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Polyethylene imine-titanium oxide embedded with amine dehydrogenase and preparation method of polyethylene imine-titanium oxide

A technology of polyethylenimine and polyethylenimine‐, which is applied in the direction of immobilization on or in the inorganic carrier, immobilization on/in the organic carrier, etc., which can solve the problem of sensitive and efficient immobilization of amine dehydrogenase and other problems, to achieve the effect of strong immobilization ability, good stability in repeated use, and low leakage rate

Active Publication Date: 2015-03-11
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, due to the sensitivity of amine dehydrogenase, the efficient immobilization of amine dehydrogenase is still less

Method used

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  • Polyethylene imine-titanium oxide embedded with amine dehydrogenase and preparation method of polyethylene imine-titanium oxide
  • Polyethylene imine-titanium oxide embedded with amine dehydrogenase and preparation method of polyethylene imine-titanium oxide
  • Polyethylene imine-titanium oxide embedded with amine dehydrogenase and preparation method of polyethylene imine-titanium oxide

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Experimental program
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Effect test

Embodiment 1

[0037] (1) According to SEQ ID NO 1, the gene sequence of amine dehydrogenase was synthesized by a commercial company (Shanghai Bioengineering Co., Ltd.), and connected to the vector pET28a to construct a plasmid.

[0038] Wherein, the designed primer sequence is as follows:

[0039] Forward primer F: (5′)GAA TTC C CA TAT GAG The underlined part of CTT AGT AGA (3′) is the recognition site for NdeI digestion;

[0040] Reverse primer R: (5')GCC CTC GAGThe underlined part of TTA GTT GCG AAT (3′) is the recognition site for XhoI digestion;

[0041] PCR amplification conditions are as follows: using Taq enzyme

[0042]

[0043] Ligation of PCR product and vector pET28a: Use NdeI and XhoI to double-enzyme digest the PCR product and vector pET28a,

[0044] After purification, react overnight at 4°C under the action of T4DNA ligase

[0045] Preparation of competent cells: Competent Cell Preparation Kit was used to complete.

[0046] Plasmid transformation: Add 0.1-10ng of t...

Embodiment 2

[0054] (1)-(2) The experimental procedures are as the steps (1)-(2) of Example 1.

[0055] (3) Prepare polyethyleneimine (PEI) solution and mixed solution: prepare linear PEI with a concentration of 25mg / ml

[0056] (molecular weight of 22KDa) solution. And amine dehydrogenase with phosphate (0.1M pH8.0) as buffer solution was added thereto to prepare a mixed solution containing amine dehydrogenase at a concentration of 0.5 mg / ml.

[0057] (4) Configuration of titanium precursor solution: configure 0.25M titanium precursor solution (Ti-BALDH), add 20ml PEI-enzyme mixture containing 0.5mg / ml amine dehydrogenase to 10ml 0.25M titanium In the precursor (Ti-BALDH), the immobilized particles were formed, washed with deionized water, resuspended in Tris-HCl (0.05M, pH7.0), and the immobilized amine dehydrogenase suspension was formed, and the embedding rate was 96.78 %, the initial enzyme activity is 0.064U / mg.

Embodiment 3

[0059] (1)-(2) The experimental procedures are as the steps (1)-(2) of Example 1.

[0060] (3) Prepare polyethyleneimine (PEI) solution and mixed solution: prepare linear PEI (molecular weight: 87 KDa) solution with a concentration of 25 mg / ml. Add amine dehydrogenase with water as a buffer solution therein to prepare a mixed solution containing amine dehydrogenase at a concentration of 0.1 mg / ml.

[0061] (4) Configuration of titanium precursor solution: configure 0.25M titanium precursor solution (Ti-BALDH), add 60ml of PEI-enzyme mixture containing amine dehydrogenase 0.1mg / ml dropwise to 10ml 0.25M titanium In the precursor (Ti-BALDH), the immobilized particles were formed, washed with deionized water, resuspended in Tris-HCl (0.05M, pH7.0), and the immobilized amine dehydrogenase suspension was formed, and the embedding rate was 75.00 %, the initial enzyme activity is 0.084U / mg.

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Abstract

The invention relates to polyethylene imine-titanium oxide embedded with amine dehydrogenase and a preparation method of the polyethylene imine-titanium oxide, and belongs to the technical field of immobilized enzymes. A polyethylene imine (PEI)-titanium oxide immobilized particle embedded with the amine dehydrogenase has a grain size of 100-600 nm, wherein polyethylene imine has catalyzing and templating double effects in the immobilization process, namely the PEI promotes the formation of titanium oxide through an electrostatic and hydrogen bonding effect between an amino group in a molecule and a titanium precursor molecule; through a polyethylene imine structure, a template can also be induced, and the formation of a titanium oxide particle are regulated and controlled. The preparation method of the polyethylene imine-titanium oxide comprises the following steps: configuring the PEI (straight chain and branched chain) as well as mixed liquid containing the amine dehydrogenase; configuring a precursor II-dihydroxybis (2-hydroxypropanoato) titante diammonium (Ti-BALDH) solution of titanium; dropwise adding the precursor solution of the titanium into the PEI and the mixed liquid containing the amine dehydrogenase, stirring and generating the PEI-titanium oxide immobilized particle embedded with the amine dehydrogenase. The polyethylene imine-titanium oxide and the preparation method thereof have the advantages that the preparation conditions are mild, the process is simple and is easy to implement, the obtained immobilized particle embedded with the amine dehydrogenase has strong immobilization capacity and good reuse stability, and the temperature stability of the amine dehydrogenase can be obviously improved.

Description

technical field [0001] The invention relates to polyethyleneimine-titanium oxide embedding amine dehydrogenase and a preparation method thereof. Background technique [0002] Chiral amines are structural units of many important biologically active molecules and are important intermediates in the synthesis of natural products and chiral drugs. Approximately 40% of optically active drugs are chiral amines. At the same time, chiral amines can be used as resolving agents, chiral auxiliary agents and chiral compounds, and are widely used in fine chemicals, pharmaceuticals and agricultural chemicals. [0003] At present, biocatalytic preparation of chiral compounds is widely used in the chemical and pharmaceutical industries, and has broad prospects. The asymmetric reductive amination of ketones and free ammonia to prepare chiral amines is currently the most coveted reaction for large-scale production. Therefore, it is very important to find ways to improve the activity and stab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/08C12N11/04
Inventor 王世珍任红方柏山
Owner XIAMEN UNIV
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