Recombinant expression strain of feruloyl esterase as well as preparation method and application of recombinant expression strain

A technology of ferulic acid esterase and ferulic acid, applied in the field of bioengineering, can solve the problems of lack of post-translational modification of eukaryotic gene expression, restriction of Escherichia coli expression system, inactive inclusion bodies, etc. Effect

Active Publication Date: 2020-08-28
FUDAN UNIV
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Problems solved by technology

The Escherichia coli expression system is the most commonly used expression system, but it also has its shortcomings. It lacks the post-translational modifications necessary for eukaryotic gene expression, and the protein often cannot be folded correctly after expression, and it is easy to form inactive inclusion bodies. And chromatography denaturation and purification, but these operations will lead to loss of protein and decrease of enzyme activity, these defects limit the application of E. coli expression system to some extent

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  • Recombinant expression strain of feruloyl esterase as well as preparation method and application of recombinant expression strain
  • Recombinant expression strain of feruloyl esterase as well as preparation method and application of recombinant expression strain
  • Recombinant expression strain of feruloyl esterase as well as preparation method and application of recombinant expression strain

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Embodiment 1

[0026] Embodiment 1, the synthesis and the amplification of the dna sequence of the ferulic acid esterase AnfaeA protein of coding Aspergillus niger source

[0027]Referring to the codon preference of Kluyveromyces marx, the nucleotide sequence of the ferulic acid esterase AnfaeA gene derived from Aspergillus niger was optimized for codons without changing the amino acid sequence. Wherein, the ferulic acid esterase AnfaeA gene nucleotide sequence derived from Aspergillus niger is shown in SEQ ID No.1, the AnfaeA gene nucleotide sequence after optimization is shown in SEQ ID No.2, and the corresponding amino acid sequence after optimization As shown in SEQ ID No.3. In this example, the gene sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0028] Using the synthesized AnfaeA DNA as a template, the AnfaeA gene was amplified by PCR using primers AnfaeA-132-F (sequence shown in SEQ ID No. 4) and AnfaeA-132-R (sequence shown in SEQ ID No. 5). The PCR process ...

Embodiment 2

[0031] Embodiment 2, construction of ferulic acid esterase AnfaeA Kluyveromyces marx recombinant expression vector derived from Aspergillus niger

[0032] Kluyveromyces marx expression vector pUKDN132 was double digested with restriction enzymes Sma I and Not I, and the digested product was subjected to 1% agarose gel electrophoresis, and the vector of about 11kb was recovered with SanPrep column DNA gel recovery kit fragment.

[0033] The AnfaeA gene fragment was connected to the vector fragment using the Gibson Assembly traceless ligation system (NEB Company, Cat. No. E2611S / L) to obtain the recombinant expression vector 132-AnfaeA of the ferulic acid esterase AnfaeA Kluyveromyces maxis derived from Aspergillus niger. The recombinant vector includes yeast autonomous replication sequence, inulinase promoter, ferulic acid esterase AnfaeA gene derived from Aspergillus niger, inulinase terminator, screening marker gene URA3 sequence, and its specific sequence is shown in SEQ ID ...

Embodiment 3

[0034] Embodiment 3, construction of Kluyveromyces marx recombinant strain FIM-AnfaeA

[0035] The yeast expression host strain used in this example is derived from Kluyveromyces marxense FIM-1 (preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, the preservation number is CGMCC No.10621, the depository unit code: CGMCC, deposit Unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, date of preservation is March 13, 2015, classification and name: Kluyveromyces marxianus), the URA3 gene was knocked out by homologous recombination, and A uracil-deficient expression host bacterium was screened by YPD containing 5-fluoroorotic acid (1.5g / L), which was named as Kluyveromyces marxense FIM-1 (Δura3).

[0036] 132-AnfaeA was transferred into FIM-1 (Δura3) by lithium acetate conversion method (World Journal of Microbiology & Biotechnology 16:653-654, 2000). Spread the transformed product on an SD plate (0...

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Abstract

The invention provides a Kluyveromyces marxianus recombinant strain capable of being used for preparing feruloyl esterase AnfaeA. The recombinant strain is constructed by cloning an aspergillus nigerferuloyl esterase AnfaeA gene with an optimized sequence to an expression vector and converting a Kluyveromyces marxianus host strain, and the yield of the feruloyl esterase obtained by high-density fermentation of the recombinant strain reaches 120000 U/ml. The invention also provides a method for preparing ferulic acid by hydrolyzing corncob powder with the feruloyl esterase. The feruloyl esterase AnfaeA prepared from the recombinant strain and xylanase have a synergistic effect, so that the corncob powder can be efficiently hydrolyzed to obtain the ferulic acid. The feruloyl esterase provided by the invention can be used for preparing phenolic substances such as ferulic acid, p-coumaric acid and the like through biodegradation of agricultural byproducts such as corncobs, rice bran, cornbran and the like, so that the feruloyl esterase has a wide application value in the fields of foods, papermaking, feeds, medicines and the like. Meanwhile, the preparation method is simple in process, high in yield and very suitable for large-scale production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for preparing an enzyme product by using a recombinant yeast strain, in particular to a Kluyveromyces marx strain recombinantly expressing ferulic acid esterase, and a method for preparing ferulic acid esterase by using the strain. And the application of the prepared ferulic acid esterase in the enzymatic preparation of ferulic acid. Background technique [0002] Ferulic esterase (feruloyl esterases, EC 3.1.1.73), also known as cinnamic esterase, is a subclass of carboxylate hydrolase. Since ferulic acid esterase can hydrolyze the ester bonds of ferulic acid and other phenolic acids interacting with hemicellulose, lignin, etc., break the dense network structure in the plant cell wall and expose the cellulose, thereby increasing the degradation rate of cellulose And release ferulic acid and other antioxidant substances, so it is widely used in food, papermaking, fee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/55C12N9/18C12P7/42C12R1/645
CPCC12N9/18C12P7/42C12Y301/01073
Inventor 吕红周峻岗余垚
Owner FUDAN UNIV
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