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Recombinant gene of glutamate dehydrogenase, acquisition method of recombinant gene, and application of glutamate dehydrogenase recombinant enzyme

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

Inactive Publication Date: 2016-11-09
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, acquisition method of recombinant gene, and application of glutamate dehydrogenase recombinant enzyme
  • Recombinant gene of glutamate dehydrogenase, acquisition method of recombinant gene, and application of glutamate dehydrogenase recombinant enzyme
  • Recombinant gene of glutamate dehydrogenase, acquisition method of recombinant gene, and application of glutamate dehydrogenase recombinant enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Step 1: Preparation of bacteria:

[0109] The Geotrichum candidum CCTCC NO:M 208167 strain was inoculated into potato juice glucose liquid medium (1000 mL of 20% potato juice, 20 g glucose, natural pH), and cultured at 28°C and 160 rpm for 18 hours, at 2.0% The inoculum amount is connected to the above medium to expand the culture, the conditions are the same, the culture time is 38h, the resulting culture is centrifuged at 5000rpm, 4℃ for 10min, the bacteria are collected, resuspended and centrifuged with sterile water, repeated 5 times, and finally Centrifuge at 8000 rpm and 4°C for 10 min to collect the wet bacteria. After induction treatment with MSM medium at 28°C and 160 rpm for 6 hours, centrifuge at 5000 rpm and 4°C for 10 minutes to collect the bacteria and wash it with sterile water 5 times. 8 000 r / m, centrifugation at 4 ℃ for 10 min, collect bacteria to extract total RNA.

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

[0111] The method for extracting total...

Embodiment 2

[0114] Step 1: Preparation of bacteria:

[0115] The same as in Example 1.

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

[0117] ①Conserved region amplification primers:

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

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

[0120] Full-length sequence amplification primers:

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

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

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

[0124]

[0125] ③PCR reaction conditions

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

[0127] Reaction conditions for the full-length sequence: pre-denaturation 95 ℃, 3 min; denaturation temperature 94 ℃, ...

Embodiment 3

[0131] Step 1: Preparation of bacteria:

[0132] The same as in Example 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 connected to pMD19-T (pMD19-T-658-GDH (conserved region) and pMD19-T-1359-GDH (full-length sequence)) and transformed E.coli DH5α competent cells, pick the positive colonies, extract the plasmid and PCR detection, 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 expression vector (pET-28as) were digested with BamHI and HindⅢ.

[0135]

[0136] (2) Place the reaction at 37 ℃ overnight, perform 1% agarose gel electrophoresis, cut the gel to recover the target band, and purify it with the Omega gel recovery kit. Then the expression vector is connected, and the target fragment after restriction digestion is connected with the expression vector....

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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 glutamate dehydrogenase, and an obtaining method and application thereof. Background technique [0002] Higher alcohols are also called fusel oils. Excessive higher alcohols can have a certain toxic effect on humans, thereby damaging human peripheral nervous system, central nervous system, digestive system and reproductive system, and also harm the mother and fetus during pregnancy. Breast cancer is also related. The anesthetic 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 it stays for a longer time, so it is harmful to health. The national standard strictly limits 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). The targeted reduc...

Claims

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Application Information

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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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