Bacteria ferulic acid decarboxylase gene and crystal structure thereof

A technology of ferulic acid decarboxylase and crystal structure, which is applied in the fields of molecular biology and applied microbiology, and can solve the problems of undiscovered public reports and the like

Inactive Publication Date: 2009-04-01
YUNNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Bacteria ferulic acid decarboxylase gene and crystal structure thereof
  • Bacteria ferulic acid decarboxylase gene and crystal structure thereof
  • Bacteria ferulic acid decarboxylase gene and crystal structure thereof

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Embodiment Construction

[0036] 1. Extraction of Bacterial Genomic DNA

[0037] 1) Inoculate Enterobacter sp.Px6-4 in liquid LB medium and culture at 37°C and 200rpm for 12 hours;

[0038] 2) Take 1 mL of the culture and centrifuge at 12,000 rpm for 2 minutes, and remove the supernatant;

[0039] 3) Wash the precipitate twice with TE buffer (10mM Tris-HCl, pH8.0; 0.1mM EDTA, pH8.0);

[0040] 4) Add 30 μL SDS (10%) and 15 μL proteinase K (20 mg / mL) after resuspending with 567 μL TE buffer, mix well and incubate at 37 ° C for 1 hour;

[0041] 5) Add 100 μL of NaCl (5M) and 80 μL of CTAB / NaCl (10% CTAB, 0.7M NaCl) in sequence, mix well and incubate at 65°C for 10 minutes;

[0042] 6) Add 700 μL of phenol: chloroform: isoamyl alcohol (25:24:1 v / v) and mix by inverting several times, then centrifuge at 5000 rpm for 5 minutes;

[0043] 7) Transfer the supernatant to a new EP tube, add 0.6 times the volume of isopropanol, and centrifuge at 12,000 rpm for 10 minutes at 4°C;

[0044] 8) The precipitate was...

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Abstract

The invention relates to an expression, purification, crystallization method, structure elucidation and the field of application technology of forulic acid decarboxylase, which belongs to the field of molecular biology and microbiology application. The invention obtains the coding area sequence of the key enzyme forulic acid decarboxylase which decomposes forulic acid to generate 4-vinyl guaiacol in Enterobacter sp.Px6-4 by the gene clone method, expresses the enzyme in a large amount in the colon bacillus ( E.coli BL21 ) by the heterogenous expressing technology, obtains the forulic acid decarboxylase pure products by the purification method and the forulic acid decarboxylase protein crystallization by the hanging-drop crystallization process, and grasps the structure and active centre of the enzyme. The invention can obtain a large amount of highly active forulic acid decarboxylase, and provide theoretical guidance for reconstructing engineering bacteria and improving the output of 4-vinyl guaiacol by analyzing the active centre of the enzyme, thus having good potential application.

Description

Technical field: [0001] The invention relates to a bacterial ferulic acid decarboxylase gene and its crystal structure, belonging to the technical fields of molecular biology and applied microbiology. Background technique: [0002] Ferulic acid (4-hydroxy-3-methoxy-2-acrylic acid) is a hydroxycinnamic acid widely present in the plant kingdom, and it is the phenolic acid with the highest content in wheat bran, which can reach 0.5-1% of its dry weight. It is mainly cross-linked with polysaccharides and lignin through ester bonds, or self-esterified or etherified to form diferulic acid. Many plants, fungi, bacteria, actinomycetes, and microalgae can decompose ferulic acid to produce vanillin. Ferulic acid, as a precursor substance for biotransformation to produce vanillin, is easy to obtain, cheap, and resistant to microorganisms. Therefore, the research on the metabolic pathway of converting ferulic acid into vanillin has great theoretical significance and production value. ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N15/70C12N9/88
Inventor 张克勤饶子和孟照辉娄智勇杨金奎顾雯
Owner YUNNAN UNIV
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