCCGCCTGGCGAAACGTCCGGCTCTGGGTCTGGGTCGTCTGGGCGGCATCTCTTCTGACGCTTCTGGTGACATCTTCCTGGCGTTCTCTACCGCGTCGCCGGGCCTGATTAACGAAAACGAAGAATCCACCATTTCCATGTTCCCGAACAACGGCCTGTCTGTTGTTTTCGAAGCGGCGGTGCAGGCGACCGAAGAAGCGATCGTTAACGCGATGGTTGCGGCGGAAACCGTTGTGGGTGCGAGCGGTCTGCAGGTTGAAGAAATGCCGGAAGATCAGCTGCGTGCTATCTTCCTGGAT(SEQ ID NO:7)

CTGATCATCAGCAGCGTTAGCGCGGCGGAACCGATCCGTGCGCGTGATCTGGGTATTCCGTTCGACGGTCAGCCGGGTTCTCTGAATGCTATTACTGATGTTGCAGGTGTTGAAGTGGGTCAAGTTACCCTGATTGATGGTGAAGGTGCACTGTTAGTGGGTTCCGGCCCGGTTCGTACTGGCGTTACCGTTATCCACCCGCGTGGCCGTAACTCCACTGACCCGGTTTTTGCTGGTTGGTTTGCTCTTAACGCTTCTGGTGAAATGACCGGTACCACTTGGCTGGAAGAACGTGGTATGGTTGACGGTCCGATTGCTATCACCAACACCCACTCTGTGGGCGTTGTTCGTGATGCGGCGGTTGCGTGGATGGTTGAACAGGGTTGGCCGGCGGATTGGCACGCGCCGGTTGTTGCCGAAACCTATGACGGTGGTCTGAACGACATCAACGGCTTCCACGTTACCCGCGAACACGCGCTGGAAGCGATGGCGAAAGCGCGTACCGGCGTTGTTGAAGAAGGCGTTGTTGGTGGTGGTACCGGTATGGTTTGCAACGGCTTCAAAGGCGGTATCGGCACTTCTAGCCGTGTTTTTGACGCACTGGGCCGTAGCTTCACCGTAGGTATCCTGGTTCAGTGCAACTATAACTGGGATGGTGAACAGGACCTGCGTATCGGCGGCAAAAACATGAGCGGTCTGCTGCCGGTTGGCAAACATTGCTTTATCTACCGTGACGTGCCGCGTCACGTAAACTGGTACCCGTACTGCGATGATAGCTCCGCGAACGATGAACTGGATAAACCGACCCGTGACGGTTCCATCATCATCATCGTGGCGACCGATGCGCCGCTGCTGCCGCACCAGCTGCGCCGCCTGGCGAAACGTCCGGCTCTGGGTCTGGGTCGTCTGGGCGGCATCTCTTCTGACTCTTCTGGTGACATCTTCCTGGCGTTCTCTACCGCGTCGCCGGGCCTGATTAACGAAAACGAAGAATCCACCATTTCCATGTTCCCGAACAACGGCCTGTCTGTTGTTTTCGAAGCGGCGGTGCAGGCGACCGAAGAAGCGATCGTTAACGCGATGGTTGCGGCGGAAACCGTTGTGGGTGCGAGCGGTCTGCAGGTTGAAGAAATGCCGGAAGATCAGCTGCGTGCTATCTTCCTGGAT(SEQ ID NO:8)

According to the embodiment of the invention, the sequence shown in SEQ ID NO. 5 is that the 928-930 th nucleotide in SEQ ID NO. 4 is mutated from GGC to GCT, and the amino acid is mutated from Gly to Ala.

According to the embodiment of the invention, the sequence shown in SEQ ID NO. 6 is that the 928-930 th nucleotide in SEQ ID NO. 4 is mutated from GGC to TCT, and the amino acid in the corresponding polypeptide is mutated from Gly to Ser.

According to the embodiment of the invention, the sequence shown in SEQ ID NO. 7 is that the 922-927 th mutation of the 928-930 th nucleotides in SEQ ID NO. 5 is TCT GAC, and the 931-936 th mutation is TCTGGT. The nucleotide mutation can make the nucleic acid more suitable for an escherichia coli expression system and better synthesize the carnosine synthase.

According to the embodiment of the invention, the sequence shown in SEQ ID NO. 8 is that the 922-927 th mutation of the 928-930 th nucleotides in SEQ ID NO. 6 is TCT GAC, and the 931-936 th mutation is TCTGGT. The nucleotide mutation can make the nucleic acid more suitable for an escherichia coli expression system and better synthesize the carnosine synthase.

In a third aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct comprises a nucleic acid as set forth in the second aspect of the invention.

In a fourth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, the recombinant cell is obtained by transforming a recipient cell with the construct proposed in the third aspect of the invention.

According to an embodiment of the invention, the recombinant cell described above has the following additional technical features:

according to an embodiment of the invention, the recombinant cell is E.coli.

In a fifth aspect of the invention, the invention features a method of increasing carnosine synthase activity. According to an embodiment of the present invention, amino acid 310 of the carnosine synthase is mutated. According to the embodiments of the present invention, the inventors have found through extensive studies that the enzyme activity of carnosine synthase can be improved by mutating the amino acid at position 310 of carnosine synthase to a specific amino acid type.

According to an embodiment of the present invention, the above method further has the following additional technical features:

according to an embodiment of the present invention, Gly at position 310 of the muscle peptide synthetase is mutated to Ala or Ser. According to the embodiment of the invention, in the chemical synthesis or biosynthesis of protein, the 310 th Gly of the muscle peptide synthetase is mutated into Ala or Ser, and the amino acid types of other sites are not changed, so that the activity of the muscle peptide synthetase can be improved.

In a sixth aspect, the invention provides a polypeptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a construct according to the third aspect of the invention, and the use of a recombinant cell according to the fourth aspect of the invention for the production of L-carnosine.

In a seventh aspect of the invention, the invention provides a method for preparing carnosine. According to an embodiment of the invention, the method comprises catalytically reacting a polypeptide as set forth in the first aspect of the invention with beta-alanine and/or a beta-alanine derivative, in order to obtain the carnosine. According to the embodiment of the invention, the polypeptide reacts with beta-alanine and/or beta-alanine derivatives and histidine to catalyze the generation of carnosine, and the conversion rate is higher than that of wild type carnosine synthetase.

According to an embodiment of the present invention, the above method further has the following additional technical features:

according to an embodiment of the invention, the polypeptide is expressed by a recombinant cell as described above. According to embodiments of the present invention, the recombinant cell, including mammalian cells, competent cells, bacteria, fungi, yeast, etc., may be specifically engineered for nucleic acid, e.g., engineered cell strains or strains that are highly efficient for producing carnosine synthase.

