KR-20260066004-A - A novel L-ligase and preparation method of L-carnosine using the same

Abstract

The present application relates to a polypeptide having carnosine synthetase activity; a polynucleotide encoding said polypeptide; a microorganism comprising one or more of said polypeptide, polynucleotide, and a vector comprising said polynucleotide; a variant polypeptide having carnosine synthetase activity; a polynucleotide encoding said variant polypeptide; a microorganism comprising one or more of said variant polypeptide, polynucleotide, and a vector comprising said polynucleotide; a composition for producing carnosine comprising one or more of said polypeptide having carnosine synthetase activity, said variant polypeptide, microorganism, and a culture thereof; a method for producing carnosine using one or more of said polypeptide having carnosine synthetase activity, said variant polypeptide, and microorganism; and a use of said polypeptide having carnosine synthetase activity, said variant polypeptide, and microorganism for carnosine production.

Inventors

• 황예지
• 이희석
• 윤병훈

Assignees

• 씨제이제일제당 (주)

Dates

Publication Date

20260512

Application Date

20260403

Claims (9)

1. A microorganism comprising one or more of a polypeptide having the amino acid sequence of SEQ ID NO. 2, a polynucleotide encoding the same, and a vector having said polynucleotide.
2. In paragraph 1, the microorganism is a microorganism that produces carnosine.
3. In claim 1, the microorganism is a microorganism in which the activity of AroP (Aromatic amino acid transport protein) protein is further enhanced.
4. In paragraph 3, the microorganism wherein the AroP protein comprises the amino acid sequence of SEQ ID NO. 19.
5. In paragraph 1, the microorganism is a microorganism of the genus Corynebacterium.
6. In claim 1, the microorganism is Corynebacterium glutamicum.
7. A composition for producing carnosine comprising one or more of the microorganism of any one of claims 1 to 6; and a culture thereof.
8. A method for producing carnosine, comprising the step of culturing a microorganism of any one of claims 1 to 6 in a culture medium.
9. A method for producing carnosine according to claim 8, wherein the method further comprises the step of recovering carnosine from one or more of the microorganism, its culture, and the medium.

Description

A novel L-ligase and preparation method of L-carnosine using the same The present application relates to a polypeptide having carnosine synthetase activity; a polynucleotide encoding said polypeptide; a microorganism comprising one or more of said polypeptide, polynucleotide, and a vector comprising said polynucleotide; a variant polypeptide having carnosine synthetase activity; a polynucleotide encoding said variant polypeptide; a microorganism comprising one or more of said variant polypeptide, polynucleotide, and a vector comprising said polynucleotide; a composition for producing carnosine comprising one or more of said polypeptide having carnosine synthetase activity, a variant polypeptide, a microorganism, and a culture thereof; a method for producing carnosine using one or more of said polypeptide having carnosine synthetase activity, a variant polypeptide, and a microorganism; and a use of said polypeptide having carnosine synthetase activity, a variant polypeptide, and a microorganism for producing carnosine. Carnosine is present in high concentrations in muscle and brain tissues and is known to have antioxidant effects and muscle fatigue-improving effects as a bioactive peptide. Carnosine production can be achieved through enzymatic conversion (US 4359416 A), but because it requires a large amount of substrate and ATP, there is a problem that it is not easy to manufacture it on a large scale industrially in terms of cost. The present application will be explained in more detail below through examples. However, the following examples are merely preferred embodiments for illustrating the present application and are therefore not intended to limit the scope of the rights of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by a person skilled in the art who is proficient in the technical field of the present application or a similar technical field. Example 1. Search and Screening of Novel Carnosine Synthesizing Enzymes The amino acid sequence of Bacillus-derived carnosine synthase was used as the query sequence, and PSI-BLAST search was performed based on the NCBI and Kegg databases. As a result, 20 candidate genes considered to be enzymes capable of synthesizing carnosine and organisms possessing said genes were selected. Among them, seven microorganisms and enzymes derived from them were selected based on similarity to the query sequence, as shown in Table 1 below. ordermicroorganismProtein registration number1Bacillus subtilis 168CAB15798.12Streptococcus pneumoniaeCVN04298.13Alkalihalobacillus haloduransMBV7318856.14Priestia megateriumWP_116516826.15Pseudomonas syringaeBAJ15424.16Pseudomonas syringae pv. phaseolicolaAAZ37741.17Bacillus pumilusGM828960.1 Example 2. Production of expression vectors into which various carnosine synthases were introduced The carnosine synthases derived from the seven species selected in Example 1 above (Bacillus subtilis, Streptococcus pneumoniae, Alkalihalobacillus halodurans, Pristia megaterium, Pseudomonas syringae, Pseudomonas syringae pv. phaseolicola, and Bacillus pumilus) each have the amino acid sequences of SEQ ID NOs. 1 to 7. Information on the genes encoding the enzymes and surrounding nucleotide sequences was obtained from the National Institutes of Health GenBank (NIH GenBank, in order, registration numbers CAB15798.1, CVN04298.1, MBV7318856.1, WP_116516826.1, BAJ15424.1, AAZ37741.1, and GM828960). Subsequently, gene fragments for vector construction were obtained by performing PCR using the results obtained through gene synthesis based on the secured sequences as templates. At this time, Solg ™ Pfu-X DNA polymerase was used as the polymerase, and under PCR amplification conditions, denaturation at 95°C for 3 minutes was followed by denaturation at 95°C for 20 seconds; annealing at 56°C for 40 seconds; and polymerization at 72°C for 2 minutes, repeated 30 times, and polymerization was performed at 72°C for 5 minutes. To amplify a gene derived from Bacillus subtilis, primers of sequence numbers 21 and 22 as shown in Table 2 below were prepared, and PCR was performed under the polymerase and PCR amplification conditions as described above, resulting in a gene fragment of 1,419 bp. To amplify the gene derived from Streptococcus pneumoniae, primers of sequence numbers 23 and 24 as shown in Table 2 below were prepared, and PCR was performed in the same manner, resulting in a gene fragment of 1,422 bp. To amplify a gene derived from Alkalihalobacillus halodurans, primers of sequence numbers 25 and 26 as shown in Table 2 below were prepared, and PCR was performed, resulting in a gene fragment of 1,419 bp. To amplify the gene derived from Pristia megaterium, primers of sequence numbers 27 and 28 as shown in Table 2 below were prepared, and PCR was performed in the same manner, resulting in a gene fragment of 1,422 bp. To amplify a gene derived from Pseudomonas syringa, primers