Method for constructing isoeugenol monooxygenase active aggregate and method for producing vanillin by transforming isoeugenol

A technology of isoeugenol and monooxygenase, applied in the direction of microbial-based methods, oxidoreductase, biochemical equipment and methods, etc., can solve the problems of high price, damage to internal organs, and less than 20t

Pending Publication Date: 2017-10-10
SHENZHEN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Studies have found that large doses of chemically synthesized vanillin can cause dizziness, vomiting, and even damage internal organs
Natural vanillin is extracted from the pods of the vanilla plant. Because the planting of vanilla is affected by climate, place of origin, and local policies, the annual output is less than 20 tons, which is far from meeting people's needs, and the price is relatively expensive. The price per kilogram Dozens of times that of chemically synthesized vanillin
The application of active enzyme aggregates combined with embedding method or cross-linked enzyme aggregates (cross-linked enzyme aggregates, CLEA) to prepare immobilized enzymes has the advantages of simple operation and high activity, which can significantly reduce the cost of protein immobilization and simplify the process. It can be successfully applied to the preliminary purification of the target protein, which is the basis for the industrial scale-up and high-throughput purification of active enzyme preparation. At present, there are no publications or reports on the construction of active aggregates of isoeugenol monooxygenase

Method used

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  • Method for constructing isoeugenol monooxygenase active aggregate and method for producing vanillin by transforming isoeugenol
  • Method for constructing isoeugenol monooxygenase active aggregate and method for producing vanillin by transforming isoeugenol
  • Method for constructing isoeugenol monooxygenase active aggregate and method for producing vanillin by transforming isoeugenol

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preparation example Construction

[0072] Preparation of commonly used solutions and media:

[0073] (1) Ampicillin (100mg / mL): Weigh 5g of ampicillin, dissolve it in sterile water, dilute to volume in a 50mL volumetric flask, filter through a disposable filter, aliquot into 1.5mL EP tubes, and store at -40°C.

[0074] (2) Kanamycin (50mg / mL): Weigh 2.5g of Kanamycin, dissolve it in sterile water, dilute it in a 50mL volumetric flask, filter it with a disposable filter, and dispense it into a 1.5mL EP tube. Store at -40°C.

[0075] (3) IPTG (isopropylthiogalactopyranoside, 0.8mol / L): Weigh 0.2g IPTG, dissolve it in sterile water, dilute it in a 10mL volumetric flask, filter it with a disposable filter, and pack it in 1.5mL EP tube, store at -40°C.

[0076] (4) Nucleic acid electrophoresis buffer solution (1×TAE): Dilute the 50×TAE stock solution 50 times with ultrapure water, and store it at room temperature until use.

[0077] (5) Preparation of 1% agarose gel: Weigh 0.25g of agarose and dissolve it in 25mL...

Embodiment 1

[0132] Take out 50 μL from the glycerol tube E.coli BL21(DE3)pET30a-IEM-18A stored at -80°C, cultivate overnight according to the above-mentioned seed culture method, and inoculate 1 mL of seed liquid into the fermentation culture containing 50 μg / mL kanamycin Base, when OD600 is 0.8, add 0.8mmol / L IPTG, induction temperature is 30°C, 200rpm, induce for 16h; centrifuge the induced fermentation broth at 10,000rpm at low temperature (below 4°C) for 15min, discard the supernatant, and collect the cells , with 0.05mol / L glycine-sodium hydroxide buffer solution of pH 10.5 to resuspend the bacteria to prepare 80g / L cell suspension.

[0133] Take 10 mL of bacteria (cell suspension), add 260 mmol / L isoeugenol, transform at 25°C and 200 rpm for 48 hours, and measure the concentration of vanillin in the final reaction solution to be 2.24 g / L.

Embodiment 2

[0135] Take out 50 μL from the glycerol tube E.coli BL21(DE3)pET30a-IEM-18A stored at -80°C, culture it for 14 hours according to the above-mentioned seed culture method, and inoculate 1 mL seed liquid into the fermentation culture containing 50 μg / mL kanamycin Base, when OD600 is 0.8, add 0.8mmol / L IPTG, induce at 30°C, 200rpm, induction time is 8h, centrifuge the induced fermentation broth at 10 000rpm at low temperature (below 4°C) for 15min, discard the supernatant, and collect the bacteria cells; resuspend the cells with 0.05mol / L pH 10.5 glycine-sodium hydroxide buffer solution, crush the cell suspension under high pressure, collect the precipitate, add 10mL 0.05mol / L pH10.5 sodium carbonate-sodium hydroxide buffer solution, gently Resuspend, slowly add 121 μL of 50% glutaraldehyde solution dropwise at low temperature (below 4° C.), keep magnetic stirring for 2 hours, and then centrifuge to collect the precipitate to prepare isoeugenol monooxygenase-immobilized enzyme.

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Abstract

The invention discloses a method for constructing an isoeugenol monooxygenase active aggregate and a method for producing vanillin by transforming isoeugenol. The construction method comprises the following steps: S1, constructing recombinant escherichia coli for producing an isoeugenol monooxygenase active aggregate, namely binding oligopeptide 18A with an amphiphilic self-assembly function with a target gene monooxygenase active aggregate to construct an isoeugenol monooxygenase-18A active aggregate, and expressing in escherichia coli to obtain recombinant escherichia coli; and S2, producing the isoeugenol monooxygenase active aggregate with the constructed recombinant escherichia coli, namely performing cultural optimization and induction on the constructed recombinant escherichia coli, centrifugally collecting the induced strain, and disrupting cells to obtain crude enzyme which is the isoeugenol monooxygenase active aggregate. The method realizes efficient production of vanillin and is convenient for industrial production and application.

Description

technical field [0001] The present invention relates to the construction of genetically engineered bacteria of isoeugenol monooxygenase and the technical field of biocatalysis, in particular to a method for constructing active aggregates of isoeugenol monooxygenase and a method for converting isoeugenol to produce vanillin . Background technique [0002] Vanillin is one of the most demanded spices in the world and is widely used in food, beverage, medicine, cosmetics and other industries. The vanillin currently on the market is divided into two types: natural vanillin and chemically synthesized vanillin. Among them, chemically synthesized vanillin accounts for more than 90% of the market share. Studies have found that large doses of chemically synthesized vanillin can cause dizziness, vomiting, and even damage internal organs. Natural vanillin is extracted from the pods of the vanilla plant. Because the planting of vanilla is affected by climate, place of origin, and loca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/04C12P7/24C12R1/19
CPCC12N9/0006C12N15/70C12P7/24C12Y101/01319
Inventor 赵丽青
Owner SHENZHEN UNIV
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