WO2022134082A1 - Solution d'extraction pour extraire un principe actif d'uréase de canavalia gladiata et procédé d'extraction d'un principe actif d'uréase - Google Patents

Solution d'extraction pour extraire un principe actif d'uréase de canavalia gladiata et procédé d'extraction d'un principe actif d'uréase Download PDF

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Publication number
WO2022134082A1
WO2022134082A1 PCT/CN2020/139654 CN2020139654W WO2022134082A1 WO 2022134082 A1 WO2022134082 A1 WO 2022134082A1 CN 2020139654 W CN2020139654 W CN 2020139654W WO 2022134082 A1 WO2022134082 A1 WO 2022134082A1
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WO
WIPO (PCT)
Prior art keywords
urease
solution
extraction
extracting
supernatant
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PCT/CN2020/139654
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English (en)
Chinese (zh)
Inventor
张梅燕
袁振华
Original Assignee
美敦力公司
张梅燕
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Publication date
Application filed by 美敦力公司, 张梅燕 filed Critical 美敦力公司
Priority to PCT/CN2020/139654 priority Critical patent/WO2022134082A1/fr
Publication of WO2022134082A1 publication Critical patent/WO2022134082A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)

Definitions

  • the present invention relates to the extraction of adsorbent materials, in particular to the extraction of urease active components.
  • Urease also known as urease, is an adsorbent material commonly used in renal dialysis treatments that catalyzes the hydrolysis of urea to release ammonia and carbon dioxide, thereby converting urea into a nitrogen source available to the organism.
  • Urease is easily soluble in water, insoluble in organic solvents such as alcohol, ether and acetone, and is unstable at normal temperature and pressure.
  • urease is also very sensitive to temperature, pH and various factors that lead to protein denaturation, and is easily affected by external conditions to change its own properties.
  • urease can also be used clinically to diagnose urea ammonia in serum and the like.
  • urease can treat urea wastewater. Therefore, the market has a great demand for urease.
  • the publication number is CN104480092A
  • the publication date is April 1, 2015
  • the title is "a method for extracting urease from concanava", which discloses the use of phosphate buffer, BTP buffer or Tris-HCL buffer A method for extracting urease using liquid as an extract.
  • the extraction rate of the urease active components in the prior art is not high.
  • One of the objectives of the present invention is to provide an extract for extracting urease active components from concanavali.
  • the inventors of the present invention have found that by using the extract in this technical solution, the urease active component can be greatly improved in the initial extraction step.
  • the extraction rate can also be greatly improved in the end.
  • the technical solution provides an extract for extracting urease active components from concanavali, the extract contains: DTT (ie DL-Dithiothreitol) with a molar concentration of 0.26-13mM.
  • the molar concentration of DTT in the extract is 5.2-13 mM.
  • concanava is used as a raw material for extracting urease.
  • the low activity concanava refers to the concanava whose activity is less than 2.5IU/mg
  • the high activity one refers to the concanava whose activity is greater than 3.2IU/mg
  • the concanava whose activity is 2.5-3.2IU/mg is called It is medium active concanava.
  • the inventors of the present case have found through extensive research that the urease extraction method in the prior art is often suitable for high-activity concanavalina, but has poor effect on low-activity concanavalina.
  • using an extract containing 0.26 mM or more DTT can make the extraction rate of urease active components reach more than 40%, and using an extract containing 5.2 mM or more DTT can make the extraction rate of urease active components It can reach more than 50%, and even in some preferred embodiments, it can make the extraction rate of urease active components reach more than 60%.
  • using an extract containing more than 0.26mM DTT can make the extraction rate of urease active components reach more than 30%, and in some preferred embodiments, using an extract containing more than 5.2mM DTT can achieve The extraction rate of the urease active component can reach more than 50%. Therefore, the extracting solution of the present invention has excellent extraction performance for both high activity and low activity concanava.
  • the extracting solution of the present invention also contains a buffer solution with a molar concentration of 25mM-200mM.
  • the pH value of the buffer solution is 6.5-8.5.
  • the buffer solution may include a phosphate buffer solution, such as NaH 2 PO 4 -Na 2 HPO 4 buffer solution, or other similar phosphate buffer solutions.
  • a phosphate buffer solution such as NaH 2 PO 4 -Na 2 HPO 4 buffer solution, or other similar phosphate buffer solutions.
  • the buffer solution may include a Tris system buffer solution, such as Tris-HCl buffer solution, Tris-citrate buffer solution, or other similar Tris system buffer solutions.
