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CN-122012643-A - Synthesis method and application of chromone compound catalyzed by benzaldehyde lyase mutant

CN122012643ACN 122012643 ACN122012643 ACN 122012643ACN-122012643-A

Abstract

The invention discloses benzaldehyde lyase derived from Pseudomonas fluorescens (Pseudomonas fluorescens) and a mutant thereof, and application of the benzaldehyde lyase and the mutant thereof serving as a catalyst in catalytic synthesis of 3-hydroxytryptone. The benzaldehyde lyase has the advantages of high reactivity, high selectivity, wide substrate range and the like, has the conversion rate of up to 99%, is favorable for realizing low-cost production, and has industrial application prospect.

Inventors

  • XIE YONGTAO
  • ZHU CHAONAN
  • ZHU XINXIN
  • LAI YINHONG
  • PAN RUI
  • ZHANG LIXIN

Assignees

  • 华东理工大学

Dates

Publication Date
20260512
Application Date
20260210

Claims (10)

  1. 1. A method for catalytic synthesis of a compound of formula 2 by benzaldehyde lyase or a mutant thereof, which has the following reaction formula: ; The benzaldehyde lyase has an amino acid sequence shown as SEQ ID NO. 1; the amino acid sequence of the benzaldehyde lyase mutant is obtained by mutating a sequence shown as SEQ ID NO. 1; Wherein X is O, S or N; n is selected from 1 or 2 or 3; R 1 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogen substituted C1-C6 alkyl, -COOC 1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkenyl-C1-C6 alkyl, when n is selected from 2 and the substituent R 1 groups are adjacent, the C atom to which the adjacent R 1 groups are attached can form a C6-C10 aryl group; R 2 is selected from hydrogen, halogen substituted C1-C6 alkyl or C1-C6 alkyl.
  2. 2. The method according to claim 1, characterized in that: Wherein X is selected from O or S; n is selected from 1 or 2; R 1 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogen substituted C1-C6 alkyl, -COOC 1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkenyl C1-C6 alkyl, when n is selected from 2 and R 1 groups are adjacent, the adjacent R 1 groups and the C atom to which they are attached may form phenyl; R 2 is selected from hydrogen or C1-6 alkyl.
  3. 3. The method according to claim 1 or 2, characterized in that: n is selected from 1 or 2; Said R 1 is selected from methyl, methoxy, ethoxy, allyl, phenyl, methyl ester, fluoro, chloro, bromo or trifluoromethyl, R 2 is selected from hydrogen or methyl; X is selected from O or S; further preferred, formula I is selected from the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 Or (b) 。
  4. 4. The method according to claim 1 or 2, wherein the amino acid sequence of the benzaldehyde lyase mutant is obtained by mutating the sequence shown in SEQ ID NO.1, the mutation comprising: The alanine at position 28 is replaced by any one of phenylalanine, glycine, serine, tryptophan and tyrosine, And/or, the glutamine at 113 th position is replaced by any one of phenylalanine, glycine, serine, tryptophan, tyrosine and alanine, And/or, the 112 th leucine is replaced by any one of phenylalanine, glycine, tyrosine, serine, tryptophan and alanine, And/or threonine at position 481 is replaced with any one of phenylalanine, glycine, serine, tryptophan, tyrosine, and alanine.
  5. 5. The method according to claim 4, wherein the catalytic reaction is selected from the group consisting of a benzaldehyde lyase mutant PfBAL Q S/A28G, and the PfBAL Q S/A28G amino acid sequence is selected from the group consisting of the amino acid sequences shown in SEQ ID No. 2.
  6. 6. The process of claim 5, wherein the catalytic reaction is a cyano conversion reaction carried out in the presence of MgSO 4 and ThDP; Preferably, the concentration of the compound of formula 1 is 30-90mmol/L, the concentration of MgSO 4 is 2.0-3.0 mmol/L, the concentration of ThDP is 0.1-0.3 mmol/L, the reaction temperature is 10-30 ℃, And/or, even more preferably, the concentration of the compound of formula 1 is 75 mmol/L, the concentration of the MgSO 4 is 2.5 mmol/L, the concentration of the ThDP is 0.15 mmol/L, and the reaction temperature is 20 ℃.
  7. 7. A benzaldehyde lyase mutant is characterized in that the amino acid of the benzaldehyde lyase mutant is selected from the amino acid sequence shown in SEQ ID No. 2.
  8. 8. An isolated nucleic acid, wherein said nucleic acid is a nucleic acid molecule encoding a benzaldehyde lyase mutant PfBAL Q S/A28G, wherein said benzaldehyde lyase mutant PfBAL Q S/A28G has an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID No. 2.
  9. 9. An expression vector containing a coding gene of a benzaldehyde lyase mutant PfBAL Q S/A28G, wherein the amino acid sequence of the benzaldehyde lyase mutant PfBAL Q S/A28G is selected from the amino acid sequence shown in SEQ ID No. 2.
  10. 10. Recombinant cells containing a coding gene of a benzaldehyde lyase mutant PfBAL Q S/A28G, wherein the amino acid sequence of the benzaldehyde lyase mutant PfBAL Q S/A28G is selected from the amino acid sequences shown in SEQ ID No. 2.

