A kind of recombinant pectin methylesterase pmea and its coding gene and application

A pectin methylesterase and gene technology, applied in the field of genetic engineering, can solve the problems of slow reaction and destruction of pectin polymerization degree and the like

Active Publication Date: 2021-08-03
GUANGDONG QIZHI BIOTECHNOLOGY CO LTD
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  • Description
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Problems solved by technology

The disadvantage of the first two methods is that the reaction is slow; the alkali method has a fast rate of ester reduction, but it will destroy the degree of polymerization of pectin; pectin methylesterase can quickly demethylate pectin molecules, which can achieve The purpose of converting oxy-pectin into low-methoxyl pectin, thus the enzymatic production of low-methoxyl pectin urgently needs to be scaled up

Method used

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  • A kind of recombinant pectin methylesterase pmea and its coding gene and application
  • A kind of recombinant pectin methylesterase pmea and its coding gene and application
  • A kind of recombinant pectin methylesterase pmea and its coding gene and application

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1: Recombinant expression of recombinant pectin methylesterase

[0044] 1. Acquisition of recombinant pectin methylesterase gene pmeA:

[0045] Refer to the amino acid sequence of Aspergillus niger pmeA published on GenBank in NCBI. Then, according to the codon preference of Candida utilis, the highest frequency codon was used to optimize the codon of the gene sequence.

[0046] In order to facilitate subsequent molecular cloning, a Bsp119I restriction site was added to the 5' end of the above sequence, and an XbaI restriction site was added to the 3' end. The optimized sequence was synthesized by General Biosystems (Anhui) Co., Ltd.

[0047] 2. The pmeA gene fragment was cloned into the pCuGAPGαAL expression vector:

[0048] (1) For the extraction of the pUC57-pmeA plasmid, refer to the operating instructions of the US-based plasmid mini-quick extraction kit.

[0049] (2) Double digestion of plasmids pUC57-pmeA and pCuGAPGαAL.

[0050] Digest pUC57-pmeA ...

Embodiment 2

[0100] Embodiment 2: Enzyme characteristic analysis of recombinant pectin methylesterase

[0101] 1. SDS-PAGE analysis and activity staining of recombinant pectin methylesterase

[0102] Inoculate the positive Candida utilis recombinant bacteria into 5 mL of YPD medium containing G418 with 2% inoculum; after culturing for 24 hours at 30°C and 200 rpm, transfer to 200 mL of YPD medium containing G418 to make the initial OD 600 0.1; after culturing for 96 hours, centrifuge at 8,000×g for 30 minutes to separate the bacteria and supernatant;

[0103] Concentrate the supernatant at 5,000×g at 4°C with a 10k ultrafiltration tube, and concentrate 100 times. Carry out protein quantification, SDS-PAGE electrophoresis and activity staining analysis on the concentrate, such as Figure 4 .

[0104] The principle of active dyeing is that after protein gel refolding, under the optimal conditions of recombinant pectin methylesterase, the recombinant enzyme will react with pectin in protei...

Embodiment 3

[0150] Embodiment 3: the high-density cultivation of recombinant Candida utilis

[0151] 1. Inoculate the recombinant Candida utilis in 5 mL of YPD medium containing G418 at an inoculum size of 2%, culture at 30°C, 200 rpm, for 24 hours;

[0152] 2. After the recombinant bacteria grow to the logarithmic phase, transfer it to 50mL YPD medium containing G418 at 2%, and after cultivating overnight, transfer it to 150mL YPDG medium containing G418 to make the initial OD 600 cultured at 0.1, 30°C, 200rpm;

[0153] 3. When the glycerin content is lower than 0.5%, add 10% glycerin to make the glycerin content 1%;

[0154] 4. After culturing for 168 hours, measure the OD 600 ;

[0155] 5. Collect the bacterial liquid, centrifuge at 5,000×g at 4°C for 30 minutes, collect the supernatant and bacterial cells, and measure the protein concentration in the supernatant and the enzyme activities of endopolygalacturonase and pectin methylesterase respectively.

[0156] The results of enzym...

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Abstract

The invention discloses a recombinant pectin methylesterase pmeA, its coding gene and application. The present invention is based on the amino acid sequence of Aspergillus niger pmeA published on GenBank, and then optimizes the codons according to the codon preference of the host bacteria, thereby obtaining a new nucleotide sequence of the pmeA gene such as SEQ ID NO.1 As shown, the amino acid sequence of the expressed recombinant pectin methylesterase is shown in SEQ ID NO.2. After the pmeA gene was connected with the pCuGAPGαAL expression vector, the recombinant plasmid pCuGAPGαAL‑pmeA was integrated into the genome of Candida utilis to realize the heterologous expression of the pmeA gene. The recombinant pectin methylesterase can efficiently catalyze the hydrolysis of methoxy group in pectin, reduce the degree of esterification of pectin, and can be used in the production of food, medicine and chemical industries.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to recombinant pectin methylesterase and its application. Background technique [0002] Pectin is abundant in plants, mainly in the form of high methoxyl pectin. For low-methoxyl pectin, there are few natural plants available for extraction. So far, the most reported at home and abroad is the extraction of low-methoxyl pectin from sunflower. However, the small planting area of ​​sunflower and the influence of temperature and season are not conducive to the commercial development of low-ester pectin. [0003] At present, the methods for producing low-ester pectin include: acid method, concentrated ammonia method, alkali method and pectin methylesterase enzymatic hydrolysis method. The disadvantage of the first two methods is that the reaction is slow; the alkali method has a fast rate of ester reduction, but it will destroy the degree of polymerization of pectin; pect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/18C12N15/81C12N1/19C12P19/04C12R1/72C12R1/84C12R1/865
CPCC12N9/18C12N15/81C12N15/815C12N2800/22C12P19/04C12Y301/01011
Inventor 刘泽寰林蒋海李帅
Owner GUANGDONG QIZHI BIOTECHNOLOGY CO LTD
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