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Method for acquiring rhodotorula glutinis phenylalanine deaminase gene sequence

A technology of phenylalanine deaminase and Rhododendron muscaria, applied in the field of gene cloning, can solve the problems of poor stability, rapid decline of enzyme activity and the like

Inactive Publication Date: 2013-04-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the enzyme is an inducible enzyme, after the enzyme activity reaches its peak in the logarithmic growth phase, the enzyme activity decreases rapidly and the stability is poor.
In order to further improve the enzyme activity and stability, it is necessary to use molecular biology methods to carry out molecular modification of the enzyme, but so far, only part of the cDNA has been obtained

Method used

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  • Method for acquiring rhodotorula glutinis phenylalanine deaminase gene sequence
  • Method for acquiring rhodotorula glutinis phenylalanine deaminase gene sequence
  • Method for acquiring rhodotorula glutinis phenylalanine deaminase gene sequence

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Effect test

Embodiment 1

[0044] Pick a single colony of Rhodotorula viscosus into 5 mL of seed medium and culture at 30°C for 24 hours, then take 50 μL of seed liquid into a 250 mL Erlenmeyer flask filled with 50 mL of medium, and culture at 30°C until OD 600= 1.0, the bacteria were collected by centrifugation, the cells were lysed by TES, the total RNA was extracted with acid phenol and chloroform, stored at -80°C for later use, and the quality of the total RNA was detected by agarose electrophoresis.

Embodiment 2

[0046] Detected by agarose gel electrophoresis, extract high-quality RNA, add reverse transcriptase and reaction buffer, react at 42°C for 90 minutes, treat at 70°C for 10 minutes, add 20 μL of sterile water to obtain cDNA. Use 5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAAC GCAGAG T-3′; 5′-CTA ATACGACTCACTA TAGGGC-3′; 5′-GGTGATGCCG TGGTTG AGGAAGTT-3′ as primers for PCR amplification, agarose gel electrophoresis to detect the amplification results, and gel recovery The target band was added with Taq enzyme, reacted at 70°C for 30 minutes, extracted with phenolform, ligated with pUCm-T, and the ligated product was transferred into E.coli JM109, and the plasmid was extracted for sequencing to obtain the sequence at the 5′ end.

Embodiment 3

[0049]

[0050]

[0051]

[0052]

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Abstract

The invention discloses a method for acquiring a rhodotorula glutinis phenylalanine deaminase gene. The method comprises the following steps of: extracting RNA (Ribose Nucleic Acid); carrying out inverse transcription; carrying out PCR (Polymerase Chain Reaction) amplification; establishing a cloning vector pUCm-T-pal; and measuring a sequence to obtain a full-length phenylalanine deaminase gene sequence. The invention belongs to the technical field of gene clone. The method disclosed by the invention has the advantages of easiness for operation and higher efficiency and success rate.

Description

Technical field: [0001] On the basis of known partial sequences, the invention adopts 5'-RACE (quick acquisition of 5' end sequence) technology to obtain the full-length gene of Rhodotorula viscosus phenylalanine deaminase, which belongs to the technical field of gene cloning. Background technique: [0002] Phenylalanine deaminase (PAL) catalyzes the deamination of L-phenylalanine (L-phe) to generate trans-cinnamic acid and ammonia at pH 8.0, and can catalyze the reverse reaction to synthesize L-phe at pH 11.0. Utilize this feature to produce essential amino acid L-phe and sweetener aspartame. PAL widely exists in plants and microorganisms, mainly including molds and yeasts, especially Rhodotorula. Among them, 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. It has been successfully used in food, pharmaceut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N15/10C12R1/645
Inventor 周哲敏朱龙宝崔文璟周丽曲娟娟
Owner JIANGNAN UNIV
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