Description

Synthesis method and application of chromone compound catalyzed by benzaldehyde lyase mutant Technical Field The invention belongs to the technical field of enzyme engineering and chemical engineering, and particularly relates to a benzaldehyde lyase mutant, in particular to a synthesis method and application of chromone compounds catalyzed by the benzaldehyde lyase mutant. Background Benzaldehyde lyase (Benzaldehyde Lyase, BAL) is a thiamine diphosphate (ThDP) -dependent enzyme originally found in pseudomonas fluorescens (Pseudomonas fluorescens). It is a multifunctional enzyme capable of catalyzing the formation of C-C bond between aldehyde substrates, cleavage reaction, etc. Benzaldehyde lyase is a multifunctional enzyme with wide industrial and scientific application prospects. The catalytic performance and the application range of the catalyst are continuously expanded by means of enzyme engineering and synthesis biology, and a new tool is provided for synthesizing green chemical and chiral compounds. Chromone is a structural skeleton in flavonoid natural products and functional molecules, exists in a large number in plants, and plays a variety of roles including maintaining growth, inhibiting dormancy, stimulating oxygen uptake and the like. Chromone has wide application in clinical medicine and functional materials, such as anticancer, antiallergic, anti-inflammatory, antiviral, etc. Among all chromone frameworks, 3-hydroxy-chromone and its derivatives are an important class, exhibiting fluorescent properties due to the excited state of the intramolecular proton transfer process, and significant fluorescent properties make its derivatives fluorescent sensors for normal and supercritical liquids, lipid membranes, DNA and proteins. In addition, 3-hydroxy-chromone has certain biological activity and potential pharmaceutical activity, for example, diosmin is a sugar-substituted chromone derivative, most commonly used for treating hemorrhoids and leg ulcers caused by poor blood flow, and for treating various vascular problems including hemorrhoids, varicose veins, venous stasis, and ocular or gingival bleeding. Apigenin is chromone derivative extracted from plant for treating cancer. Yellow esters, known as antimuscarinics, are a muscle relaxant used in the treatment of the bladder and urinary system. Khellin is a furanyl cyclic chromone derivative, called furanyl pigment, which acts primarily as vasodilator. Chromen-one is a folk herb used for treating various diseases including kidney stones, psoriasis, vitiligo, bronchial asthma, coronary artery disease and renal colic. However, the current synthesis methods of 3-hydroxy-chromones are limited, and mainly depend on the synthesis methods which generally depend on oxidation of chromone type substrates or oxidation cyclization of o-hydroxyphenylenamine ketone, and these oxidation methods have the problem that the substrate synthesis route is long, organic peracid or hydrogen peroxide is required as an oxidant, and the synthesis is carried out in an organic solvent. Therefore, there is a need to mine biocatalytic synthesis processes with high efficiency, high selectivity, and more environmental protection. In view of this, the present invention has been made. Disclosure of Invention In a first aspect, the present invention provides a method for catalytic synthesis of a compound of formula 2 by benzaldehyde lyase or a mutant thereof, which has the following reaction formula: ; The benzaldehyde lyase has an amino acid sequence shown as SEQ ID NO. 1; the amino acid sequence of the benzaldehyde lyase mutant is obtained by mutating a sequence shown as SEQ ID NO. 1; Wherein X is O, S or N; n is selected from 1 or 2 or 3; R 1 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogen substituted C1-C6 alkyl, -COOC 1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkenyl-C1-C6 alkyl, when n is selected from 2 and the substituent R 1 groups are adjacent, the C atom to which the adjacent R 1 groups are attached can form a C6-C10 aryl group; R 2 is selected from hydrogen, halogen substituted C1-C6 alkyl or C1-C6 alkyl. Further, wherein X is selected from O or S, n is selected from 1 or 2; R 1 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogen substituted C1-C6 alkyl, -COOC 1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkenyl C1-C6 alkyl, when n is selected from 2 and R 1 groups are adjacent, the adjacent R 1 groups and the C atom to which they are attached may form phenyl; R 2 is selected from hydrogen or C1-6 alkyl. Still further, n is selected from 1 or 2, and R 1 is selected from methyl, methoxy, ethoxy, allyl, phenyl, methyl ester, fluoro, chloro, bromo or trifluoromethyl, R 2 is selected from hydrogen or methyl; X is selected from O or S; further preferred, formula I is selected from the following compounds: 、、、、、、、、、、、、、、、、、、、、 Or (b) 。 In a second aspect, the invention provides a ThDP-dependent enzyme, i.e