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Isoeugenol monooxygenase mutant and application thereof

A technology of isoeugenol and monooxygenase, applied in the biological field, can solve problems affecting industrial application and the like, and achieve the effect of high catalytic activity

Active Publication Date: 2017-05-31
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the production of vanillin by biocatalytic technology is not only efficient, low-carbon and environmentally friendly, but also highly competitive, the activity of isoeugenol monooxygenase used to produce vanillin by biocatalytic technology is inhibited by vanillin, which affects the production of vanillin. Industrial application of technology

Method used

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  • Isoeugenol monooxygenase mutant and application thereof
  • Isoeugenol monooxygenase mutant and application thereof
  • Isoeugenol monooxygenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Site-directed mutagenesis of isoeugenol monooxygenase site 120

[0058] For the technique of site-directed mutagenesis, please refer to the description of Ho et al. Gene. 1989, 77:51-33. The specific process is as follows:

[0059] In order to mutate Nsn (N) at position 120 in the parental amino acid sequence to Ile (I) to obtain mutant N 120I, the plasmid pET30a-ISO was used as a template to design primer pairs Nsn120-F and Nsn120-R (see Table 1) .

[0060] The F-NR fragment was amplified with the primer pair NdeI-F and N120-R, and the NF-R fragment was amplified with the primer pair Nsn120-F and XhoI-R. The amplification reaction conditions were: 25 μL Max DNA polymerase (TaKaRa, Japan), 2 μL pET21a-ISO, and 1.5 μL primer NdeI-F and 1.5 μL primer Nsn120-R (or, 1.5 μL primer Nsn120-F and 1.5 μL primer XhoI -R), adjust the reaction volume to 50 microliters with sterile water. The PCR amplification reaction program was: 98°C for 2 minutes, 30 cycles: 98°C ...

Embodiment 2

[0063] Example 2: Site-directed mutagenesis of isoeugenol monooxygenase site 121

[0064] The technique of site-directed mutagenesis can refer to the description of Ho et al. Gene. 1989, 77:51-33. The specific process is as follows:

[0065] In order to mutate Thr(T) at position 121 in the parental amino acid sequence to Pro(P) to obtain mutant T120P, the plasmid pET30a-ISO was used as a template to design primer pairs Thr121-F and Thr121-R (see Table 1).

[0066]The F-TR fragment was amplified with the primer pair NdeI-F and Thr121-R, and the TF-R fragment was amplified with the primer pair Thr121-F and XhoI-R. The amplification reaction conditions were: 25 μL Max DNA polymerase (TaKaRa, Japan), 2 μL pET21a-ISO, and 1.5 μL primer NdeI-F and 1.5 μL primer Thr121-R (or, 1.5 μL primer Thr121-F and 1.5 μL primer XhoI -R), adjust the reaction volume to 50 microliters with sterile water. The PCR amplification reaction program was: 98°C for 2 minutes, 30 cycles: 98°C for 10 secon...

Embodiment 3

[0069] Example 3: Site-directed mutation of isoeugenol monooxygenase site 281

[0070] The technique of site-directed mutagenesis can refer to the description of Ho et al. Gene. 1989, 77:51-33. The specific process is as follows:

[0071] In order to mutate Phe(F) at position 281 in the parental amino acid sequence to Gln(Q) to obtain mutant F281Q, the plasmid pET30a-ISO was used as a template to design primer pairs Phe281-F and Phe281-R (see Table 1).

[0072] The primer pair NdeI-F and Phe281-R was used to amplify the F-FR fragment, and the primer pair Phe281-F and XhoI-R was used to amplify the FF-R fragment. The amplification reaction conditions were: 25 μL Max DNA polymerase (TaKaRa, Japan), 2 μL pET21a-ISO, and 1.5 μL primer NdeI-F and 1.5 μL primer Phe281-R (or, 1.5 μL primer Phe281-F and 1.5 μL primer XhoI -R), adjust the reaction volume to 50 microliters with sterile water. The PCR amplification reaction program was: 98°C for 2 minutes, 30 cycles: 98°C for 10 secon...

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Abstract

The invention discloses an isoeugenol monooxygenase mutant and application thereof. The isoeugenol monooxygenase mutant comprises an amino acid sequence as shown in SEQ 2, and the amino acid sequence has at least one mutant selected from a 120th site, a 121st site, a 281st site, a 298th site and a 470th site. The isoeugenol monooxygenase mutant with high catalytic activity is obtained by carrying out site-specific mutagenesis on an isoeugenol monooxygenase gene; the isoeugenol monooxygenase mutant can be efficiently catalyzed to generate vanillin by using isoeugenol as a substrate; by using the isoeugenol as the substrate, the isoeugenol monooxygenase mutant has the isoeugenol monooxygenase catalytic activity which is at least 117 percent higher than that of a parent.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an isoeugenol monooxygenase mutant and application thereof. Background technique [0002] The main functions of vanillin include the following points: (1) Food additives: vanillin contains strong milk fragrance and mysterious orchid fragrance. It is known as the "Queen of Natural Flavors" and is a very important food additive industry. It is mainly used as a flavor enhancer, flavoring agent, and fixative in ice cream, biscuits, candies, beverages, etc., especially in dairy products containing milk flavor; (2) Intermediates and important raw materials of drugs : For example, 3,4,5-trimethoxybenzaldehyde (TMB) can be synthesized by biotransformation from vanillin, and TMB is used for the sulfonamide synergist trimethoprim (TMP); it can be condensed with glycine to form a lactone , vanillin is the intermediate of the synthetic drug L-dopa for the treatment of Parkinson's disease; and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P7/24
CPCC12N9/0006C12P7/24C12Y101/01319
Inventor 赵丽青
Owner SHENZHEN UNIV
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