WO2019084713A1 - Urease purification from beans - Google Patents

Urease purification from beans Download PDF

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Publication number
WO2019084713A1
WO2019084713A1 PCT/CN2017/108319 CN2017108319W WO2019084713A1 WO 2019084713 A1 WO2019084713 A1 WO 2019084713A1 CN 2017108319 W CN2017108319 W CN 2017108319W WO 2019084713 A1 WO2019084713 A1 WO 2019084713A1
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WIPO (PCT)
Prior art keywords
urease
solution
extracted
liquid
extracting
Prior art date
Application number
PCT/CN2017/108319
Other languages
French (fr)
Inventor
Anyuan YIN
Zhenrong ZHOU
Original Assignee
Medtronic Inc.
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Publication date
Application filed by Medtronic Inc. filed Critical Medtronic Inc.
Priority to PCT/CN2017/108319 priority Critical patent/WO2019084713A1/en
Publication of WO2019084713A1 publication Critical patent/WO2019084713A1/en

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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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01005—Urease (3.5.1.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • the invention relates to a method of extracting, separating and purifying urease from jack beans or other organisms such as soy beans, peas etc.
  • Urea-degrading enzymes ( "urease” ) , functionally, belong to the superfamily of amidohydrolases and phosphotriestrerases. It is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia.
  • Urease is used as an important biologic agent, which has been applied in medical, agriculture, environment, farming, clinic diagnostic and so on.
  • Jack bean is a common source ofurease.
  • the vast majority of the mass of jack beans is made up of materials other than urease, including other proteins, fats and lipids.
  • Jack bean-based urease was first purified in 1926, and several researchers have published purification methods in literature since then.
  • a method of extracting urease from jack beans was disclosed in CN104480092A, namely, the urease was obtained from jack bean meal via the extraction with 2ME and acetone, settled down, neutralization, microfiltration, ultrafiltration and lyophilization. However, the process is still completed and toxic agent is used.
  • a method of urease extraction and purification from jack beans or other organisms was disclosed in WO2016018465A1.
  • the method includes steps of defatting the nature sources of urease, extracting the urease from the impurities and further purifying the extracted urease.
  • the present invention is directed to provide a method of extracting urease from beans and purifying the extracted urease in an efficient and low-cost way.
  • the present invention is also directed to provide a method of extracting urease from beans and purifying the extracted urease to obtain high activity urease.
  • the present invention is also directed to provide a method of extracting urease from beans and purifying the extracted urease to obtain high yield urease.
  • the first aspect of the invention is drawn to a method of extraction and purifying urease from beans.
  • the method can comprise the steps of:
  • the extracted urease solution is adjusted to be alkaline (i.e. the pH of the extracted urease solution is greater than 7) to obtain a purified urease liquid and then the purified urease liquid is adjusted to be acidic (i.e. the pH of the purified urease liquid is less than 7) .
  • pH adjustments a much more purified urease can be obtained.
  • the hulls of the beans can be removed prior to the step of extracting the urease from beans.
  • the beans can be ground to make bean meal prior to the step of extracting urease from the beans.
  • the beans can be ground to 80 meshes or above to make bean meal prior to the step of extracting urease from the beans.
  • the pH of extracting solution can be 7 ⁇ 8.
  • the extracting solution can be an aqueous solution.
  • the extracting solution can be acetone aqueous solution, namely, can consist of water and acetone, wherein the mass percent of acetone in the extracting solution can be 10%to 50%.
  • the extracting solution can consist of water, acetone, EDTA (Ethylene Diamine Tetraacetic Acid) and DTT (DL-Dithiothreitol) .
  • the acetone can be replaced by but not limited to methanol, ethanol, or petroleum ether.
  • the extracting solution can consist of water, 10wt. % ⁇ 50wt. %of acetone, 0.01wt. % ⁇ 0.5wt. %of EDTA and 0 ⁇ 0.1wt. %of DTT.
  • the extracting solution can consist of water, EDTA and DTT.
  • the extracting solution can consist of water, 0.01wt. % ⁇ 0.5wt. %of EDTA and 0 ⁇ 0.1wt. %of DTT.
  • the extracting solution can consist of water, acetone, EDTA, DTT and buffer solution.
  • the extracting solution can consist of water, 10wt. % ⁇ 50wt. %of acetone, 0.01wt. % ⁇ 0.5wt. %of EDTA, 0 ⁇ 0.1wt. %of DTT and 45wt. % ⁇ 90wt. %of buffer solution.
  • the extracting solution can consist of water, EDTA, DTT and buffer solution.
  • the extracting solution can consist of water, 0.01wt. % ⁇ 0.5wt. %%of EDTA, 0 ⁇ 0.1wt. %of DTT and 45wt. % ⁇ 90wt. %of buffer solution.
  • the buffer solution can be at least any one of phosphate buffer solution, Tris buffer solution or Bis-tris propane buffer solution.
  • the mass ratio between the beans and the extracting solution can be 1/20 to 1/1.
  • the step of extracting urease from the beans can comprise mixing the beans, particularly bean meal, with the extracting solution to create a mixture
  • the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic, which provide energy to break the bean cell to release the urease.
  • the extracting solution is an environmentally friendly extraction solution (without any organic solvent) , which is developed to extract the urease from the bean meal.
  • the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic for 15 ⁇ 60 minutes.
  • the step of extracting urease from the beans can further comprise mechanically separating the mixture to obtain the extracted urease solution which contains urease.
  • the pH of the extracted urease solution can be adjusted to greater than 7 and not greater than 9.
  • the step of adjusting the pH of the extracted urease solution to greater than 7 can be followed by a step of heat-treatment.
  • the heat-treatment can comprise heating the extracted urease solution at a temperature of 30 ⁇ 50 degree centigrade.
  • the purified urease liquid can be obtained by mechanical separation.
  • the pH of the purified urease liquid can be adjusted to 3 ⁇ 6.
  • the step of adjusting the pH of the extracted urease liquid to less than 7 can be followed by a step of aging the urease liquid for at least 12 hours.
