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Recombinant yarrowia lipolytica for expressing myrosinase TGG4 and application of recombinant yarrowia lipolytica

A technology of myrosinase and TGG4, which is applied in the field of functional enzymes and can solve the problems of cumbersome steps, high price and high production cost

Active Publication Date: 2021-06-04
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of this method is high, the steps are cumbersome, and the loss of radishin is large
In addition, exogenous enzymes can also be used for the preparation of radish, but these exogenous enzymes are generally purified from plants and are expensive
So in general, the preparation cost of radishine is relatively high, so it is of great significance to develop a simple and efficient preparation method of radishine, which can simplify the preparation process of radishine and reduce its cost

Method used

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  • Recombinant yarrowia lipolytica for expressing myrosinase TGG4 and application of recombinant yarrowia lipolytica
  • Recombinant yarrowia lipolytica for expressing myrosinase TGG4 and application of recombinant yarrowia lipolytica
  • Recombinant yarrowia lipolytica for expressing myrosinase TGG4 and application of recombinant yarrowia lipolytica

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of embodiment 1 myrosinase gene TGG4

[0039] The myrosinase gene TGG4 of the present invention is obtained by whole gene synthesis, and the synthesis unit is Sangon Biotech Group (Shanghai) Co., Ltd., and the synthesis address is Qingdao Branch Factory. After the inventor obtained the gene fragment, the sequence contained 1467 base sequences, as shown in SEQ ID NO.1, and encoded 489 amino acid sequences, as shown in SEQ ID NO.2.

[0040] Using the synthesized gene fragment as a template, primers for seamless connection were designed on the upstream and downstream of the myrosinase gene, and the TGG4 gene fragment was amplified by PCR.

[0041] The sequences of the primers are as follows:

[0042] Upstream primer: 5'-CGGCCGTTCTGGCCTCCCAGAAGGTTTGTAACCCAG-3', as shown in SEQ ID NO.3;

[0043] Downstream primer: 5'-CTCTAAGTTCCTTGCGAAGATGAGCAAAG-3', as shown in SEQ ID NO.4.

[0044] The green fluorescent protein GFP was connected behind the TGG4 gene, and primers...

Embodiment 2

[0051] The expression vector construction of embodiment 2 myrosinase gene

[0052] The gene fragment was connected with the pINA1314 cloning vector using seamless cloning technology, and the connection product was transferred into E.coli DH5α competent cells, and spread on a solid plate of (LB) medium containing 50 μg / m L kanamycin. After culturing in a 37°C incubator for 12-16 hours, pick a single clone into LB liquid medium containing 50 μg / mL kanamycin, culture overnight on a 37°C shaker with a rotation speed of 220 rpm, sequence after positive verification, and name it pINA1314- TGG4-GFP.

Embodiment 3

[0053] The recombinant plasmid of embodiment 3 myrosinase gene and the construction of engineering bacteria

[0054] The recombinant plasmids with correct sequencing were extracted, primers were designed to linearize the plasmids, and transformed into host Y.lipolyticaPO1g competent cells, and the constructed engineering bacteria were grown on uracil-deficient plates.

[0055] The sequences of the primers are as follows:

[0056] Upstream primer: 5'-TCTACTGAACGGTGATCCCCACCGGA-3', as shown in SEQ ID NO.7;

[0057] Downstream primer: 5'-ATCTCATGCTGGAGTTCTTCGCCCAC-3', as shown in SEQ ID NO.8.

[0058] The PCR reaction system is: 2×PCR Buffer 25 μl, dNTP 10 μl, primers 1.5 μl, template 1 μl, KOD Fx enzyme 1 μl, sterile water 10 μl, total system 50 μl.

[0059] The PCR reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 95°C for 20 s, annealing at 60°C for 30 s, extension at 72°C for 300 s, 30 cycles of reaction, and post-extension at 72°C for 10 min.

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Abstract

The invention discloses recombinant yarrowia lipolytica for expressing myrosinase TGG4. A genome of the recombinant yarrowia lipolytica contains a gene for coding the myrosinase TGG4. The gene for coding the myrosinase TGG4 is as shown in SEQ ID NO. 1; and the myrosinase TGG4 is as shown in SEQ ID NO. 2. The recombinant yarrowia lipolytica is applied to the preparation of myrosinase TGG4, applied to preparation of sulforaphane, and applied to degradation of glucoraphenin. The invention also discloses an enzyme preparation which is prepared by culturing the recombinant yarrowia lipolytica and collecting thalli. The food-grade yarrowia lipolytica provided by the invention can efficiently express the myrosinase TGG4, and can catalyze the hydrolysis of glucoraphenin in radish seeds in vitro to generate sulforaphene. The myrosinase TGG4 is attached to or embedded in the surfaces of the cells, the repeated utilization of the myrosinase can be realized by recycling the cells, and high-efficiency and multi-batch repeated preparation is carried out.

Description

technical field [0001] The invention relates to a recombinant Yarrowia lipolytica expressing myrosinase TGG4, an enzyme preparation, and an application in preparing rataxin, belonging to the technical field of functional enzymes. Background technique [0002] Rathamin is one of the substances with the strongest anti-cancer activity in nature. It also has strong effects in anti-inflammation, anti-oxidation, and obesity suppression. It is expected to become a new type of anti-cancer specific drug. The content of radhamin in cruciferous plants in nature is very small, and it needs myrosinase to catalyze the hydrolysis of the substrate radishin, which is extremely unstable. The steps in the extraction and purification process are cumbersome, the loss is very large, and the cost is high. Therefore, a high-efficiency method is sought. The method of preparation of radish is crucial. [0003] At present, the preparation of radishin mainly uses cruciferous plants, such as radish see...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N9/24C12N15/56C12N15/81C12P13/00C12R1/645
CPCC12N9/2402C12N15/815C12P13/00C12Y302/01147
Inventor 毛相朝姜宏王丽丽薛长湖
Owner OCEAN UNIV OF CHINA
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