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Ferulaic acid decarboxylase crystal structure and analysis method thereof

A technology of ferulic acid decarboxylase and crystal structure, which is applied in the field of enzyme engineering and can solve the problems of incomparability of FDC, inability to identify substrate linking residues, and short sequence.

Active Publication Date: 2014-06-18
WUXI NEWWAY FERMENTATION TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the amino acid sequence of Enterobacter sp.Px6-4FDC (168 amino acid residues) has only 19.0% identity with yeast FDC (503 amino acid residues), and the sequence is shorter
Therefore, the structure of FDC from Enterobacter is not comparable to that from yeast and cannot be used to identify substrate-linking residues for yeast-derived FDC

Method used

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  • Ferulaic acid decarboxylase crystal structure and analysis method thereof
  • Ferulaic acid decarboxylase crystal structure and analysis method thereof
  • Ferulaic acid decarboxylase crystal structure and analysis method thereof

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Effect test

Embodiment 1

[0049] The structure determination of embodiment 1 ferulic acid decarboxylase

[0050] Direct freezing of vapor flowing liquid N2 at 100K, two single crystals can be obtained directly from the mother liquor, using the X-ray source 191Dbeamline equipped with a CCD detector (ADSCQUANTUM315r) of Argonne National Laboratory can be directly obtained at 412.87 Crystal data at mm and 378.90 mm. exist , 0.3° and 1.0° amplitude indicators (P1211 and C121) and measurements with HKL3000 (Minor et al., 2006) can obtain crystal pictures (600frames and 180frames). Table 1 lists the parameters and data refinement statistics of the unit cell crystals obtained by induction. The FDC in this example comes from the yeast Saccharomyces cerevisiae (strain ATCC204508 / S288c).

[0051] Initial FDC (P121, ) structure can be based on the putative aromatic acid decarboxylase (PDB: 4IWS) structure (Jacewicz et al., "Structural insights into the UbiD protein family from the crystal structure of PA025...

Embodiment 2

[0057] The crystal space structure characteristic of embodiment 2FDC protein

[0058] Such as figure 1 As shown, the asymmetric unit of FDC crystal is composed of eight monomers. Among them, six monomers form three dimers in sequence (that is, monomer A and monomer H, monomer B and monomer F, monomer C and monomer E), and the other two monomers correspond to The asymmetric chaperones form dimers. The experimental map proves the rationality of the molecular structure of the above protein.

[0059] One dimer can be identified among the asymmetric units in FDC crystals ( figure 2 ). This dimer assembly is mainly maintained by the interaction of amino acid residues belonging to the two α-helical subunits. Regulation of dimers is accomplished through salt bridges and hydrophobic interactions. Using Sephacryl S-200 to analyze the results of gel chromatography, combined with the results of SDS-PAGE, it can be known that FDC is a dimeric protein with a molecular weight of 112.0...

Embodiment 3

[0063] Application of embodiment 3 ferulic acid decarboxylase

[0064] In the system for producing syringic acid, introduce syringic acid decarboxylase (Cinnamic Acid Decarboxylase, CDC; also known as ferulic acid decarboxylase, Ferulic Acid Decarboxylase, FDC) to obtain styrene.

[0065] The introduction of phenylalanine ammonia lyase (PAL) and syringate decarboxylase (CDC) genes into phenylalanine-producing Escherichia coli successfully achieved the biosynthesis of styrene. The above scheme proves that renewable biomass is used as raw material Styrene can be produced.

[0066] Figure 8 The artificially designed synthetic pathway shown is mainly composed of three parts. First, through the overexpression of three key enzymes, the efficient conversion from glucose to phenylalanine was achieved, and a large amount of phenylalanine (phenylalanine) was accumulated in the cells. Second, excess phenylalanine was converted to cinnamate by overexpressing plant phenylalanine ammoni...

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Abstract

The invention discloses a ferulaic acid decarboxylase crystal structure and an analysis method thereof, belonging to the technical field of enzyme engineering. An amino acid sequence of the ferulaic acid decarboxylase is shown in SEQ ID NO.1 described in the specification, and the ferulaic acid decarboxylase has the molecular weight of 112KDa; the crystal structure consists of 8 asymmetric monomers, wherein the monomer A (1) and the monomer H (8) form a dimmer, the monomer B (2) and the monomer F (6) form a dimmer, the monomer C (3) and the monomer E (5) form a dimmer, the monomer D (4) and the asymmetric partner thereof form a dimmer, the monomer G (7) and the asymmetric partner thereof for a dimmer, and the cell parameter is as shown in the specification. The structure information of the ferulaic acid decarboxylase provided by the invention can be used for improving the yield of styrene, improving the enzymatic activity, changing the specificity of a substrate, improving the pH and the thermal stability of the enzyme, constructing a mutant library of the ferulaic acid decarboxylase, producing an analogue of trans-cinnamic acid, removing a feedback inhibition hamper, reducing diffusion and accumulation of intermediates, and the like.

Description

technical field [0001] The invention relates to a ferulic acid decarboxylase crystal structure and an analysis method thereof, belonging to the technical field of enzyme engineering. Background technique [0002] Today, approximately 10% of fossil fuels are used annually to produce materials such as plastics, rubber and resins. Since the invention of plastic some 70 years ago, these "universal" materials have been used in all aspects of modern life. However, due to the non-renewable nature of fossil fuels and the increasing attention to environmental issues caused by the accumulation of carbon dioxide, it is becoming more and more important to find renewable energy sources that can replace fossil fuels such as oil, natural gas, and coal. Research in this area is directly related to economic competitiveness and national security. The development of new technologies for preparing these polymers is an urgent task in the world of academic research and industrial applications. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88
CPCC07K2299/00C12N9/88
Inventor 余晓丹
Owner WUXI NEWWAY FERMENTATION TECH RES INST
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