Method of immobilizing enzyme by means of coordination of graphene oxide and metal ions
A technology of metal ions and immobilized enzymes, which is applied in the direction of immobilization on or in the inorganic carrier, can solve the problems of low loading capacity, low immobilization rate, poor stability and reusability of immobilized enzymes, and achieve a high loading area Effects of large size, high immobilization rate, pH stability, and enhanced stability for repeated use
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Embodiment 1
[0036] 1) Preparation of crude enzyme solution:
[0037] Construction of a recombinant expression strain that can express the target enzyme with the His-tag tag: the original sequence of the gene of formate dehydrogenase (FDH) comes from Candida boidinii (190-1284nucleotide, Genebank ID: AF004096.1), as shown in SEQ ID NO.1 ; According to the above-mentioned original sequence, design primers (as shown in table 1) and adopt PCR method to construct the formate dehydrogenase gene that 5 ' end and 3 ' end respectively have NdeI and Xhol restriction site, PCR synthetic process is by Shanghai Sheng Gong Biological Engineering Technology Service Co., Ltd. completed:
[0038] Table 1 Primers used for FDH gene amplification
[0039] Primer name
Corresponds to the number in the sequence listing
Sequence (5'-3')
FDH-P1
SEQ ID NO.2
GGAATTC CATATG AAAATTGTCCTGGTCCTGT
FDH-P2
SEQ ID NO.3
CCG CTCGAG TTACTTTTTTATCGTGTTTGCCAT
[0040] A...
Embodiment 2
[0052] 1)-4) The experimental steps are as the steps 1)-4) of Example 1.
[0053] 5) Preparation of graphene oxide-metal ion immobilized carrier: adding ZnCl to the graphene oxide solution 2 Solution, so that the final concentration after coordination equilibrium in the solution is 10 mM: shake in a constant temperature oscillator (4° C., 1 h), and obtain a graphene oxide-metal ion suspension.
[0054] 6) Preparation of graphene oxide-metal coordination-immobilized enzyme: in the graphene oxide-metal ion suspension obtained in step 5), add the formate dehydrogenase solution obtained in step 2) and make its final concentration 10 mg / mL, shake in a constant temperature shaker (4°C, 1h) to form immobilized particles, wash with pure water three times and resuspend with Tris-HCl buffer solution (0.1M, pH 10) to obtain immobilized enzyme GO-Zn- FDH, the recovery rate of enzyme activity can reach 84.28%. Figure 6 shown.
[0055] 7) Determination of the temperature stability of t...
Embodiment 3
[0058] 1)-4) The experimental steps are as the steps 1)-4) of Example 1.
[0059] 5) Preparation of graphene oxide-metal ion immobilized carrier: adding MgCl to the graphene oxide solution 2 Solution, so that the final concentration after coordination equilibrium in the solution is 0.1 mM: shake in a constant temperature oscillator (4° C., 1 h), and obtain a graphene oxide-metal ion suspension.
[0060] 6) Preparation of graphene oxide-metal coordination immobilized enzyme: add the formate dehydrogenase solution obtained in step 2) to the graphene oxide-metal ion suspension obtained in step 5) and make the final concentration 0.1mg / mL, shake in a constant temperature shaker (4°C, 1h) to form immobilized particles, wash with pure water three times and resuspend with Tris-HCl buffer solution (0.05M, pH 11.5) to obtain the immobilized enzyme GO-Mg -FDH, the recovery rate of enzyme activity can reach 83.10%. Figure 9 shown.
[0061] 7) Determination of the temperature stabili...
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