  • the step of aging the urease liquid for at least 12 hours can be followed by a step of mechanical separation for obtaining the urease precipitates.
  • the step of mechanical separation can be followed by the step of drying the urease precipitates by spry drying, lyophilization or supercritical drying.
  • the urease is very sensitive to temperature and moisture. To obtain the final urease, the proper drying method for removing the additional moisture with lower temperature is needed to make sure the final sample is stable. Herein, spray drying, lyophilization or supercritical approach can be used.
  • the second aspect of the invention is drawn to a method of extracting and purifying urease from beans.
  • the method of extracting and purifying urease from beans can comprise the steps of:
  • extracting urease from the beans with an extracting solution to obtain an extracted urease solution wherein the extracting solution is an aqueous solution or an extracting solution consisting of water and at least any one of acetone, EDTA, DTT and buffer solution; and purifying the extracted urease solution to obtain purified urease.
  • the hulls of the beans can be removed prior to the step of extracting urease from the beans.
  • the beans can be ground to make bean meal prior to the step of extracting urease from the beans.
  • the pH of extracting solution can be 7 ⁇ 8.
  • the extracting solution can be an aqueous solution.
  • the extracting solution can consist of water and acetone.
  • the mass percent of acetone in the extracting solution can be 10% ⁇ 50%.
  • the extracting solution can consist of water, acetone, EDTA and DTT.
  • the acetone can be replaced by but not limited to methanol, ethanol, petroleum ether.
  • the extracting solution can consist of water, 10wt. % ⁇ 50wt. %of acetone, 0.01wt. % ⁇ 0.5wt. %of EDTA and 0 ⁇ 0.1wt. %of DTT.
  • the extracting solution can consist of water, EDTA and DTT.
  • the extracting solution can consist of water, 0.01wt. % ⁇ 0.5wt. %of EDTA and 0 ⁇ 0.1wt. %of DTT.
  • the extracting solution can consists of water, acetone, EDTA, DTT and buffer solution.
  • the extracting solution can consist of water, 10wt. % ⁇ 50wt. %of acetone, 0.01wt. % ⁇ 0.5wt. %of EDTA, 0 ⁇ 0.1wt. %of DTT and 45wt. % ⁇ 90wt. %of buffer solution.
  • the extracting solution can consist of water, EDTA, DTT and buffer solution.
  • the extracting solution can consist of water, 0.01wt. % ⁇ 0.5wt. %of EDTA, 0 ⁇ 0.1wt. %of DTT and 45wt. % ⁇ 90wt. %of buffer solution.
  • the buffer solution can be at least any one of phosphate buffer solution, Tris buffer solution or Bis-tris propane buffer solution.
  • the step of extracting urease from the beans can comprise mixing the beans with the extracting solution to create a mixture.
  • the mass ratio between the beans and the extracting solution can be 1/20 to 1/1.
  • the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring, or by magnetic stirring or by ultrasonic.
  • the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic for 15 ⁇ 60 minutes.
  • the step of extracting urease from the beans can further comprise mechanically separating the mixture to obtain the extracted urease solution.
  • the step of purifying the extracted urease solution can comprise:
  • the pH of the extracted urease solution can be adjusted to greater than 7 and not greater than 9.
  • the step of adjusting the pH of the extracted urease solution to greater than 7 can be followed by a step of heat-treatment.
  • the heat-treatment can comprise heating the extracted urease solution at a temperature of 30 ⁇ 50 degree centigrade.
  • the purified urease liquid can be obtained by mechanical separation.
  • the pH of the purified urease liquid can be adjusted to 3 ⁇ 6.
  • the step of aging the urease liquid for at least 12 hours can be followed by a step of mechanical separation for obtaining the urease precipitates.
  • the step of mechanical separation is followed by the step of drying the urease precipitates by spry drying, lyophilization or supercritical drying.
  • the purified urease is urease with activity of no less than 300 IU/mg and/or yield of no less than 50%.
  • aging refers to keeping the solution for a certain time under the specified conditions.
  • aqueous solution refers to water solution not containing organic solvent.
  • beans refers to jack beans, soy beans, or peas, or any other similar organisms. Particularly, the beans can be jack beans.
  • Bean meal refers to beans that have been ground to fine particles.
  • defat or “defatting” refers to a process of removing lipids from a mixture of natural sources of urease as described herein including organic materials, generally containing ureases. Once the lipids have been removed, the material is “defatted. "
  • hull refers to the dry outer covering of the seeds.
  • “Jack beans” are the seeds of the plant canavalia ensiformis.
  • “Lyophilization” refers to a process by which material is preserved by cooling the material to a temperature below the triple point of water and then reducing the pressure to cause the water present to sublimate.
  • Mechanical separation or “mechanically separating” means any method of separating a liquid and a solid including filtration, decanting and centrifuging.
  • Rotovap is a device used in chemical laboratories for the efficient and gentle removal of solvents from samples by evaporation.
  • Supernatant liquid is the liquid lying above a solid residue.
  • the present invention is directed to provide a method of extracting urease from beans and purifying the extracted urease in an efficient and low-cost way. Besides, the present invention is also directed to provide a method of extracting urease from beans and purifying the extracted urease to obtain high activity and/or high yield urease.
  • the method can comprise the steps of:
  • the hulls of the beans can be removed prior to the step of extracting the urease from beans.
  • the beans can be ground to a certain size (e.g. 80 meshes or above) to make bean meal prior to the step of extracting urease from the beans.
  • the step of extracting urease from the beans can comprise mixing the beans, particularly mixing the above-mentioned bean meal, with the extracting solution to create a mixture.
  • the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic for a certain period of time, such as for 15 ⁇ 60 minutes.
  • the extracting solution acts on bean meal for extracting urease.
  • the step of extracting urease from the beans with an extracting solution can be repeated for more than one times, such as three times, for a higher yield.
  • the pH of extracting solution can preferably be 7 ⁇ 8.
  • the extracting solution is an environmentally friendly extraction solution (without any organic solvent) , which is developed to extract the urease from the bean meal.
  • the mass ratio between the beans and the extracting solution can be 1/20 to 1/1.