According to an embodiment of the invention, said process comprises subjecting said E.coli to a fermentation treatment in the presence of said beta-alanine and/or beta-alanine derivative, so as to obtain said carnosine.

According to an embodiment of the invention, the beta-alanine methyl ester salt is beta-alanine methyl ester hydrochloride. According to the embodiment of the invention, the carnosine synthetase provided by the invention takes beta-alanine methyl ester or derivative and histidine as substrates, and can efficiently produce carnosine.

According to an embodiment of the invention, the fermentation treatment is carried out in a fermentation medium comprising histidine.

According to an embodiment of the invention, the molar ratio of histidine to beta-alanine and/or beta-alanine derivative is (1-12): 5, preferably 1: 1.

According to an embodiment of the present invention, the fermentation treatment is performed at a temperature of 25 to 35 degrees celsius, preferably 30 degrees celsius, for 3 to 6 hours, preferably 4 hours, and more preferably 4.5 hours.

According to an embodiment of the invention, the initial pH of the fermentation treatment is 6-8, preferably 7.

In an eighth aspect of the invention, the invention provides a method for preparing carnosine. According to an embodiment of the invention, the method comprises: carrying out fermentation treatment on the escherichia coli in a fermentation medium for 3-6 hours, wherein the fermentation medium comprises L-histidine and beta-alanine methyl ester hydrochloride, the temperature of the fermentation treatment is 25-35 ℃, and the initial pH of the fermentation treatment is 6-8; wherein the molar ratio of the L-histidine to the beta-alanine methyl ester hydrochloride is (1-12): 5, preferably 1: 1.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows the enzymatic activities of different mutants according to an embodiment of the present invention;

FIG. 2 shows the results of enzyme activity detection of a 310-site saturation mutant according to an embodiment of the present invention;

FIG. 3 shows the results of enzyme activity measurements at different concentrations of beta-alanine methyl ester hydrochloride according to the example of the present invention;

FIG. 4 shows the results of enzyme activity at different initial pH values according to an embodiment of the present invention;

FIG. 5 shows the results of enzyme activities at different incubation temperatures according to an embodiment of the present invention;

FIG. 6 shows the results of enzyme activities at different incubation times according to examples of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

The term "nucleic acid" as used herein refers to an organic polymer composed of two or more monomers (including nucleotides, nucleosides, or analogs thereof), including, but not limited to, single-or double-stranded, sense or antisense deoxyribonucleic acid (DNA) of any length, including siRNA. The term "nucleotide" refers to any of a variety of compounds consisting of ribose or deoxyribose bonded to a purine or pyrimidine base and to a phosphate group, and they are the basic building blocks of nucleic acids. The term "nucleoside" refers to a compound consisting of a purine or pyrimidine base in combination with deoxyribose or ribose (e.g., guanosine or adenosine), and which is present in nucleic acids.

It is understood that "gene" is included within the meaning of "nucleic acid" described herein, and that the nucleic acids described herein also include "vector" or "plasmid".

It is understood that the amino acid mutations of the polypeptides described herein, which correspond to nucleotide sequences having a plurality of kinds, i.e., according to the codon rules, one amino acid can correspond to a plurality of codons, and further correspond to a plurality of combinations of deoxyribonucleotide sequences, e.g., Ala corresponds to a codon of GCU, GCC, GCA, GCG, which different codons correspond to different deoxyribonucleotide sequences, but eventually can correspond to the formation of Ala amino acids.

The term "construct" as used in the present invention refers to a genetic vector comprising a specific nucleic acid sequence and capable of transferring the nucleic acid sequence of interest into a host cell to obtain a recombinant cell. According to embodiments of the present invention, the form of the construct is not limited, and includes, but is not limited to, at least one of a plasmid, a bacteriophage, an artificial chromosome, a Cosmid (Cosmid), and a virus, preferably a plasmid. The plasmid is used as a genetic carrier, has the characteristics of simple operation, capability of carrying larger fragments and convenience for operation and treatment. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid, and may be either single-stranded or double-stranded. The skilled person can select as desired. The term "nucleic acid" used in the present invention may be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation. For constructs used to construct recombinant cells, it is preferred that the nucleic acid be DNA, as DNA is more stable and easier to manipulate than RNA.

Polypeptides

According to a specific embodiment of the present invention, the catalytic activity of the carnosine synthase can be inactivated by subjecting the wild-type carnosine synthase to the following site mutations: W133D, Y142N, Y142K, S86Q or D218E; the G310A mutation can greatly improve the enzyme activity; after double-site mutation of S86N/G310A or R270G/G310A, the double-mutant enzymes have no catalytic activity on carnosine, which indicates that the regulation of the enzyme activity by R270 in wild type carnosine synthetase is also important.

According to an embodiment of the invention, the polypeptide has p.gly310ala or p.gly310ser as compared to wild type carnosine synthetase SEQ ID NO: 1. The inventor finds that when the amino acid at position 310 of the wild type carnosine synthetase is mutated from glycine to alanine, the activity of the carnosine synthetase can be greatly improved, and the enzyme activity can be improved by 26%; the activity of the wild-type carnosine synthetase can also be improved when the 310 th amino acid is mutated from glycine to alanine to serine.

According to an embodiment of the invention, the polypeptide has the amino acid sequence shown in SEQ ID No. 2 or 3 or an amino acid sequence with at least 95% identity compared to the amino acid sequence shown in SEQ ID No. 2 or 3, further the polypeptide has the amino acid sequence shown in SEQ ID No. 2 or 3 or an amino acid sequence with 95%, 96%, 97%, 98%, 99%, 100% identity compared to the amino acid sequence shown in SEQ ID No. 2 or 3.

Nucleic acids

In a second aspect of the invention, the invention features an isolated nucleic acid. According to an embodiment of the invention, the isolated nucleic acid has at least one of the following combinations of mutations compared to the wild-type carnosine synthase gene SEQ ID NO: 4: c.929G > C and c.930C > T; and c.928G > Cc.929G > C and c.930C > T.

According to an embodiment of the invention, the isolated nucleic acid described above has the following additional technical features:

according to an embodiment of the invention, the nucleic acid has the nucleotide sequence as set forth in any one of SEQ ID NO 5 to 7 or a nucleotide sequence having at least 95% homology with the nucleotide sequence as set forth in any one of SEQ ID NO 5 to 7.

