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Feruloyl esterase and preparing method and application thereof

A technology of ferulic acid esterase and screening method, which is applied in the field of new ferulic acid esterase and its preparation, and the field of preparation of ferulic acid esterase, which can solve the problems that no ferulic acid esterase gene has been found, microorganisms cannot be cultivated, etc. , to achieve good thermal stability and high enzyme activity

Active Publication Date: 2019-04-19
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional ferulic acid esterase screening method is based on the purification and screening of cultivable microorganisms, with the ability to degrade specific substrates as the screening criterion, but more than 99% of microorganisms in nature cannot be cultivated under laboratory conditions, so far Numerous undiscovered ferulic esterase genes still exist in nature

Method used

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  • Feruloyl esterase and preparing method and application thereof
  • Feruloyl esterase and preparing method and application thereof
  • Feruloyl esterase and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of soil metagenomic library, screening of positive clones and identification of corresponding ferulic acid esterase gene

[0054] Construction of the soil metagenomic library: Weigh 10 g of the soil sample, add CTAB extract, shake and mix at 37°C for 45 minutes. Add appropriate amount of lysozyme and proteinase K to the components to lyse cells and remove proteins. Add 2.5mL of 20% SDS (g / 100mL), bathe in water at 65°C for 2h, then add 3mL of pre-cooled chloroform, mix well and centrifuge to collect the supernatant. Add an equal volume of pre-cooled phenol: chloroform: isoamyl alcohol (25:24:1 volume ratio) solution, invert and mix well and centrifuge, take the upper aqueous phase and add an equal volume of chloroform: isoamyl alcohol (24:1 volume ratio) , centrifuge again, take the water phase and add 0.6 times the volume of pre-cooled isopropanol, ethanol precipitation in a water bath at room temperature for 1 h, and centrifuge to collect the ...

Embodiment 2

[0058] Embodiment 2: Molecular cloning and expression purification of ferulic acid esterase gene

[0059] Molecular cloning of the ferulic esterase gene: Amplification of the ferulic esterase gene dlfae4fae-F / NcoI:5'-CATG using the following primers CCATGG ATGACGATGGATACG-3' (SEQ ID No. 3) and fae-R / XhoI 5'-CCG CTCGAG TACACTCGCATACACC-3' (SEQ ID No.4) (the restriction sites of NcoI and XhoI are underlined). PCR reaction system (50 μL): 20 μl of ultrapure water, 25 μL of 2×Taq Master Mix, 2 μL of upstream and downstream primers (10 μM), 1 μL of DNA template (the positive clone plasmid DNA prepared in Example 1). PCR reaction conditions: 95°C for 5min; 35 cycles of 95°C for 30s, 56°C for 30s, 72°C for 1min; 72°C for 10min. The PCR product is electrophoresed and recovered by tapping the rubber to obtain a purified ferulic acid esterase gene PCR product. The PCR product was subjected to double enzyme digestion, and the digestion time was 3h. The enzyme digestion system (100 ...

Embodiment 3

[0061] Embodiment 3: Enzymatic property characterization of ferulic acid esterase DLFae4

[0062] Determination of enzyme activity: Take 1 mL of Gly-NaOH buffer solution with pH 8.6, add 5 μmol of methyl ferulate solution, add 3 μL of recombinant enzyme, react at 50 ° C, and use HPLC to measure the degradation of the substrate at 320 nm.

[0063] Definition of enzyme activity: under the reaction conditions of 50°C and pH 8.6, the amount of enzyme required to degrade methyl ferulate to generate 1 μmol ferulic acid per minute is defined as 1 enzyme activity unit (U).

[0064] Optimum pH and pH stability analysis: at 37°C, measure the enzyme activity at different pH (3.0-11.0), and determine the optimum pH of the enzyme according to the size of the enzyme activity ( Figure 4 a). Add 3 μL of recombinant enzymes to buffers with different pHs, incubate at 4°C for 3 h, measure the remaining enzyme activity, and determine the stability of the enzyme ( Figure 4 b).

[0065] Optimu...

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Abstract

The invention provides feruloyl esterase and a preparing method and application thereof. A feruloyl esterase gene coming from a soil macro gene library have the nucleotide sequence and amino acid sequence shown in SEQ ID NO.1 and SEQ ID NO.2. The gene contains a tetrapeptide SXXK sequence motif which is rarely seen, and after the esterase gene is inserted into plasmid pET28a(+), the gene is transformed into escherichia coli BL21(DE3) to achieve heterogeneous expression. The molecular weight of purified recombinase (DLFae4) is 38.3 kDa. Besides, it is put forward for the first time that novel feruloyl esterase can hydrolyze penbritin, penicillin, cefazolin and other lactam antibiotics. As is shown by site-directed mutagenesis experiments, a catalysis triplet of DLFae4 is composed of serine(S11), histidine (H74) and aspartic acid (D302), and the mutation of any of serine (S11), histidine (H74) and aspartic acid (D302) can cause loss of the catalysis capability of DLFae4. DLFae4 has a high hydrolytic activity on methyl ferulate and has good heat stability. In the presence of cellulase, DLFae4 can obviously increase the amount of ferulic acid released from destarched wheat bran. Due to peculiar activities and enzymatic characteristics of novel feruloyl esterase, novel feruloyl esterase can be applied to feed, paper making, food, pharmacy and other fields.

Description

technical field [0001] The invention belongs to the field of bioengineering, including a novel ferulic acid esterase and its preparation method and application, and specifically relates to the macrogene screening technology of ferulic acid esterase, the recombination and expression of the novel ferulic acid esterase gene, and ferulic acid esterase gene. Preparation and application of ferulic acid esterase. Background technique [0002] Ferulic esterase (feruloyl esterase, FAE, EC 3.1.1.73), also known as cinnamic esterase, belongs to the subclass of carboxylesterase in the hydrolase family, which can hydrolyze the ester bond between carbohydrates and phenolic acids, And release phenolic acids, such as ferulic acid (ferulic acid), caffeic acid (caffeic acid), coumaric acid (p-coumalic acid), etc., these phenolic acids have anti-oxidation, anti-bacterial and anti-inflammatory, anti-cancer, anti-thrombotic, anti-arterial Atherosclerosis and other good physiological functions. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/18C12Y301/01073
Inventor 辛志宏南放吴盛露邱佳容姜俊伟
Owner NANJING AGRICULTURAL UNIVERSITY
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