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R-transaminase derived from pseudonocardia ammoxidation and synthesis method of R-transaminase

A pseudo-Nocardian and synthetic method technology, applied in the field of production of chiral amines and unnatural amino acids, can solve the problems of high energy consumption, high pollution, etc., achieve broad substrate spectrum, simple preparation method, and easy operation Effect

Pending Publication Date: 2021-03-19
JIANGSU OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In a word, the use of R-transaminase to produce optically pure chiral amines and unnatural amino acids can avoid the problems of high temperature and pressure, high energy consumption and high pollution in traditional chemical processes. The R-transaminase of Streptomonas and its application in the production of chiral amines and unnatural amino acids, this R-transaminase is named after PamAT, is a kind of IV type transaminase with (R) stereoselectivity, the specific invention obtained Stereoselective enzymes are the key to distinguish them from chemical catalysts. They conform to the concept of green production and are conducive to subsequent research, transformation and promotion of industrial applications.

Method used

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  • R-transaminase derived from pseudonocardia ammoxidation and synthesis method of R-transaminase
  • R-transaminase derived from pseudonocardia ammoxidation and synthesis method of R-transaminase
  • R-transaminase derived from pseudonocardia ammoxidation and synthesis method of R-transaminase

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Experimental program
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Effect test

Embodiment 1

[0025] Embodiment one: prepare R-transaminase:

[0026] (1) The construction of the R-transaminase PamAT carrier derived from Pseudonocardia ammoxidans:

[0027] The R-transaminase PamAT gene sequence of the present invention is derived from the GeneBank database wp-093355841.1. On the premise of not changing the amino acid sequence, the codon of the transaminase gene derived from Pseudomonas ammoniaoxidans is first optimized, and the restriction endonuclease is used to The enzymes Nco I and Hind III were connected (Sangon Bioengineering (Shanghai) Co., Ltd.) to the pET-28b vector, and the plasmid map is as follows figure 1 As shown, the nucleic acid sequence of R-transaminase is shown in SEQ NO.1, and the amino acid sequence is shown in SEQ NO.2. The pET-28b vector is transformed into the competent cells of E. coli DH5α strain, and the shaker is recovered and coated Incubate overnight in an LB culture dish containing kana at a final concentration of 50 μg / ml in an incubator ...

Embodiment 2

[0044] Example 2: PamAT catalyzes donor D-serine and acceptor pyruvate to generate D-alanine and 3-hydroxypyruvate:

[0045] Using 50mmol / L D-serine, 50mmol / L pyruvate, 2mmol / L PLP, and deionized water as raw materials, prepare a transamination reaction solution, adjust the pH to 7.5, take 490μl of the transamination reaction solution, and add 10μl of PamAT enzyme solution , under the condition of 30° C., reacted for 5 h, and measured the amount of the product D-alanine generated by the reacted mixture according to the above method (3).

Embodiment 3

[0046] Example 3: PamAT catalyzes the generation of D-serine from the donor D-alanine and the acceptor 3-hydroxypyruvate:

[0047] Use 50mmol / L D-alanine, 50mmol / L 3-hydroxypyruvate, 2mmol / L PLP, and deionized water as raw materials to prepare a transamination reaction solution, adjust the pH to 7.5, take 490μl of the transamination reaction solution, and add 10μl of The PamAT enzyme solution was reacted at 30° C. for 5 h, and the reaction mixture was measured according to the method in (3) above to determine the amount of D-serine produced.

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Abstract

The invention discloses R-transaminase derived from pseudonocardia ammoxidation and a synthesis method thereof. The R-transaminase is named as PamAT, the nucleotide sequence of a gene is shown as SEQNO.1, the amino acid sequence is shown as SEQ NO.2, and the R-transaminase contains the amino acid sequence shown in SEQ NO.2 or is provided with at least 90% of identity with the SEQ NO.2 or is subjected to substitution, deletion or addition of one or more amino acids; and the high stereoselectivity means that the content of one stereoisomer is at least about 1.1 times that of the other stereoisomer. The R-transaminase has high stereoselectivity, has a wide substrate spectrum and has great application potential in the aspect of biological manufacturing of chiral amine and non-natural amino acid.

Description

technical field [0001] The invention relates to the field of biocatalysis, and mainly relates to a preparation method of R-transaminase derived from Psendonocardia ammonioxydans and its application in the production of chiral amines and unnatural amino acids. Background technique [0002] Unnatural amino acids and chiral amines are key intermediates or key chiral modules in the synthesis of many fine chemicals and pharmaceuticals, such as: sitagliptin, paclitaxel, cisplatin, glufosinate; in addition, polypeptides containing unnatural amino acids It is more stable than natural peptides and still maintains its biological activity in the presence of proteases, so unnatural amino acids will play an irreplaceable role in the development of new drugs, antibodies and artificial proteins that are highly stable and will not be rejected by the human body . However, the difference between unnatural amino acids and natural amino acids is that the existing chiral amines and unnatural am...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P13/00C12P13/06
CPCC12N9/1096C12N15/70C12P13/001C12P13/06
Inventor 李婷婷程艺卢辰许恒皓罗志丹司鑫鑫陈科奇霍一君吴佳懿
Owner JIANGSU OCEAN UNIV
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