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Recombinant gene of glutamate dehydrogenase and its acquisition method and application

A glutamate dehydrogenase and recombinant gene technology, applied in the field of glutamate dehydrogenase recombinant gene and its acquisition, can solve the problems of unsuitability for industrial production, low enzyme yield and high cost, and achieve low production cost, High degradation efficiency and short production time

Inactive Publication Date: 2019-11-01
NORTHWESTERN POLYTECHNICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzyme production in the bacteria is low, time-consuming and costly, not suitable for industrial production

Method used

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  • Recombinant gene of glutamate dehydrogenase and its acquisition method and application
  • Recombinant gene of glutamate dehydrogenase and its acquisition method and application
  • Recombinant gene of glutamate dehydrogenase and its acquisition method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Step 1: Cell preparation:

[0109] Inoculate Geotrichum candidum CCTCC NO: M 208167 strain in potato juice glucose liquid medium (20% potato juice 1000mL, glucose 20 g, natural pH), culture at 28°C, 160 rpm for 18 hours, press 2.0% The inoculum amount was inserted into the above-mentioned culture medium to expand the culture, the conditions were the same as above, and the culture time was 38h. The obtained culture was centrifuged at 5000rpm and 4°C for 10min, and the bacteria were collected, resuspended and centrifuged with sterile water, repeated 5 times, and finally Centrifuge at 8000rpm and 4°C for 10min to collect the wet cells, induce treatment with MSM medium at 28°C and 160rpm for 6h, then centrifuge at 5000rpm and 4°C for 10min to collect the cells, wash them with sterile water for 5 times, and finally Centrifuge at 8 000 r / m for 10 min at 4°C to collect the cells and extract total RNA.

[0110] Step 2: Extraction of bacterial total RNA:

[0111] The total RNA...

Embodiment 2

[0114] Step 1: Cell preparation:

[0115] With embodiment 1.

[0116] Step 2: Cloning of conserved regions and full-length sequences:

[0117] ① Conserved region amplification primers:

[0118] GDH-F1 5'- GGATC CACGCCGCTCAAGGTC-3' BamHI

[0119] GDH-R1 5'- AAGCTT TACCAAGAAAATCACCGTGGTC-3' HindⅢ

[0120] Primers for full-length sequence amplification:

[0121] GDH-F2 5'-CG GGATCC ATCAAAATGGTCCAGCCTTCC-3' BamHI

[0122] GDH-R2 5'-CCC AAGCTT TTACCAGAAATCACCGTGGTCG-3' HindⅢ

[0123] ② PCR amplification system for the conserved region and full-length sequence of glutamate dehydrogenase gene

[0124]

[0125] ③PCR reaction conditions

[0126] Reaction conditions in the conserved region: pre-denaturation at 95 °C for 5 min; denaturation temperature at 94 °C for 30 s; annealing temperature at 55.6 °C and 58.0 °C for 30 s; extension temperature at 72 °C for 1:30 min; after 35 cycles, at 72 °C Extend for 10 min.

[0127] Reaction conditions for the full-length sequen...

Embodiment 3

[0131] Step 1: Cell preparation:

[0132] With embodiment 1.

[0133] Step 2: Expression of the conserved region and full-length sequence of the enzyme gene:

[0134] (1) After the PCR amplification product is recovered, it is ligated with pMD19-T (pMD19-T-658-GDH (conserved region) and pMD19-T-1359-GDH (full-length sequence)) and transformed E. coli DH5α competent cells, pick positive colonies, extract plasmids for PCR detection, and the positive clones have been sequenced. Then the recombinant T vector (pMD19-T-658-GDH (conserved region) and pMD19-T-1359-GDH (full-length sequence)) and the expression vector (pET-28as) were digested with BamHI and HindIII.

[0135]

[0136] (2) React overnight at 37 °C, perform 1% agarose gel electrophoresis, cut the gel to recover the target band, and purify it with the Omega Gel Recovery Kit. Then connect the expression vector, and connect the target fragment and the expression vector after digestion.

[0137]

[0138] Ligation ...

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Abstract

The invention relates to a recombinant gene of glutamate dehydrogenase, an acquisition method of the recombinant gene, and an application of glutamate dehydrogenase recombinant enzyme. At present, the degradation of higher alcohols in food is poor in specificity, the degradation of the higher alcohols under acidic conditions is restricted, the yield of enzymes in thalli is low, time consuming is long, cost is high and industrial production can not performed. Conserved regions and full-length sequences of glutamate dehydrogenase genes in galactomyces geotrichum T3d329TF strain which are capable of efficiently degrading the higher alcohols under the acidic conditions are subjected to cloning and expression, and recombinant enzyme activity verification is performed. The recombinant enzyme capable of degrading n-hexyl alcohol and isopentanol under the acidic conditions is used as a catalyst for directionally reducing the content of higher alcohols in foods, especially in acidic foods, and the application is safe and environmentally friendly, high in controllability, low in production cost, and suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to a recombinant gene of an enzyme, in particular to a recombinant gene of glutamic acid dehydrogenase and its obtaining method and application. Background technique [0002] Higher alcohols are also called fusel oils. Excessive higher alcohols will have a certain toxic effect on people, thereby damaging the peripheral nervous system, central nervous system, digestive system and reproductive system of people, and also harmful to the mother and fetus during pregnancy, and Breast cancer is also relevant. The anesthesia effect of higher alcohol on the human nervous system is more than ten times that of ethanol. Its oxidation rate in the body is slower than that of ethanol, and the residence time is longer, so it is harmful to health. National standards strictly limit the content of fusel oil in liquor. At present, n-hexanol has been listed as a substance that can cause harm to the human body (HSDB, Hazardous Substances Data Bank). ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/10C12N15/70A23L29/00
CPCC12N9/0016C12Y104/01002
Inventor 师俊玲朱静徐晓光
Owner NORTHWESTERN POLYTECHNICAL UNIV
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