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CN-121975783-A - PH responsive metal ligand-mutant metal dependent enzyme complex and preparation method and application thereof

CN121975783ACN 121975783 ACN121975783 ACN 121975783ACN-121975783-A

Abstract

The invention relates to the field of biocatalysis and enzyme engineering, and discloses a pH response type metal ligand-mutation metal dependent enzyme complex, and a preparation method and application thereof. The complex is formed by compounding mutant metal dependent enzyme and pH response type metal-ligand material, wherein the mutant metal dependent enzyme makes the amino acid residue in the metal dependent enzyme reduce the repulsive interaction of the amino acid residue on target metal ions or attract the target metal ions mutually in a metal ion transport channel of the metal dependent enzyme through directional mutation, the pH response type metal-ligand material is formed by coordination of the target metal ions and a multidentate organic ligand, and the target metal ions can be complexed or released by adjusting the pH. The invention can greatly improve the efficiency of metal ions entering an enzyme active center, realize the on-demand release and cyclic complexation of the metal ions, and can realize the recycling through the pH callback, thereby effectively reducing the cost and having excellent catalytic performance and environmental protection.

Inventors

  • WEI JUNNAN
  • ZHOU BO
  • ZHANG JINGWEN
  • LI XIUJUAN
  • CUI HAIYANG

Assignees

  • 南京师范大学

Dates

Publication Date
20260505
Application Date
20260121

Claims (10)

  1. 1. The pH response type metal ligand-mutation metal dependent enzyme complex is characterized in that the complex is formed by complexing a mutation metal dependent enzyme with a pH response type metal-ligand material, wherein the mutation metal dependent enzyme reduces the repulsive interaction of an amino acid residue in the metal dependent enzyme on target metal ions or attracts the amino acid residue to the target metal ions in a metal ion transport channel of the metal dependent enzyme through directional mutation, and the pH response type metal-ligand material is formed by complexing target metal ions with a polydentate organic ligand through coordination and can complex or release the target metal ions through adjusting pH.
  2. 2. The complex of claim 1, wherein the amino acid residues are residues that exhibit rejection of the target metal ion that can be screened out using short-range coulombic interactions; preferably, the short-range coulombic interaction energy is between 0.5 and 2 kcal/mol.
  3. 3. The complex of claim 1 or 2, wherein the metal-dependent enzyme is selected from at least one of a sugar isomerase, a dehydrogenase, and an oxidoreductase.
  4. 4. The composite of claim 1 or 2, wherein the target metal ion is selected from at least one of Co 2+ 、Mn 2+ 、Zn 2+ 、Ni 2+ 、Cu 2+ 、Fe 2+ and Fe 3+ .
  5. 5. The complex of claim 1 or 2, wherein the polydentate organic ligand is selected from at least one of citric acid, EDTA, and phthalic acid.
  6. 6. A method for preparing a pH-responsive metal ligand-mutant metal-dependent enzyme complex, comprising the steps of: s1, preparing mutant metal dependent enzyme, namely carrying out directional mutation on amino acid residues with strong rejection to target metal ions in the metal dependent enzyme, so that the rejection to the target metal ions in a metal ion transport channel of the metal dependent enzyme is reduced or the amino acid residues are mutually attracted with the target metal ions, and obtaining the mutant metal dependent enzyme; the preparation of the pH response type metal-ligand material precursor liquid comprises the steps of mixing a metal salt solution containing target metal ions with a ligand solution containing a multi-tooth organic ligand to obtain the pH response type metal-ligand material precursor liquid; S2, assembling a complex, namely mixing the mutant metal-dependent enzyme with the pH response type metal-ligand material precursor liquid to obtain the mixture II.
  7. 7. The method according to claim 6, wherein the amino acid residue having a strong repulsive interaction is a residue exhibiting a repulsive interaction with a target metal ion that can be screened out using short-range coulomb interaction; And/or the metal-dependent enzyme is selected from any one of a sugar isomerase, a dehydrogenase, and an oxidoreductase; And/or the target metal ion is selected from at least one of Co 2+ 、Mn 2+ 、Zn 2+ 、Ni 2+ 、Cu 2+ 、Fe 2+ and Fe 3+ ; And/or the polydentate organic ligand is selected from at least one of citric acid, EDTA, and phthalic acid.
  8. 8. The method according to claim 6 or 7, wherein the conditions of mixing II comprise a time of 5-15 min and a rotation speed of 5000-10000 rpm; and/or the molar ratio of the metal salt to the polydentate organic ligand is 1:0.8-1.2; And/or the mutant metal-dependent enzyme is used in an amount of 1.8 to 3 mg relative to the target metal ion of 1 mg.
  9. 9. Use of the pH-responsive metal ligand-mutant metal-dependent enzyme complex of any one of claims 1-5 or the pH-responsive metal ligand-mutant metal-dependent enzyme complex prepared by the method of any one of claims 6-8 to catalyze the epoxidation of cyclohexene to cyclohexane oxide.
  10. 10. Use of a pH-responsive metal ligand-mutant metal-dependent enzyme complex according to any one of claims 1-5 or prepared by a method according to any one of claims 6-8 for catalyzing the production of L-alanine from DL-alanine or for catalyzing the production of D-fructose from D-glucose solution.