  • Tris system buffer solution such as Tris-HCl buffer solution, Tris-citrate buffer solution, or other similar Tris system buffer solutions.
  • the molarity of the phosphate buffer solution may be 50-200 mM, and further the molarity of the phosphate buffer solution may be 50 mM-150 mM.
  • the pH value of the phosphate buffer solution may be 6.5-8.3, and the pH value of the Tris system buffer solution may be 7.1-8.5.
  • Na2HPO4 - citrate buffer solution its molarity can be 25mM-200mM, its pH value can be 6.5-8.3.
  • the KH 2 PO4-NaOH buffer solution may have a molar concentration of 25mM-200mM, and a pH value of 6.5-8.3.
  • the molar concentration of Tris-HCl buffer solution can be 25mM-200mM, and its pH value can be 7.1-8.5.
  • Tris-citrate buffer solution, its molar concentration can be 25mM-200mM, and its pH value can be 7.1-8.5.
  • the extracting solution of the present invention also contains: at least one of ethanol and acetone, wherein the mass percentage concentration of ethanol and/or acetone is 25-35%.
  • the extraction solution of the present invention can obtain the claimed extraction rate without EDTA, while the extraction solution in the prior art often contains EDTA.
  • the present invention also provides an extract for extracting urease active components from concanavali, which is composed of the following: (1) DTT; (2) buffer solution; (3) ethanol and At least one of acetone; wherein the molar concentration of DTT is 0.26-13 mM.
  • the molar concentration of DTT in the extract is 5.2-13 mM.
  • the molar concentration of the buffer solution is 25mM-200mM.
  • the pH value of the buffer solution is 6.5-8.5.
  • the buffer solution may include a phosphate buffer solution, such as NaH 2 PO 4 -Na 2 HPO 4 buffer solution, or other similar phosphate buffer solutions.
  • a phosphate buffer solution such as NaH 2 PO 4 -Na 2 HPO 4 buffer solution, or other similar phosphate buffer solutions.
  • the buffer solution may include a Tris system buffer solution, such as Tris-HCl buffer solution, Tris-citrate buffer solution, or other similar Tris system buffer solutions.
  • Tris system buffer solution such as Tris-HCl buffer solution, Tris-citrate buffer solution, or other similar Tris system buffer solutions.
  • the molarity of the phosphate buffer solution may be 50-200 mM, and further the molarity of the phosphate buffer solution may be 50 mM-150 mM.
  • the pH value of the phosphate buffer solution may be 6.5-8.3, and the pH value of the Tris system buffer solution may be 7.1-8.5.
  • Na2HPO4 - citrate buffer solution its molarity can be 25mM-200mM, its pH value can be 6.5-8.3.
  • the KH 2 PO4-NaOH buffer solution may have a molar concentration of 25mM-200mM, and a pH value of 6.5-8.3.
  • the molar concentration of Tris-HCl buffer solution can be 25mM-200mM, and its pH value can be 7.1-8.5.
  • Tris-citrate buffer solution, its molar concentration can be 25mM-200mM, and its pH value can be 7.1-8.5.
  • the mass percentage concentration of ethanol and/or acetone is 25-35%.
  • the extraction rate of the urease active component extracted from the concanavalina is ⁇ 30%.
  • the extraction rate of the urease active component extracted from the concanava is ⁇ 40%.
  • the extraction rate of the urease active component extracted from the concanavalina is ⁇ 50%.
  • the extraction rate of the urease active component extracted from the concanavalina is ⁇ 60%.
  • the extraction rate of the urease active ingredient refers to the content of the urease active ingredient in the solution containing the urease active ingredient (that is, the first supernatant liquid described below) and the raw material of the urease active ingredient. ratio of active ingredient content.
  • the content of the active ingredient of urease can be obtained by multiplying the concentration of the active ingredient in the solution (for example, the unit parameter is IU/ml) by the volume of the solution (for example, the unit parameter is ml), and the content of the active ingredient in the concanava can be obtained by the concave bean
  • the activity value of for example, the unit parameter is IU/mg
  • the mass of the bean for example, the unit parameter is mg
  • Another object of the present invention is to provide a method for extracting urease active components from concanava, which can greatly improve the extraction rate of urease.
  • the present invention provides a method for extracting urease active component from concanavalin, which comprises the steps:
  • a first supernatant containing the urease active component is obtained.
  • the temperature of the extraction solution is 18-40°C.
  • the temperature of the extraction solution is 31-35°C.
  • the stirring and soaking time is ⁇ 1h.
  • the stirring and soaking time is ⁇ 2h.
  • Another object of the present invention is to provide a method for extracting urease from concanavalinum.
  • the method adopts a specific extracting solution, so that the concanavalina treated with the extracting solution has a higher urease extraction rate, so that the final prepared Urease has a high yield.
  • the present invention provides a kind of method for extracting urease from concanavali, which comprises the steps:
  • Urease was extracted from the first supernatant.
  • the step of extracting urease from the first supernatant comprises:
  • step (a) the pH value of the first supernatant is adjusted to 7.5-9.
  • step (b) acetone or ethanol is added to make the mass percentage concentration reach 30-40%.
  • step (c) the pH value of the second supernatant is adjusted to 5.0-6.0.
  • step (b) the stirring time can be 30-45min.
  • step (c) stand for 16-20h.
  • step (e) the solution is adjusted to neutrality by using Tris solution as an alkaline solution.
  • step (g) urease is obtained by freeze-drying or chromatography.
  • the extraction solution of the present invention for extracting urease active components from concanavalinas adopts the above technical scheme, so that the extraction rate of urease active components is greatly improved.
  • using an extract containing 0.26 mM or more DTT can make the extraction rate of urease active components reach more than 40%
  • using an extract containing 5.2 mM or more DTT can make the extraction rate of urease active components reach more than 50%.
  • the extraction rate of urease active components can reach more than 30% by using the extract containing DTT above 0.26mM, and in some preferred embodiments, using DTT containing more than 5.2mM and using phosphoric acid
  • the extraction solution of the salt buffer solution can make the extraction rate of urease active components reach more than 50%.
  • the extraction rate of urease active components will vary with the buffer solution. increased with an increase in pH.
  • the addition of DTT can help improve the extraction rate of urease active components under the same extraction conditions, compared with no addition of DTT, for example,
  • the extraction rate of the active ingredient of low activity canava urease can be increased by at least 24%, and the extraction rate of active ingredient of high activity canava urease can be increased by at least 18%. Therefore, the extracting solution of the present invention has excellent extraction performance for both high activity and low activity concanava.
  • the method for extracting urease active components of the present invention also has the above beneficial effects.
  • the extraction rate of the urease active component can be greatly improved in the initial extraction step, and the yield of urease can also be greatly improved in the end.
  • Urease is obtained from the third supernatant by lyophilization or chromatography.
  • Table 1 lists the components of the extracts employed in Examples 1-79 of the present invention.
  • Table 2 lists the process parameters of each step of extracting urease active components and urease in Examples 1-79 of the present invention.
  • the use of the extract of the present invention for extracting urease active components from concanava can greatly improve the extraction rate of urease active components.
  • the reason is that 0.26mM-13mM of DTT is added to the extract.
  • the addition of 0.26mM-13mM DTT is very effective in improving the extraction rate of urease active components of low activity and high activity concanava beans, especially for the extraction rate of urease active components of low activity concanavalis. Effect.
  • the extraction rate of urease active components can reach at least 30% or more by using the extract containing more than 0.26mM DTT, while using the extract containing more than 5.2mM DTT
  • the extraction rate of the urease active component can also be made to reach at least 30% or more, and when a more appropriate buffer solution, such as a phosphate buffer solution, is used, the extraction rate of the urease active component can reach more than 50%.
  • the extraction rate of urease active components can reach at least 40% by using the extract containing more than 0.26mM DTT, and using the extract containing more than 5.2mM DTT can make the extraction of urease active components The rate reaches more than 50%, while the use contains.
  • the extraction rate of the urease active component can reach more than 60% by using the DTT-containing extract.
  • Table 1 specifically lists all extraction conditions (including extraction process, buffer type, concentration, pH value) , auxiliary types and concentrations, etc.) are all the same, the extraction rate of the urease active ingredient without DTT, and the relative improvement rate of the extraction rate of the urease active ingredient due to the addition of DTT compared to the absence of DTT.
  • Table 1 although the use of different buffer solutions will make the extraction rate of urease active components different, for example, for the buffer solution of Tris system, the extraction rate of urease active components will vary with the pH value of the buffer solution. rise and rise.
  • DTT can help to improve the extraction rate of urease active components on the original basis, compared with no addition of DTT.
  • adding DTT can increase the extraction rate of its urease active component by at least 24%, compared with no DTT.
  • adding DTT can increase the extraction rate of its urease active component by at least 18%, compared with not adding DTT.

