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Novel ferulic acid esterase and applications thereof

A ferulic acid esterase, a new type of technology, applied in the field of genetic engineering, can solve problems such as defects in enzymatic properties and restrictions on the wide application of ferulic acid esterase

Inactive Publication Date: 2012-10-10
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The various industrial applications mentioned above require ferulic acid esterase to act on different substrates at different pH values ​​and temperatures. The enzymatic properties of ferulic acid esterase still have defects in application, which limits the wide application of ferulic acid esterase

Method used

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  • Novel ferulic acid esterase and applications thereof
  • Novel ferulic acid esterase and applications thereof
  • Novel ferulic acid esterase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Establishment of metagenomic library and acquisition of positive clones, gene cloning and expression

[0040] 1. Extraction of total DNA

[0041] Weigh 6g sample, add 13.5ml DNA extraction buffer (0.1M Tris, 0.1M EDTA-Na, 0.1M Na 3 PO 4 , 1.5M NaCl, 1% CTAB, pH 8.0), shake vigorously for 3-5min, add 200μL lysozyme (100mg / ml), invert repeatedly 5-6 times, 37℃ water bath for 30min, add 1.5ml 20% SDS, 65℃ Water bath for 1h (during this period, turn it upside down several times every 15min), centrifuge at 8000r / min for 5min, take the supernatant, extract twice with an equal volume of chloroform, centrifuge at 16000r / min for 10min, take the supernatant, add 0.6 times the volume of isopropyl Alcohol, place at room temperature for 2h, centrifuge at 20000r / min for 20min, discard the supernatant, add 5mL of pre-cooled 70% ethanol to the pellet, centrifuge at 20000r / min for 10min, collect the DNA precipitate, air-dry, and dissolve with an appropriate amount of TE buff...

Embodiment 2

[0062] Embodiment 2 recombinant ferulic acid esterase Est27 enzyme activity assay

[0063] 1. Determination of enzyme activity

[0064] The invention uses p-nitrophenol ferulate as a substrate to detect the enzyme activity of ferulic acid esterase. The reaction system was 500 μl, which consisted of 50 μl of 10 mM p-nitrophenol ferulate stock solution (dissolved in DMSO), 440 μl of 0.1 mM potassium phosphate buffer containing 2.5% (v / v) Triton X-100 ( pH 6.8) and 10 μl enzyme solution. The reaction system was reacted at 40° C. for 5 minutes, and then the absorbance of p-nitrophenol released during the process was measured at a wavelength of 405 nm, and a blank control without adding enzyme solution was made. Determine its concentration according to the standard curve of p-nitrophenol. The enzyme activity unit is defined as: under the reaction conditions, the amount of enzyme required to catalyze 1 μmol of p-nitrophenol ferulate per minute is defined as 1 enzyme activity unit...

Embodiment 3

[0072] Example 3 Research on the enzymatic properties of recombinant ferulic acid esterase Est27

[0073] 1. Optimum reaction temperature and thermal stability of recombinant ferulic acid esterase Est27

[0074] After carrying out the enzymatic reaction of the crude enzyme liquid of recombinant ferulic acid esterase Est27 at 30-65° C., its enzyme activity was measured according to the above-mentioned method to obtain its optimum reaction temperature (recorded as 100% when the enzyme activity is the highest). In 100mM phosphate buffer (pH6.8), incubate the ferulic acid esterase Est27 enzyme solution at different temperatures (40-60°C), take out the enzyme solution at regular intervals and measure the residual enzyme activity to treat for 0min The enzyme activity of the enzyme solution is 100%. The test results are attached image 3As shown, the optimal reaction temperature of the recombinant protein Est27 is 40°C. The enzyme is stable at 40°C and has a half-life of 72h at 45...

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Abstract

The invention discloses a novel ferulic acid esterase whose amino acid sequence is shown in SEQ ID NO.3; the invention further discloses a DNA of the novel ferulic acid esterase, and a nucleotide sequence of the DNA is shown in SEQ ID NO.1. The invention also discloses an expression vector containing the DNA of the above novel ferulic acid esterase, a recombined ferulic acid esterase, a preparation method of the recombined ferulic acid esterase, and applications of the recombined ferulic acid esterase in degrading plant cell walls. The novel ferulic acid esterase and the recombined ferulic acid esterase of the invention have high efficient soluable expression in an Escherichia coli expression system. The recombined ferulic acid esterase can release 16.3 plus or minus 0.9mu. g of ferulic acid from 20mg of discarded starch wheat bran, and the released ferulic acid is 16% of the total amount of the ferulic acid.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a novel ferulic acid esterase obtained from seabed mud samples by using a metagenomic library screening method, and an application of the ferulic acid esterase. Background technique [0002] Lignocellulose (such as straw, bran, etc.) is the most abundant renewable resource on the earth, and its production amount is as high as 5×10 10 tons, and can be regenerated year after year through photosynthesis, thus gaining more and more attention. The main components of lignocellulose are cellulose, hemicellulose and lignin, and the enzymatic hydrolysis of lignocellulose is mainly through the joint action of cellulase, xylanase and ligninase. However, phenolic acids (such as ferulic acid and p-coumaric acid) rich in plant cell walls form cross-links between lignin, hemicellulose, and hemicellulose and lignin in the form of ester bonds. , thus forming a hard skeleton struct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/10C12N15/63C12N15/70C12P7/42
Inventor 刘玉焕桑姝丽范新炯
Owner SUN YAT SEN UNIV
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