Constructs and recombinant cells

According to an embodiment of the invention, the recombinant cell is E.coli.

According to some embodiments of the invention, the recombinant cells of the invention are capable of efficiently producing carnosine.

According to the embodiment of the present invention, the kind of the recipient cell is not particularly limited, and may be, for example, an escherichia coli cell, a mammalian cell, and preferably, the recipient cell is derived from an escherichia coli cell.

Method for increasing carnosine synthase activity

According to an embodiment of the present invention, Gly at position 310 of the muscle peptide synthetase is mutated to Ala or Ser. According to the embodiment of the invention, in the chemical synthesis or biosynthesis of protein, the 310 th Gly of the muscle peptide synthetase is mutated into Ala or Ser, and the amino acid types of other sites are not changed, so that the activity of the muscle peptide synthetase can be improved.

Method for preparing carnosine

In yet another aspect of the invention, the invention provides a method for preparing carnosine. According to an embodiment of the invention, the method comprises catalytically reacting a polypeptide as set forth in the first aspect of the invention with beta-alanine and/or a beta-alanine derivative, in order to obtain the carnosine. According to the embodiment of the invention, the polypeptide reacts with beta-alanine and/or beta-alanine derivatives and histidine to catalyze the generation of carnosine, and the conversion rate is higher than that of wild type carnosine synthetase.

According to an embodiment of the invention, the polypeptide is expressed by a recombinant cell as described above. According to embodiments of the present invention, the recombinant cell, including mammalian cells, competent cells, bacteria, fungi, yeast, etc., may be specifically engineered for nucleic acid, e.g., engineered cell strains or strains that are highly efficient for producing carnosine synthase.

According to an embodiment of the invention, the fermentation treatment is carried out in a fermentation medium comprising histidine.

According to an embodiment of the invention, the molar ratio of histidine to beta-alanine and/or beta-alanine derivative is (1-12): 5, preferably 1: 1.

According to a particular embodiment of the invention, said histidine is L-histidine.

According to a particular embodiment of the invention, the carnosine is L-carnosine.

According to the embodiment of the invention, the fermentation treatment is carried out for 3-6 hours at the temperature of 25-35 ℃, preferably 30 ℃.

According to an embodiment of the invention, the initial pH of the fermentation treatment is 6-8, preferably 7.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

Materials and methods

Strains and culture conditions

The wild-type DNA sequence (SEQ ID NO:4) for expressing L-carnosine synthetase was synthesized by Shanghai Biotechnology Ltd and inserted into pET-26b plasmid via NcoI and XhoI cleavage sites to give a recombinant plasmid pET26 b-WTCAR. The plasmid was transformed into E.coli BL21(DE3) for expression. The recombinant strain was cultured overnight at 37 ℃ and 200rpm in LB medium containing 40. mu.g/ml kanamycin. Then, 2mL of the inoculum was inoculated into 200mL of fresh LB medium containing 40. mu.g/mL. However, when the OD600 value was about 0.6 to 0.8, the expression was induced overnight at 16 ℃ by adding 0.02mM IPTG.

Aminopeptidase modeling with homologous sequences

Sequence similarity searches were performed in the Protein Database (PDB) using the BLAST program. The x-ray crystal structure (PDB code:1b65) of a protein similar to the L-aminopeptidase sequence of the wild type (SEQ ID NO:1) was used as a template. A three-dimensional model of L-aminopeptidase was generated by sequence alignment using Modeller 9v 21.

Matching of substrates to amino acids

The resulting model was then superimposed on the x-ray crystal structure (PDB code:3nfb) of aminopeptidase of sphingomyelina (Sphingosinella xenopeptiytica). The substrate in 3nfb was then extracted to the corresponding active site of the L-aminopeptidase using PyMol v0.99 software. In the virtual screening, the distance between the active site and the selected site is less than or equal to that of the active siteThe amino acid residue of (1).

Computer-assisted saturation mutagenesis

A computer-simulated saturation mutation method is used for further improving the activity of the L-carnosine synthetase. Specifically, each amino acid residue recognized at the active site was mutated to other types of amino acids, and the conformation of L-carnosine synthetase after binding to a substrate was predicted using AutoDock Vina. Subsequently, point mutation was performed by PCR using pET26 b-WTCCar as a template and primers used for the mutation are shown in Table 1, and high fidelity Q5 DNA polymerase (New England Biolabs, Beijing, China) was used to construct a mutant plasmid. And was digested with DpNI at 37 ℃ for 2h to remove the remaining plasmid. The constructed mutant plasmids W133D, Y142N, Y142K, S86Q, D218E, G310A and double mutant plasmids S86N/G310A and R270G/G310A. Saturation mutagenesis was also performed on G310 and the primers used are shown in table 2. All plasmids above were verified by sequencing.

Table 1: primers used for mutant construction

Table 2: primers for G310 saturation mutation

Enzyme activity detection

In order to screen the best enzyme, the enzyme activity was measured by whole cell reaction. Cells inducing expression were centrifuged at 5000rpm for 5min and the bacterial pellet was washed twice with Na2CO3/NaHCO3 buffer (pH 10.0) and subsequently suspended in this buffer to adjust the OD600 to 20. The substrate concentration was set at 10mM beta-alanine-amide and 50mM L-histidine (Heyland et al 2010). The reaction was carried out at 30 ℃ and 200rpm and stopped by adding 0.3M HCl. Sample concentrations were determined using High Performance Liquid Chromatography (HPLC). Two additional beta-alanine donors, beta-alanine methyl ester hydrochloride (beta-AlaOMe) and beta-alanine ethyl ester hydrochloride (beta-AlaOEt), were also used as substrates for the reaction instead of beta-alanine-amide. The enzyme activity is defined as 100 percent according to the conversion rate of beta-alanine amide

The purified enzyme was used for the determination of the enzyme activity of the mutant proteins. All the his-tagged enzymes were purified by the kit using Ni-agarose (CoWin Biosciences co., beijing, china). The enzymatic reaction system included 10mM beta-alanine methyl ester hydrochloride, 50mM L-histidine, and enzyme dissolved in Na2CO3/NaHCO3 buffer (pH 10) to a final concentration of 74 μ g/mL. The reaction was carried out in a 30 ℃ water bath, quenched by the addition of 50 μ L of 6M hydrochloric acid and analyzed by HPLC.

Example 1

Construction of recombinant plasmid

1. The gene codon of the beta-aminopeptidase sequence found from the deep-sea metagenome library is optimized aiming at an Escherichia coli system, and a related gene (SEQ ID NO:4) is synthesized.

