Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Novel feruloyl esterase, coding gene and application thereof

A ferulic acid esterase and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of non-cultivation, limited application, limited development and utilization of new ferulic acid esterase, etc., to achieve easy digestion and absorption , high enzyme activity, the effect of improving feed utilization

Active Publication Date: 2018-07-31
NANJING AGRICULTURAL UNIVERSITY
View PDF12 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The methods for studying FAE generally include direct fermentation extraction method and heterologous expression method. esters, etc.) as the substrate to screen the active strains producing FAE, and obtain FAE from the fermentation product of the strain, however, most of the wild strains have very low ability to produce FAE, such as the enzyme activity of Aspergillus niger after fermentation is only 0.01-0.096U / mL greatly limits the application of the enzyme in industry; the latter is to molecularly clone the gene of FAE and express it in a suitable host through genetic engineering methods, and then separate and purify to obtain the enzyme. Compared with the direct fermentation extraction method, The heterologous expression method can significantly improve the yield and activity of the enzyme. At present, a variety of FAEs have been successfully expressed in heterologous hosts (such as Escherichia coli, filamentous fungi, Pichia pastoris, etc.)
[0004] However, studies have shown that only a very small number of microorganisms can be cultured under the existing experimental conditions, and more than 99% of the microorganisms are not cultivable, which greatly limits the development and utilization of new ferulic acid esterases

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
  • Novel feruloyl esterase, coding gene and application thereof
  • Novel feruloyl esterase, coding gene and application thereof
  • Novel feruloyl esterase, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Screening of ferulic acid esterase gene in soil metagenomic library

[0052] 1. Primary screening by plate method

[0053] Prepare FAE screening medium, add 1.5% (v / v) methyl ferulate (dissolved in dimethylformamide, 10% w / v) in LB liquid medium, after high temperature and high pressure sterilization, in suitable temperature (50-60°C), add ampicillin with a final concentration of 100mg / L, shake vigorously to make the methyl ferulate evenly distributed, and immediately pour the plate on the ultra-clean bench. Spread the cosmid library bacterial solution on each screening plate, culture at 37°C for 1-2 days, and observe the formation of transparent circles around the colonies ( figure 1 ).

[0054] 2. Double screening

[0055] After primary screening, the clones were inoculated in LB liquid medium, cultured overnight at 37°C, and centrifuged at 12,000g for 8 minutes to collect the bacteria. The cells were washed three times with sterile water and resuspend...

Embodiment 2

[0060] Embodiment 2: Cloning of ferulic acid esterase gene

[0061] 1. PCR amplification

[0062] Using primer fae-f(5'-CATG CCATGG GCATGCGTGCAGGGGGGAG-3') and fae-r(5'-CCC AAGCTT CCGGCCGCTCAGCCAGT-3') amplifies the ferulic acid esterase gene fae-xuan, and the underlines of the upstream and downstream primers represent the enzyme cleavage sites of NcoI and HindIII respectively. PCR reaction system (25 μL): ultrapure water 9.5 μL, Mix 12.5 μL, upstream and downstream primers 1 μL, positive subcloning plasmid DNA 1 μL. PCR reaction conditions: 94°C for 4min; 35 cycles of 94°C for 30s, 60°C for 45s, 72°C for 1min; 72°C for 10min. The PCR product was electrophoresed and recovered by tapping the gel to obtain a purified PCR product. 2. Digestion

[0063] The purified and recovered PCR product was subjected to double enzyme digestion for 3 hours. The digestion system is: NcoI 5 μL, HindIII 5 μL, 10×K Buffer 10 μL, 0.1% BSA 10 μL, PCR product 5 μg, sterile water to 100 μL. Th...

Embodiment 3

[0069] Example 3: Heterologous expression and purification of ferulic acid esterase FAE-Xuan

[0070] 1. Conversion

[0071] Take 10 μL of the pET28a-fae-xuan plasmid obtained in Example 2 and add it to 100 μL of Escherichia coli BL21 (DE3) competent cells, incubate on ice for 30 minutes, heat shock in a water bath at 42°C for 90 seconds, and add 900 μL of LB liquid culture after 2 minutes of ice bathing Base, 150rpm, shake at 37°C for 45min. The culture was centrifuged at 2500 g for 5 min, and 600 μL of the supernatant was removed. The bacteria were resuspended with the remaining culture medium and spread on an LB plate containing kanamycin. After culturing overnight at 37°C, a single colony was picked. Escherichia coli BL21(DE3) containing pET28a-fae-xuan was thus obtained.

[0072] 2. Express

[0073] The recombinant bacteria were inoculated into 5 mL LB liquid medium containing kanamycin, and cultured at 37° C. and 180 rpm for 12 hours. Inoculate 1.5mL of bacterial liq...

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

No PUM Login to View More

Abstract

The invention provides a novel feruloyl esterase gene from soil. The nucleotide sequence and the amino acid sequence of the novel feruloyl esterase gene are respectively shown in SEQ ID NO.1 and SEQ ID NO.2. The esterase gene is heterologously expressed in escherichia coli BL21(DE3), and the molecular weight of purified recombinase (FAEXuan) is 29kDa. The catalytic activity of the FAEXuan for a substrate (ferulic acid methyl ester) is highest, the enzyme activity is 40U / mg, the optimum temperature is 30 DEG C, and the optimum pH is 5.0. After reaction for 4 hours under the pH of 3.0-10.0, theferuloyl esterase can keep the activity still by 75% or more, so that stronger pH stability is shown. The feruloyl esterase FAEXuan has better tolerance to metal ions and organic solvent. The substrate utilization preference and the phylogenetic analysis show that the FAEXuan belongs A-type feruloyl esterase. Due to good enzymatic properties, the FAEXuan has wide application prospect in industrialproduction of the fields of foods, pharmacy and feed and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for obtaining a novel ferulic acid esterase from soil samples by using a metagenomic library function screening method, its coding gene and its application. Background technique [0002] Feruloyl esterase (Feruloyl esterase, FAE), also known as cinnamate esterase, is a subclass of carboxylic acid hydrolase, which plays a key role in the degradation of plant cell walls, and can make ferulic acid and the monosaccharides combined with it or oligosaccharides are released. The ferulic acid released has many good physiological functions, such as: anti-cancer, anti-thrombosis, anti-atherosclerosis and scavenging free radicals. In addition, FAE also has broad application prospects in food, pharmaceutical, paper and feed industries. [0003] FAEs come from a wide range of sources and generally exist in plants, fungi and bacteria. The FAE content in plants is g...

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
IPC IPC(8): C12N9/18C12N15/55C12N15/10C12N15/70C12N15/11
CPCC12N9/18C12N15/1086C12N15/70C12Y301/01073
Inventor 辛志宏李宣宣郭佳胡伊旻南放姜俊伟杨雨蒙吴盛露
Owner NANJING AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com