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Abstract

Solution d'extraction pour extraire un principe actif d'uréase de Canavalia gladiata, la solution d'extraction contenant du DTT avec une concentration molaire de 0,26 à 13 mM. En outre, la présente invention concerne également un procédé d'extraction d'un principe actif d'uréase de Canavalia gladiata, et un procédé d'extraction d'uréase de Canavalia gladiate. Le taux d'extraction du principe actif d'uréase est considérablement amélioré par l'utilisation de la solution technique. La solution d'extraction a une excellente performance d'extraction à la fois pour le gladiate de Canavalia ayant une activité élevée et le gladiate de Canavalia ayant une faible activité.
PCT/CN2020/139654 2020-12-25 2020-12-25 Solution d'extraction pour extraire un principe actif d'uréase de canavalia gladiata et procédé d'extraction d'un principe actif d'uréase WO2022134082A1 (fr)

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PCT/CN2020/139654 WO2022134082A1 (fr) 2020-12-25 2020-12-25 Solution d'extraction pour extraire un principe actif d'uréase de canavalia gladiata et procédé d'extraction d'un principe actif d'uréase

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN85104238A (zh) * 1985-04-10 1986-10-08 华南师范大学 处理尿素废水用的固定化脲酶制法
CN104480092A (zh) * 2014-12-24 2015-04-01 陕西嘉禾植物化工有限责任公司 一种从刀豆中提取尿素酶的方法
WO2016018465A1 (fr) * 2014-07-30 2016-02-04 Medtronic, Inc. Purification de l'uréase issue de pois sabre ou d'autres organismes
WO2019084713A1 (fr) * 2017-10-30 2019-05-09 Medtronic Inc. Purification d'uréase à partir de haricots
CN109735521A (zh) * 2019-03-18 2019-05-10 中国石油大学(华东) 一种洋刀豆中脲酶的结晶提取方法
CN111171108A (zh) * 2019-12-27 2020-05-19 中国热带农业科学院南亚热带作物研究所 一种澳洲坚果叶片总蛋白质的高效提取方法
CN112094888A (zh) * 2020-11-09 2020-12-18 湖南大地同年生物科技有限公司 一种多功能缓冲液及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN85104238A (zh) * 1985-04-10 1986-10-08 华南师范大学 处理尿素废水用的固定化脲酶制法
WO2016018465A1 (fr) * 2014-07-30 2016-02-04 Medtronic, Inc. Purification de l'uréase issue de pois sabre ou d'autres organismes
CN104480092A (zh) * 2014-12-24 2015-04-01 陕西嘉禾植物化工有限责任公司 一种从刀豆中提取尿素酶的方法
WO2019084713A1 (fr) * 2017-10-30 2019-05-09 Medtronic Inc. Purification d'uréase à partir de haricots
CN109735521A (zh) * 2019-03-18 2019-05-10 中国石油大学(华东) 一种洋刀豆中脲酶的结晶提取方法
CN111171108A (zh) * 2019-12-27 2020-05-19 中国热带农业科学院南亚热带作物研究所 一种澳洲坚果叶片总蛋白质的高效提取方法
CN112094888A (zh) * 2020-11-09 2020-12-18 湖南大地同年生物科技有限公司 一种多功能缓冲液及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KLOTH R. H, HYMOWITZ T: "Re-evaluation of the inheritance of urease in soybean seed", CROP SCIENCE, vol. 25, no. 2, 31 March 1985 (1985-03-31), US , pages 352 - 354, XP055946120, ISSN: 0011-183X *

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