  • the extracting solution preferably is an aqueous solution or an extracting solution consisting of water and at least any one of acetone, EDTA, DTT and buffer solution.
  • the extracting solution can be an aqueous solution.
  • the extracting solution can consist of water and acetone.
  • the mass percent of acetone in the extracting solution can be 10%to 50%.
  • the extracting solution can consist of water, acetone, EDTA (Ethylene Diamine Tetraacetic Acid) and DTT (DL-Dithiothreitol) .
  • the extracting solution can consist of water, 10wt. % ⁇ 50wt. %of acetone, 0.01wt. % ⁇ 0.5wt. %of EDTA and 0 ⁇ 0.1wt. %of DTT.
  • the acetone can be replaced by, but not limited to, methanol, ethanol, or petroleum ether.
  • the extracting solution can consist of water, EDTA and DTT.
  • the extracting solution can consist of water, 0.01wt. % ⁇ 0.5wt. %of EDTA and 0 ⁇ 0.1wt. %of DTT.
  • the extracting solution can consist of water, acetone, EDTA, DTT and buffer solution.
  • the extracting solution can consist of water, 10wt. % ⁇ 50wt. %of acetone, 0.01wt. % ⁇ 0.5wt. %of EDTA, 0 ⁇ 0.1wt. %of DTT and 45wt. % ⁇ 90wt. %of buffer solution.
  • the extracting solution can consist of water, EDTA, DTT and buffer solution.
  • the extracting solution can consist of water, 0.01wt. % ⁇ 0.5wt. %%of EDTA, 0 ⁇ 0.1wt. %of DTT and 45wt. % ⁇ 90wt. %of buffer solution.
  • the buffer solution can be at least any one of phosphate buffer solution, Tris buffer solution or Bis-tris propane buffer solution.
  • the step of extracting urease from the beans can further comprise mechanically separating the mixture to obtain a supernatant liquid (i.e. the extracted urease solution) which contains urease.
  • the extracted urease solution is purified.
  • the step of purifying the extracted urease solution can comprise the steps of:
  • the pH of the extracted urease solution can be adjusted to greater than 7 and not greater than 9.
  • the step of adjusting the pH of the extracted urease solution to greater than 7, particularly to greater than 7 and not greater than 9, can be followed by a step of heat-treatment.
  • the alkaline extracted urease solution the pH of which has been was adjusted to greater than 7, particularly to greater than 7 and not greater than 9, can directly be centrifuged to obtain a purified urease liquid without any heat-treatment in some other embodiments, resulting in degraded effects in view of those technical solutions having a step of heat-treatment.
  • the heat-treatment can comprise heating the extracted urease solution at a temperature of 30 ⁇ 50 degree centigrade.
  • the heat-treatment can comprise heating the extracted urease solution at a temperature (e.g. any temperature of 30 ⁇ 50 degree centigrade) for a certain period of time, such as for 30 ⁇ 50 minutes.
  • a temperature e.g. any temperature of 30 ⁇ 50 degree centigrade
  • a step of separation is performed to obtain a purified urease liquid.
  • the step of mechanical separation can comprise centrifugation. That is, the alkaline extracted urease solution, the pH of which has been adjusted to greater than 7, particularly to greater than 7 and not greater than 9, was centrifuged to obtain a purified urease liquid.
  • the pH of the purified urease liquid can be adjusted to 3 ⁇ 6.
  • the step of adjusting the pH of the extracted urease liquid to less than 7, particularly to 3 ⁇ 6, can be followed by a step of aging the urease liquid for at least 12 hours.
  • the step of aging the urease liquid for at least 12 hours can be followed by a step of mechanical separation for obtaining urease precipitates.
  • the step of mechanical separation can comprise centrifugation. That is, the aged urease liquid is centrifuged to obtain the urease precipitates.
  • the step of mechanical separation can be followed by a step of drying the urease precipitates by spry drying, lyophilization or supercritical drying to obtain purified urease.
  • a step of drying the urease precipitates by spry drying, lyophilization or supercritical drying to obtain purified urease.
  • the urease is very sensitive to temperature and moisture, it is needed to adopt proper drying method for removing the additional moisture from urease with lower temperature in order to make sure the final urease is stable.
  • spray drying, lyophilization or supercritical approach can be used.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 30 minutes.
  • the extracting solution consisted of 10%acetone, 88%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 3 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. After that, the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 306 IU/mg and a yield of 56%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.01%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 4 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 505 IU/mg and a yield of 71%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 4 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 590 IU/mg and a yield of 82%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.5%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 4 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 509 IU/mg and a yield of 62%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 3 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 301 IU/mg and a yield of 51%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.1%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 5 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 530 IU/mg and a yield of 67%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 50%acetone, 40%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 5 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 509 IU/mg and a yield of 61%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 45 minutes.
  • the extracting solution consisted of 32%ethanol, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 3 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 326 IU/mg and a yield of 51%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 60 minutes.
