A kind of polyethyleneimine-titanium oxide embedding amine dehydrogenase and its preparation method
A technology of polyethyleneimine and amine dehydrogenase, which is applied in the direction of immobilization on or in the inorganic carrier, on/in the organic carrier, etc., which can solve the problem of sensitivity of amine dehydrogenase, less efficient immobilization, etc. problems, to achieve the effects of strong immobilization ability, good repeated use stability and low leakage rate
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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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