2. Taking the gene synthesized in the step 1, carrying out double enzyme digestion by using restriction enzymes NcoI and XhoI, and recovering an enzyme digestion product.

3. The plasmid pET-26b was digested simultaneously with restriction enzymes NcoI and XhoI, and the vector backbone was recovered.

4. And (3) connecting the enzyme digestion product in the step (2) with the vector skeleton in the step (3) to obtain a recombinant plasmid pET26 b-WTCCar. According to the sequencing result, the structure of the recombinant plasmid pET26 b-WTCHA is described as follows: the DNA molecule shown in SEQ ID NO 4 was inserted between the NcoI and XhoI cleavage sites of the pET-26b plasmid. The DNA molecule shown in SEQ ID NO. 4 encodes the wild-type protein shown in SEQ ID NO. 1.

5. The recombinant plasmid pET26 b-WTCH template is adopted, and a primer pair consisting of W133D-F and W133D-R is adopted to carry out single-point mutation, so that the recombinant plasmid pET26 b-WTCH (W133D) is obtained. The recombinant plasmid pET26 b-WTCCar (W133D) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (W133D) is different only in that the nucleotide at position 397-399 of the DNA molecule shown in the sequence 4 is mutated from "TGG" to "GAC". The mutated DNA molecule encodes the W133D mutant protein.

6. The recombinant plasmid pET26 b-WTCHA template is adopted, and a primer pair consisting of Y142N-F and Y142N-R is adopted to carry out single-point mutation, so that the recombinant plasmid pET26 b-WTCHA (Y142N) is obtained. The recombinant plasmid pET26 b-WTCHA (Y142N) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (Y142N) is different only in that the 424-bit and 426-bit nucleotides of the DNA molecule shown in the sequence 4 are mutated from "TAT" to "AAC". The mutated DNA molecule encodes the Y142N mutant protein.

7. The recombinant plasmid pET26 b-WTCHA template is adopted, and a primer pair consisting of Y142K-F and Y142K-R is adopted to carry out single-point mutation, so that the recombinant plasmid pET26 b-WTCHA (Y142K) is obtained. The recombinant plasmid pET26 b-WTCHA (Y142K) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (Y142K) is different only in that the 424-426 th nucleotide of the DNA molecule shown in the sequence 4 is mutated from "TAT" to "AAA". The mutated DNA molecule encodes the Y142K mutant protein.

8. The recombinant plasmid pET26 b-WTCH template is adopted, and a primer pair consisting of S86Q-F and S86Q-R is adopted to carry out single-point mutation, so that the recombinant plasmid pET26 b-WTCH (S86Q) is obtained. The recombinant plasmid pET26 b-WTCHA (S86Q) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCH, the recombinant plasmid pET26 b-WTCH (S86Q) is different only in that the nucleotide at position 256-258 of the DNA molecule shown in the sequence 4 is mutated from "TCT" to "CAG". The mutated DNA molecule encodes the S86Q mutant protein.

9. The recombinant plasmid pET26 b-WTCH template is adopted, and a primer pair consisting of D218E-F and D218E-R is adopted to carry out single-point mutation, so that the recombinant plasmid pET26 b-WTCH (D218E) is obtained. The recombinant plasmid pET26 b-WTCHA (D218E) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (D218E) is different only in that the nucleotides 652-654 of the DNA molecule shown in the sequence 4 are mutated from "GAT" to "GAA". The mutated DNA molecule encodes the D218E mutant protein.

10. The recombinant plasmid pET26 b-WTCH template is adopted, and a primer pair consisting of D218E-F and D218E-R is adopted to carry out single-point mutation, so that the recombinant plasmid pET26 b-WTCH (D218E) is obtained. The recombinant plasmid pET26 b-WTCHA (D218E) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (D218E) is different only in that the nucleotides 652-654 of the DNA molecule shown in the sequence 4 are mutated from "GAT" to "GAA". The mutated DNA molecule encodes the D218E mutant protein.

11. The recombinant plasmid pET26 b-WTCH template is adopted, and a primer pair consisting of G310A-F and G310A-R is adopted to carry out single-point mutation, so that the recombinant plasmid pET26 b-WTCH (G310A) is obtained. The recombinant plasmid pET26 b-WTCHA (G310A) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310A) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "GCT". The mutated DNA molecule encodes the G310A mutant protein.

12. The recombinant plasmid pET26 b-WTCHA (G310A) is used as a template, and a primer pair consisting of S86NG310A-F and S86NG310A-R is adopted for carrying out two-point mutation to obtain the recombinant plasmid pET26 b-WTCHA (S86NG 310A). The sequencing of the recombinant plasmid pET26 b-WTCH (S86NG310A) shows that compared with the recombinant plasmid pET26 b-WTCH, the recombinant plasmid pET26 b-WTCH (S86NG310A) only has the difference that the 928- bit 930 nucleotide of the DNA molecule shown in the sequence 2 is mutated from 'GGC' to 'GCT', the 256- bit 258 nucleotide of the DNA molecule shown in the sequence 4 is replaced from 'TCT' to 'AAC', and the mutated DNA molecule codes the S86NG310A mutant protein.

13. The recombinant plasmid pET26 b-WTCHA (G310A) is used as a template, and a primer pair consisting of R270GG310A-F and R270GG310A-R is adopted for carrying out two-point mutation to obtain the recombinant plasmid pET26 b-WTCHA (R270GG 310A). The sequencing result of the recombinant plasmid pET26 b-WTCH (R270GG310A) shows that compared with the recombinant plasmid pET26 b-WTCH, the recombinant plasmid pET26 b-WTCH (S86NG310A) only has the difference that the nucleotides 928 and 930 of the DNA molecule shown in the sequence 4 are mutated from 'GGC' to 'GCT', the nucleotides 808 and 810 of the DNA molecule shown in the sequence 4 are replaced from 'ARG' to 'GGT', and the mutated DNA molecule encodes the S86NG310A mutant protein.

14. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310K-f and carnosine G310K-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310K). The recombinant plasmid pET26 b-WTCHA (G310K) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310K) is different only in that the 928-rd 930 th nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "AAA". The mutated DNA molecule encodes the G310K mutant protein.

15. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310P-f and carnosine G310P-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310P). The recombinant plasmid pET26 b-WTCHA (G310P) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310P) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "CCG". The mutated DNA molecule encodes the G310P mutant protein.

16. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310R-f and carnosine G310R-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310R). The recombinant plasmid pET26 b-WTCHA (G310R) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310R) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "CGT". The mutated DNA molecule encodes the G310R mutant protein.

17. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310T-f and carnosine G310T-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310T). The recombinant plasmid pET26 b-WTCHA (G310T) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310T) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "ACC". The mutated DNA molecule encodes the G310T mutant protein.

18. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310D-f and carnosine G310D-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310D). The recombinant plasmid pET26 b-WTCHA (G310D) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310D) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "GAC". The mutated DNA molecule encodes the G310D mutant protein.

19. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310S-f and carnosine G310S-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310S). The recombinant plasmid pET26 b-WTCHA (G310S) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310S) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "GTC". The mutated DNA molecule encodes the G310S mutant protein.

20. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310I-f and carnosine G310I-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310I). The recombinant plasmid pET26 b-WTCHA (G310I) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCH, the recombinant plasmid pET26 b-WTCH (G310I) is different only in that the 928-rd 930 th nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "ATC". The mutated DNA molecule encodes the G310I mutant protein.

21. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310V-f and carnosine G310V-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310V). The recombinant plasmid pET26 b-WTCHA (G310V) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310V) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "GTT". The mutated DNA molecule encodes the G310V mutant protein.

22. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310L-f and carnosine G310L-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310L). The recombinant plasmid pET26 b-WTCHA (G310L) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310L) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "CTG". The mutated DNA molecule encodes the G310L mutant protein.

23. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310H-f and carnosine G310H-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310H). The recombinant plasmid pET26 b-WTCHA (G310H) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310H) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "CAC". The mutated DNA molecule encodes the G310H mutant protein.

24. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310Q-f and carnosine G310Q-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310Q). The recombinant plasmid pET26 b-WTCHA (G310Q) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310Q) is different only in that the 928-rd 930 th nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "CAG". The mutated DNA molecule encodes the G310Q mutant protein.

25. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310F-f and carnosine G310F-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310H). The recombinant plasmid pET26 b-WTCHA (G310F) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCH, the recombinant plasmid pET26 b-WTCH (G310F) is different only in that the 928-rd 930 th nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "TTC". The mutated DNA molecule encodes the G310F mutant protein.

26. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310N-f and carnosine G310N-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310H). The recombinant plasmid pET26 b-WTCHA (G310N) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310N) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "AAC". The mutated DNA molecule encodes the G310N mutant protein.

27. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310E-f and carnosine G310E-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310E). The recombinant plasmid pET26 b-WTCHA (G310E) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310E) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "GAA". The mutated DNA molecule encodes the G310E mutant protein.

28. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310M-f and carnosine G310M-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310M). The recombinant plasmid pET26 b-WTCHA (G310M) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310M) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "ATG". The mutated DNA molecule encodes the G310M mutant protein.

29. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310W-f and carnosine G310W-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310W). The recombinant plasmid pET26 b-WTCHA (G310W) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCH, the recombinant plasmid pET26 b-WTCH (G310W) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "TGG". The mutated DNA molecule encodes the G310W mutant protein.

30. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310C-f and carnosine G310C-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310C). The recombinant plasmid pET26 b-WTCHA (G310C) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCH, the recombinant plasmid pET26 b-WTCH (G310C) is different only in that the 928-bit 930 nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "TGC". The mutated DNA molecule encodes the G310C mutant protein.

31. The recombinant plasmid pET26 b-WTCHA template is adopted to carry out single point mutation by adopting a primer pair consisting of carnosine G310Y-f and carnosine G310Y-r, thus obtaining the recombinant plasmid pET26 b-WTCHA (G310Y). The recombinant plasmid pET26 b-WTCHA (G310Y) is sequenced, and the sequencing result shows that: compared with the recombinant plasmid pET26 b-WTCHA, the recombinant plasmid pET26 b-WTCHA (G310Y) is different only in that the 928-rd 930 th nucleotide of the DNA molecule shown in the sequence 4 is mutated from "GGC" to "TAC". The mutated DNA molecule encodes the G310Y mutant protein.

Second, construction of recombinant bacteria

The recombinant plasmid pET26 b-WTCHA was introduced into E.coli BL21(DE3) to obtain recombinant strain WT.

The recombinant plasmid pET26 b-WTCHA (W133D) was introduced into E.coli BL21(DE3) to obtain a recombinant strain W133D.

The recombinant plasmid pET26 b-WTCHA (Y142N) was introduced into E.coli BL21(DE3) to obtain a recombinant strain Y142N.

The recombinant plasmid pET26 b-WTCHA (Y142K) was introduced into E.coli BL21(DE3) to obtain a recombinant strain Y142K.

The recombinant plasmid pET26 b-WTCHA (S86Q) was introduced into E.coli BL21(DE3) to obtain a recombinant strain S86Q.

The recombinant plasmid pET26 b-WTCHA (D218E) was introduced into E.coli BL21(DE3) to obtain a recombinant strain D218E.

The recombinant plasmid pET26 b-WTCHA (G310A) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310A.

The recombinant plasmid pET26 b-WTCHA (S86NG310A) was introduced into E.coli BL21(DE3) to obtain a recombinant strain S86NG 310A.

The recombinant plasmid pET26 b-WTCHA (R270GG310A) was introduced into E.coli BL21(DE3) to obtain a recombinant strain R270GG 310A.

The recombinant plasmid pET26 b-WTCHA (G310K) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310K.

The recombinant plasmid pET26 b-WTCHA (G310P) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310P.

The recombinant plasmid pET26 b-WTCHA (G310R) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310R.

The recombinant plasmid pET26 b-WTCHA (G310T) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310T.

The recombinant plasmid pET26 b-WTCHA (G310D) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310D.

The recombinant plasmid pET26 b-WTCHA (G310S) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310S.

The recombinant plasmid pET26 b-WTCHA (G310I) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310I.

The recombinant plasmid pET26 b-WTCHA (G310V) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310V.

The recombinant plasmid pET26 b-WTCHA (G310L) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310L.

The recombinant plasmid pET26 b-WTCHA (G310H) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310H.

The recombinant plasmid pET26 b-WTCHA (G310Q) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310Q.

The recombinant plasmid pET26 b-WTCHA (G310F) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310F.

The recombinant plasmid pET26 b-WTCHA (G310N) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310N.

The recombinant plasmid pET26 b-WTCHA (G310M) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310M.

The recombinant plasmid pET26 b-WTCHA (G310W) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310W.

The recombinant plasmid pET26 b-WTCHA (G310C) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310C.

The recombinant plasmid pET26 b-WTCHA (G310Y) was introduced into E.coli BL21(DE3) to obtain recombinant strain G310Y.

