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Phenylalanine dehydrogenase for catalytic preparation of non-natural amino acid and application of phenylalanine dehydrogenase

A technology of phenylalanine dehydrogenase and unnatural amino acid, applied in the field of molecular biology, can solve the problems of low catalytic activity of natural amino acid dehydrogenase, and achieve the effects of good industrial application prospect, convenient operation and high yield

Active Publication Date: 2019-12-20
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for substrate ketones with large sterically hindered aromatic group side chains and cycloalkyl side chains, the catalytic activity of the genetically engineered amino acid dehydrogenase obtained through genetic modification or the natural amino acid dehydrogenase obtained by screening is relatively low. Low

Method used

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  • Phenylalanine dehydrogenase for catalytic preparation of non-natural amino acid and application of phenylalanine dehydrogenase
  • Phenylalanine dehydrogenase for catalytic preparation of non-natural amino acid and application of phenylalanine dehydrogenase
  • Phenylalanine dehydrogenase for catalytic preparation of non-natural amino acid and application of phenylalanine dehydrogenase

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Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1: Construction of amino acid dehydrogenase mutant gene:

[0023] (1) In order to improve the catalytic activity of the amino acid dehydrogenase derived from Bacillus nanhaiensis (CGMCC NO.8969) (its amino acid sequence is SEQ D NO.07, and its encoding nucleotide sequence is SEQ ID NO.08), construct C - The amino acid sequence of the mutant with 4 amino acids reduced at the end is SEQ D NO.01, and the specific steps are as follows: use PCR to construct phenylalanine depletion sites with NdeI and Xhol restriction sites at the 5' end and 3' end respectively. Hydrogenase gene, PCR synthesis process was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd. like figure 1 As shown, after the PCR amplification product was identified by 1% agarose gel electrophoresis, the PheDH gene fragment was recovered from the gel, and double-digested with NdeI and XhoI enzymes, and the digested product was recovered, and the same double-digested pET-28a The ...

Embodiment 2

[0031] (1) In order to improve the activity of phenylalanine dehydrogenase derived from Bacillus nanhaiensis to catalyze unnatural amino acids, construct a mutant with 8 amino acids at the C-terminus, the sequence is shown in SEQ ID NO.02; the specific steps are as follows: The phenylalanine dehydrogenase gene with NdeI and Xhol restriction sites at the 5' end and 3' end was constructed by PCR method, and the PCR synthesis process was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd. like figure 1 As shown, after the PCR amplification product was identified by 1% agarose gel electrophoresis, the PheDH gene fragment was recovered from the gel, and double-digested with NdeI and XhoI enzymes, and the digested product was recovered, and the same double-digested pET-28a The plasmid (with His-tag tag) was ligated, and the ligated plasmid was transformed into Escherichia coli BL21(DE3) to obtain the pET28a-PheDH plasmid. The above plasmid was transformed into ...

Embodiment 3

[0036] (1) In order to improve the activity of phenylalanine dehydrogenase derived from Bacillus nanhaiensis to catalyze unnatural amino acids, construct a mutant with 12 amino acids at the C-terminus, the sequence is shown in SEQ ID NO.03; the specific steps are as follows:

[0037] The PCR method was used to construct the phenylalanine dehydrogenase gene with NdeI and Xhol restriction sites at the 5' end and 3' end respectively, and the PCR synthesis process was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd. like figure 1 As shown, after the PCR amplification product was identified by 1% agarose gel electrophoresis, the PheDH gene fragment was recovered from the gel, and double-digested with NdeI and XhoI enzymes, and the digested product was recovered, and the same double-digested pET-28a The plasmid (with His-tag tag) was ligated, and the ligated plasmid was transformed into Escherichia coli BL21(DE3) to obtain the pET28a-PheDH plasmid. The above...

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Abstract

The invention discloses phenylalanine dehydrogenase for catalytic preparation of non-natural amino acid and application of the phenylalanine dehydrogenase. Amino acid sequences of the phenylalanine dehydrogenase are shown in SEQ ID NO.01, SEQ ID NO.02 and SEQ ID NO.03 or SEQ ID NO.04. According to the phenylalanine dehydrogenase, the phenylalanine dehydrogenase obtained from a marine strain Bacillus nanhaiensis (CGMCC NO.8969) is subjected to C terminal area modification, the obtained variant amino acid dehydrogenase can catalyze a series of non-natural amino acid substrates, the ability of the phenylalanine dehydrogenase to catalyze the non-natural amino acid with large hydrophobic groups is improved, and a substrate spectrum is expanded.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a phenylalanine dehydrogenase for catalyzing the preparation of unnatural amino acids and an application thereof. Background technique [0002] Chiral amino acids, including natural amino acids and unnatural amino acids, are key intermediates for the synthesis of fine chemicals such as chiral drugs, chiral pesticides and chiral food additives. For example, D-phenylglycine is an important intermediate of β-lactam semi-synthetic antibiotics, and as an important side chain of ampicillin and ampicillin, the market prospect is broad. L-Phenylalanine (L-Homophenylalanine), that is (S)-2-amino-4-phenylbutyric acid is a non-natural chiral α-amino acid, the amino acid is 20 anti- Hypertension drugs Angiotensin inhibitor Pril drugs, such as benazepril, cilazapril, lirazapril, enalapril, denazepril, and captopril, are used to treat high blood pressure and various Ang...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12P13/04C12P13/22
CPCC12N9/0018C12P13/04C12P13/222C12Y104/0102
Inventor 王世珍王世燕任红
Owner XIAMEN UNIV
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