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Novel feruloyl esterase and preparation method and application thereof

A ferulic acid esterase, a new type of technology, applied in the field of bioengineering to achieve the effect of high enzymatic activity

Active Publication Date: 2019-03-01
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, some studies have pointed out that more than 99% of the microorganisms in the environment are difficult to obtain by using conventional microbial culture methods in the laboratory.

Method used

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  • Novel feruloyl esterase and preparation method and application thereof
  • Novel feruloyl esterase and preparation method and application thereof
  • Novel feruloyl esterase and preparation 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 2.5 mL of 20% SDS, bathe in water at 65°C for 2 h, then add 3 mL 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) solution, invert and mix well, then centrifuge, take the upper aqueous phase and add an equal volume of 24:1 (chloroform: isoamyl alcohol), and centrifuge again. Take the aqueous 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 precipitate. Finally, 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 bds4hFv / BamHI using the following primers

[0060] 5'-GCTGGATCCATGCCATATATTTCCACC-3' (SEQ ID No. 3) and hRv / HindIII

[0061] 5'-GCTAAGCTTTGGCTTTTGAATTAATTG-3' (SEQ ID No. 4) (restriction sites of BamHI and HindIII 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. PCR reaction conditions: 95°C for 5min; 95°C for 5min, 56°C for 30s, 72°C 1min, 35 cycles; 10min at 72°C. The PCR product was electrophoresed and recovered by tapping the gel to obtain the 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 μL) was: 5 μL of BamHI, 5 μL of HindIII, 10 μL o...

Embodiment 3

[0063] Embodiment 3: Enzymatic property characterization of ferulic acid esterase BDS4

[0064] Determination of enzyme activity: Take 1 mL of Tris-HCl buffer solution with pH 8.0, add 5 μmol of methyl ferulate solution, add 20 μg of enzyme solution (about 10 μL), react at 37 ° C, and measure the absorbance value at 320 nm by HPLC.

[0065] Definition of enzyme activity: under the reaction conditions of 37°C and pH 8.0, 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).

[0066] 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). The enzyme solution was added to buffer solutions with different pHs, placed at 4°C for 1 hour, and the remaining enzyme activity was measured to determine the stability of the enzyme ( Figure 4 b).

[006...

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Abstract

The invention provides a feruloyl esterase gene derived from a soil macrogene library. The nucleotide sequence and amino acid sequence of the feruloyl esterase gene are shown as SEQ ID NO.1 and SEQ IDNO.2. The esterase gene is inserted into a plasmid pET-28a(+) and transformed into escherichia coli BL21 (DE3) to achieve heterologous expression. The molecular weight of a purified recombinant enzyme (BDS4) is 38.8 kDa. In addition, it is proposed for the first time that novel feruloyl esterase can hydrolyze various plasticizers such as dimethyl phthalate, diethyl phthalate and dibutyl phthalate. It is shown through site-directed mutagenesis experiments that a catalytic triplet of BDS4 consists of serine (S158), aspartic acid (D256) and histidine (H286), and the mutation of any of the threeamino acids can lead to the loss of catalytic capability of BDS4. In the presence of xylanase, BDS4 can significantly increase the amount of ferulic acid released from de-starched wheat bran. The novel feruloyl esterase can be applied in the fields of feed, paper making, food, pharmacy and the like due to the unique activity and enzymatic properties of the novel feruloyl esterase.

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 acid (ferulic acid), chemical name 3-methoxy-4-hydroxycinnamic acid, has good physiological functions such as anti-oxidation, antibacterial and anti-inflammatory, anti-cancer, anti-thrombotic, anti-atherosclerosis, etc. Currently, ferulic acid It has been widely used in industries such as medicine and cosmetics, among which trans-ferulic acid has been allowed as a food additive in the United States and Japan. Ferulic acid widely exists in food raw materials, such as wheat bran, corncobs, beet residues, brewer's grains, etc., but ferul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70A62D3/02A62D101/28
CPCA62D3/02A62D2101/28C12N9/18C12N15/70C12Y301/01073
Inventor 辛志宏吴盛露姜俊伟张月琦南放乔贝贝邱佳容李珊
Owner NANJING AGRICULTURAL UNIVERSITY
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