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Aspergillus cristatus feruloyl esterase gene as well as engineering bacteria and application of thereof

A ferulic acid esterase and gene technology, applied in the field of genetic engineering, can solve problems such as low enzyme activity

Inactive Publication Date: 2018-08-10
SHANDONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on ferulic acid esterase mainly focuses on the optimization of the original strain culture conditions to increase the enzyme activity or enzyme production. Some researchers also use genetic engineering technology to improve the enzyme activity of ferulic acid esterase, but the enzyme still generally low

Method used

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  • Aspergillus cristatus feruloyl esterase gene as well as engineering bacteria and application of thereof
  • Aspergillus cristatus feruloyl esterase gene as well as engineering bacteria and application of thereof
  • Aspergillus cristatus feruloyl esterase gene as well as engineering bacteria and application of thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: the PCR amplification of aspergillus coronis feruloesterase gene

[0032] Total RNA was extracted from 5 mL of Aspergillus coronis culture medium with strain number 3.6088 in the General Microbiology Center (CGMCC) of China Committee for Culture Collection of Microbial Cultures, and the RNA extraction kit was used to extract the total RNA. The extracted total RNA was used as a template to carry out reverse transcription with a reverse transcription kit to obtain cDNA. The reverse transcription steps refer to the instructions of the reverse transcription kit from Takara Company.

[0033] The nucleotide sequences of ferulic acid esterase genes of different strains of the same genus were retrieved from GenBank, and a pair of primers fae-F and fae-R were designed. The primer sequences are as follows:

[0034] fae-F: 5'-ATGAAACTCTTGACTGCAACA-3'SEQ ID NO.3;

[0035] fae-R: 5'-CTACCAGGAACAGGCTCC-3' SEQ ID NO.4;

[0036] The cDNA was used as a template for ampl...

Embodiment 2

[0038] Example 2: Expression and purification of Aspergillus coronis feruloesterase gene in Pichia pastoris SMD1168H

[0039] Primers fae-A-F and fae-A-R were designed according to the sequence shown in SEQ ID No.1, respectively introduced into the EcoRI and XbaI restriction sites (shown underlined) that can be inserted into the pGAPZα-A plasmid (purchased from Invitrogen, USA), and the sequences are as follows

[0040] fae-A-F: 5'-GCG GAATTC ATGAAACTCTTGACTGCAACA-3' SEQ ID NO. 5;

[0041] fae-A-R: 5'-GCG TCTAGA AACTACCAGGAACAGGCTCC-3' SEQ ID NO. 6;

[0042] Using the reverse-transcribed Aspergillus coronis cDNA as a template, the above primers were used for PCR amplification. PCR amplification conditions and PCR product recovery method were the same as in Example 1. After the PCR product was recovered, EcoRI and XbaI (NEB, USA) were added to digest at 37°C for 1 hour for DNA purification, and the pGAPZα-A plasmid vector was treated with the same digestion method. The p...

Embodiment 3

[0051] Example 3: Recombinant Aspergillus coronis ferulic acid esterase catalyzes the production of ferulic acid in vitro

[0052] The eluate of recombinant Aspergillus coronis feruloesterase obtained in Example 2 is added in a 10kD ultrafiltration tube, centrifuged at 6000rpm until the unfiltered solution volume is 1 / 10 of the original volume, and 50mmol of the filtered volume is added / L Tris·HCl (pH7.0) buffer. Repeat the above steps 2 times to remove imidazole and NaCl in the eluent, and finally collect the concentrated ferulic acid esterase solution. The concentration of ferulic acid esterase in the obtained ferulic acid esterase solution was determined by SDS-PAGE electrophoresis and Bradford method.

[0053] Take 10 g of starch-free wheat bran and mix with 200 ml of 50 mmol / LTris·HCl (pH 5.0) buffer solution, and stir evenly to prepare a wheat bran suspension. Take 1 mg of the above-mentioned recombinant ferulic acid esterase and 1 mg of cellulase, mix with the above-...

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Abstract

The invention belongs to the field of genetic engineering, and in particular relates to an aspergillus cristatus feruloyl esterase gene as well as engineering bacteria and application thereof. The nucleotide sequence of the aspergillus cristatus feruloyl esterase gene is shown in SEQ ID No. 1. Feruloyl esterase encoded by the aspergillus cristatus feruloyl esterase gene has an amino acid sequenceshown in SEQ ID No. 2. The aspergillus cristatus feruloyl esterase gene provided by the invention can be used for in vitro generation of ferulic acid. Tests prove that the release rate of the ferulicacid in de-starch wheat bran suspension is 75.3%; the aspergillus cristatus feruloyl esterase gene enables the release rate of the ferulic acid to be much higher than other feruloyl esterase genes; therefore, a new enzyme source is provided for production of ferulic acid.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an Aspergillus coronis ferulic acid esterase gene, engineering bacteria and applications thereof. Background technique [0002] The chemical name of Ferulic Acid is 4-hydroxy-3-methoxycinnamic acid, which is one of the derivatives of cinnamic acid. It is ubiquitous in plant cells and combines with polysaccharides, proteins, and lignin through ester bonds. Become the skeleton of the cell wall, making the entire cell wall hard. Ferulic acid is a recognized natural antioxidant, and it is also an internationally recognized anti-cancer substance in recent years. It has various pharmacological effects such as inhibiting platelet aggregation and anti-atherosclerosis. In addition, ferulic acid has a strong long-wave ultraviolet absorption function. At present, it is widely used in food, medicine and cosmetic industries. [0003] Ferulic acid esterases (Ferulic acid estera...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/18C12N15/81C12N1/19C12P7/42C12R1/84
CPCC12N9/18C12N15/815C12P7/42
Inventor 朱希强张芳芳张金华刘飞薛佳俊袁超
Owner SHANDONG UNIV
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