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L-glutamate oxidase gene from Streptomyces griseoflavus as well as preparation method and application of L-glutamate oxidase gene

A technology of glutamic acid oxidase and Streptomyces griseus, applied in the field of microbial genetic engineering, can solve the problems of limited and unclear LGOX genes, low enzyme production capacity, etc., and achieve improved catalytic activity, reduced costs, and strong specificity Effect

Inactive Publication Date: 2017-01-04
上海瑞丰农业科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] All the LGOX found so far are derived from Streptomyces. However, there are great differences in the molecular size, specific activity, specificity for glutamic acid, and optimum pH value of LGOX among different isolated strains. LGOX genes are limited and the mechanisms responsible for these differences are poorly understood
In addition, because the production of LGOX is relatively lagging behind, most of the L-glutamate oxidase-producing bacteria screened so far have very low enzyme-producing ability, even after mutagenesis, the enzyme-producing ability is not high. Therefore, a new type of LGOX gene was obtained and the gene Engineering production becomes the fundamental way to solve the source of the enzyme

Method used

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  • L-glutamate oxidase gene from Streptomyces griseoflavus as well as preparation method and application of L-glutamate oxidase gene
  • L-glutamate oxidase gene from Streptomyces griseoflavus as well as preparation method and application of L-glutamate oxidase gene
  • L-glutamate oxidase gene from Streptomyces griseoflavus as well as preparation method and application of L-glutamate oxidase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Separation of strains

[0073] Weigh 1 g of soil sample, add 1 ml of 0.85% (w / v) sodium chloride solution, shake and mix at 5000 rpm, centrifuge gently at 3000 rpm, pour off the supernatant, and then add 0.85% (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 (Gaoshi No. 1 medium: soluble starch 20g / L, KNO 3 1g / L, K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, NaCl 0.5g / L, FeSO 4 ·7H 2 (00.01g / L, agar 20g / L, pH=7.4), cultivated for 4 days at 30°C. According to the shape and size of the colonies, the colonies were selected and respectively inserted into the control and color-developing plates (the plates were all Gaoshi No. 1 medium). .5), 0.07% 4-aminoantipyridine, 0.8% L-glutamic acid, 0.1% phenol, 2000U / L horseradish peroxidase. Cultivate at 30°C for 3 days, take a color-developing plate, place the filter pap...

Embodiment 2

[0074] Example 2 Total DNA extraction and strain identification

[0075]The isolated bacterial strain was inoculated in 10mL liquid medium (yeast powder 3g / L, peptone 20g / L, glucose 10g / L, soluble starch 5g / L, soybean peptone 5g / L, MgSO 4 ·7H 2 (0.2 g / L, pH=7.0), cultured at 30° C. for 3 days, and the culture solution was centrifuged at 7000 r / min for 6 minutes to obtain bacterial precipitates.

[0076] Take 0.15 g of fresh bacteria in a mortar (pre-cooled at -20°C), grind repeatedly with liquid nitrogen until it becomes a fine powder, quickly put it into a 1.5mL centrifuge tube, add 550 μL of TE buffer, and add 20% ( w / v) 30 μL of SDS solution, mediate and shake for 5 minutes, then add 20 μL of proteinase K at 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 invert and mix, centrifuge at 10000r / min for 10 minutes; draw the supernatant into a new centrifuge tu...

Embodiment 3

[0078] Example 3 Acquisition of the L-glutamic acid oxidase gene of Streptomyces grisea and flavum

[0079] Primers were designed according to the conserved box sequence of L-glutamic acid oxidase: Primer A: 5'-CACCTGGATCCACGTCAACG-3' and Primer B: 5'-GTGTAGAAGACCTCGACGCG-3' as amplification primers, using the total DNA as a template to obtain a 1179bp 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). Ligated overnight at 4°C, transformed efficiently in DH5α competent cells, obtained positive clones, and sequenced.

[0080] Based on the obtained 1179bp PCR fragment, the Genome Walking Kit (Dalian Bao Biological Company) was used to obtain the unknown sequences on both sides, and the complete open reading frame was searched to obtain the complete sequence of the L-gl...

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Abstract

The invention relates to an L-glutamate oxidase gene from Streptomyces griseoflavus as well as a preparation method and application of the L-glutamate oxidase gene. The nucleotide sequence of the L-glutamate oxidase gene is represented by SEQ ID No.1, the total length is 2004bp, and the amino acid sequence of an encoded protein of the L-glutamate oxidase gene is represented by SEQ ID No.2. A Streptomyces griseoflavus stain for producing L-glutamate oxidase is screened from soil, a full-length sequence of the L-glutamate oxidase gene is obtained from the strain, the L-glutamate oxidase gene with the nucleotide sequence represented by SEQ ID No.1 is obtained, and the L-glutamate oxidase gene is synthesized by virtue of a gene synthetic method, so that the cost for extracting L-glutamate oxidase from wild fungi is greatly lowered, and the catalytic activity of L-glutamate oxidase is increased; and meanwhile, a L-glutamate oxidase substrate has very strong specificity, is capable of specifically catalyzing L-glutamate and can be applied to the content detection of L-glutamate and the fields of food industries and clinical biochemistry 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 griseoflavus and a preparation method and application thereof. Background technique [0002] The determination of L-glutamic acid is very important in the production of monosodium glutamate, food industry and clinical testing. Chromatographic and enzymatic methods are commonly used for the detection of glutamate. Chromatography is difficult to promote due to the limitation of equipment and cumbersome pretreatment. Enzyme methods are commonly used to detect glutamic acid dehydrogenase and glutamic acid decarboxylase, but their substrate specificity is not strong, and expensive coenzymes are required. reaction. L-glutamate oxidase (LGOX) is a flavoproteinase with flavin adenine dinucleotide (FAD) as the prosthetic group, which can specifically catalyze the oxidation of glutamate to α-ketoglutarate , ammoni...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/10C12Q1/26
Inventor 王丽娟姚泉洪彭日荷王荣谈田永生丁卫星严培兰王波孙斌
Owner 上海瑞丰农业科技有限公司
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