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L-glutamate oxidase gene from Afghanistan streptomyces and application thereof

A technology of glutamate oxidase and Streptomyces, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of low enzyme production capacity and low enzyme production capacity, and achieve high specificity and reduce extraction costs. Effect

Inactive Publication Date: 2015-06-24
上海瑞丰农业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The research on finding L-glutamate oxidase producing bacteria is in the ascendant so far, however, the enzyme producing ability of the L-glutamate oxidase producing bacteria screened so far is very low, even after mutagenesis, the enzyme producing ability is not high , so the realization of genetic engineering production becomes the fundamental way to solve the source of the enzyme

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  • L-glutamate oxidase gene from Afghanistan streptomyces and application thereof
  • L-glutamate oxidase gene from Afghanistan streptomyces and application thereof
  • L-glutamate oxidase gene from Afghanistan streptomyces and application thereof

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Embodiment 1

[0035] Embodiment 1, strain separation

[0036] Weigh 1 g of soil sample, add 1 ml of 0.9% (w / v) sodium chloride solution, shake and mix at 5000 rpm, centrifuge gently at 3000 rpm, pour off the supernatant, and then add 0.9% (w / v) chlorine Sodium chloride solution 1ml, shake and mix at 5000 rpm, let it stand on ice for 10min, draw 100μL solution and spread it on Gaoshi No. 1 medium containing 50ppm potassium dichromate, 30 ℃ cultured for 4 days. According to the shape and size of the colonies, the colonies were picked and respectively inserted into the control and color development plates (both in the plates, Gaoshi No. 1 medium), 30 ℃ Cultivate for 3 days under low temperature, take the color developing plate, put the filter paper soaked in the color developing solution on the colony, mark the colony with fast color development and deep red color on the control plate corresponding to the colony, pick the colony and cultivate it in Gaoshi No. 1 Separation on dashed base, 30 ...

Embodiment 2

[0037] Embodiment 2, total DNA extraction and strain identification

[0038] The isolated bacterial strain was inoculated in 20mL liquid medium (yeast powder 3g / L, peptone 20g / L, glucose 10g / L, soluble starch 5g / L, soybean peptone 5g / L, MgSO 4 ·7H 2 (00.2g / L, pH=7.0), 30 ℃ After 3 days of cultivation, the culture solution was centrifuged at a speed of 8000r / min for 5 minutes to obtain bacterial pellets.

[0039] Take 0.15g of fresh bacteria in a mortar (-20 ℃ pre-cooled), ground repeatedly with liquid nitrogen until fine powder, quickly put into a 1.5mL centrifuge tube, added 550μL of TE buffer, 65 ℃30 μL of preheated 20% (w / v) SDS solution, mediate and shake for 5 minutes, then add 20 μL of proteinase K with a concentration of 20 mg / mL, mix well, bathe in water at 37°C for 1 hour, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, v / v) extraction, gently inverted and mixed, centrifuged at 10000r / min for 10 minutes; pipette the supernatant into a new cent...

Embodiment 3

[0043] Embodiment 3, acquisition of the L-glutamate oxidase gene of Streptomyces afghanistan

[0044] Primers were designed according to the conserved box sequence of L-glutamic acid oxidase: Primer A: 5'-AGTACGCGGAGGCCGGCGCCAT-3' and Primer B: 5'-GCTGAACTCCAGCAGCACCTTGGT-3' as amplification primers, using the total DNA as a template to obtain a 984bp PCR fragments. The PCR reaction conditions were: 94°C for 30s, 55°C for 30s, 72°C for 90s, and 30 cycles of amplification. The fragment was recovered with 1% agarose gel, and 10 μl was directly connected to the T / A cloning vector (Dalian Bao Biological Company). 4 ℃ After ligation overnight, high-efficiency transformation in DH5α competent cells, positive clones were obtained and sequenced. Based on this sequence, the Genome Walking method was used to obtain the unknown sequences on both sides, and the complete open reading frame was searched, so as to obtain the complete sequence of the L-glutamic acid oxidase gene.

[0045]...

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Abstract

The invention relates to an L-glutamate oxidase gene from Afghanistan streptomyces and an application thereof and in particular relates to a recombinant bacterium which is obtained by screening Afghanistan streptomyces capable of generating L-glutamate oxidase from soil, cloning and coding the L-glutamate oxidase gene from the bacterium by a homological cloning method and artificially synthesizing the gene and recombining the gene in an escherichia coli and has the activity of L-glutamate oxidase. The nucleotide sequence of the L-glutamate oxidase gene is SEQ ID No 1, the full length of the L-glutamate oxidase gene is 2073bp, and a coding protein sequence of the L-glutamate oxidase gene is SEQ ID No 2. L-glutamate oxidase coded by the gene can be used for specifically catalyzing L-glutamic acid, can serve as an enzyme for detecting the content of L-glutamic acid and is applied to the fields of the food industry and clinical biochemical detection.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to an L-glutamic acid oxidase gene from Streptomyces amylase chromogenes and its preparation method and application. Background technique [0002] In 1983, Japanese Kamei et al first discovered an enzyme that could catalyze the oxidation of glutamic acid to produce α-ketoglutarate and hydrogen peroxide from the extract of wheat bran culture medium of Streptomyces violascens. It is a typical oxidase rather than a dehydrogenase. NAD+ and NADP+ cannot be used as the electron acceptor of the enzyme, so it is named L-Glutamate oxidase (LGOX). The enzyme has a molecular weight of about 60KD and has a characteristic absorption spectrum (absorption peak of 490nm) of flavoprotein (FAD). Each mole of enzyme contains 1 mole of FAD (Chemical & pharmaceutical bulletin, 1983, 31, 1307-1314). Substrate specificity studies show that it can efficiently catalyze the oxidation...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/10C12N15/70C12Q1/68C12R1/465
Inventor 彭日荷姚泉洪王荣谈王丽娟田永生朱波严培兰丁卫星高建杰王波
Owner 上海瑞丰农业科技有限公司
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