Method for synthetizing L-carnosine by one-step method and truncated L-carnosine synthetase

A technology for synthesizing enzymes and carnosine, applied in biochemical equipment and methods, peptides, enzymes, etc., can solve the problems of difficult extraction and purification, low yield of L-carnosine, complex reaction products, etc., and achieve easy large-scale production and wide sources. , the effect of simple operation

Active Publication Date: 2019-06-07
苏州百因诺生物科技有限公司
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  • Application Information

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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 aminopepti...

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  • Method for synthetizing L-carnosine by one-step method and truncated L-carnosine synthetase
  • Method for synthetizing L-carnosine by one-step method and truncated L-carnosine synthetase

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Embodiment Construction

[0023] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0024] The preparation method of carnosine: using β-alanine, L-histidine, ATP and polyphosphate as raw materials, MgCl 2 As an activator, add L-carnosine synthetase and polyphosphate kinase to catalyze the synthesis of carnosine through enzymatic reaction coupling at pH 6.5-8.5, 30-45°C, such as figure 1 shown. Specific steps:

[0025] 1. Construction of recombinant truncated L-carnosine synthetase strain

[0026] 1.1: Extract the amino acid sequence from 480 to 930 of carnosine synthase1 encoded by the gene accession number GU453679 in Genbank, and add MERKT peptide to the amino acid to form a truncated L-carnosine synthase amino acid sequence, such as SEQ ID NO As shown in .3, after codon optimization, an NcoI restriction site is added to the 5' end of the optimized sequence, and an XhoI restriction site is added to the 3' end to...

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Abstract

The invention discloses a method for synthetizing L-carnosine by a one-step method and truncated L-carnosine synthetase. According to the method, beta-alanine, L-histidine, ATP and polyphosphate are used as raw materials, MgCl2 is used as an activator, L-carnosine synthetase and polyphosphate kinase are added, and through an enzymatic reaction under the condition of pH being 6.5-8.5 and the temperature being 30-45 DEG C, the carnosine is coupled, catalyzed and synthetized. According to the method disclosed by the invention, escherichia coli is used for respectively expressing the truncated L-carnosine synthetase (of which the amino acid sequence is as shown in SEQID No.3) and the polyphosphate kinase, through purification, mixing is performed, a dual-enzyme coupling system is formed, and the L-carnosine synthetase in truncated expression can catalyze the L-carnosine synthetized from beta-alanine and L-histidine; besides, accompanied with ATP dephosphorylation, ADP is formed, the polyphosphate kinase catalyzes polyphosphate transphosphorylation groups, and ATP is formed for the ADP, so that circulation and regeneration of the ATP can be realized; and through the dual-enzyme couplingsystem, a reaction is performed under the appropriate reaction condition, so that carnosine is obtained. The method disclosed by the invention has the advantages that the raw materials are low in cost, the enzymatic conversion time is short, the operation is simple and convenient, and the production cost is low.

Description

technical field [0001] The invention relates to a preparation method of carnosine, in particular to a method for synthesizing L-carnosine in one step, 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 various physiological activities, the active peptide and its derivatives hav...

Claims

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Application Information

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IPC IPC(8): C07K5/062C12P21/02C12N9/00
Inventor 朱益波王明崇陈颖武国寒高桂兰赵成相
Owner 苏州百因诺生物科技有限公司
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