Description

PH responsive metal ligand-mutant metal dependent enzyme complex and preparation method and application thereof Technical Field The invention relates to the technical field of biocatalysis and enzyme engineering, in particular to a pH response type metal ligand-mutation metal dependent enzyme complex, a preparation method and application thereof. Background The metal dependent enzyme is an enzyme which needs specific metal ions as cofactors in the catalytic process, and has wide application in the fields of amino acid synthesis, drug intermediate synthesis, fine chemical preparation and other biological manufacturing. The catalytic activity of the enzyme is highly dependent on the effective supply of metal ions, and the conventional technology generally provides sufficient metal ions for the enzyme by directly adding metal salts into a reaction system or optimizing a buffer system and adopting external environment regulation modes such as an immobilized carrier, and meanwhile, the metal coordination site or the residue near the active center of the enzyme can be modified by means of site-directed mutagenesis, directed evolution and the like so as to improve the metal binding capacity or the catalytic efficiency. However, the prior art still has obvious defects that the metal ions are in a non-directional diffusion state in the solution and can really reach the vicinity of an enzyme active site and participate in catalysis, the proportion of the metal ions is extremely low, excessive addition of metal salts is needed for achieving an expected catalysis effect, the cost of raw materials is increased, the burden of subsequent separation and purification and environmental pressure are increased, the conventional immobilized carrier can only provide structural support for enzymes, the reversible storage and on-demand release functions of the metal ions under mild conditions are lacked, local high-concentration metal ion microenvironment cannot be built around the enzymes, the catalysis requirement of the enzymes is difficult to be matched, the conventional enzyme modification technology focuses on the metal coordination site or the residue near the active center, the system optimization for the metal ion transport channel in the enzyme is less, the rejection of the residue in the channel to the metal ions is not considered, the efficiency of the metal ions entering the active center is limited, the external metal supply mode and the internal structure modification of the enzyme are mutually independent, the synergistic effect is not formed, the design of realizing the recycling of the enzymes and the metal ions is lacked through simple regulation and control means, the reusability of the reaction system is poor, and the production energy consumption and the cost are further improved. Particularly in the specific catalysis scenes such as resolution of amino acid racemate, epoxidation of cyclohexene and the like, the problems are more remarkable that excessive use of metal salt can lead to reduced product purity and increased separation difficulty, and the one-time use mode of enzyme and metal ion can lead to production flow not having economy and environmental protection. Therefore, developing a technical scheme that can realize the recycling of enzymes and metal ions by cooperatively optimizing external metal supply and internal enzyme transport channels and utilizing a mild regulation and control means, and further remarkably improve the catalytic activity of metal-dependent enzymes under lower total metal usage becomes a key problem to be solved in the field. Disclosure of Invention The invention aims to solve the problems that the prior art has low metal utilization efficiency, the external metal supply and the mutation transformation of the metal dependent enzyme lack synergy and the recycling of the enzyme and the metal ion can not be realized, and provides a pH response type metal ligand-mutation type metal dependent enzyme compound, a preparation method and application thereof, wherein the compound can realize the reversible complexation and release of the metal ion under the mild pH condition through the synergistic effect of a pH response type metal-ligand material and the directional mutation type metal dependent enzyme, the method has the advantages that an instantaneous high-concentration metal ion microenvironment is constructed around the enzyme, the efficiency of metal ions entering an enzyme active center can be improved through a low-rejection metal transport channel of the metal-dependent enzyme, meanwhile, the cyclic regeneration and the repeated use of the compound can be realized through pH regulation, the catalytic activity is obviously improved under the condition of low total metal ion consumption, and the method has the advantages of economy, stability and environmental friendliness, and is suitable for multiple biological manufacturing scenes such as amino acid synthesis, drug intermedi