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Method for catalytically synthesizing L-piperidine acid by using ornithine cyclization deaminase

A technology of pipecolic acid and ornithine, which is applied in the field of biocatalysis, can solve the problems of low enzyme activity/catalytic efficiency, and great difference

Active Publication Date: 2021-10-19
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, it has been proved by experiments that the actual catalytic reaction efficiency of the enzymes reported in the above-mentioned patent documents for L-lysine conversion is quite different, obviously low , far below the enzyme activity / catalytic efficiency requirements for industrial applications

Method used

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  • Method for catalytically synthesizing L-piperidine acid by using ornithine cyclization deaminase
  • Method for catalytically synthesizing L-piperidine acid by using ornithine cyclization deaminase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of recombinant Escherichia coli expressing wild-type ornithine cyclodeaminase

[0054] For the wild-type ornithine cyclodeaminase SEQ ID NO: 1 derived from Streptomyces melanosporofaciens, an optimized codon sequence SEQ ID NO: 2 suitable for expression in E. The restriction endonuclease sites NdeI and XhoI were designed, and subcloned into the pSH plasmid (including NdeI / XhoI sites) constructed by Zhejiang Huarui Biotechnology Co., Ltd. to obtain the recombinant plasmid pSH-OCD. The recombinant plasmid pSH-OCD was transformed into the expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli expressing wild-type ornithine cyclizing deaminase (OCD), abbreviated as Ocd.

Embodiment 2

[0055] Embodiment 2: the construction of error-prone PCR and random mutation library

[0056] Using the base sequence of SEQ ID NO:2 as a template, a primer pair Ocd-Nde1-F / Ocd-Xho1-R was designed, and an error-prone PCR technique was used to construct a random mutant library.

[0057] Forward primer Ocd-Nde1-F: 5'-ATATACATATGGAAACCACCGTGC-3',

[0058] Reverse primer Ocd-Xho1-R: 5'-GTCGACTTACTGAAAGCTATACGGG-3'.

[0059] 50μL error-prone PCR reaction system includes: 50ng plasmid template pSH-OCD, 5μL DMSO, 30pmol pair of primers Ocd-Nde1-F and Ocd-Xho1-R, 1X Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (Thermo Fisher Scientific).

[0060] The PCR reaction conditions were: 95°C for 5min; 94°C for 30s, 60°C for 30s, 72°C for 2min / kbp; 30 cycles; 72°C for 10min. The 1kb random mutation fragment was recovered from the gel as a large primer, and MegaPrimer PCR was performed with KOD-pl...

Embodiment 3

[0062] Example 3: Screening of Random Mutant Library

[0063] The transformants in the mutant library were selected and inoculated into a 96-well deep-well culture plate containing 700 μL LB medium containing 100 μg / mL kanamycin, cultured at 37°C for 6 h, and then added with a final concentration of 0.1 mM IPTG, Cool down to 25°C and incubate overnight. Centrifuge at 5000 rpm for 10 min, discard the supernatant, add 200 μL of 50 mM Tris-HCl (pH 7.5), resuspend the bacteria, and use for the determination of OCD enzyme activity.

[0064] The assay protocol for mutant OCD enzyme activity, that is, L-lysine conversion, is as follows:

[0065] Reaction system: 400mM L-lysine, 150mM, pH7.0 potassium phosphate buffer, 0.1% triton X-100, 20mM FeSO 4 , adjust pH to 7.5, bacterial concentration 15v / v%;

[0066] Reaction conditions: 37°C, 150rpm, 24h;

[0067] Termination conditions: 90°C, 5min.

[0068] After the reaction was terminated, it was centrifuged at 13000rpm for 5min, and...

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Abstract

The invention provides a method for preparing L-piperidine acid by using ornithine cyclization deaminase. The method comprises the following steps: taking L-lysine as a substrate, and catalyzing cyclization deamination reaction by using ornithine cyclization deaminase SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 to obtain the L-piperidine acid.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for preparing L-pipericolic acid catalyzed by ornithine cyclodeaminase. Background technique [0002] L-pipecolic acid (CAS3105-95-1, L-pipecolic acid, referred to as PA) is a kind of imino acid, which exists in free form in most plants, especially in leguminous plants. As an important rigid cyclic non-protein amino acid, it can not only limit the conformation of polypeptides, but also serve as a multifunctional backbone in the synthesis library of different compounds, and can be widely used in the synthesis of many chiral drugs, pipecolic acid and its derivatives Is a class of widely used pharmaceutical intermediates. For example, the new-generation local anesthetic Ropivacaine, the antipsychotic drug Thioridazine, the immunosuppressant Rapamycin, and the antitumor antibiotic Sandramycin are all based on Pipecolic acid or its derivatives are the main ...

Claims

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Application Information

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IPC IPC(8): C12P17/12C12N9/88C12N15/60C12N1/21C12R1/19
CPCC12P17/12C12N9/88C12Y403/01012
Inventor 范文超高书良杨海锋黎肖平
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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