Immobilized maleic acid cis-trans isomerase and preparation method and application thereof

A technology of cis-trans isomerization and immobilization of enzymes, applied in the fields of enzymology, enzyme engineering, and biochemical industry, can solve the problems of difficulty in development and application, reduced activity, high price, etc., to reduce the complexity of operation and reduce the amount of enzyme. Loss, production cost saving effect

Active Publication Date: 2015-10-07
JIANGNAN UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, due to the limitations of high price, poor stability, and difficult recovery after reaction, it is difficult to be developed and applied. However, compared with free enzymes, immobilized enzymes have higher stability and are easy to separate in the reaction system. Reusable, conducive to the realization of automatic production and other advantages
[0004] Conventional immobilization methods include adsorption method, covalent bonding method, embedding method, etc. During the adsorption process, there will be various interactions between the material and the enzyme molecule, and some reactions will cause the enzyme molecule to form an unstable structure and thus inactivate the enzyme. Both adsorption and covalent cross-linking can reduce and avoid the leakage of enzymes, but the activity of enzymes may be reduced or even inactivated after binding to the two-dimensional surface of the material.

Method used

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  • Immobilized maleic acid cis-trans isomerase and preparation method and application thereof
  • Immobilized maleic acid cis-trans isomerase and preparation method and application thereof
  • Immobilized maleic acid cis-trans isomerase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1R5

[0051] Example 1 Construction of R5 short peptide and maleic acid cis-trans isomerase fusion protein engineering bacteria

[0052]Primer F: 5'-CCATATGTCCTCCAAGAAATCGGGATCCTACTCGGGATCCAAGGGTTCCAAGCGTCGCATCTTGATGAGCAACCACTACCGCATCGGCCAGATC-3' and primer R: 5'-GCTGTCCACCAGTCATGCTAGCCATATGTATATCTCC-3' were designed according to the R5 short peptide gene sequence shown in SEQ ID NO.1.

[0053] Using primer F and primer R as primers, the recombinant plasmid pET24a-maiA carrying the gene encoding maleic acid cis-trans isomerase [Wang Ya, Cui Wenjing, Zhou Li, Liu Zhongmei, Zhou Zhemin. Serratia marcescens Malay Expression, purification and enzymatic properties of acid cis-trans isomerase [J]. Journal of Food and Biotechnology, Vol. 33, No. 11, 2014, 1204-1209] as template, PCR reverse amplification, PCR conditions: 94 ℃ pre-denaturation 5min; 98°C denaturation for 10s, 68°C extension for 6min 30s, 30 cycles; 72°C for 10min.

[0054] After the PCR product was purified, it was digeste...

Embodiment 2

[0055] Induced expression of embodiment 2 fusion protein

[0056] The pET24a-R5-MaiA plasmid was transformed into E.coli BL21 to obtain the E.coli BL21 / pET24a-R5-MaiA recombinant strain. E.coli BL21 / pET24a-R5-MaiA recombinant strain was cultured in LB medium containing kanapenicillin at 37°C for 8 hours, and 0.3mL culture solution was put into 30mL LB medium and cultivated to OD at 37°C 600 When the concentration was 0.6, IPTG was added to a final concentration of 0.2 mM, and the bacterial cells were collected by centrifugation after induction at 20°C for 20 h, the cell wall was broken by ultrasonic, and electrophoresed by SDS-PAGE ( figure 2 ) obtained a 29kDa size band, indicating that the fusion enzyme was successfully expressed in Escherichia coli. The specific enzyme activity of the purified recombinant enzyme R5-MaiA was 42U / ml, and that of the original enzyme MaiA was 48U / ml. The activity of the recombinant enzyme R5-MaiA did not decrease significantly after fusion, w...

Embodiment 3

[0057] Embodiment 3 Preparation of immobilized maleate cis-trans isomerase

[0058] Take the fermentation broth of the fusion engineered bacteria and centrifuge to obtain the cells, wash twice with buffer, and concentrate the cells to OD 600 =30-40, after ultrasonic crushing of the bacteria, centrifuge at 12000rpm for 10min, slowly add ammonium sulfate powder to the broken supernatant and keep stirring until the saturation reaches 60%, continue stirring for 30min, and slowly add glutaraldehyde to the final concentration of 0.05%-0.2% (v / v) and stirring continuously at room temperature for 1 h to obtain cross-linked enzyme aggregate particles, and the cross-linked enzyme aggregates obtained by centrifugation were washed 3 times with buffer, and the cross-linked enzyme aggregates and Mix the silicon reaction solution and methyl orthosilicate (ratio: 1:8:1) and stir vigorously for 30s, let stand for 5min, centrifuge at 5000rpm for 5min, collect the immobilized enzyme, and wash wi...

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Abstract

The invention discloses immobilized maleic acid cis-trans isomerase and a preparation method and application thereof, and belongs to the technical fields of enzymology and enzyme engineering and the field of biochemical industries. According to the immobilized maleic acid cis-trans isomerase and the preparation method and application thereof, R5 peptide fragments are connected to the N ends of the maleic acid cis-trans isomerase, R5-MaiA is crosslinked through glutaraldehyde, embedding is conducted on the crosslinked R5-MaiA by utilizing silicon reaction liquid, and immobilization of the maleic acid cis-trans isomerase is completed. The immobilized maleic acid cis-trans isomerase (R5-MI-CLEA-Si) with high stability is obtained through the method which is low in cost and easy to operate, the optimum reaction temperature of the immobilized isomerase is 55 DEG C, and the optimum reaction pH is 7.7; on the condition that the temperature is 55 DEG C, the half-life period of the immobilized isomerase is 4 h, the half-life period of the free isomerase is 0.5 h, the stability of the immobilized isomerase is eight times of that of the free isomerase, and after a catalytic reaction is repeated for eight times, the enzyme activity of the immobilized isomerase can be still retained at 75%.

Description

technical field [0001] The invention relates to an immobilized maleate cis-trans isomerase and its preparation method and application, belonging to the technical fields of enzymology and enzyme engineering and the field of biochemical industry. Background technique [0002] At present, the industrial production of fumaric acid is mainly based on chemical synthesis, including furfural oxidation and maleic anhydride isomerization. However, the chemical synthesis method needs to use petroleum resources, and petroleum is a non-renewable resource, which increases the production cost and has the disadvantages of heavy pollution. With the development of technology, biosynthesis methods based on microbial fermentation and enzymatic conversion are gradually replacing chemical synthesis, but microbial fermentation will produce a large number of fermentation by-products, which is not conducive to the separation and purification of products. The enzymatic method has the advantages of h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/90C12N11/04C12P7/46C12R1/19
Inventor 周哲敏刘文茂周丽崔文璟刘中美
Owner JIANGNAN UNIV
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