  • the extracting solution consisted of 32%petroleum, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 3 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 308 IU/mg and a yield of 54%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 32%acetone, 65%tris, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 4 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 568 IU/mg and a yield of 69%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 32%acetone, 65%BTP (Bis-tris propane) , 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for 4 times for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 557 IU/mg and a yield of 67%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process was not repeated.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 469 IU/mg and a yield of 50%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process was not repeated.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 478 IU/mg and a yield of 67%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction was repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 598 IU/mg and a yield of 69%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 10 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 457 IU/mg and a yield of 53%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 20 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 443 IU/mg and a yield of 51%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 7.1. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 322IU/mg and a yield of 52%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 30 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 446 IU/mg and a yield of 53%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 12 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 598 IU/mg and a yield of 50%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 20 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 599 IU/mg and a yield of 68%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 50 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 462 IU/mg and a yield of 53%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 9.0. And the extracted urease solution was heated at a temperature of 50 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 325 IU/mg and a yield of 59%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 349 IU/mg and a yield of 51%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 6. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 375 IU/mg and a yield of 55%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1169 IU/mg and a yield of 86%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by supercritical drying to obtain purified urease with an activity of 667IU/mg and a yield of 62%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65% phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by rotovap to obtain purified urease with an activity of 505 IU/mg and a yield of 59%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 100%water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 356 IU/mg and a yield of 59%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone and68%water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 446 IU/mg and a yield of 62%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 67.65%water, 0.05%DTT and 0.3%EDTA and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 506 IU/mg and a yield of 61%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 99.65%water, 0.05%DTT and 0.3%EDTA and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 407 IU/mg and a yield of 54%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 1 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray to obtain purified urease with an activity of 460 IU/mg and a yield of 55%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 3 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray to obtain purified urease with an activity of 557 IU/mg and a yield of 63%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 15 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.5.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray to obtain purified urease with an activity of 450IU/mg and a yield of 50%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.2.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1092 IU/mg and a yield of 78%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1099 IU/mg and a yield of 81%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1172 IU/mg and a yield of 83%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.8.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1120 IU/mg and a yield of 80%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 15%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 853 IU/mg and a yield of 62%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 20%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 921 IU/mg and a yield of 65%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 40%acetone, 55%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 915 IU/mg and a yield of 64%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 45%acetone, 50%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 821 IU/mg and a yield of 60%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1098 IU/mg and a yield of 79%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.4%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1123 IU/mg and a yield of 81%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.025%DTT, 0.1%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 760 IU/mg and a yield of 62%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.075%DTT, 0.1%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 805 IU/mg and a yield of 57%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 55%phosphate, 0.025%DTT, 0.1%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 453 IU/mg and a yield of 55%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 23%acetone, 75%phosphate, 0.025%DTT, 0.1%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 472 IU/mg and a yield of 61%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 12%acetone, 85%phosphate, 0.025%DTT, 0.1%EDTA and water and was with a pH of 7.6.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 477 IU/mg and a yield of 58%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 55%phosphate, 0.075%DTT, 0.1%EDTA and water and was with a pH of 7.2.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 7.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 805 IU/mg and a yield of 57%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 55%phosphate, 0.075%DTT, 0.1%EDTA and water and was with a pH of 7.2.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 7.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 352 IU/mg and a yield of 58%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 55%phosphate, 0.075%DTT, 0.1%EDTA and water and was with a pH of 7.2.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.0. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 408 IU/mg and a yield of 61%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 55% phosphate, 0.075%DTT, 0.1%EDTA and water and was with a pH of 7.2.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 455 IU/mg and a yield of 66%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 55%phosphate, 0.075%DTT, 0.1%EDTA and water and was with a pH of 7.2.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.8. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 404 IU/mg and a yield of 61%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 23%acetone, 75%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3.2. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 358 IU/mg and a yield of 52%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 23%acetone, 75%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 379 IU/mg and a yield of 58%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3.6. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 426 IU/mg and a yield of 60%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3.8. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 564 IU/mg and a yield of 59%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 4.0. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 589 IU/mg and a yield of 62%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 4.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 565 IU/mg and a yield of 61%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.0. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 656 IU/mg and a yield of 68%.
  • Fresh beans were de-hulled and ground to make bean meal.
  • Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes.
  • the extracting solution consisted of 32%acetone, 65%phosphate, 0.05%DTT, 0.3%EDTA and water and was with a pH of 7.4.
  • the mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution.
  • the extraction process can be repeated for twice for a higher yield.
  • the pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.5. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 934 IU/mg and a yield of 75%.

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Abstract

A method for obtaining purified urease from beans, involves extracting urease from the beans with an extracting solution to obtain an extracted urease solution; adjusting the pH of the extracted urease solution to greater than 7; obtaining a purified urease liquid; adjusting the pH of the purified urease liquid to less than 7; and obtaining purified urease by drying urease precipitates obtained from the purified urease liquid.

Description

UREASE PURIFICATION FROM BEANS FIELD OF THE INVENTION
The invention relates to a method of extracting, separating and purifying urease from jack beans or other organisms such as soy beans, peas etc.
BACKGROUND
Urea-degrading enzymes ( "urease" ) , functionally, belong to the superfamily of amidohydrolases and phosphotriestrerases. It is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia.
Urease is used as an important biologic agent, which has been applied in medical, agriculture, environment, farming, clinic diagnostic and so on.
Jack bean is a common source ofurease. The vast majority of the mass of jack beans is made up of materials other than urease, including other proteins, fats and lipids. Jack bean-based urease was first purified in 1926, and several researchers have published purification methods in literature since then.
Known methods of extracting and purifying urease from jack beans, such as the method of Sung, et al, Proc. Natl. Sci. Counc. B. ROC, Vol. 13, No. 4, 1989, pp. 250-257, suffer from low purity of the urease recovered. Sung describes an extraction from jack bean meal using 20％acetone after stirring at 20℃for 5 minutes, followed by alkalifying the extract with heat treatment in the presence of 31-35％acetone, centrifuging the mixture, and then precipitating the urease by acidifying the extract. However, other proteins besides urease are present in the recovered material.
A method of extracting urease from jack beans was disclosed in CN104480092A, namely, the urease was obtained from jack bean meal via the extraction with 2ME and acetone, settled down, neutralization, microfiltration, ultrafiltration and lyophilization. However, the process is still completed and toxic agent is used.
A method of urease extraction and purification from jack beans or other organisms was disclosed in WO2016018465A1. The method includes steps of defatting the nature sources of urease, extracting the urease from the impurities and further purifying the extracted urease.
However, none of these processes were designed to produce urease in high volume and at low cost. Thus a cost effective and scalable process of purifying the urease from jack beans is needed.
Therefore, a method of extracting and purifying urease from jack beans efficiently and costly is desired.
SUMMARY OF THE INVENTION
The present invention is directed to provide a method of extracting urease from beans and purifying the extracted urease in an efficient and low-cost way.
The present invention is also directed to provide a method of extracting urease from beans and purifying the extracted urease to obtain high activity urease.
The present invention is also directed to provide a method of extracting urease from beans and purifying the extracted urease to obtain high yield urease.
