Extraction method for ferulic acid esterase

A technology of ferulic acid esterase and enzyme liquid, applied in the direction of hydrolytic enzymes, etc., can solve the problem that the digestion utilization rate does not exceed 50%, and achieve the effect of high practical application value

Inactive Publication Date: 2008-07-09
CHINA AGRI UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

About 30% to 80% of the dry matter of straw and forage grass is composed of cell walls, h

Method used

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  • Extraction method for ferulic acid esterase
  • Extraction method for ferulic acid esterase

Examples

Experimental program
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Embodiment 1

[0022] The processing of embodiment 1 beef cattle rumen liquid

[0023] In the early morning when the cattle were fasting, 1200mL of rumen fluid was extracted from the rumen through the rumen cannula of the cattle, filtered through four layers of medical gauze, and the filtrate was centrifuged at 20,000×g at 4°C for 30 minutes to remove bacterial precipitates, and 1000mL of the supernatant was collected for supernatant. Filtration and other next procedures.

[0024] Take the supernatant and first pass it through the ultrafiltration membrane ultrafiltration with a molecular weight cut-off of 100KDa, collect the part 100KDa, and prepare To obtain a higher-purity enzyme solution, use ice to control the temperature of the enzyme solution at 5°C, and the enzyme recovery rate can be as high as 55%.

[0025] Then, 100 mL of the concentrate sample obtained by ultrafiltration with a molecular weight between 10-100 KDa was heated at 60°C for 30 minutes, and centrifuged at 10,000×g at 4...

Embodiment 2

[0026] The processing of embodiment 2 concentrate samples

[0027] Dialyze 100mL of the concentrated solution with a molecular weight > 100KDa and the concentrated solution with a molecular weight between 10-100KDa to remove denatured proteins, concentrate, add an equal volume of 200mM sodium phosphate buffer (pH7) and 2 times the volume of deionized water, and mix well. Take 0.8mL samples with molecular weight > 100KDa and separate them with strong anion exchange chromatography column Q Sepharose Fast Flow column at a speed of 2mL / min, using 50mM sodium phosphate buffer (pH7) as solution A, 50mM sodium phosphate buffer containing 1M NaCl Solution (pH7) is B solution. Unbound substances were eluted with 30mL A solution, and bound proteins were eluted with a five-step gradient: 30mL 10% B solution, 30mL 25% B solution, 30mL 50% B solution, 30mL 75% B solution, 100mL B solution to elute bound proteins, Collect the fraction with ferulic acid esterase activity, dialyze, concentra...

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Abstract

The method relates to a method for extracting ferulic acid esterase from rumen fluid or culture solution which produces the ferulic acid esterase, the method comprises following steps: filtering centrifugal, ultrafiltration enrichment, heating, ion-exchange chromatography, hydrophobic interaction chromatography and molecular sieve chromatography. The invention extracts various proteins which are provided with ferulic acid esterase activity from the rumen fluid or the culture solution whose components are more complex and ferulic acid esterase species are more according to different physicochemical natures of different ferulic acid esterase through organic combination of various separating and purifying steps and obtains esterase with high activity. Various ferulic acid esterase can be extracted through using the method of the invention and thereby the invention reach better production performances and has higher practical value in the feed industry.

Description

technical field [0001] The invention relates to a method for extracting ferulic acid esterase, in particular to a method for extracting ferulic acid esterase from rumen fluid or ferulic acid-producing bacteria culture liquid. Background technique [0002] Ferulic acid esterase (E.C.3.1.1.73, also known as cinnamic acid esterase, cinnamic acid hydrolase) is a class of enzymes that hydrolyze the ester bond between hydroxycinnamic acid and sugar in the plant cell wall, and the main reaction it catalyzes is : polysaccharide-ferulic acid ester+H 2 O→ferulic acid + polysaccharides (including some derivatives of ferulic acid). [0003] In recent years, the role of ferulic esterase in feed digestion has attracted increasing attention. About 30% to 80% of the dry matter of straw and forage grass is cell wall components, however, the digestion and utilization of these cell walls by ruminants does not exceed 50%. Therefore, the cell wall becomes a bottleneck factor for ruminants to ...

Claims

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Application Information

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IPC IPC(8): C12N9/18
Inventor 杨红建岳群
Owner CHINA AGRI UNIV
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