Escherichia coli for producing isoeugenol monooxygenase and construction method and application of Escherichia coli

A technology of Escherichia coli and isoeugenol, which is applied in the field of Escherichia coli producing isoeugenol monooxygenase and its construction

Active Publication Date: 2014-09-10
中科阿尔诺(深圳)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports of recombinant bacteria transforming isoeugenol to produce vanillin

Method used

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  • Escherichia coli for producing isoeugenol monooxygenase and construction method and application of Escherichia coli
  • Escherichia coli for producing isoeugenol monooxygenase and construction method and application of Escherichia coli
  • Escherichia coli for producing isoeugenol monooxygenase and construction method and application of Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Acquisition and preservation of engineering bacteria

[0032] 1. Construction of engineering bacteria

[0033] 1. Take soil samples around Wenshan Lake in Shenzhen University to Spin Kit for Soil (soil genomic DNA extraction kit) extracts the whole genome DNA of soil.

[0034] 2. According to the known gene sequence related to isoeugenol monooxygenase, design PCR primers, including upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2.

[0035] The sequence of SEQ ID NO.1 is as follows: 5'-atgctacatatggcaacgtttgaccgcaat-3'

[0036] The sequence of SEQ ID NO.2 is as follows: 5'-cttatctctcgaggttcttagactgccaac-3'

[0037] 3. Using the whole genome DNA as a template, carry out PCR amplification with the PCR primers designed in step 2 (PCR amplification adopts a conventional method: 10 μL of 5*FastPfu buffer, 0.5 μL of template DNA, 1 μL of primer SEQ ID NO.1, and 1 μL of SEQ ID NO.1. ID NO.2 is 1 μL, dNTPS 1.25 μL, Transtart FastPfu DNA polymerase 1 ...

Embodiment 2

[0043] Embodiment two: application engineering bacterium produces isoeugenol monooxygenase

[0044] 1. Preparation of culture medium

[0045] Seed culture medium (pH7.0): Take 10g tryptone, 5g yeast extract and 5g sodium chloride, dissolve in water and dilute to 1L; sterilize at 121°C for 30min.

[0046] Fermentation medium (pH 7.0): Take 10g tryptone, 15g yeast extract, 5g sodium chloride, 10g glycerin, 10mM potassium dihydrogen phosphate and 65mM dipotassium hydrogen phosphate, dissolve in water and dilute to 1L; extinguish at 121°C Bacteria 30min.

[0047] 2. Application of engineering bacteria to produce isoeugenol monooxygenase

[0048] 1. Inoculate Escherichia coli BL21(DE3) IEM-PP into the seed medium, shake culture at 35°C (100r / min) to OD 600nm =3, namely the seed solution.

[0049] 2. Use a pipette to inoculate the seed solution in step 1 into 40 mL fermentation medium (250 mL shake flask can be used) at an inoculum size of 1% (v / v) to obtain OD 600nm =0.1 ferme...

Embodiment 3

[0054] In a 50mL Erlenmeyer flask, add 0.4g of isoeugenol, 1g of isoeugenol monooxygenase, add 9mL of glycine / NaOH buffer solution with a pH of 10.4 and a glycine concentration of 100mM, DMSO1mL, cover the mouth with two layers of gauze , transformed at 28° C. and 200 rpm for 48 h on a shaking table, and the concentration of vanillin in the final reaction solution was determined to be 2.4 g / L.

[0055] Determination method of vanillin:

[0056] Determination of vanillin and isoeugenol by high performance liquid chromatography (HPLC).

[0057] Sample treatment: Add the same volume of ethanol to the reaction solution to precipitate the protein (isoeugenol monooxygenase), dissolve the substrate (isoeugenol) and product (vanillin), centrifuge at 10,000r / min for 5min, and then Dilute it 10-20 times with ethanol (10 times in this example), filter through a filter membrane, and wait for the test.

[0058] Chromatographic column: Lichrospher100RP-18 column (250mm×4mm×5μm);

[0059]...

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PUM

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Abstract

The invention discloses an Escherichia coli for producing isoeugenol monooxygenase and a construction method and an application of Escherichia coli. The Escherichia coli is named as BL21 (DE3) IEM-PP, and the preservation number is CGMCC No. 8918. The construction method comprises the steps of amplifying whole genome DNA of soil as a template by a PCR (polymerase chain reaction) technology under the action of PCR primers to obtain fragments of which the lengths are 1438bp, carrying out gel extraction and recovery, purifying and linking with a T-vector pET21 (a) to obtain the recombinant strain BL21 (DE3) IEM-PP cloning vector recombinant plasmid PET21 (a)-IEM. The Escherichia coli can be applied in producing vanillin by the biotransformation of isoeugenol via the recombinant strain.

Description

technical field [0001] The invention relates to the construction of genetically engineered bacteria of isoeugenol monooxygenase and the technical field of biocatalysis, in particular to an Escherichia coli for producing isoeugenol monooxygenase, its construction method and application. Background technique [0002] Vanillin is one of the most widely used spices in the industry. It is widely used in the food industry. It can be used as the main raw material for aroma modification and fragrance fixation in food, toothpaste, soap, and tobacco; it can be used as an important raw material or intermediate in pharmaceutical and chemical industries. It can be used in the manufacture of commonly used drugs for treating hypertension, heart disease, skin diseases, and eliminating bad breath and diuresis; it can be used as a chemical auxiliary in the chemical industry, and can be used as an anti-hardening agent for plastic products and for metals such as Ni, Cr, and Cd. Electroplating b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/02C12P7/24C12R1/19
Inventor 赵丽青马田田吴序栎李小曼宋江宁
Owner 中科阿尔诺(深圳)生物科技有限公司
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