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Histidine ammonia lyase and application thereof

A technology of histidine ammonia-lyase and application, applied in the field of biocatalysis, can solve the problems of high production cost, affecting product yield, large amount of enzyme, etc., and achieve the effect of reducing production cost and improving production efficiency

Active Publication Date: 2017-05-31
湖州颐盛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual production and application, the catalytic activity of phenylalanine ammonia lyase (ilPAL) from Idiomarinaloihiensis is still low, resulting in a large amount of enzyme used in the catalytic reaction, which affects the product yield, and the production cost is still relatively high. high

Method used

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  • Histidine ammonia lyase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of psHAL expression strain

[0040] The protein sequence of histidine ammonia lyase psHAL derived from Pseudoalteromonas sp.P1-26 (NCBI accession number: KPZ73722, SEQ ID NO: 1) was optimized for codon expression in Escherichia coli, and the gene sequence was fully synthesized (SEQ ID NO: 2). The synthetic gene was connected to GenScript standard vector pUC57 by Nanjing GenScript Company to obtain plasmid pUC57-psHAL.

[0041] Lyophilized plasmid pUC57-psHAL (4 μg) (from GenScript) was dissolved in 40 μl ddH 2 In O, it is used as a template to amplify the psHAL gene fragment, so that both ends of the gene have restriction sites NdeI and BamHI. Primers were designed as follows:

[0042] psHAL-NdeI-F: GGAATTCCATATGAGCGAGCACAGCACCAC;

[0043] psHAL-BamHI-R: CGGGATCCTTACGCATACAGGCTCCAAC.

[0044] The PCR reaction system includes: 1X KOD neo plus buffer, 0.2mM dNTP, 25mM MgSO 4 , psHAL-NdeI-F and psHAL-BamHI-R 50pmol each, pUC57-psHAL 50ng, KOD n...

Embodiment 2

[0047] Embodiment 2 Construction of ilPAL expression strain

[0048] Referring to the method disclosed in patent CN101517089A, a strain expressing phenylalanine ammonia-lyase ilPAL derived from Idiomarina loihiensis was constructed, and the obtained strain was named Top10 / pSE420-ilPAL.

Embodiment 3

[0049] Example 3 Construction of RgPAL expression strains

[0050] Referring to the method disclosed in patent CN101517089A, a strain expressing R. glutinis-derived phenylalanine ammonia-lyase RgPAL was constructed, and the obtained strain was named DH5α / pUC57-RgPAL.

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Abstract

The invention discloses histidine ammonia lyase and application thereof. An amino acid sequence of the histidine ammonia lyase is SEQ ID NO:1, the histidine ammonia lyase can be applied to producing L-phenylalanine compound, and the histidine ammonia lyase can efficiently catalyze cinnamic acid compound and amino donor to be converted into the L-phenylalanine compound. Compared with phenylalanine ammonia lyase in the prior art, the histidine ammonia lyase disclosed by the invention has higher catalystic activity, reduces production cost and has an industrialized application prospect.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, relates to a kind of histidine ammonia lyase and its application, specifically, relates to the production of L-phenylalanine by the histidine ammonia lyase whose amino acid sequence is SEQ ID NO:1 use in compounds. Background technique [0002] L-phenylalanine is one of the essential amino acids. It is used as a nutritional enhancer, an amino acid infusion and a component of compound amino acid preparations. It is mainly used as a synthetic raw material for the sweetener aspartame in the food industry. L-phenylalanine compounds such as 3-bromophenyl-L-alanine and (S)-2-chlorophenylalanine are important drug intermediates, of which (S)-2-chlorophenylalanine Amino acid is the main raw material for the production of perindopril, an angiotensin I converting enzyme inhibitor (ACE) for the treatment of hypertension. [0003] WO2006 / 069799A1 discloses a process for preparing enantiomeric phenylal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22C12P13/04C12R1/19
CPCC12N9/88C12P13/04C12P13/22C12Y403/01003
Inventor 陶荣盛王博杨晟王亚夫朱傅赟孙梁栋沈青徐明潘震华
Owner 湖州颐盛生物科技有限公司