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Optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant thallus, surface display system and application

A technology of zearalenone and surface display carrier, applied in the field of genetic engineering, can solve the problems of less than 15%, no literature extraction and purification and enzymatic properties, difficult to remove strains in the later stage, etc., and achieves the effect of reducing costs.

Pending Publication Date: 2021-03-02
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the report of Delfina Popiel et al., the ZEN degradation efficiency of living bacteria containing the original gene of zearalenone degrading enzyme ZHD-P invading Trichoderma is very low. After 4 days of cultivation, the degradation rate of ZEN is less than 15%. After 6 days of cultivation, the degradation rate of ZEN reached about 50%.
Detoxification of living bacteria by using the invading Trichoderma containing the original gene of zearalenone-degrading enzyme ZHD-P has problems such as long culture time of the strain, low degradation activity and difficulty in removing the strain itself in the later stage, which is of little significance for practical application.
At present, there is no experiment to prove that the original gene of the zearalenone degrading enzyme ZHD-P can be expressed heterologously in other hosts, and there is no literature on the extraction, purification and enzymatic properties of the enzyme.

Method used

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  • Optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant thallus, surface display system and application
  • Optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant thallus, surface display system and application
  • Optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant thallus, surface display system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062]Example 1. Construction of recombinant expression vector

[0063]1. Artificial synthesis of gene sequence

[0064]The optimized nucleotide sequence shown in SEQ ID NO. 1 was entrusted to Nanjing GenScript Biotechnology Co., Ltd. to artificially synthesize the gene according to conventional techniques in the field, insert the gene into the plasmid vector pUC57, and store it for later use.

[0065]2. Amplification of gene sequence and linearization of expression vector

[0066]Use primer zhd-pF (as shown in SEQ ID NO.3, Primer means primer, the same below) and primer zhd-pR (as shown in SEQ ID NO.4) to amplify the nucleus shown in SEQ ID NO.1 Nucleotide sequence, and named it zhd-pDNA fragment.

[0067]The plasmid pET22b(+) was digested with NdeI and XhoI, and the digested products were recovered by agarose electrophoresis, and the linearized expression vector pET22b(+) was obtained.

[0068]3. Construction of recombinant expression vector

[0069]Using NEBuilder HiFi DNA Assembly Cloning Kit, conne...

Embodiment 2

[0073]Example 2. Expression and purification of target protein

[0074]1. IPTG induced expression of recombinant bacteria

[0075]The positive recombinant bacteria BL21 / pET22b(+)-zhd-p prepared in step 4 of the above example 1 was inoculated into 10mL LB liquid medium (containing 100mg / L Amp), cultured at 37°C for 8-10h, then 1mL was transferred Receive 100mL LB liquid medium (containing 100mg / L Amp). Incubate at 37°C, 200 rpm, for 2 hours, measure OD600, and when OD600 exceeds 0.7A (no more than 0.8A), add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1mmol / L, Incubate at 22°C, 200 rpm, for 16-22 hours. The control bacteria prepared in step 4 of Example 1 were cultured using the same procedure.

[0076]2. Preparation of crude enzyme solution

[0077]After the induction is over, put the fermentation broth in two 100mL centrifuge tubes, centrifuge at 4℃, 8000rpm for 5min, pour out the supernatant, add 20mL TE buffer, resuspend the bacteria, 4℃, 8000rpm, centrifuge 5min, ...

Embodiment 3

[0088]Example 3. Test for degradation of zearalenone by recombinant zearalenone degrading enzyme ZHD-P

[0089]The protein concentration of the recombinant zearalenone ZHD-P pure enzyme solution in step 4 of Example 2 was determined to be 1.3891 mg / mL. The recombinant zearalenone degrading enzyme ZHD-P pure enzyme solution in step 4 of Example 2 was diluted 4 times with Buffer A solution (0.5M Nacl, 20Mm Tris, adjusted to pH 8.0 with HCl solution). The protein concentration of the enzyme solution was 0.3473 mg / mL. The diluted enzyme solution is tested for enzyme activity. The diluted enzyme solution is recorded as the diluted enzyme solution.

[0090]Using zearalenone as the substrate, the degradation rate of the substrate by the zearalenone degrading enzyme ZHD-P was determined. The determination method is: 5μL of diluted enzyme solution, 5.0μL of 1.0mg / ml ZEN and 240.0μL of 50mmol / L Tris-HCl (pH 7.5). After mixing, react for 30min at 45℃, and then add 250μL of methanol to stop. reaction...

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Abstract

The invention discloses an optimized zearalenone degrading enzyme ZHD-P encoding gene, a recombinant thallus, a surface display system and application. The nucleotide sequence of the zearalenone degrading enzyme encoding gene is as shown in SEQ ID NO. 1. The coding gene can realize heterologous high-efficiency expression in escherichia coli. The recombinant zearalenone degrading enzyme has the advantages of being high in enzyme activity, wide in temperature and pH application range, good in tolerance and the like, zearalenone (ZEN) can be efficiently degraded, and the recombinant zearalenone degrading enzyme has good industrial application prospects. Zearalenone degrading enzyme ZHD-P is displayed on the surface of escherichia coli BL21 cells in a positioned mode, detection of whole-cell enzyme activity shows that display expression of zearalenone degrading enzyme ZHD-P on the surfaces of the cells is successfully achieved, and the whole-cell catalyst which does not need to be purified, can be repeatedly used and is low in cost is obtained. The method is used for biodegradation of zearalenone, and has a good industrial application prospect.

Description

Technical field[0001]The invention relates to the field of genetic engineering, in particular to an optimized zearalenone degrading enzyme ZHD-P coding gene, recombinant bacteria, surface display system and application.Background technique[0002]Zearalenone (Zearalenone, ZEN, ZEA), also known as F-2 toxin, is an estrogenic mycotoxin with a dihydroxybenzoic acid lactone structure. It has strong heat resistance and can be treated at 110°C for 1 hour. Completely destroyed. Studies have shown that ZEN enters the food chain through contaminated grain, agricultural and sideline products and feed. After being absorbed by animals, it causes estrogen syndrome in livestock, resulting in excessive estrogen in livestock, causing infertility, abortion and stillbirth. Its carcinogenicity seriously endangers the health of livestock and humans.[0003]ZEN is the most widely polluted mycotoxin in the world. The Fusarium bacteria that can produce ZEN include Fusarium graminearum, Fusarium triline, Fusar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/70C12N1/21C12N9/18C12N15/66A23L5/20
CPCC12N9/18C12N15/70C12N15/66A23L5/25C12N2800/22
Inventor 陈书容潘力王斌
Owner SOUTH CHINA UNIV OF TECH
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