Method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of gene in coliform strain

A technology of phenylalanine deaminase and Rhododendron muscaria, which is applied in the fields of enzymology and enzyme engineering, can solve the problems of high production cost, long-term inability of bacterial cells, repeated use, poor stability and the like

Inactive Publication Date: 2013-11-27
JIANGNAN UNIV
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Problems solved by technology

However, since the enzyme is an induced enzyme, after the enzyme activity reaches its peak in the logarithmic phase of growth, the enzyme activity decreases rapidly and the stability is poor. The bacteria cannot be reused for a long time, resulting in high production costs.

Method used

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  • Method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of gene in coliform strain
  • Method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of gene in coliform strain
  • Method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of gene in coliform strain

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Experimental program
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Effect test

Embodiment 1

[0063] Insert Rhodotorula viscosus into a 250mL Erlenmeyer flask containing 50mL medium, and culture at 30°C for 18-20h to the logarithmic phase of growth (OD 600 =1.0), using acidic phenol to extract total RNA, using specific primers 5'-AAGCAGTG GTATCAACGCAGAGTACGGGGG-3' and 5'-(T) 25 VN (N=A, C, G, T; V=A, G, C)-3' and reverse transcription to obtain cDNA.

Embodiment 2

[0065] Using primer F: 5′-GGAATTCCATATGATGGCCCCCTCCGTCGACTC GATC-3′; primer R: 5′-CGGAATTCCTAGTATGGTCTACGTCCAAAGG-3′), PCR conditions: pre-denaturation at 94°C for 4min, denaturation at 94°C for 1min, annealing at 58°C for 30sec, extension at 72°C for 2min, 25 loops. The PCR product was gel-cut and recovered, ligated with the pUCm-T vector, transformed into E.coli JM109, picked a single colony, and extracted the plasmid to obtain the cloning vector pUCm-T-pal. Nde 1 and EcoR 1 were used to double-digest pUCm-T-pal and the empty vector pET-28a(+), respectively, after gel recovery, they were ligated at 16°C overnight, and then transformed into E.coli JM109. Pick positive recombinants on the LB plate, extract the recombinant plasmids and carry out PCR and double enzyme digestion to verify the constructed expression vector pET-28a(+)-pal, transfer pET-28a(+)-pal into E.coli JMBL21, pick positive Single colony, cultured to OD at 37°C 600 When it was 0.6, IPTG was added to a final...

Embodiment 4

[0068]

[0069]

[0070]

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Abstract

The invention discloses a method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of the gene in a coliform strain, and belongs to the technical field of enzymology and enzyme engineering. Pure recombinase is obtained through gene cloning, recombinant bacterium construction, induced expression and recombinase purification, and the specific activity of the pure recombinase is 3U / mg. The method can be used for producing a great amount of phenylalanine ammonia lyase (PAL) in relatively short time, to meet industrial production demand.

Description

Technical field: [0001] The invention relates to a gene cloning of Rhodotorula viscosus phenylalanine deaminase and a high-efficiency expression method in Escherichia coli, belonging to the technical field of enzymology and enzyme engineering. Background technique: [0002] Rhodotorula glutinis can grow on cheap raw materials such as corn flour, molasses, molasses, soybean meal, monosodium glutamate wastewater and other industrial wastes, and it is easy to achieve large-scale cultivation, and has high phenylalanine deaminase activity. It is used industrially to produce L-phe and the sweetener aspartame. However, since the enzyme is an induced enzyme, after the enzyme activity reaches its peak in the logarithmic phase of growth, the enzyme activity decreases rapidly and the stability is poor. The bacteria cannot be reused for a long time, resulting in high production costs. In order to improve enzyme activity and stability, it is necessary to use molecular biological means t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/88C12R1/645
Inventor 周哲敏朱龙宝崔文璟周丽张川
Owner JIANGNAN UNIV
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