Example 2 Whole cell catalysis and enzyme Activity assays of wild-type and respective mutant proteins

Expression of proteins

Each recombinant bacterium prepared in example 1 was used to prepare each protein fused with His6 tag at its N-terminus, and the specific steps were as follows

1. The recombinant strain was inoculated into liquid LB medium containing 40. mu.g/mL kanamycin and cultured with shaking at 37 ℃ and 200rpm until OD600nm became 0.6-0.8.

2. After completion of step 1, isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to the system to a concentration of 0.02mM, and cultured at 16 ℃ for 8 hours with shaking at 200 rpm.

3. After completion of step 2, cells were collected by centrifugation.

4. The cells obtained in step 3 were suspended in binding buffer, then sonicated (10% power, 3 sec for 5 sec, total time 10 min), then centrifuged at 12000g for 20 min and the supernatant collected.

Binding buffer (ph 7.8): containing 20mM Tris-HCl, 500mM sodium chloride and 10mM imidazole, the balance being water.

5. And 4, taking the supernatant obtained in the step 4, and purifying the target protein by adopting nickel affinity chromatography.

The column for nickel affinity chromatography was: Ni-Agarose affinity chromatography column (15 mL).

And (3) purification process: sampling 2mL of supernatant; washing 15 column volumes with binding buffer; collecting 5 column volumes of elution buffer solution for washing, and collecting elution peaks.

Elution buffer (ph 7.8): containing 20mM Tris-HCl, 500mM sodium chloride and 500mM imidazole, the balance being water.

6. The post-column solution obtained in step 5 was taken out, and the solvent system of the protein was replaced with Na2CO3/NaHCO3 buffer (pH 10) using a desalting column of GE Healthcare to obtain a protein solution.

The solution obtained by performing the above steps on the recombinant bacteria obtained in the construction of the recombinant bacteria of example 1 was named as a corresponding recombinant bacteria solution. For example: the solution obtained by carrying out the above steps on the recombinant bacterium W133D is named as W133D solution.

Protein concentration in each solution was determined by the Bradford method using bovine serum albumin as a standard.

II, enzyme activity detection

And (4) taking each cell solution prepared in the step one as a solution to be detected, and detecting the enzyme activity of the cell solution as the L-carnosine synthase.

Reaction system (1.0 ml): containing 10mM beta-alanine methyl ester hydrochloride, 50mM L-histidine, enzyme at a final concentration of 74mg/ml, and the balance Na2CO3/NaHCO3 buffer (pH 10.0).

Reaction conditions are as follows: the reaction was carried out at 30 ℃ and 200 rpm.

After 4.5h of reaction, 0.3M HCl was added to complete the reaction.

L-carnosine in the reaction product was quantified by High Performance Liquid Chromatography (HPLC).

HPLC detection conditions: the stationary phase was a NH2 column (200mm 4.6mm,5 xm) and the mobile phase was 44% acetonitrile and 56% 40mm k2HPO4 solution (pH 6.3, pH adjusted with phosphoric acid). The measurement parameters of the L-carnosine are that the flow rate is 1.0mL/min, the ultraviolet detection wavelength is 210nm, the column temperature is 25 ℃, and the injection volume is 10 mu L. The assay was validated using an L-carnosine standard and the concentration in the sample was quantified using a linear regression curve.

The results for the different mutants are shown in FIG. 1. In FIG. 1, the abscissa represents the different mutants. Almost all other mutants lost catalytic activity, indicating that the activity of the enzyme is very sensitive to binding pocket structure. However, the G310A mutant showed 26% improvement in L-carnosine production over the wild type.

The result of the 310 site saturation mutation is shown in FIG. 2, and in FIG. 2, the enzyme activity of the G310A mutant is highest, and G310S times.

Example 3 influence of the G310A mutant on the Whole cell catalytic Synthesis of L-carnosine

In order to further improve the enzyme activity of G310A and simplify the process flow, L-carnosine is synthesized by the G310A mutant through whole-cell catalysis, and the reaction conditions are optimized.

First, preparing a whole cell extract

The whole cell extract was prepared using G310A prepared in example 1, with the following specific steps:

1. the recombinant strain was inoculated into liquid LB medium containing 40. mu.g/mL kanamycin and cultured with shaking at 37 ℃ and 200rpm until OD600nm became 0.6-0.8.

2. After completion of step 1, isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to the system to a concentration of 0.02mM, and cultured at 16 ℃ for 8 hours with shaking at 200 rpm.

3. After completion of step 2, cells were collected by centrifugation.

4. The cells obtained in step 3 were washed twice with Na2CO3/NaHCO3 buffer (pH 10.0), and then suspended in this buffer, to obtain a whole-cell extract.

Enzymatic activity detection of di-and different beta-alanine methyl ester hydrochlorates

And (4) taking the whole cell extracts prepared in the step one as solutions to be detected respectively, and detecting the enzyme activity of the whole cell extracts as L-carnosine synthase.

Reaction system (1.0 ml): each of the bacterial solutions contained 10,20,30,50,100mM β -alanine methyl ester hydrochloride, 50mM L-histidine, final OD600 ═ 20, and the balance Na2CO3/NaHCO3 buffer (pH 10.0).

Reaction conditions are as follows: the initial pH was 7.0, 30 ℃ and the reaction was carried out at 200 rpm.

After 6 hours of reaction, 0.3M HCl was added to complete the reaction.

L-carnosine in the reaction product was quantified by High Performance Liquid Chromatography (HPLC).

The results are shown in FIG. 3. In FIG. 3, the abscissa is increasing concentrations of beta-alanine methyl ester hydrochloride and the ordinate is L-carnosine production. The highest L-carnosine production was 2.29g/L at a concentration of 50mM beta-alanine methyl ester hydrochloride.

Enzyme activity detection at different culture temperatures

The culture temperature was changed in the culture conditions, and set to: the detection of enzyme activity at different culture temperatures is the same as the detection of enzyme activity of different beta-alanine methyl ester hydrochlorides at 25 ℃,30 ℃, 37 ℃ and 45 ℃ in other culture modes, the result is shown in figure 5, the 30 ℃ is the optimal temperature of the reaction, and the yield of the L-carnosine is 0.31 g/L.

Enzyme activity detection at four different culture times

The culture time was changed in the culture conditions and set to: 1h, 4.5h, 7.5h and 16h, and other culture modes are the same as the 'different beta-alanine methyl ester hydrochloride enzyme activity detection', the enzyme activity under different culture time is detected, the result is shown in figure 6, 4.5h is the optimal time for reaction, and the yield of L-carnosine is 0.87 g/L.

Five, detection of enzyme activity of different initial culture pH values

The initial pH was varied in the culture conditions and set to: 6.8, 7.2, 7.6, 8 and 8.9, the culture time is set to be 4.5h, other culture modes are the same as the 'different beta-alanine methyl ester hydrochloride enzyme activity detection', the enzyme activity under different pH values is detected, the result is shown in figure 4, 6.8 is the optimal pH value of the reaction, and the yield of the L-carnosine is 1.16 g/L.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> university of agriculture in China

<120> isolated polypeptide, nucleic acid and uses thereof

<130> BI3210110

<160> 60

<170> PatentIn version 3.5

<210> 1

<211> 389

<212> PRT

<213> Artificial Sequence

<220>

<223> wild-type carnosine synthetase

<400> 1

Leu Ile Ile Ser Ser Val Ser Ala Ala Glu Pro Ile Arg Ala Arg Asp

1 5 10 15

Leu Gly Ile Pro Phe Asp Gly Gln Pro Gly Ser Leu Asn Ala Ile Thr

20 25 30

Asp Val Ala Gly Val Glu Val Gly Gln Val Thr Leu Ile Asp Gly Glu

35 40 45

Gly Ala Leu Leu Val Gly Ser Gly Pro Val Arg Thr Gly Val Thr Val

50 55 60

Ile His Pro Arg Gly Arg Asn Ser Thr Asp Pro Val Phe Ala Gly Trp

65 70 75 80

Phe Ala Leu Asn Ala Ser Gly Glu Met Thr Gly Thr Thr Trp Leu Glu

85 90 95

Glu Arg Gly Met Val Asp Gly Pro Ile Ala Ile Thr Asn Thr His Ser

100 105 110

Val Gly Val Val Arg Asp Ala Ala Val Ala Trp Met Val Glu Gln Gly

115 120 125

Trp Pro Ala Asp Trp His Ala Pro Val Val Ala Glu Thr Tyr Asp Gly

130 135 140

Gly Leu Asn Asp Ile Asn Gly Phe His Val Thr Arg Glu His Ala Leu

145 150 155 160

Glu Ala Met Ala Lys Ala Arg Thr Gly Val Val Glu Glu Gly Val Val

165 170 175

Gly Gly Gly Thr Gly Met Val Cys Asn Gly Phe Lys Gly Gly Ile Gly

180 185 190

Thr Ser Ser Arg Val Phe Asp Ala Leu Gly Arg Ser Phe Thr Val Gly

195 200 205

Ile Leu Val Gln Cys Asn Tyr Asn Trp Asp Gly Glu Gln Asp Leu Arg

210 215 220

Ile Gly Gly Lys Asn Met Ser Gly Leu Leu Pro Val Gly Lys His Cys

225 230 235 240

Phe Ile Tyr Arg Asp Val Pro Arg His Val Asn Trp Tyr Pro Tyr Cys

245 250 255

Asp Asp Ser Ser Ala Asn Asp Glu Leu Asp Lys Pro Thr Arg Asp Gly

260 265 270

Ser Ile Ile Ile Ile Val Ala Thr Asp Ala Pro Leu Leu Pro His Gln

275 280 285

Leu Arg Arg Leu Ala Lys Arg Pro Ala Leu Gly Leu Gly Arg Leu Gly

290 295 300

Gly Ile Ser Ser Asp Gly Ser Gly Asp Ile Phe Leu Ala Phe Ser Thr

305 310 315 320

Ala Ser Pro Gly Leu Ile Asn Glu Asn Glu Glu Ser Thr Ile Ser Met

325 330 335

Phe Pro Asn Asn Gly Leu Ser Val Val Phe Glu Ala Ala Val Gln Ala

340 345 350

Thr Glu Glu Ala Ile Val Asn Ala Met Val Ala Ala Glu Thr Val Val

355 360 365

Gly Ala Ser Gly Leu Gln Val Glu Glu Met Pro Glu Asp Gln Leu Arg

370 375 380

Ala Ile Phe Leu Asp

385

<210> 2

<211> 389

<212> PRT

<213> Artificial Sequence

<220>

<223> carnosine synthase mutant

<400> 2

Leu Ile Ile Ser Ser Val Ser Ala Ala Glu Pro Ile Arg Ala Arg Asp

1 5 10 15

Leu Gly Ile Pro Phe Asp Gly Gln Pro Gly Ser Leu Asn Ala Ile Thr

20 25 30

Asp Val Ala Gly Val Glu Val Gly Gln Val Thr Leu Ile Asp Gly Glu

35 40 45

Gly Ala Leu Leu Val Gly Ser Gly Pro Val Arg Thr Gly Val Thr Val

50 55 60

Ile His Pro Arg Gly Arg Asn Ser Thr Asp Pro Val Phe Ala Gly Trp

65 70 75 80

Phe Ala Leu Asn Ala Ser Gly Glu Met Thr Gly Thr Thr Trp Leu Glu

85 90 95

Glu Arg Gly Met Val Asp Gly Pro Ile Ala Ile Thr Asn Thr His Ser

100 105 110

Val Gly Val Val Arg Asp Ala Ala Val Ala Trp Met Val Glu Gln Gly

115 120 125

Trp Pro Ala Asp Trp His Ala Pro Val Val Ala Glu Thr Tyr Asp Gly

130 135 140

Gly Leu Asn Asp Ile Asn Gly Phe His Val Thr Arg Glu His Ala Leu

145 150 155 160

Glu Ala Met Ala Lys Ala Arg Thr Gly Val Val Glu Glu Gly Val Val

165 170 175

Gly Gly Gly Thr Gly Met Val Cys Asn Gly Phe Lys Gly Gly Ile Gly

180 185 190

Thr Ser Ser Arg Val Phe Asp Ala Leu Gly Arg Ser Phe Thr Val Gly

195 200 205

Ile Leu Val Gln Cys Asn Tyr Asn Trp Asp Gly Glu Gln Asp Leu Arg

210 215 220

Ile Gly Gly Lys Asn Met Ser Gly Leu Leu Pro Val Gly Lys His Cys

225 230 235 240

Phe Ile Tyr Arg Asp Val Pro Arg His Val Asn Trp Tyr Pro Tyr Cys

245 250 255

Asp Asp Ser Ser Ala Asn Asp Glu Leu Asp Lys Pro Thr Arg Asp Gly

260 265 270

