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Enzyme for synthesizing and metabolizing xanthine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme

A Chinese hairpin, synthetic metabolism technology, applied in the field of guanine deaminase, can solve the problems of rare research on genes and proteins, and achieve the effects of major application prospects, high expression, and expanded production

Active Publication Date: 2012-09-26
ZHEJIANG UNIV OF TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the purine nucleoside producing bacteria used are mainly Bacillus subtilis, and Cordyceps sinensis, which is an important anabolic purine nucleoside, only stays in the analysis of metabolite components and efficacy research. Studies on related genes and proteins in the nucleoside synthetic metabolic pathway are still rare

Method used

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  • Enzyme for synthesizing and metabolizing xanthine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme
  • Enzyme for synthesizing and metabolizing xanthine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme
  • Enzyme for synthesizing and metabolizing xanthine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Cultivation of "Bailing" producing fungus Cordyceps sinensis

[0028] Strain source: Firstly, natural Cordyceps sinensis was collected from Qinghai and brought back to Hangzhou for isolation and screening. The strain L0106 was obtained, and the strain was identified as Hirsutella Sinensis. The strain was preserved in China for typical culture The collection number is CCTCC No: M 2011278, which has been disclosed in the prior patent application CN102373190A.

[0029] Inoculate the strain on the slant, the medium formula (this is the liquid formula before solidification, and then make the slant according to the following ratio) is glucose 2.0% (w / v, 1% means 100mL medium contains 1g , The same below), corn flour 1.0%, potato juice 0.5%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm pupa powder 2.0%, peptone 1.0%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05% , Agar powder 1.0%, the remainder is water, cultured at 12~16℃ for 25 days; then the ...

Embodiment 2

[0030] Example 2: Extraction of total RNA from "Bailing" producing strain Cordyceps sinensis

[0031] Extract total RNA with TRIzol reagent, the specific steps are as follows: 1) Liquid nitrogen grinding: Take 1g of fresh bacteria into a mortar, add liquid nitrogen repeatedly to fully grind it to powder, and distribute it into pre-cooled 1.5mL centrifuge tubes. Add 1mL of TRIzol reagent, mix well, and let stand on ice for 5 minutes to separate the nucleic acid protein complex completely. 2) RNA isolation: add 0.2mL chloroform, vigorously shake and mix for 15s, let stand on ice for 2~3min, centrifuge at 4℃, 12000rpm for 15min, layer into layers, take the upper water phase, about 600μL. 3) RNA precipitation: add 500μL of isopropanol, let stand on ice for 10min, centrifuge at 4℃, 12000rpm for 10min, discard the supernatant. 4) RNA washing: add 1mL of 75% (v / v) ethanol, suspend the precipitate, let stand on ice for 10 minutes, centrifuge at 4°C, 7500 rpm for 15 minutes; repeat the a...

Embodiment 3

[0032] Example 3: Sequencing of RNA samples of "Bailing" producing strain Cordyceps sinensis

[0033] After extracting the total RNA of the sample, the mRNA is enriched with magnetic beads with Oligo (dT). Add fragmentation buffer to interrupt mRNA into short fragments (200~700bp), use mRNA as template, synthesize the first cDNA strand with random hexamers, then synthesize the second cDNA strand, and then go through QiaQuick PCR After the kit is purified and eluted with EB buffer, the ends are repaired, polyA is added and the sequencing adapters are added, then fragment size selection is performed by agarose gel electrophoresis, and finally PCR amplification is performed. The established sequencing library is performed with Illumina GA IIx Sequencing. The original image data obtained by sequencing is converted into sequence data by base calling, namely raw data or raw reads. Remove the reads containing only the adaptor sequence from the original sequencing reads and prepare the...

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Abstract

The invention relates to a guanine deaminase for synthesizing and metabolizing an xanthine from guanine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis from 'Bailing' production strain, a gene coding the enzyme and application of the guanine deaminase. The guanine deaminase comprises the proteins shown in SEQ ID No. 1 and SEQ ID No. 2, and the coding gene of the guanine deaminase corresponds to the nucleotide sequence shown in SEQ ID No. 3 and SEQ ID No.4. According to the invention, the metabolizing way for synthesizing the xanthine from the guanine is researched in detail from the principle, comprising: the cloned DNA of the nucleotide sequence provided by the invention can be transferred to an engineering strain by transduction, transformation, and combined transfer; the expression of the gene is biologically synthesized by regulating the xanthine, the host xanthine is given high expressivity, an effective way is provided to increase the yield of the xanthine, and the invention has a great application prospect.

Description

(1) Technical field [0001] The invention relates to a guanine deaminase from the "Bailing" producing strain Cordyceps sinensis, which participates in the synthesis and metabolism of xanthine from guanine, a gene encoding this enzyme and its application. (2) Background technology [0002] Cordyceps sinensis (Berk.) Sacc. is a complex of Cordyceps sinensis (Berk.) Sacc. parasitic on the Lepidoptera Hepialus armoricanus Oberthur larvae and on the larval carcass. ). Cordyceps sinensis is a kind of cherished traditional fungal medicinal resource. It has the characteristics of diverse metabolites and biological activities. It has shown great application and development prospects in the field of biomedicine. Cordyceps has attracted much attention for its wide and obvious medicinal effects, and is highly respected worldwide. Chinese medicine believes that Cordyceps enters the lung and kidney two meridians, which can not only invigorate lung yin, but also invigorate kidney yang, and cur...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N1/15C12N1/19C12N1/21C12P17/18
Inventor 郑裕国李邦良吴晖柳志强许静陈丽芳许峰薛亚平袁水金王鸿艳
Owner ZHEJIANG UNIV OF TECH
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