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Method using biological method to prepare raspberry ketone

A technology of raspberry ketone and biological method is applied in the field of bioengineering, and can solve the problems of relying on the supply of anisaldehyde raw materials, and achieve the effects of increasing yield and increasing conversion efficiency.

Active Publication Date: 2018-11-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The aroma of raspberry ketone synthesized from anisaldehyde is similar to that of naturally extracted raspberry ketone, and can be used in the formulation of high-end daily chemical flavors and food flavors, but this semi-synthetic method depends on the supply of anisaldehyde raw materials

Method used

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  • Method using biological method to prepare raspberry ketone
  • Method using biological method to prepare raspberry ketone
  • Method using biological method to prepare raspberry ketone

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Embodiment 1

[0032] The nucleotide sequence of the gene 4CL encoding the 4-coumaroyl-CoA ligase is shown in SEQ ID NO.1, which is derived from the plant parsley (Petroselinum crispum) and codon-optimized; encoding the benzyl The nucleotide sequence of the gene BAS of acetone synthase is shown in SEQ ID NO.2, derived from the plant Rheum palmatum (Rheum palmatum) and through codon optimization; the nucleoside of the gene RiRZS1 encoding the benzyl acetone reductase The acid sequence is shown as SEQ ID NO.3, which is derived from the plant Raspberry ketone and has been codon optimized.

[0033] Construction of plasmid pACYC-BAS-4CL:

[0034]Use primers A1 (SEQ ID No.4) and A2 (SEQ ID No.5) to clone gene 4CL2 (SEQ ID NO.1 encoding 4-coumaroyl-CoA ligase) by PCR: use primer B1 (SEQ ID No. 5) by PCR No.6) and B2 (SEQ ID No.7) were cloned to obtain the gene BAS (SEQ ID NO.2 encoding benzylacetone synthase). First, the recombinant plasmid pACYC-4CL was obtained by one-step cloning with EcoRI an...

Embodiment 2

[0044] Construction of plasmid pET-RZS1:

[0045] Select pET-Duet-1 as the expression vector of RiRZS1 gene. The gene RiRZS1 (SEQ ID NO.3 encoding benzylacetone reductase) was cloned by PCR using primers C1 (SEQ ID NO.8) and C2 (SEQ ID NO.9). After the vector pET-Duet-1 was digested with BglII and KpnI restriction endonucleases, the RiRZS1 gene was inserted into the corresponding restriction site of the plasmid pET-Duet-1 by a one-step cloning method to obtain the recombinant plasmid pET-RZS1 . The PCR reaction conditions were as follows: 95°C for 5 min; 98°C for 10 s, 55°C for 5 s, 72°C for 5 s / kb (30 cycles); 72°C for 10 min, and then verified by 1% agarose gel electrophoresis and recovered the PCR amplification product. One-step cloning specific method Mix fragment (0.04-0.08ng / bp) and vector (0.005-0.01ng / bp) in the same PCR tube, add 2-4μl of recombinase buffer and 1-2μl of recombinase, add ddH2O Make up to 10-20 μl and react at 37°C for 30 minutes to obtain the recomb...

Embodiment 3

[0051] Construction of raspberry ketone producing strain Mu1:

[0052] Plasmids pACYC-BAS-4CL2 and pET-RZS1 were simultaneously introduced into Escherichia coli BL21(DE3) by heat shock method, and the resulting positive strain was named Mu1. The antibiotics in the medium were chloramphenicol (25-50mg / mL) and ampicillin (50-100mg / mL). Among them, the basic operation of thermal shock method:

[0053] 1) Thaw the Escherichia coli competent cells stored at -80°C in ice for 10 minutes;

[0054] 2) Add 0.1ng-10ng of the corresponding plasmid to E. coli competent cells, mix gently and place in ice for 30min;

[0055] 3) Heat shock in a water bath at 42°C for 45-90 seconds, and immediately place it in ice for 1-2 minutes;

[0056] 4) Add 890 μl of LB medium;

[0057] 5) Place in a shaker at 37°C, 180rmp-300rmp, and incubate for 1 hour;

[0058] 6) Take an appropriate amount of bacterial solution to coat the plate, and place the plate upside down in a 37°C incubator overnight;

...

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PUM

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Abstract

The invention discloses a method using a biological method to prepare raspberry ketone. The method includes: simultaneously expressing plant-source 4-coumaryl coenzyme A ligase, benzyl acetone synthase and benzyl acetone reductase in Escherichia coli in an induced manner. The method has the advantages that the benzyl acetone reductase (RiRZS1) is added to the metabolic pathway of biological raspberry ketone synthesis, so that the conversion efficiency of the intermediate product 4-hydroxybenzal acetone is increased; in addition, the yield of the shaking-flask-fermentation raspberry ketone reaches 70.62mg / L, and the yield of the raspberry ketone is evidently increased by the biological synthesis method.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for preparing raspberry ketone by a biological method. Background technique [0002] Raspberry ketone, also known as raspberry ketone, is the main aroma component of raspberry fruit and has a characteristic sweet fruit aroma. Found in fruits and vegetables such as raspberries, blackberries, grapes and rhubarb. The use of raspberry ketone is diverse. As a flavor essence, modifier or fixative, it is widely used in daily flavors, food flavors, daily chemical flavors, tobacco flavors and cosmetics. In addition, raspberry ketone, as a fine chemical intermediate, can be used in the synthesis of drugs, dyes and pesticides. Nowadays, raspberry ketone has become a spice with extremely high economic value after vanillin in the spice industry. [0003] There are three main methods of preparing raspberry ketone, plant extraction, chemical synthesis and biosynth...

Claims

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Application Information

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IPC IPC(8): C12P7/26C12N15/70C12N15/53C12N1/21C12R1/19
CPCC12N9/0004C12N15/70C12P7/26
Inventor 郑璞王程程陈鹏程
Owner JIANGNAN UNIV
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