Ser Ile Ile Ile Ile Val Ala Thr Asp Ala Pro Leu Leu Pro His Gln

275 280 285

Leu Arg Arg Leu Ala Lys Arg Pro Ala Leu Gly Leu Gly Arg Leu Gly

290 295 300

Gly Ile Ser Ser Asp Ala Ser Gly Asp Ile Phe Leu Ala Phe Ser Thr

305 310 315 320

Ala Ser Pro Gly Leu Ile Asn Glu Asn Glu Glu Ser Thr Ile Ser Met

325 330 335

Phe Pro Asn Asn Gly Leu Ser Val Val Phe Glu Ala Ala Val Gln Ala

340 345 350

Thr Glu Glu Ala Ile Val Asn Ala Met Val Ala Ala Glu Thr Val Val

355 360 365

Gly Ala Ser Gly Leu Gln Val Glu Glu Met Pro Glu Asp Gln Leu Arg

370 375 380

Ala Ile Phe Leu Asp

385

<210> 3

<211> 389

<212> PRT

<213> Artificial Sequence

<220>

<223> carnosine synthase mutant

<400> 3

Leu Ile Ile Ser Ser Val Ser Ala Ala Glu Pro Ile Arg Ala Arg Asp

1 5 10 15

Leu Gly Ile Pro Phe Asp Gly Gln Pro Gly Ser Leu Asn Ala Ile Thr

20 25 30

Asp Val Ala Gly Val Glu Val Gly Gln Val Thr Leu Ile Asp Gly Glu

35 40 45

Gly Ala Leu Leu Val Gly Ser Gly Pro Val Arg Thr Gly Val Thr Val

50 55 60

Ile His Pro Arg Gly Arg Asn Ser Thr Asp Pro Val Phe Ala Gly Trp

65 70 75 80

Phe Ala Leu Asn Ala Ser Gly Glu Met Thr Gly Thr Thr Trp Leu Glu

85 90 95

Glu Arg Gly Met Val Asp Gly Pro Ile Ala Ile Thr Asn Thr His Ser

100 105 110

Val Gly Val Val Arg Asp Ala Ala Val Ala Trp Met Val Glu Gln Gly

115 120 125

Trp Pro Ala Asp Trp His Ala Pro Val Val Ala Glu Thr Tyr Asp Gly

130 135 140

Gly Leu Asn Asp Ile Asn Gly Phe His Val Thr Arg Glu His Ala Leu

145 150 155 160

Glu Ala Met Ala Lys Ala Arg Thr Gly Val Val Glu Glu Gly Val Val

165 170 175

Gly Gly Gly Thr Gly Met Val Cys Asn Gly Phe Lys Gly Gly Ile Gly

180 185 190

Thr Ser Ser Arg Val Phe Asp Ala Leu Gly Arg Ser Phe Thr Val Gly

195 200 205

Ile Leu Val Gln Cys Asn Tyr Asn Trp Asp Gly Glu Gln Asp Leu Arg

210 215 220

Ile Gly Gly Lys Asn Met Ser Gly Leu Leu Pro Val Gly Lys His Cys

225 230 235 240

Phe Ile Tyr Arg Asp Val Pro Arg His Val Asn Trp Tyr Pro Tyr Cys

245 250 255

Asp Asp Ser Ser Ala Asn Asp Glu Leu Asp Lys Pro Thr Arg Asp Gly

260 265 270

Ser Ile Ile Ile Ile Val Ala Thr Asp Ala Pro Leu Leu Pro His Gln

275 280 285

Leu Arg Arg Leu Ala Lys Arg Pro Ala Leu Gly Leu Gly Arg Leu Gly

290 295 300

Gly Ile Ser Ser Asp Ser Ser Gly Asp Ile Phe Leu Ala Phe Ser Thr

305 310 315 320

Ala Ser Pro Gly Leu Ile Asn Glu Asn Glu Glu Ser Thr Ile Ser Met

325 330 335

Phe Pro Asn Asn Gly Leu Ser Val Val Phe Glu Ala Ala Val Gln Ala

340 345 350

Thr Glu Glu Ala Ile Val Asn Ala Met Val Ala Ala Glu Thr Val Val

355 360 365

Gly Ala Ser Gly Leu Gln Val Glu Glu Met Pro Glu Asp Gln Leu Arg

370 375 380

Ala Ile Phe Leu Asp

385

<210> 4

<211> 1167

<212> DNA

<213> Artificial Sequence

<220>

<223> wild-type carnosine synthase gene

<400> 4

ctgatcatca gcagcgttag cgcggcggaa ccgatccgtg cgcgtgatct gggtattccg 60

ttcgacggtc agccgggttc tctgaatgct attactgatg ttgcaggtgt tgaagtgggt 120

caagttaccc tgattgatgg tgaaggtgca ctgttagtgg gttccggccc ggttcgtact 180

ggcgttaccg ttatccaccc gcgtggccgt aactccactg acccggtttt tgctggttgg 240

tttgctctta acgcttctgg tgaaatgacc ggtaccactt ggctggaaga acgtggtatg 300

gttgacggtc cgattgctat caccaacacc cactctgtgg gcgttgttcg tgatgcggcg 360

gttgcgtgga tggttgaaca gggttggccg gcggattggc acgcgccggt tgttgccgaa 420

acctatgacg gtggtctgaa cgacatcaac ggcttccacg ttacccgcga acacgcgctg 480

gaagcgatgg cgaaagcgcg taccggcgtt gttgaagaag gcgttgttgg tggtggtacc 540

ggtatggttt gcaacggctt caaaggcggt atcggcactt ctagccgtgt ttttgacgca 600

ctgggccgta gcttcaccgt aggtatcctg gttcagtgca actataactg ggatggtgaa 660

caggacctgc gtatcggcgg caaaaacatg agcggtctgc tgccggttgg caaacattgc 720

tttatctacc gtgacgtgcc gcgtcacgta aactggtacc cgtactgcga tgatagctcc 780

gcgaacgatg aactggataa accgacccgt gacggttcca tcatcatcat cgtggcgacc 840

gatgcgccgc tgctgccgca ccagctgcgc cgcctggcga aacgtccggc tctgggtctg 900

ggtcgtctgg gcggcatctc ttccgatggc tctggcgaca tcttcctggc gttctctacc 960

gcgtcgccgg gcctgattaa cgaaaacgaa gaatccacca tttccatgtt cccgaacaac 1020

ggcctgtctg ttgttttcga agcggcggtg caggcgaccg aagaagcgat cgttaacgcg 1080

atggttgcgg cggaaaccgt tgtgggtgcg agcggtctgc aggttgaaga aatgccggaa 1140

gatcagctgc gtgctatctt cctggat 1167

<210> 5

<211> 1167

<212> DNA

<213> Artificial Sequence

<220>

<223> mutant carnosine synthase Gene

<400> 5