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A novel ferulic acid esterase and its coding gene and application

A ferulic acid esterase and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of limited application, non-cultivation, and limited development and utilization of new ferulic acid esterase, and achieve strong pH stability The effect of good resistance, good tolerance and broad application prospects

Active Publication Date: 2022-06-24
NANJING AGRICULTURAL UNIVERSITY
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  • 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

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  • A novel ferulic acid esterase and its coding gene and application
  • A novel ferulic acid esterase and its coding gene and application
  • A novel ferulic acid esterase and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0052] 1. Preliminary screening by plate method

[0053] To prepare the FAE screening medium, add 1.5% (v / v) methyl ferulate (dissolved in dimethylformamide, 10% w / v) to the LB liquid medium. Add ampicillin with a final concentration of 100 mg / L at a temperature (50-60 °C), shake vigorously to make the methyl ferulate evenly distributed, and immediately pour the plate on the ultra-clean table. Cosmid library bacterial solution was coated on each screening plate, cultured at 37°C for 1-2 days, and the formation of a transparent circle around the colony was observed ( figure 1 ).

[0054] 2. Rescreening

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

Embodiment 2

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

[0061] 1. PCR amplification

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

[0063] The purified and recovered PCR product was subjected to double digestion, and the digestion time was 3h. The digestion system was as follows: NcoI 5 μL, HindIII 5 μL, 10×K Buffer 10 μL, 0.1% BSA 10 μL, PC...

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 min, heat shock in a water bath at 42°C for 90 s, and add 900 μL LB liquid culture after ice bathing for 2 min. base, 150rpm, 37°C shaking culture for 45min. The culture was centrifuged at 2500 g for 5 min, 600 μL of supernatant was removed, the bacteria were resuspended with the remaining medium and spread on LB plates containing kanamycin, and single colonies were picked after overnight culture at 37°C. Thus, Escherichia coli BL21(DE3) containing pET28a-fae-xuan was 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 h. Inoculate 1.5 mL of bacterial liquid into 150 mL of fresh LB...

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Abstract

The present invention provides a novel ferulic acid esterase gene derived from soil, the nucleotide sequence and amino acid sequence of which are respectively shown in SEQ ID NO.1 and SEQ ID NO.2. The esterase gene was heterologously expressed in Escherichia coli BL21(DE3), and the purified recombinant enzyme (FAE‑Xuan) had a molecular weight of 29 kDa. FAE‑Xuan has the highest catalytic activity on the substrate methyl ferulate, with an enzyme activity of 40U / mg, its optimum temperature is 30℃, and its optimum pH is 5.0. After reacting at pH 3.0‑10.0 for 4 hours, the enzyme can still maintain more than 75% of its activity, showing strong pH stability. The novel ferulic acid esterase FAE‑Xuan has good tolerance to metal ions and organic solvents. Substrate utilization preference and phylogenetic analysis showed that FAE‑Xuan belongs to type A ferulic acid esterase. These good enzymatic properties of FAE-Xuan in the present invention make it have wide application prospects in the industrial production in the fields of food, pharmacy and feed.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for obtaining a novel ferulic acid esterase from soil samples by using a method for functional screening of a metagenome library, its encoding gene and its application. Background technique [0002] 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 its combined monosaccharide possible. or oligosaccharides are released. The released ferulic acid has many good physiological functions, such as: anti-cancer, anti-thrombotic, anti-atherosclerosis and free radical scavenging. In addition, FAE also has broad application prospects in the food, pharmaceutical, paper and feed industries. [0003] FAEs come from a wide range of sources and are commonly found in plants, fungi and bacteria. The content of FAEs in plants ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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
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