Method of synthesizing L-carnosine using L-amino acid ligase by one-step method

A technology of ligating enzymes and amino acids, applied in the biological field, can solve the problems of complex reaction products, difficult extraction and purification, etc., and achieve the effects of wide sources, easy large-scale production, and simple operation

Active Publication Date: 2019-04-09
苏州百因诺生物科技有限公司
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Problems solved by technology

This method still needs to methylate β-alanine, and the L-carnosine synthesized in the reaction solution will be degraded into β-alanine and L-histidine by aminopeptidase, and at the same time, aminopeptidase will form tripeptide , leading to complex reaction products and difficult extraction and purification

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  • Method of synthesizing L-carnosine using L-amino acid ligase by one-step method

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Embodiment Construction

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0023] The invention utilizes Escherichia coli to separately express L-amino acid ligase and polyphosphate kinase, which are purified and then mixed to form a dual-enzyme coupling system. L-amino acid ligase catalyzes the synthesis of L-carnosine from β-alanine and L-histidine, and at the same time, ATP is dephosphorylated to form ADP. Polyphosphate kinase catalyzes the transphosphorylation of polyphosphate to ADP to form ATP. In order to realize the recycling of ATP, such as figure 1 shown. Specific steps:

[0024] 1. Construction of recombinant L-amino acid ligase strain

[0025] 1.1: According to the L-amino acid ligase gene sequence whose GI number is GI: 1094636766, after codon optimization, an NdeI restriction site is added to the 5' end of the optimized sequence, and an XhoI restriction site is added to the 3' end to form SE...

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Abstract

The invention discloses a method of synthesizing L-carnosine using L-amino acid ligase by a one-step method. The method comprises the steps of taking beta-alanine, L-histidine, ATP (adenosine triphosphate) and polyphosphate as raw materials, and MgCl2 as an activator, adding L-amino acid ligase and polyphosphate kinase, and synthesizing L-carnosine through an enzymatic reaction and coupled coupleunder conditions of a pH of 6.5-8.5 and a temperature of 30-45 DEG C. According to the method, a sequence optimized L-amino acid ligase gene is successfully expressed in escherichia coli, and beta alanine and L-histidine can be catalyzed to synthesize L-carnosine at one step. A substrate is not required to be methylated or subjected to group protection; ATP required by a catalytic reaction is continuously regenerated by allowing polyphosphate kinase to catalyze phosphorylation of ADP (adenosine diphosphate); cyclic regeneration of ATP can be achieved only by consuming a small amount of ADP; and a renewable raw material, sodium hexametaphosphate, is wide in source, low in cost, simple to operate and easy in large-scale production.

Description

technical field [0001] The invention relates to a method for synthesizing carnosine, in particular to a method for synthesizing L-carnosine in one step by using L-amino acid ligase, and belongs to the field of biotechnology. 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 various physiological activities, the active peptide and its de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/10C12N15/70C12N9/12C12N9/00
CPCC12N9/1229C12N9/93C12N15/70C12N2800/22C12P17/10C12Y207/04001C12Y603/02028
Inventor 朱益波张璐孙安迪包承润邹锦涛于志勇
Owner 苏州百因诺生物科技有限公司
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