Mutated L-amino acid ligase and process for preparing L-glutamic acid-L-trp-trp by adopting enzyme catalysis method

A tryptophan dipeptide and amino acid technology, applied in the field of biochemistry, can solve the problems of not finding ligases, etc., and achieve the effects of reducing production costs, improving yield and production efficiency, and fast connection

Active Publication Date: 2020-02-11
SHENZHEN READLINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many literatures have reported that some LaIs are active on L-glutamic acid and L-trypto

Method used

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  • Mutated L-amino acid ligase and process for preparing L-glutamic acid-L-trp-trp by adopting enzyme catalysis method
  • Mutated L-amino acid ligase and process for preparing L-glutamic acid-L-trp-trp by adopting enzyme catalysis method
  • Mutated L-amino acid ligase and process for preparing L-glutamic acid-L-trp-trp by adopting enzyme catalysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of L-glutamic acid-L-tryptophan dipeptide (LalM-1&PPK1-ADK) catalyzed by liquid enzyme

[0044] Obtain wet cells containing amino acid ligase LalM-1 (H276G, W332K, M334D, L12A, Y75S and W76L) or ATP regeneration enzyme PPK1-ADK according to the method in the specific embodiment; mix in 500ml 50mM Tris pH 8.0 buffer (Buffer A), after stirring evenly, the cells are broken by high pressure, and the cell wall is removed by high-speed centrifugation (16000rpm, 45min), and the collected supernatant and crude enzyme solution are directly subjected to the following catalytic reactions:

[0045] Add 11.1 grams of L-glutamic acid (75mM), 14.3 grams of L-tryptophan (70mM), and 2.8 grams of adenosine triphosphate disodium in 1000ml of trishydrochloride (Tris.HCl) solution containing 100mM pH 8.0 Salt ATP (5mM), 10.3 grams of polyphosphoric acid (Sigma, 25 poly, 100mM monophosphoric acid), 0.9 grams of magnesium chloride (10mM), 1.5 grams of potassium chloride...

Embodiment 2

[0049] Example 2: Preparation of L-glutamic acid-L-tryptophan dipeptide (LalM-2&PPK2-ADK) catalyzed by liquid enzyme

[0050] Obtain wet cells containing amino acid ligase LalM-2 (H276S, W332N, M334S, L12S, Y75G and W76V) or ATP regeneration enzyme PPK2-ADK according to the method in the specific embodiment; mix in 500ml 50mM Tris pH8.0 buffer In (buffer solution A), after stirring evenly, the cells are broken by high pressure, and the cell wall is removed by high-speed centrifugation (16000rpm, 45min), and the collected supernatant and crude enzyme solution are directly subjected to the following catalytic reactions:

[0051] Similar to Example 1, but the reaction concentration is slightly lower, 7.4 grams of L-glutamic acid (50 mM), 13.4 grams of L-glutamic acid (13.4 grams of L- Tryptophan (47mM), 2.8g adenosine triphosphate disodium salt ATP (5mM), 6.7g polyphosphoric acid (Sigma, 25 poly, 65mM monophosphate), 0.9g magnesium chloride (10mM), 1.5g potassium chloride (20mM);...

Embodiment 3

[0053] Example 3: Preparation of L-glutamic acid-L-tryptophan dipeptide (LalM-3&PPK1-ADK) catalyzed by liquid enzyme

[0054] Obtain the wet cell containing amino acid ligase LalM-3 (N108H, L110A, L182F, G331D) or ATP regeneration enzyme PPK1-ADK with reference to the method in the specific embodiment; Mix in the damping fluid of 500ml 50mM Tris pH 8.0 (buffer A ), after stirring evenly, the cells are broken by high pressure, and the cell wall is removed by high-speed centrifugation (16000rpm, 45min), and the clear liquid crude enzyme solution collected directly carries out the following catalytic reactions:

[0055] Similar to Examples 1 and 2, but the amount of enzyme is more; 7.4 grams of L-glutamic acid (50 mM), 13.4 grams of L-glutamic acid (50 mM), and 13.4 grams of L-tryptophan (47mM), 2.8g adenosine triphosphate disodium salt ATP (5mM), 6.7g polyphosphoric acid (Sigma, 25 poly, 65mM monophosphate), 0.9g magnesium chloride (10mM), 1.5g potassium chloride (20mM ); after...

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Abstract

The invention relates to the technical field of biochemistry, and discloses mutated L-amino acid ligase and a process for preparing L-glutamic acid-L-trp-trp by adopting an enzyme catalysis method. Based on wild type L-amino acid ligase, the mutated L-amino acid ligase has site mutation of H276G/S, W332K/N/Q/S, M334D/S, L12A/S, Y75S/G and W76L/V. According to the mutated L-amino acid ligase and the process for preparing the L-glutamic acid-L-trp-trp by adopting the enzyme catalysis method disclosed by the invention, based on a known-reported research on non-specific L-amino acid ligase, systematical amino acid replacement on binding sites of the activity of a substrate of the mutated L-amino acid ligase is carried out, and finally, quick connection of L-glutamic acid and L-tryptophan is realkzed; and meanwhile, through combination of cyclic regeneration of coenzyme ATP and catalysis of immobilized enzyme, on one hand, the production cost is reduced; and on the other hand, the quality of trp-trp products and the stability of production of the trp-trp products are improved preferably, and therefore, an application requirement is met.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to a mutated L-amino acid ligase and a process for preparing L-glutamic acid-L-tryptophan dipeptide by an enzyme-catalyzed method. Background technique [0002] L-glutamic acid-L-tryptophan (Oglufanide) is a dipeptide formed by connecting two basic amino acids L-glutamic acid and L-tryptophan through an amide bond at the Alpha position. Its molecular formula is C16H19N3O5 and its molecular weight is 333.3, CAS No. 122933-59-9. The dipeptide is an immunomodulator (immunomodulator) used in the treatment of chronic hepatitis C (Hepatitis C) infection. It is also used to regulate the human immune response in the clinical treatment of cancer. It is also a Drugs that inhibit angiogenesis. [0003] L-glutamic acid-L-tryptophan is mainly prepared by chemical synthesis, and there are also a small amount of enzymatic and fermentation methods on the market. Most of the chemical synthes...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12P21/02C12R1/125
CPCC07K5/06104C12N9/93C12P21/02C12Y603/02
Inventor 于铁妹黄俊华刘文龙潘俊锋刘建
Owner SHENZHEN READLINE BIOTECH CO LTD
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