A method for immobilizing ferulic esterase with magnetic nanomaterials

A technology of ferulic acid esterase and magnetic nanometer, which is applied in the direction of being fixed on or in an inorganic carrier, can solve the problems of non-reusable and expensive ferulic acid esterase, and achieves easy reuse and thermal stability. Improve and fix the effect of simple process

Inactive Publication Date: 2018-06-19
SHANDONG SHUANGLINGCHUN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for immobilizing ferulic acid esterase with magnetic nanomaterials with high enzyme activity, so as to overcome the problems that existing ferulic acid esterases are expensive and cannot be reused.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Degradation of p-nitrophenol ferulic acid ester by immobilized ferulic acid esterase:

[0018] p-nitrophenol ferulic acid ester is degraded into p-nitrophenol by ferulic acid esterase or immobilized ferulic acid esterase, and p-nitrophenol has a strong characteristic absorption at 410 nm, which can be measured by UV-Vis spectrophotometry meter for testing. p-Nitrophenol Ferulic Acid is a designed simulated substrate, which is a non-commercialized compound. Ferulic acid and p-Nitrophenol are used as raw materials to synthesize p-Nitrophenol Ferulic Acid in one step. The product was purified by recrystallization four times.

[0019] Buffer solution configuration: take 68.5mL 0.1mol / L NaH 2 PO 4 and 31.5mL 0.1mol / L Na 2 HPO 4 , mix well, then add 2.5mL Triton X-100, shake well, dissolve and set aside for use.

[0020] Preparation of 10 mmol / L p-nitrophenol ferulic acid ester (or p-nitrophenol) in dimethyl sulfoxide (DMSO) solution: weigh 0.1 mmol of p-nitrophenol fer...

Embodiment 2

[0033] Degradation of ethyl ferulate to ferulic acid by immobilized ferulic acid esterase:

[0034] The degradation product ferulic acid has a characteristic absorption at 322 nm. The standard curve of ferulic acid is determined by HPLC, and the concentration of ferulic acid obtained by the degradation of ethyl ferulic acid by immobilized ferulic acid esterase is determined by using this curve, so as to calculate the concentration of ferulic acid Activity and degradation rate.

[0035] Enzyme activity assay method: Dissolve ethyl ferulate in PB buffer (pH6.5, concentration 0.02M) to obtain 0.1mg / mL ethyl ferulate solution, take 250μL ferulic acid esterase or immobilized ferulic acid Esterase was placed in a centrifuge tube, incubated at 45°C for 5 minutes, then 250 μL of ethyl ferulic acid solution was added, reacted for 10 minutes, and then 500 μL of 50% glacial acetic acid aqueous solution was added to terminate the reaction, and then centrifuged at 7500 rpm for 10 minutes, ...

Embodiment 3

[0047] Degradation of wheat bran by immobilized ferulic acid esterase:

[0048] Wheat bran was soaked in 0.3% (v / w) sodium acetate solution, treated at 95°C for 30 minutes, then dried in an oven at 105°C to constant weight, and passed through a 50-mesh sieve to obtain starch-free wheat bran. Degradation of starchy wheat bran by immobilized ferulic acid esterase.

[0049] The total ferulic acid content in starchy wheat bran was determined by alkaline hydrolysis and defined as 100%. Measure 0.25g of starchy wheat bran, add it to 1mol / L NaOH solution, and incubate at 40°C, 120rpm in a shaker for 10h. The mixture was then centrifuged at 10000 rpm for 10 min. The supernatant was adjusted to pH 5.0 with glacial acetic acid, and then its ferulic acid content was measured by HPLC.

[0050] Free ferulic acid esterase and an equal amount of immobilized ferulic acid esterase (both containing the same amount of enzyme) were added to PB buffer (0.2M, pH 6.0) containing 0.25g starchy whe...

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Abstract

The invention discloses a method for immobilizing ferulic acid esterase with magnetic nanomaterials, which comprises the preparation of Fe2O3 immobilized carrier magnetic nanomaterials; mixing the immobilized carrier magnetic nanomaterials with ferulic esterase solution; and preparing on magnetic equipment Immobilized ferulic acid esterase. The preparation process of the invention is simple, the reaction conditions are mild, and the utilization rate of repeated regeneration is high. The prepared immobilized ferulic acid esterase maintains more than 90% of the equivalent free enzyme activity, and the thermal stability is improved. The enzyme activity of ferulic acid esterase can preserve more than 80%.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzyme preparation, and in particular relates to a method for immobilizing ferulic esterase with magnetic nanometer materials. Background technique [0002] Ferulic esterase, also known as cinnamic esterase, belongs to the subclass of carboxylesterase, which is derived from fungi, bacteria and yeast, and can hydrolyze the cross-linked albino between hemicellulose and between hemicellulose and lignin. Ferulic acid ester linkages, such as oligoferulic acid esters, methyl ferulic acid esters, and ester linkages in polysaccharide ferulic acid esterases, play an important role in the degradation of lignocellulosic fibers, such as promoting hemicellulose, lignin The separation from cellulose is beneficial to the further utilization of cellulose, so it has broad application prospects in the food, feed and paper industries. [0003] However, ferulic acid esterase is expensive, and the use of ferulic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14
Inventor 张松刘新利和富明
Owner SHANDONG SHUANGLINGCHUN BIOLOGICAL TECH
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