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CN-121975767-A - High-temperature-resistant feruloyl esterase liquid as well as preparation method and application thereof

CN121975767ACN 121975767 ACN121975767 ACN 121975767ACN-121975767-A

Abstract

The invention relates to a high temperature resistant ferulic acid esterase liquid and a preparation method and application thereof, comprising the following steps of (1) constructing a reaction system, suspending recombinant microorganism thalli expressing the high temperature resistant ferulic acid esterase in a buffer solution with pH of 7.0-9.0, (2) carrying out ultrasonic pyrolysis and thermal purification, heating the reaction system to 70-85 ℃ under the condition of ultrasonic breaking, preserving heat for 1-8h, utilizing ultrasonic to destroy the structure of the microorganism thalli so as to release intracellular high temperature resistant ferulic acid esterase, utilizing the difference of the thermal stability of the high temperature resistant ferulic acid esterase and host hybrid proteins to denature and precipitate the host hybrid proteins, and (3) obtaining the enzyme liquid, namely obtaining the reaction liquid after ultrasonic and thermal treatment in the step (2), and centrifugally collecting supernatant to obtain the high temperature resistant ferulic acid esterase liquid.

Inventors

  • YANG SHENG
  • Lin Miaofang
  • LIU HONGBIN
  • Wei Canxian
  • Dang Mengyao

Assignees

  • 宁德师范学院

Dates

Publication Date
20260505
Application Date
20260310

Claims (8)

  1. 1. The preparation method of the high-temperature-resistant feruloyl esterase liquid is characterized by comprising the following steps of: (1) Constructing a reaction system, namely suspending recombinant microorganism thalli expressing high-temperature resistant feruloyl esterase in a buffer solution with pH of 7.0-9.0; (2) Ultrasonic pyrolysis and thermal purification, namely heating the reaction system to 70-85 ℃ under the condition of no chemical pyrolysis reagent, and preserving heat for 1-8 hours, utilizing ultrasonic to destroy the structure of microbial thalli to release intracellular high-temperature resistant feruloyl esterase, and simultaneously utilizing the thermal stability difference between the high-temperature resistant feruloyl esterase and host hybrid protein to denature and precipitate the host hybrid protein, wherein the high-temperature resistant feruloyl esterase maintains catalytic activity under the condition of more than 60 ℃ after the heat treatment of the step (2); (3) Obtaining enzyme liquid, namely centrifugally collecting supernatant fluid from the reaction system treated in the step (2) to obtain the high-temperature-resistant feruloyl esterase liquid; the feruloyl esterase is selected from any one of the following: (a) A protein having an amino acid sequence shown in SEQ ID NO. 2; (b) The protein has a mutant which is substituted, deleted or added by one or a plurality of amino acid residues and has the derivative protein with feruloyl esterase activity, and the heat inactivation half life under the condition of 75 ℃ is not less than 10 hours.
  2. 2. The method according to claim 1, wherein the heating temperature in the step (2) is 75 ℃ and the holding time is 3 to 8 hours.
  3. 3. The method of claim 1, wherein the buffer is phosphate buffer or Tris-HCl buffer.
  4. 4. The method of claim 1, wherein the mutant is an F56Y mutant or an R164Q mutant.
  5. 5. A method for producing ferulic acid from plant materials using a high temperature resistant feruloyl esterase, comprising: (1) Preparing a high temperature resistant feruloyl esterase solution by the method of any of claims 1 to 4; (2) Pretreating, namely suspending the plant raw material rich in ferulic acid in the reaction liquid, adding xylanase, and carrying out enzymolysis pretreatment at 50 ℃ to integrate reaction so as to partially hydrolyze the arabinoxylan structure in the plant raw material; (3) Catalyzing with feruloyl esterase, namely adding the high-temperature resistant feruloyl esterase liquid prepared in the step (1), increasing the temperature to 70-85 ℃ and keeping for 1-8h; (4) Separating the product, separating the reaction system, and collecting the liquid phase containing the free ferulic acid.
  6. 6. The method of claim 5, wherein the plant material is de-starched wheat bran or corn bran.
  7. 7. A feruloyl esterase preparation, characterized in that it is prepared by the method of any of claims 1-4.
  8. 8. Use of the feruloyl esterase preparation of claim 7 in the preparation of a food additive, a cosmetic raw material or a pharmaceutical intermediate.

