Recombinant expression strain, preparation method and application of ferulic acid esterase

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, inability to fold correctly, and reduced enzyme activity, and achieve the effect of good practical application value.

Active Publication Date: 2021-05-11
FUDAN UNIV
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant expression strain, preparation method and application of ferulic acid esterase
  • Recombinant expression strain, preparation method and application of ferulic acid esterase
  • Recombinant expression strain, preparation method and application of ferulic acid esterase

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
release amountaaaaaaaaaa
Login to view more

Abstract

The invention provides a recombinant strain of Kluyveromyces marx that can be used to prepare ferulic acid esterase AnfaeA, which clones the sequence-optimized Aspergillus niger ferulic acid esterase AnfaeA gene into an expression vector and transforms Kluyveromyces maximus The ferulic acid esterase production of the recombinant strain reaches 120000U / ml through high-density fermentation. The present invention also provides a method for preparing ferulic acid by using ferulic acid esterase to hydrolyze corn cob powder, using the synergistic effect of ferulic acid esterase AnfaeA and xylanase produced by the aforementioned recombinant strains to efficiently hydrolyze corn The core powder obtains ferulic acid. The ferulic acid esterase provided by the present invention can be used to biodegrade agricultural by-products such as corn cobs, rice bran and corn bran to prepare phenolic substances such as ferulic acid and p-coumaric acid, so it is widely used in food, papermaking, feed, medicine and other fields. All have wide application value, and meanwhile, the preparation method has simple process and high yield, and is 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap