# US 10,865,225 B2 · Engineered alanyl-glutamine dipeptide biosynthetic enzyme and application thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,225 B2 - Document ID: US10865225B2 - Status: Granted - Kind: B2 - Application number: 16/455,502 - Filed: 2019-06-27 - Published: 2019-10-17 - Granted: 2020-12-15 - Earliest claimed: 2016-12-30 - Assignees: Innobio Corporation Limited - Applicants: Innobio Corporation Limited - Inventors: Wenjie Yuan; Chao Fan; Yimin Li; Hao Hong; Wenzhong Wu - Web page: https://patentgrep.com/patent/US10865225B2 ## CPC - C07K 5/06: Dipeptides - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C12N 1/16: Yeasts; Culture media therefor - C12N 1/20: Bacteria; Culture media therefor - C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)} - C12N 2500/32: Amino acids - C12N 2500/34: Sugars - C12R 2001/19: Escherichia coli - C12R 2001/865: Saccharomyces cerevisiae - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract Provided is a gene encoding alanyl-glutamine dipeptide biosynthetic enzyme and the application thereof. The nucleotide sequence of said gene is shown in SEQ ID NO: 1. Further, provided is an amino acid sequence encoded by the gene, and provided recombinant vector, recombinant E. coli, recombinant S. cerevisiae, and a self-flocculating recombinant S. cerevisiae containing the gene. Furthermore, provided is a method for carrying out bioconversion to synthesize alanyl-glutamine dipeptide by using said recombinant microorganisms. The application of the gene and recombinant batteries has the characteristics of high molar conversion rate, fast production rate and repeated recycling of the bacteria. It is a good choice for efficient, environmentally friendly and economical production of alanyl-glutamine dipeptide. In addition, through the recycling process, it is expected to reduce the cost of industrial production, solve the problems of difficult enzyme recovery, low utilization rate and unstable enzyme activity. ## Claims ### Claim 1 A gene encoding alanyl-glutamine dipeptide synthetase, comprising the nucleotide sequence of SEQ ID NO: 1. ### Claim 2 (depends on 1) An alanyl-glutamine dipeptide synthetase, encoded by the gene of claim 1, comprising the amino acid sequence of SEQ ID NO: 2. ### Claim 3 (depends on 1) A recombinant E. coli. obtained by transfecting the gene of claim 1 into Escherichia coli. ### Claim 4 (depends on 3) A biosynthesis method of alanyl-glutamine dipeptide, comprising a step of transforming a substrate using the recombinant E. coli. of claim 3, wherein the substrate comprises a carboxyl component and an amine component, the carboxyl component is selected from the group consisting of amino acid esters and amino acid amides, the amine component is selected from the group consisting of amino acids, C-protected amino acids and amines. ### Claim 5 (depends on 4) The method according to claim 4, further comprising the following steps: (1) dissolving the substrate L-alanine methyl ester hydrochloride and L-glutamine in a buffer solution, and adjusting the pH to 8.0-10.0; (2) adding the recombinant E. coli to a system obtained in step (1) to react, with the reaction temperature of 15 to 40° C., and pH of the reaction system of 8.0 to 10.0; (3) collecting the reaction solution and centrifuging to separate bacterial cells and terminate the reaction at the reaction end point when pH is not lowered any longer. ### Claim 6 (depends on 5) The method according to claim 5, further comprising a step of recycling the bacterial cells of recombinant E. coli. ### Claim 7 (depends on 1) A recombinant S. cerevisiae, comprising a gene of claim 1. ### Claim 8 (depends on 7) The recombinant S. cerevisiae according to claim 7, further comprising an exogenous flocculating gene FLO1. ### Claim 9 (depends on 7) A biosynthesis method of alanyl-glutamine dipeptide, comprising the step of transforming a substrate using the recombinant S. cerevisiae of claim 7, wherein the substrate comprises a carboxyl component and an amine component, the carboxyl component is selected from the group consisting of amino acid esters and amino acid amides, the amine component is selected from the group consisting of amino acids, C-protected amino acids and amines. ### Claim 10 (depends on 9) The method according to claim 9, further comprising the following steps: (1) dissolving the substrate L-alanine methyl ester hydrochloride and L-glutamine in a solvent system, and adjusting the pH to 8.0-9.0; (2) adding the recombinant S. cerevisiae to a system obtained in step (1) to react, with the reaction temperature of 10 to 40° C., and pH of the reaction system of 8.0 to 9.0; (3) collecting the reaction solution and centrifuging to separate yeast cells and terminate the reaction at the reaction end point when pH is not lowered any longer. ### Claim 11 (depends on 10) The method according to claim 10, further comprising a step of recycling recombinant S. cerevisiae.