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Method for immobilization of phenylalanine dehydrogenase by chemical reduced graphene oxide

A technology of phenylalanine dehydrogenase and graphene, applied in the direction of oxidoreductase, microbial-based methods, biochemical equipment and methods, etc., to achieve the effect of maintaining enzyme activity, high immobilization rate, and enhancing effect

Inactive Publication Date: 2019-03-12
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no method for immobilizing phenylalanine dehydrogenase using reduced graphene oxide

Method used

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  • Method for immobilization of phenylalanine dehydrogenase by chemical reduced graphene oxide
  • Method for immobilization of phenylalanine dehydrogenase by chemical reduced graphene oxide
  • Method for immobilization of phenylalanine dehydrogenase by chemical reduced graphene oxide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Preparation of crude enzyme solution:

[0032] Construction of a recombinant expression strain capable of expressing phenylalanine dehydrogenase with a His-tag tag: the original sequence of the phenylalanine dehydrogenase (PheDH) gene is from Bacillus nanhaiensis, as shown in SEQ ID NO.1; according to the above Original sequence, design primers as shown in SEQ ID NO.2 and SEQ ID NO.3, use PCR method to construct phenylalanine dehydrogenase with NdeI and Xhol restriction sites at the 5' end and 3' end respectively The gene and PCR synthesis process were completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd. After the PCR amplified product was identified by 1% agarose gel electrophoresis, the PheDH gene fragment was recovered from the gel, and NdeI and XhoI enzymes were used to perform double digestion, and the digested product was recovered, and the same double-digested pET-28a plasmid (with with His-tag tag) for ligation, and the ligated plasmid w...

Embodiment 2

[0043] (1)-(4) The experimental procedures are as the steps (1)-(4) of Example 1.

[0044] (5) Preparation of reduced graphene oxide immobilized phenylalanine dehydrogenase: add the phenylalanine with His-tag label obtained in step (2) to the reduced graphene oxide suspension obtained in step (3) Pure enzyme solution of acid dehydrogenase, and make the final concentration of phenylalanine dehydrogenase with His-tag tag to 0.4116mg / mL, adjust the concentration of NaCl to 1M, shake in a constant temperature shaker at 4°C for 4h, and obtain immobilized Liase 1M-CRGO-PheDH, the recovery rate of enzyme activity can reach 42.15%. figure 2 shown. Reduced graphene oxide immobilized enzyme load capacity is 0.409mg / mg, as attached to the instructions image 3 shown.

[0045] (6) Determination of the temperature stability of the immobilized enzyme: keep the free enzyme and the immobilized enzyme 1M-CRGO-PheDH in a 40°C water bath for 3 hours, and measure the enzyme activity every 30 ...

Embodiment 3

[0047] (1)-(4) The experimental procedures are as the steps (1)-(4) of Example 1.

[0048] (5) Preparation of reduced graphene oxide immobilized phenylalanine dehydrogenase: add the phenylalanine with His-tag label obtained in step (2) to the reduced graphene oxide suspension obtained in step (3) Pure enzyme solution of acid dehydrogenase, and make the final concentration of phenylalanine dehydrogenase with His-tag tag to 0.4116mg / mL, adjust the NaCl concentration to 2M, oscillate in a constant temperature shaker at 4°C for 4h, and get immobilized Liase 2M-CRGO-PheDH, the recovery rate of enzyme activity can reach 46.44%. figure 2 shown. The load of reduced graphene oxide immobilized enzyme is 0.410mg / mg, as shown in the attached image 3 shown.

[0049] (6) Determination of the temperature stability of the immobilized enzyme: keep the free enzyme and the immobilized enzyme 2M-CRGO-PheDH in a 40°C water bath for 3 hours, and measure the enzyme activity every 30 minutes dur...

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Abstract

The invention discloses a method for immobilization of phenylalanine dehydrogenase by chemical reduced graphene oxide, and belongs to the technical field of immobilized enzyme. The chemical reduced graphene oxide is stable in structure, is good in biocompatibility, and is a good enzyme immobilization carrier. The surface of the chemical reduced graphene oxide with a sheet-layer structure is lack of groups such as an epoxy group, a carbonyl group and a carboxyl group, is great in load area, and has hydrophobicity. The immobilization of phenylalanine dehydrogenase by the chemical reduced graphene oxide mainly has a hydrophobic effect; and in an immobilization process, the degree of reduction and salt concentration of the chemical reduced graphene oxide have influences on immobilization of enzyme by the chemical reduced graphene oxide. The obtained immobilized phenylalanine dehydrogenase is obviously improved in pH stability and repeated use stability, and meanwhile, has relatively high enzyme activity recovery rate. The method disclosed by the invention has the advantages that the process is simple; the preparation conditions are mild; and the immobilization rate is high.

Description

technical field [0001] The invention relates to a method for immobilizing phenylalanine dehydrogenase by using reduced graphene oxide, belonging to the field of enzyme immobilization. Background technique [0002] Phenylalanine dehydrogenase (Phenylalanine Dehydrogenase, PheDH) can catalyze L-phenylalanine in the coenzyme NAD + In the presence of oxidative deamination to form phenylpyruvate, it can also reversely catalyze the synthesis of L-phenylalanine from phenylpyruvate, ammonia and coenzyme NADH. PheDH can be used to detect phenylketonuria (PKU), the detection process is convenient and fast, and the interference of endogenous fluorescence and antibiotics can be avoided. [0003] Due to the poor stability of free phenylalanine dehydrogenase, it can be improved by immobilization. The immobilized enzyme can be recovered and reused, and has good stability. The performance of the immobilized enzyme depends on the nature of the carrier material used for the immobilized enz...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N9/06C12R1/19
CPCC12N9/0018C12N11/14C12Y104/0102
Inventor 王世珍姚光晓霍鹤宇
Owner XIAMEN UNIV
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