Whole-cell one-step method for synthesizing L-carnosine

A whole-cell, carnosine technology, applied in the field of carnosine synthesis, can solve problems such as unfavorable large-scale application, low L-carnosine yield, low L-carnosine yield, etc., to solve the problem of enzyme separation and purification, high cost and molar conversion rate High, simple reaction system effect

Active Publication Date: 2019-04-12
苏州百因诺生物科技有限公司
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Problems solved by technology

However, there is a certain degree of inhibition in the activity of organic relative enzymes, and the solubility of amino acid substrates in organic phases is also low. Various reasons have resulted in very low yields based on aminopeptidase synthesis of L-carnosine (the best result is 3.7g/L, Microbial Biotechnology, 2010, 3(1):74-83.)
In addition, aminopeptidase will form tripeptides, resulting in complex reac

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  • Whole-cell one-step method for synthesizing L-carnosine
  • Whole-cell one-step method for synthesizing L-carnosine

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Embodiment Construction

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0028] The L-carnosine biosynthetic pathway of the present invention is briefly described as such figure 1 As shown, it specifically includes the following steps:

[0029] 1. Construction of amino acid fatty acyltransferase expression vector and Escherichia coli

[0030] According to the known amino acid fatty acyl nucleotide sequence (GenBank: AB610978.1), the gene fragment (SEQ ID NO.1) encoding amino acids 21-619 was obtained by gene synthesis after Jcat codon optimization. MscI and XhoI restriction sites were added to the 5' and 3' ends of the fragment, respectively.

[0031] After the synthetic gene fragment and pET22b vector were digested by MscI and XhoI respectively, the target fragment was recovered by gel, the target fragment was ligated, and the ligated product was transformed into E.coli BL21(DE3), cultured overnight on ...

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Abstract

The present invention discloses a whole-cell one-step method for synthesizing L-carnosine. The whole-cell one-step method comprises the following steps: 1) a recombinant vector is constructed, the recombinant vector contains an amino acid fatty acyltransferase gene having a nucleotide sequence shown in SEQ ID NO.1, and an amino acid sequence encoded by the gene is shown in SEQ ID NO.2; 2) the genehaving the nucleotide sequence shown in the SEQ ID NO.1 is transferred into Escherichia coli to obtain recombinant Escherichia coli; 3) substrates of beta-alanine methyl ester and L-histidine are dissolved in a buffer solution, and pH is 7.5-9.5; and 4) the recombinant Escherichia coli is added to the buffer solution for reaction at a reaction temperature of 25-42 DEG C. Recombinant plasmids andrecombinant engineering bacteria of the gene are utilized to directly catalyze synthesis of the L-carnosine in a catalytic system, and the method improves conversion rate of the L-carnosine, and at the same time solves problems of complicated separation and purification of enzymes and high cost of an aminopeptidase catalytic process.

Description

technical field [0001] The invention relates to a method for synthesizing carnosine, in particular to a method for efficiently synthesizing L-carnosine by directly using recombinant Escherichia coli as a whole-cell catalyst, and belongs to the technical field of biological production. Background technique [0002] L-carnosine (β-alanyl-L-histidine) and its analogues (such as homocarnosine and anserine) are natural active dipeptides widely present in the brain, muscle and other important tissues of mammals. Since the discovery of this active peptide for more than 100 years, a large number of studies have found or proved that L-carnosine has significant anti-oxidation, elimination of intracellular free radicals, anti-aging and other activities, and it is clinically used for hypertension, heart disease, etc. adjuvant therapy for disease, senile cataract, ulcer, anti-tumor, and promotion of wound healing. Due to its strong antioxidant activity, low toxic and side effects and va...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/54C12P17/10
CPCC12N9/1029C12N15/70C12N2800/22C12P17/10
Inventor 朱益波王志杰孙启武赖淑涵彭鑫成楚银凤
Owner 苏州百因诺生物科技有限公司
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