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Enzyme from Cordyceps sinensis Hirsutella sinensis for participating in anabolism to obtain xanthylic acid and application of enzyme

A xanthine nucleotide and inosine nucleotide technology, applied in the field of IMP dehydrogenase, can solve the problems of rare research on genes and proteins, and achieve great application prospects, high expression, and the effect of expanding production

Inactive Publication Date: 2012-10-03
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • 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 from Cordyceps sinensis Hirsutella sinensis for participating in anabolism to obtain xanthylic acid and application of enzyme
  • Enzyme from Cordyceps sinensis Hirsutella sinensis for participating in anabolism to obtain xanthylic acid and application of enzyme
  • Enzyme from Cordyceps sinensis Hirsutella sinensis for participating in anabolism to obtain xanthylic acid and application of enzyme

Examples

Experimental program
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Effect test

Embodiment 1

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

[0028] Source of the strain: Firstly, natural Cordyceps sinensis was collected from Qinghai, and brought back to Hangzhou for isolation and screening, and the L0106 strain was obtained, and the strain was identified as Hirsutella Sinensis, which was preserved in a typical culture in China Objects Collection Center, the deposit number is CCTCC No: M 2011278, which has been disclosed in the previous patent application CN102373190A.

[0029] Inoculate the strain on the slant, and the medium formula (this is the liquid formula before solidification, prepared according to the following proportions before making the slant) is glucose 2.0% (w / v, 1% means that 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 chrysalis powder 2.0%, peptone 1.0%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05% , agar powder 1...

Embodiment 2

[0030] Example 2: Extraction of total RNA of "Bailing" production fungus Cordyceps sinensis

[0031]The total RNA was extracted with TRIzol reagent, and the steps were as follows: 1) Grinding with liquid nitrogen: Take 1 g of fresh bacteria and put it into a mortar, add liquid nitrogen repeatedly to grind until powdery, and divide into pre-cooled 1.5mL centrifuge tubes, Add 1mL TRIzol reagent, mix well, and let stand on ice for 5min to completely separate the nucleic acid-protein complex. 2) RNA isolation: add 0.2mL chloroform, shake vigorously for 15s, let stand on ice for 2~3min, centrifuge at 12000rpm at 4°C for 15min, separate the layers, and take the upper aqueous phase, about 600μL. 3) RNA precipitation: add 500 μL of isopropanol, let stand on ice for 10 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes, and discard the supernatant. 4) RNA washing: add 1 mL of 75% (v / v) ethanol, suspend the pellet, let stand on ice for 10 min, centrifuge at 4°C, 7500 rpm for 15 min...

Embodiment 3

[0032] Example 3: Sequencing of the RNA sample of "Bailing" production fungus Cordyceps sinensis

[0033] After extracting the total RNA from the sample, the mRNA was enriched with Oligo(dT) magnetic beads. Add fragmentation buffer to break mRNA into short fragments (200~700bp), use mRNA as a template, use six base random primers (random hexamers) to synthesize the first cDNA strand, then synthesize the second cDNA strand, and then pass through QiaQuick PCR After the kit is purified and eluted with EB buffer, end repair is performed, polyA is added and sequencing adapters are connected, and then agarose gel electrophoresis is used for fragment size selection, and finally PCR amplification is performed. The built sequencing library is carried out with Illumina GA IIx sequencing. The original image data obtained by sequencing is converted into sequence data through base calling, that is, raw data or raw reads. The reads containing only the adapter sequence in the original sequ...

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Abstract

The invention relates to an inosine monophosphate (IMP) dehydrogenase from 'Bailing' producing strain Cordyceps sinensis Hirsutella sinensis for participating in anabolism on IMP to obtain a xanthylic acid, a gene for encoding the dehydrogenase and application of the dehydrogenase. The IMP dehydrogenase comprises a protein shown by SEQ ID No.1 and a protein shown by SEQ ID No.2, and the coding gene of the protein shown by SEQ ID No.1 and the coding gene of the protein shown by SEQ ID No.2 respectively correspond to a nucleotide sequence shown by SEQ ID No.3 and a nucleotide sequence shown by SEQ ID No.4. According to the invention, the metabolic pathway of the IMP to synthetize the xanthylic acid is traversed from the principle; the cloning deoxyribonucleic acids (DNA) of the nucleotide sequences provided by the invention can be transferred into engineering bacteria through a transduction method, a transformation method and a conjugal transfer method; and through regulating the expression of the biosynthetic gene of the xanthylic acid, the high expressivity of a host xanthylic acid is endowed, and an effective way for increasing the yield of the xanthylic acid is provided. The IMP dehydrogenase has a great application prospect.

Description

(1) Technical field [0001] The invention relates to an IMP dehydrogenase from the "Bailing" production fungus Cordyceps sinensis and participates in the synthesis and metabolism of xanthine nucleotides from hypoxanthine nucleotides, the gene encoding the enzyme and its application. (2) Background technology [0002] Cordyceps sinensis (Berk.) Sacc. is a complex of Cordyceps sinensis parasitizing on the larvae of Lepidoptera (Lepidoptera) bat moth (Hepialus armoricanus Oberthur) larvae and larval corpses (including subunits and worm bodies ). Cordyceps sinensis is a kind of precious traditional fungal medicinal resources, which has the characteristics of diverse metabolites and biological activities, and has shown great application and development prospects in the field of biomedicine. Cordyceps sinensis has attracted much attention for its extensive and obvious medicinal effects, and is highly respected all over the world. Chinese medicine believes that Cordyceps sinensis ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12P19/32C12N15/70C12N1/21C12R1/19
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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