The first aspect of the invention is drawn to a method of extraction and purifying urease from beans. In any embodiment of the first aspect of the invention, the method can comprise the steps of:
extracting urease from the beans with an extracting solution to obtain an extracted urease solution；
adjusting the pH of the extracted urease solution to greater than 7；
obtaining a purified urease liquid by separation；
adjusting the pH of the purified urease liquid to less than 7； and
obtaining purified urease by drying urease precipitates obtained from the purified urease liquid.
According to the invention, after extracting urease from the beans with an extracting solution to obtain an extracted urease solution, further purification from the extracted urease solution was carried via pH adjustments. The urease can be precipitated under the acidic condition； however a number of protein can be firstly precipitated under the basic condition to remove the impurities. Therefore, the extracted urease solution is adjusted to be alkaline (i.e. the pH of the extracted urease solution is greater than 7) to obtain a purified urease liquid and then the purified urease liquid is adjusted to be acidic (i.e. the pH of the purified urease liquid is less than 7) . After pH adjustments, a much more purified urease can be obtained.
In any embodiment of the first aspect of the invention, the hulls of the beans can be removed prior to the step of extracting the urease from beans.
In any embodiment of the first aspect of the invention, the beans can be ground to make bean meal prior to the step of extracting urease from the beans.
In any embodiment of the first aspect of the invention, the beans can be ground to 80 meshes or above to make bean meal prior to the step of extracting urease from the beans.
In any embodiment of the first aspect of the invention, the pH of extracting solution can be 7～8.
In any embodiment of the first aspect of the invention, the extracting solution can be an aqueous solution.
In any embodiment of the first aspect of the invention, the extracting solution can be acetone  aqueous solution, namely, can consist of water and acetone, wherein the mass percent of acetone in the extracting solution can be 10％to 50％.
In any embodiment of the first aspect of the invention, the extracting solution can consist of water, acetone, EDTA (Ethylene Diamine Tetraacetic Acid) and DTT (DL-Dithiothreitol) .
In any embodiment of the first aspect of the invention, the acetone can be replaced by but not limited to methanol, ethanol, or petroleum ether.
In any embodiment of the first aspect of the invention, the extracting solution can consist of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
In any embodiment of the first aspect of the invention, the extracting solution can consist of water, EDTA and DTT.
In any embodiment of the first aspect of the invention, the extracting solution can consist of water, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
In any embodiment of the first aspect of the invention, the extracting solution can consist of water, acetone, EDTA, DTT and buffer solution.
In any embodiment of the first aspect of the invention, the extracting solution can consist of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution.
In any embodiment of the first aspect of the invention, the extracting solution can consist of water, EDTA, DTT and buffer solution.
In any embodiment of the first aspect of the invention, the extracting solution can consist of water, 0.01wt. ％～0.5wt. ％％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution.
In any embodiment of the first aspect of the invention, the buffer solution can be at least any one of phosphate buffer solution, Tris buffer solution or Bis-tris propane buffer solution.
In any embodiment of the first aspect of the invention, the mass ratio between the beans and the extracting solution can be 1/20 to 1/1.
In any embodiment of the first aspect of the invention, the step of extracting urease from the beans can comprise mixing the beans, particularly bean meal, with the extracting solution to create a mixture
In any embodiment of the first aspect of the invention, the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic, which provide energy to break the bean cell to release the urease.
Particularly, the extracting solution is an environmentally friendly extraction solution (without any organic solvent) , which is developed to extract the urease from the bean meal.
In any embodiment of the first aspect of the invention, the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic for 15～60 minutes.
In any embodiment of the first aspect of the invention, the step of extracting urease from the beans can further comprise mechanically separating the mixture to obtain the extracted urease solution which contains urease.
In any embodiment of the first aspect of the invention, the pH of the extracted urease solution can be adjusted to greater than 7 and not greater than 9.
In any embodiment of the first aspect of the invention, the step of adjusting the pH of the extracted urease solution to greater than 7 can be followed by a step of heat-treatment.
In any embodiment of the first aspect of the invention, the heat-treatment can comprise heating the extracted urease solution at a temperature of 30～50 degree centigrade.
In any embodiment of the first aspect of the invention, the purified urease liquid can be obtained by mechanical separation.
In any embodiment of the first aspect of the invention, the pH of the purified urease liquid can be adjusted to 3～6.
In any embodiment of the first aspect of the invention, the step of adjusting the pH of the extracted urease liquid to less than 7 can be followed by a step of aging the urease liquid for at least 12 hours.
In any embodiment of the first aspect of the invention, the step of aging the urease liquid for at least 12 hours can be followed by a step of mechanical separation for obtaining the urease precipitates.
In any embodiment of the first aspect of the invention, the step of mechanical separation can be followed by the step of drying the urease precipitates by spry drying, lyophilization or supercritical drying.
The urease is very sensitive to temperature and moisture. To obtain the final urease, the proper drying method for removing the additional moisture with lower temperature is needed to make sure the final sample is stable. Herein, spray drying, lyophilization or supercritical approach can be used.
It can be known that any other drying methods known by a person skilled in the art can be used for drying the urease precipitates.
In any embodiment of the first aspect of the invention, the purified urease with activity of no less than 300 IU/mg and/or yield of no less than 50％can be obtained.
The second aspect of the invention is drawn to a method of extracting and purifying urease from beans. In any embodiment of the second aspect of the invention, the method of extracting and purifying urease from beans can comprise the steps of:
extracting urease from the beans with an extracting solution to obtain an extracted urease solution, wherein the extracting solution is an aqueous solution or an extracting solution consisting of water and at least any one of acetone, EDTA, DTT and buffer solution； and purifying the extracted urease solution to obtain purified urease.
In any embodiment of the second aspect of the invention, the hulls of the beans can be removed prior to the step of extracting urease from the beans.
In any embodiment of the second aspect of the invention, wherein the beans can be ground to make bean meal prior to the step of extracting urease from the beans.
In any embodiment of the second aspect of the invention, the pH of extracting solution can be 7～8.
In any embodiment of the second aspect of the invention, the extracting solution can be an aqueous solution.
In any embodiment of the second aspect of the invention the extracting solution can consist of water and acetone. Particularly, the mass percent of acetone in the extracting solution can be 10％～50％.
In any embodiment of the second aspect of the invention, the extracting solution can consist of water, acetone, EDTA and DTT.
In any embodiment of the second aspect of the invention, the acetone can be replaced by but not limited to methanol, ethanol, petroleum ether.
In any embodiment of the second aspect of the invention, the extracting solution can consist of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
In any embodiment of the second aspect of the invention, the extracting solution can consist of water, EDTA and DTT.
In any embodiment of the second aspect of the invention, the extracting solution can consist of water, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
In any embodiment of the second aspect of the invention, the extracting solution can consists of water, acetone, EDTA, DTT and buffer solution.
In any embodiment of the second aspect of the invention, the extracting solution can consist of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA, 0～0.1wt. ％of DTT and  45wt. ％～90wt. ％of buffer solution.
In any embodiment of the second aspect of the invention, the extracting solution can consist of water, EDTA, DTT and buffer solution.
In any embodiment of the second aspect of the invention, the extracting solution can consist of water, 0.01wt. ％～0.5wt. ％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution.
In any embodiment of the second aspect of the invention, the buffer solution can be at least any one of phosphate buffer solution, Tris buffer solution or Bis-tris propane buffer solution.
In any embodiment of the second aspect of the invention, the step of extracting urease from the beans can comprise mixing the beans with the extracting solution to create a mixture.
In any embodiment of the second aspect of the invention, the mass ratio between the beans and the extracting solution can be 1/20 to 1/1.
In any embodiment of the second aspect of the invention, the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring, or by magnetic stirring or by ultrasonic.
In any embodiment of the second aspect of the invention, the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic for 15～60 minutes.
In any embodiment of the second aspect of the invention, the step of extracting urease from the beans can further comprise mechanically separating the mixture to obtain the extracted urease solution.
In any embodiment of the second aspect of the invention, the step of purifying the extracted urease solution can comprise:
adjusting the pH of the extracted urease solution to greater than 7；
obtaining a purified urease liquid by separation；
adjusting the pH of the purified urease liquid to less than 7； and
obtaining purified urease by drying urease precipitates obtained from the purified urease liquid.
In any embodiment of the second aspect of the invention, the pH of the extracted urease solution can be adjusted to greater than 7 and not greater than 9.
In any embodiment of the second aspect of the invention, the step of adjusting the pH of the extracted urease solution to greater than 7 can be followed by a step of heat-treatment.
In any embodiment of the second aspect of the invention, the heat-treatment can comprise heating the extracted urease solution at a temperature of 30～50 degree centigrade.
In any embodiment of the second aspect of the invention, the purified urease liquid can be obtained by mechanical separation.
In any embodiment of the second aspect of the invention, the pH of the purified urease liquid can be adjusted to 3～6.
In any embodiment of the second aspect of the invention, the step of aging the urease liquid for at least 12 hours can be followed by a step of mechanical separation for obtaining the urease precipitates.
In any embodiment of the second aspect of the invention, the step of mechanical separation is followed by the step of drying the urease precipitates by spry drying, lyophilization or supercritical drying.
In any embodiment of the second aspect of the invention, the purified urease is urease with activity of no less than 300 IU/mg and/or yield of no less than 50％.
Any embodiment of the first aspect of the invention can be introduced into any embodiment of the second aspect of the invention.
This Summary is provided to introduce a selection of concepts, in a simplified form, that are further described below in the Detailed Description. This Summary is not intended to be used in any way to limit the scope of the claimed subject matter. Rather, the claimed subject matter is defined by the language set forth in the Claims of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the relevant art.
The articles "a" and "an" used herein refer to one or more than one (i.e., to at least one) of the grammatical object of the article.
The term "aging" refers to keeping the solution for a certain time under the specified conditions.
The term “aqueous solution” refers to water solution not containing organic solvent.
The term “beans” refers to jack beans, soy beans, or peas, or any other similar organisms. Particularly, the beans can be jack beans.
"Bean meal" refers to beans that have been ground to fine particles.
The term "comprising" includes, but is not limited to, whatever follows the word "comprising" . Thus, use of the term indicates that the listed elements are required or mandatory but that other elements are optional and may or may not be present.
The term "consisting of" includes and is limited to whatever follows the phrase "consisting of" . Thus, the phrase indicates that the limited elements are required or mandatory and that no other elements may be present.
The terms "defat" or "defatting" refers to a process of removing lipids from a mixture of natural sources of urease as described herein including organic materials, generally containing ureases. Once the lipids have been removed, the material is "defatted. "
The term "hull" , as used in relation to seeds, refers to the dry outer covering of the seeds.
"Jack beans" are the seeds of the plant canavalia ensiformis.
"Lyophilization" refers to a process by which material is preserved by cooling the material to a temperature below the triple point of water and then reducing the pressure to cause the water present to sublimate.
"Mechanical separation" or "mechanically separating" means any method of separating a liquid and a solid including filtration, decanting and centrifuging.
"Rotovap" is a device used in chemical laboratories for the efficient and gentle removal of solvents from samples by evaporation.
"Supernatant liquid" is the liquid lying above a solid residue.
The present invention is directed to provide a method of extracting urease from beans and purifying the extracted urease in an efficient and low-cost way. Besides, the present invention is also directed to provide a method of extracting urease from beans and purifying the extracted urease to obtain high activity and/or high yield urease.
Thus, according to the first or second aspect of the invention, the method can comprise the steps of:
extracting urease from the beans with an extracting solution to obtain an extracted urease solution； and
purifying the extracted urease solution to obtain purified urease.
In any embodiment of the first or second aspect of the invention, the hulls of the beans can be removed prior to the step of extracting the urease from beans.
In any embodiment of the first or second aspect of the invention, the beans can be ground to a certain size (e.g. 80 meshes or above) to make bean meal prior to the step of extracting urease from the beans.
In any embodiment of the first or second aspect of the invention, the step of extracting urease from the beans can comprise mixing the beans, particularly mixing the above-mentioned bean meal, with the extracting solution to create a mixture.
