Method for synthesizing 5-hydroxy beta-indolyl alanine by taking beta-indolyl alanine as substrate and application of 5-hydroxy beta-indolyl alanine

A technology of indolylalanine and indolylalanine protein, which is applied in the biological field, can solve the problems of high price and difficult storage of BH4, and achieve the effect of high synthesis yield and increased yield

Active Publication Date: 2022-08-09
河北维达康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the hydroxylation of β-indolylalanine to 5-hydroxy β-indolylalanine requires the participation of BH4 as a cofactor, and BH4 is difficult to store and expensive, which makes industrial production by adding additional BH4 5-Hydroxybeta-indolylalanine made impossible

Method used

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  • Method for synthesizing 5-hydroxy beta-indolyl alanine by taking beta-indolyl alanine as substrate and application of 5-hydroxy beta-indolyl alanine
  • Method for synthesizing 5-hydroxy beta-indolyl alanine by taking beta-indolyl alanine as substrate and application of 5-hydroxy beta-indolyl alanine
  • Method for synthesizing 5-hydroxy beta-indolyl alanine by taking beta-indolyl alanine as substrate and application of 5-hydroxy beta-indolyl alanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction and screening of single BH4 regeneration system engineering bacteria

[0047] Tandem expression of key enzyme gene TPH2 in β-indolylalanine hydroxylation pathway; key enzyme FOLE, PTPS and SPR in BH4 synthesis pathway.

[0048] The TPH2, FOLE, PTPS and SPR genes were synthesized and inserted between the NdeI and XhoI sites of the vector plasmid pET28a(+) to obtain pET28a-TPH2, pET28a-FOLE, pET28a-PTPS and pET28a-SPR plasmids.

[0049] Using F1 and R1 as primers, the pET28a-TPH2 plasmid was used as the template to clone the pET28a plasmid vector containing the TPH2 gene; using F2 and R2 as the primers, the plasmid pET28a-FOLE was used as the template to clone to obtain the FOLE gene; using F3 and R3 as the primers, the plasmid pET28a was cloned - PTPS was used as template to clone to obtain PTPS gene; F4 and R4 were used as primers, plasmid pET28a-SPR was used as template to clone to obtain SPR gene.

[0050] The above fragments were ligated toget...

Embodiment 2

[0066] Embodiment 2 Construction and screening of double BH4 regeneration system engineering bacteria

[0067] The above-mentioned BH4 regeneration key genes QDPR and DHFR were further combined to form a double BH4 regeneration system, and the effect of screening the engineering bacteria of these different gene combinations to synthesize 5-hydroxyβ-indolylalanine was evaluated. Specifically, a double BH4 regeneration system was constructed by taking the PCD gene shown in SEQ ID NO: 5 and the QDPR gene shown in SEQ ID NO: 8 as examples.

[0068] Based on the double BH4 regeneration system, the synthesis route of 5-hydroxyβ-indolylalanine using β-indolylalanine as substrate is as follows: Figure 4 shown.

[0069] Tandem expression of key genes PCD, QDPR and different DHFRs in the dual BH4 regeneration system:

[0070] DHFR genes from different sources were selected and synthesized by codon optimization to obtain F1-F5 (the sequences are shown in SEQ ID NOs: 11-15 in sequence)...

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Abstract

The invention provides a method for synthesizing 5-hydroxy beta-indolyl alanine by taking beta-indolyl alanine as a substrate and application of the 5-hydroxy beta-indolyl alanine. The 5-hydroxyl beta-indolyl alanine high-producing strain provided by the invention is used for a method for synthesizing 5-hydroxyl beta-indolyl alanine by a biological method by taking beta-indolyl alanine as a substrate, and the synthesis yield of the 5-hydroxyl beta-indolyl alanine is high. Through construction of a BH4 regeneration system and excavation of a new BH4 regeneration enzyme, the regeneration capacity of an engineering strain BH4 is enhanced, and especially the yield of biosynthesized 5-hydroxyl beta-indolyl alanine is remarkably increased under the simultaneous action of a double BH4 regeneration system.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for synthesizing 5-hydroxy β-indolyl alanine using β-indolyl alanine as a substrate and application thereof. Background technique [0002] 5-Hydroxyβ-indolylalanine is widely present in the seeds of leguminous plants, among which the seeds of the African plant Ghana have the highest content. The basic process route of extracting and purifying 5-hydroxy β-indolylalanine from Ghana seeds is: Ghana seeds crushing - extraction - filtration and centrifugation - physical decolorization - vacuum concentration - crystallization, recrystallization - vacuum drying. However, as the number of African Ghana trees continues to decrease, it is becoming more and more difficult to obtain 5-hydroxyβ-indolylalanine by traditional extraction methods. In recent years, the market demand for 5-hydroxy β-indolylalanine is increasing, and it is difficult to meet the market demand by relying on natu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/06C12N15/53C12N15/55C12N15/60C12N15/70C12N1/21C12R1/19
CPCC12P13/06C12N9/0071C12Y114/16004C12N9/78C12Y305/04016C12N9/88C12N9/0006C12Y101/01153C12N9/0028C12Y105/01034C12N9/003C12N15/70C12N1/20Y02A50/30
Inventor 赵云现杨志彬胡江林崔金旺赵凯展全乐
Owner 河北维达康生物科技有限公司
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