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Preparation method of recombinant feruloyl esterase

A technology of ferulic acid esterase and recombinant plasmid, which is applied in the field of preparation of recombinant ferulic acid esterase, can solve the problem of difficult to meet the requirements of industrial production, complicated separation and extraction process of ferulic acid esterase, ferulic acid esterase enzyme activity and low ferulic acid esterase. The expression amount does not meet the needs of the industry and other problems, so as to achieve the effect of improving the expression efficiency and expression amount

Inactive Publication Date: 2015-03-25
HUAQIAO UNIVERSITY
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  • Abstract
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  • Application Information

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Problems solved by technology

[0003] So far, more than 40 kinds of ferulic acid esterases have been isolated from bacteria and fungi, but the enzyme activity and expression level of ferulic acid esterases reported so far cannot meet the needs of industry
Secondly, the separation and extraction process of ferulic acid esterase is relatively complicated, and it is difficult to meet the requirements of industrial production.

Method used

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  • Preparation method of recombinant feruloyl esterase
  • Preparation method of recombinant feruloyl esterase
  • Preparation method of recombinant feruloyl esterase

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: Preparation of recombinant ferulic acid esterase

[0032] 1) Obtaining the target gene fae: screening the amino acid sequence of ferulic acid esterase O42807 with high enzymatic activity in the UniProt database, which was isolated from Aspergillus niger (Aspergillus niger) CBS120.49 by De Vries RP et al. It belongs to extracellular enzyme; the amino acid sequence of ferulic acid esterase O42807 is shown in SEQ ID NO.2, according to the mature peptide sequence of O42087 (as shown in SEQ ID NO.2, 22-281 is the mature peptide sequence) The gene sequence of ferulic acid esterase was designed by standard codon optimization of Pichia pastoris, and HindⅢ and EcoRI enzyme cleavage sites were respectively introduced into the two ends of the gene sequence, and the gene sequence shown in SEQ ID NO.1 was chemically synthesized and named as Fae;

[0033] 2) Construct the pAO815-fae expression vector: use HindⅢ and EcoRI to double-digest the synthetic gene sequence fae...

Embodiment 2

[0036] Example 2: Identification of GS115 / pPIC9K-fae

[0037] Extract the genomic DNA of the GS115 / pPIC9K-fae obtained in Example 1, utilize universal primer 5'AOX and 3'AOX to carry out PCR identification, the result is as follows image 3 As shown, GS115 / pPIC9K-fae was constructed successfully.

Embodiment 3

[0038] Example 3: Determination of the expression level of GS115 / pPIC9K-fae

[0039] For the induced expression of recombinant yeast GS115 / pPIC9K-fae, refer to Invitrogen’s Pichia pastoris operation and expression manual. Every 12h or 24h of methanol induction, the fermentation supernatant was taken for SDS-PAGE analysis to determine the expression of recombinant ferulic acid esterase. Such as Figure 4 As shown, a clear band was obtained at 42kDa, and there were almost no mixed bands, preliminarily indicating that fae was successfully expressed. Utilize Bradford method to measure recombinant ferulic acid esterase protein content, HPLC method measures enzyme activity, and assay result is as follows Figure 5 As shown, the enzyme activity is 4.7U / mL, and the specific enzyme activity is 31.4U / mg.

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Abstract

The invention discloses a preparation method of recombinant feruloyl esterase. The feruloyl esterase O42807 is adopted, after codon optimization, the gene fae of the feruloyl esterase O42807 is manually synthesized, A pAO815-fae expression vector is established, a pPIC9K-fae expression vector is established and the pPIC9K-fae expression vector is transformed to pichia pastoris GS115 for expression, and then the recombinant feruloyl esterase is obtained. SDS-PAGE analysis shows that the fermentation liquid supernatant of the recombinant feruloyl esterase is a single stripe, the apparent molecular weight is 42kDa, the enzyme activity is 4.7U / mL, the enzymatic specific activity is 31.4U / mg, the optimal reaction temperature is 50 DEG C and the optimal pH is 5.0-5.5. According to the preparation method of the recombinant feruloyl esterase, an efficiently expressed recombinant feruloyl esterase transformant is obtained, and the expression efficiency and the expression quantity of the recombinant feruloyl esterase transformant are greatly improved in contrast with feruloyl esterase O42807.

Description

technical field [0001] The invention relates to a preparation method of recombinant ferulic acid esterase. Background technique [0002] In plant cell walls, phenolic acids (such as ferulic acid, caffeic acid, p-coumaric acid, etc.) The bonds are connected with lignin to form a dense network structure. Ferulic acid esterase (EC 3.1.1.73, Ferul ic acid esterases, FAE), also known as cinnamic esterase, can hydrolyze the ester bond in ferulic acid ester, oligosaccharide ferulic acid ester and polysaccharide ferulic acid ester , releasing functional substances such as ferulic acid. Ferulic acid esterase can assist xylanase and ligninase to destroy this dense network structure, making the cell wall structure loose, thereby promoting the complete degradation of lignocellulose. Ferulic acid esterase plays an important role in both industry and agriculture. [0003] So far, more than 40 kinds of ferulic acid esterases have been isolated from bacteria and fungi, but the enzyme ac...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/81
CPCC12N9/18C12Y301/01073
Inventor 李夏兰张光亚陈云华陈培钦葛慧华
Owner HUAQIAO UNIVERSITY
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