Description

High-temperature-resistant feruloyl esterase liquid as well as preparation method and application thereof Technical Field The invention relates to a high-temperature resistant feruloyl esterase liquid, and a preparation method and application thereof, and belongs to the technical field of feruloyl esterase preparation. Background Ferulic acid (Ferulicacid) is widely used in plant cell walls as a phenolic acid compound having important value in the fields of foods, cosmetics and medicines. Because ferulic acid is mainly combined with structures such as arabinoxylan through ester bonds, the ferulic acid is difficult to directly extract, and enzymatic hydrolysis becomes a research hot spot due to mild conditions and high selectivity. However, the industrial application of the existing feruloyl esterases faces a significant bottleneck: The preparation cost is high, the acquisition of intracellular enzymes usually requires high-energy consumption mechanical disruption (such as high-pressure homogenization), and complicated chromatographic purification is further required to remove host proteins, so that the process is complicated. The heat stability is insufficient, most of feruloyl esterase from sources have an optimal temperature lower than 60 ℃, cannot adapt to an industrially efficient high-temperature reaction system, and limit the cooperative application of the feruloyl esterase and heat-resistant xylanase. The process is discontinuous, the traditional 'enzyme preparation-purification-application' sectional process has low efficiency, and the large-scale green manufacture of the ferulic acid is limited. Therefore, development of a feruloyl esterase technical system which is high-temperature resistant and can be prepared and applied at low cost through a simple process is urgent. Disclosure of Invention The invention aims to solve the problems of high cell disruption energy consumption, complex purification steps and intolerance of an enzyme preparation in the prior art, and provides a high-temperature-resistant feruloyl esterase liquid and a preparation method and application thereof. The invention provides a preparation method of a high-temperature resistant feruloyl esterase liquid, which comprises the following steps: (1) Constructing a reaction system, namely suspending recombinant microorganism thalli expressing high-temperature resistant feruloyl esterase in a buffer solution with pH of 7.0-9.0; (2) Ultrasonic cracking and heat purification, namely crushing the reaction system by ultrasonic waves and heating to 70-85 ℃ and preserving heat for 1-8 hours under the condition of no addition of a chemical cracking reagent, utilizing ultrasonic waves to cause structural destruction of microbial thalli so as to release intracellular high-temperature resistant feruloyl esterase, and simultaneously utilizing the heat stability difference between the high-temperature resistant feruloyl esterase and host hybrid proteins to denature and precipitate the host hybrid proteins; (3) Obtaining enzyme liquid, namely centrifugally collecting supernatant fluid from the reaction system treated in the step (2) to obtain the high-temperature-resistant feruloyl esterase liquid; the feruloyl esterase is selected from any one of the following: (a) An amino acid sequence shown as SEQ ID NO. 2; (b) The protein has a mutant which is substituted, deleted or added by one or a plurality of amino acid residues and has the derivative protein with feruloyl esterase activity, and the heat inactivation half life under the condition of 75 ℃ is not less than 10 hours. In the method, after ultrasonic crushing in a buffer system, under the conditions, a certain amount of host-derived soluble proteins are contained in the supernatant, wherein part of the proteins are thermally inactivated, and the high-temperature resistant feruloyl esterase can keep good structural stability and catalytic activity under the conditions, so that enough effective enzyme activity is kept in the obtained supernatant. The obtained enzyme-containing supernatant can be directly used for enzymatic conversion reaction of the starch-removed wheat bran without chromatography purification or immobilization, and can realize effective release of the combined ferulic acid in the plant raw material under the synergistic effect of the enzyme-containing supernatant and the commercial xylanase. In some embodiments, the heating temperature in step (2) is 75 ℃ and the incubation time is 3-8 hours. In some embodiments, the buffer is phosphate buffer or Tris-HCl buffer. In some embodiments, the mutant is an F56Y mutant or an R164Q mutant. A method for producing ferulic acid from plant materials using a high temperature resistant ferulic acid esterase, comprising: (1) Preparing high temperature resistant feruloyl esterase liquid by the method; (2) Pretreating, namely suspending the plant raw material rich in ferulic acid in the reaction liquid, adding xylanase, and carrying out enzy