In any embodiment of the first or second aspect of the invention, the step of mixing the beans with the extracting solution can comprise agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic for a certain period of time, such as for 15～60 minutes.
During the step of step of extracting urease from the beans, the extracting solution acts on bean meal for extracting urease.
In any embodiment of the first or second aspect of the invention, the step of extracting urease from the beans with an extracting solution can be repeated for more than one times, such as three times, for a higher yield.
In any embodiment of the first or second aspect of the invention, the pH of extracting solution can preferably be 7～8. The extracting solution is an environmentally friendly extraction solution (without any organic solvent) , which is developed to extract the urease from the bean meal.
In any embodiment of the first or second aspect of the invention, the mass ratio between the beans and the extracting solution can be 1/20 to 1/1.
In any embodiment of the first or second aspect of the invention, the extracting solution preferably is an aqueous solution or an extracting solution consisting of water and at least any one of acetone, EDTA, DTT and buffer solution.
In some embodiments of the first or second aspect of the invention, the extracting solution can be an aqueous solution.
In some embodiments of the first or second aspect of the invention, the extracting solution can consist of water and acetone. Particularly, the mass percent of acetone in the extracting solution can be 10％to 50％.
In some embodiments of the first or second aspect of the invention, the extracting solution can consist of water, acetone, EDTA (Ethylene Diamine Tetraacetic Acid) and DTT (DL-Dithiothreitol) . Particularly, the extracting solution can consist of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
However, in other embodiments of the first or second aspect of the invention, the acetone can be replaced by, but not limited to, methanol, ethanol, or petroleum ether.
In some embodiments of the first or second aspect of the invention, the extracting solution can consist of water, EDTA and DTT. Particularly, the extracting solution can consist of water, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
In some embodiments of the first or second aspect of the invention, the extracting solution can consist of water, acetone, EDTA, DTT and buffer solution. Particularly, the extracting solution can consist of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution.
In some embodiments of the first or second aspect of the invention, the extracting solution can consist of water, EDTA, DTT and buffer solution. Particularly, the extracting solution can consist of water, 0.01wt. ％～0.5wt. ％％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution. And in Particular, the buffer solution can be at least any one of phosphate buffer solution, Tris buffer solution or Bis-tris propane buffer solution.
After sufficient reaction between the extracting solution and the bean meal, in any embodiment of the first or second aspect of the invention, the step of extracting urease from the beans can further comprise mechanically separating the mixture to obtain a supernatant liquid (i.e. the extracted urease solution) which contains urease.
After obtaining the extracted urease solution, the extracted urease solution is purified. In any embodiment of the first or second aspect of the invention, the step of purifying the extracted urease solution can comprise the steps of:
adjusting the pH of the extracted urease solution to greater than 7；
obtaining a purified urease liquid；
adjusting the pH of the purified urease liquid to less than 7；
obtaining purified urease by drying urease precipitates obtained from the purified urease liquid.
In any embodiment of the first or second aspect of the invention, the pH of the extracted urease solution can be adjusted to greater than 7 and not greater than 9.
In any embodiments of the first or second aspect of the invention, the step of adjusting the pH of the extracted urease solution to greater than 7, particularly to greater than 7 and not greater than 9, can be followed by a step of heat-treatment.
It should be known that the alkaline extracted urease solution, the pH of which has been was adjusted to greater than 7, particularly to greater than 7 and not greater than 9, can directly be centrifuged to obtain a purified urease liquid without any heat-treatment in some other embodiments, resulting in degraded effects in view of those technical solutions having a step of heat-treatment.
In any embodiment of the first or second aspect of the invention, the heat-treatment can comprise heating the extracted urease solution at a temperature of 30～50 degree centigrade.
In any embodiment of the first or second aspect of the invention, the heat-treatment can comprise heating the extracted urease solution at a temperature (e.g. any temperature of 30～50 degree centigrade) for a certain period of time, such as for 30～50 minutes.
In any embodiments of the first or second aspect of the invention, a step of separation, particularly a mechanical separation, is performed to obtain a purified urease liquid.
In any embodiments of the first or second aspect of the invention, the step of mechanical separation can comprise centrifugation. That is, the alkaline extracted urease solution, the pH of which has been adjusted to greater than 7, particularly to greater than 7 and not greater than 9, was centrifuged to obtain a purified urease liquid.
In any embodiment of the first or second aspect of the invention, the pH of the purified urease liquid can be adjusted to 3～6.
In any embodiment of the first or second aspect of the invention, the step of adjusting the pH of the extracted urease liquid to less than 7, particularly to 3～6, can be followed by a step of aging the urease liquid for at least 12 hours.
In any embodiment of the first or second aspect of the invention, the step of aging the urease liquid for at least 12 hours can be followed by a step of mechanical separation for obtaining urease precipitates.
In any embodiments of the first or second aspect of the invention, the step of mechanical separation can comprise centrifugation. That is, the aged urease liquid is centrifuged to obtain the urease precipitates.
In any embodiment of the first or second aspect of the invention, the step of mechanical separation can be followed by a step of drying the urease precipitates by spry drying, lyophilization or supercritical drying to obtain purified urease. Because the urease is very sensitive to temperature and moisture, it is needed to adopt proper drying method for removing the additional moisture from urease with lower temperature in order to make sure the final urease is stable. Herein, spray drying, lyophilization or supercritical approach can be used.
In any embodiment of the first or second aspect of the invention, the purified urease with activity of no less than 300 IU/mg and/or yield of no less than 50％can be obtained.
The following examples were performed to further explain the invention and verify the effects of the invention. It should be understood that all percentages (％) described below represent mass percentages (wt. ％) .
Example 1
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 30 minutes. The extracting solution consisted of 10％acetone, 88％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 3 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution  was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. After that, the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 306 IU/mg and a yield of 56％.
Example 2
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.01％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 4 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 505 IU/mg and a yield of 71％.
Example 3
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 4 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 590 IU/mg and a yield of 82％.
