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Rhodotorula glutinis phenylalanine deaminase gene and application thereof

A technology of phenylalanine deaminase and gene, which is applied in Rhodotorula viscosus phenylalanine deaminase gene and its application field, can solve the problems of high GC content and difficulty in reverse transcription, and improve enzyme activity Effect

Inactive Publication Date: 2014-11-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high GC content of the 5' end sequence of the gene, it is easy to form a hairpin structure, and the traditional reverse transcription is difficult to succeed

Method used

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  • Rhodotorula glutinis phenylalanine deaminase gene and application thereof
  • Rhodotorula glutinis phenylalanine deaminase gene and application thereof
  • Rhodotorula glutinis phenylalanine deaminase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Extraction of Rhodotorula viscosus total RNA

[0020] (1) Using the strain Rhodotorula glutinis (Rhodotorula glutinis) CCTCC NO:M2011490 used for industrial production as the starting strain, pick a single colony from the Rhodotorula glutinis plate and put it into 5 mL of seed medium, culture it at 30°C for 24 hours, and take 50 μL of Put the seed solution into a 250mL Erlenmeyer flask filled with 50mL medium, and culture at 30°C for 18-20h to OD 600 =1.0, collect the bacterial cells by centrifugation at 1500 g for 5 min, wash the bacterial cells twice with cold water, and discard the supernatant.

[0021] (2) Resuspend the cells with 400 μL of TES (10mM Tris-HCl, 10mM EDTA, 0.5% SDS, pH7.5) buffer, add 400 μL of acidic phenol, shake vigorously for 10 sec, incubate at 65°C for 1 hour, and vortex from time to time The device oscillates briefly.

[0022] (3) Incubate on ice for 5 minutes, and centrifuge at 12000g for 5 minutes at 4°C.

[0023] (4) Transfer th...

Embodiment 2

[0028] Example 2 Reverse transcription to obtain cDNA

[0029] (1) Add 50ng total RNA and 1μL 5'-CDS(5'-(T) to a 0.5mL centrifuge tube 25 VN (N = A, C, G, T; V = A, G, C)-3') and 1 μL SMARTer II A Oligonucleotide (5'-AAGCAGTGGTATCAACGCAGAGTACGGGGG-3'), add water to 4.75 μL and mix well.

[0030] (2) Incubate at 72° C. for 3 minutes, then incubate at 42° C. for 2 minutes, and centrifuge at 1500 g for 10 sec.

[0031] (3) Add 2μL of 5×buffer (250Mm Tris-HCl, 375mM KCl, 30mM MgCl 2 ), 1 μL DTT (20mM), 1 μL dNTP (10mM), 0.25 μL RNase inhibitor and 1 μL SMARTer TM For reverse transcriptase, incubate at 42°C for 90 minutes, denature at 70°C for 10 minutes, cool to room temperature and add 20 μL sterile water to obtain cDNA.

Embodiment 3

[0032] Example 3 Obtaining the sequence of the 5' end of pal

[0033] (1) Add 1 μL cDNA, 5 μL Mix primer (5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'; 5'-CTAATACGACTCACTATAGGGC-3'), 1 μL GSP-specific primer (5'-GGTGATGCCG TGGTTG AGGAAGTT-3') to a 0.25 mL centrifuge tube , 5μL 10×buffer, 5μL dNTP (2mM), 0.5μL pfu DNA polymerase, 3μL MgSO 4 (25mM), add deionized water to 50μL.

[0034] (2) Perform PCR amplification, PCR amplification conditions: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 1 min, annealing at 58°C for 30 sec, extension at 72°C for 60 sec, 25 cycles, and detection by agarose gel electrophoresis.

[0035] (3) Add 5 μL of PCR product, 1 μL of 10×Taq buffer, 1 μL of dATP (2mM), 1 μL of Taq enzyme (5U / μL) into a 0.25 mL centrifuge tube, add water to 10 μL, react at 70°C for 30 minutes, extract with phenolform, and precipitate with ethanol , aseptic operation typhoon for 15 minutes until the alcohol evaporates completely, and resuspended with...

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Abstract

The invention discloses a rhodotorula glutinis phenylalanine deaminase gene and an application thereof, and belongs to the field of biotechnology. Cloning of the phenylalanine deaminase gene plays an important role in biosynthesis of L-phenylalanine by an enzyme method. The phenylalanine deaminase gene is obtained by extracting rhodotorula glutinis RNA and carrying out reverse transcription to obtain the full-length phenylalanine deaminase gene, wherein the full length of the gene is 2121 bp, and 705 amino acids are encoded. Phenylalanine deaminase gene resources are further expanded, and a scientific basis is provided for molecular modification of phenylalanine deaminase and improvement of the stability and catalytic activity of enzymes.

Description

technical field [0001] The invention relates to a rhodotorula viscosus phenylalanine deaminase gene and application thereof, belonging to the field of biotechnology. 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. This feature is used industrially to produce the essential amino acid L-phe and the 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, pharmaceutical and other industrial fields. However, since the enzyme is an inducible enzyme, after t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/10C12N15/70C12N1/21C12P13/22C12P7/40C12R1/645
Inventor 周哲敏朱龙宝崔文璟周丽
Owner JIANGNAN UNIV
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