Example 4
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.5％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 4 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which  were dried by spray drying to obtain purified urease with an activity of 509 IU/mg and a yield of 62％.
Example 5
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 3 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 301 IU/mg and a yield of 51％.
Example 6
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.1％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 5 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 530 IU/mg and a yield of 67％.
Example 7
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes. The extracting solution consisted of 50％acetone, 40％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 5 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 509 IU/mg and a yield of 61％.
Example 8
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 45 minutes. The extracting solution consisted of 32％ethanol, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 3 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 326 IU/mg and a yield of 51％.
Example 9
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 60 minutes. The extracting solution consisted of 32％petroleum, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 3 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 308 IU/mg and a yield of 54％.
Example 10
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes. The extracting solution consisted of 32％acetone, 65％tris, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 4 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 568 IU/mg and a yield of 69％.
Example 11
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic  for 15 minutes. The extracting solution consisted of 32％acetone, 65％BTP (Bis-tris propane) , 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for 4 times for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 557 IU/mg and a yield of 67％.
Example 12
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by ultrasonic for 15 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process was not repeated.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 469 IU/mg and a yield of 50％.
Example 13
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process was not repeated.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 478 IU/mg and a yield of 67％.
Example 14
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The  extraction was repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 598 IU/mg and a yield of 69％.
Example 15
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 10 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 457 IU/mg and a yield of 53％.
Example 16
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 20 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 443 IU/mg and a yield of 51％.
Example 17
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 7.1. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a  purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 322IU/mg and a yield of 52％.
Example 18
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 30 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 446 IU/mg and a yield of 53％.
Example 19
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 12 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 598 IU/mg and a yield of 50％.
Example 20
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 20 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 599 IU/mg and a yield  of 68％.
Example 21
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 50 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 462 IU/mg and a yield of 53％.
Example 22
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 9.0. And the extracted urease solution was heated at a temperature of 50 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 325 IU/mg and a yield of 59％.
Example 23
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 349 IU/mg and a yield of 51％.
Example 24
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 6. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray drying to obtain purified urease with an activity of 375 IU/mg and a yield of 55％.
Example 25
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1169 IU/mg and a yield of 86％.
Example 26
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by supercritical drying to obtain purified urease with an activity of 667IU/mg and a yield of 62％.
Example 27
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％ phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by rotovap to obtain purified urease with an activity of 505 IU/mg and a yield of 59％.
Example 28
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 100％water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 356 IU/mg and a yield of 59％.
Example 29
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone and68％water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 446 IU/mg and a yield of 62％.
Example 30
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 67.65％water, 0.05％DTT and 0.3％EDTA and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution  was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 506 IU/mg and a yield of 61％.
Example 31
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 99.65％water, 0.05％DTT and 0.3％EDTA and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 407 IU/mg and a yield of 54％.
Example 32
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 1 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray to obtain purified urease with an activity of 460 IU/mg and a yield of 55％. 
Example 33
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 3 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray to obtain purified urease with an activity of 557 IU/mg and a yield of 63％.
Example 34
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 15 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.5. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by spray to obtain purified urease with an activity of 450IU/mg and a yield of 50％.
Example 35
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.2. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1092 IU/mg and a yield of 78％.
Example 36
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1099 IU/mg and a yield of 81％.
Example 37
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by  mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1172 IU/mg and a yield of 83％.
Example 38
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.8. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1120 IU/mg and a yield of 80％.
Example 39
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 15％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 853 IU/mg and a yield of 62％.
Example 40
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 20％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The  extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 921 IU/mg and a yield of 65％.
Example 41
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 40％acetone, 55％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 915 IU/mg and a yield of 64％.
Example 42
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 45％acetone, 50％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 821 IU/mg and a yield of 60％.
Example 43
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a  purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1098 IU/mg and a yield of 79％.
Example 44
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.4％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 1123 IU/mg and a yield of 81％.
Example 45
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.025％DTT, 0.1％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 760 IU/mg and a yield of 62％.
Example 46
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.075％DTT, 0.1％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 805 IU/mg and a yield  of 57％.
Example 47
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 55％phosphate, 0.025％DTT, 0.1％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 453 IU/mg and a yield of 55％.
Example 48
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 23％acetone, 75％phosphate, 0.025％DTT, 0.1％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 472 IU/mg and a yield of 61％.
Example 49
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 12％acetone, 85％phosphate, 0.025％DTT, 0.1％EDTA and water and was with a pH of 7.6. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 477 IU/mg and a yield of 58％.
Example 50
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 55％phosphate, 0.075％DTT, 0.1％EDTA and water and was with a pH of 7.2. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 7.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 805 IU/mg and a yield of 57％.
Example 51
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 55％phosphate, 0.075％DTT, 0.1％EDTA and water and was with a pH of 7.2. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 7.5. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 352 IU/mg and a yield of 58％.
Example 52
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 55％phosphate, 0.075％DTT, 0.1％EDTA and water and was with a pH of 7.2. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.0. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 408 IU/mg and a yield of 61％.
Example 53
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 55％ phosphate, 0.075％DTT, 0.1％EDTA and water and was with a pH of 7.2. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 455 IU/mg and a yield of 66％.
Example 54
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 55％phosphate, 0.075％DTT, 0.1％EDTA and water and was with a pH of 7.2. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.8. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 404 IU/mg and a yield of 61％.
Example 55
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 23％acetone, 75％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3.2. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 358 IU/mg and a yield of 52％.
Example 56
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 23％acetone, 75％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 379 IU/mg and a yield of 58％.
Example 57
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3.6. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 426 IU/mg and a yield of 60％.
Example 58
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 3.8. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 564 IU/mg and a yield of 59％.
Example 59
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 4.0. The acid  urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 589 IU/mg and a yield of 62％.
Example 60
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 4.4. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 565 IU/mg and a yield of 61％.
Example 61
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.0. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 656 IU/mg and a yield of 68％.
Example 62
Fresh beans were de-hulled and ground to make bean meal. Bean meal was placed in an extraction solution in the mass ratio of 1: 5 to create a mixture which was agitated by mechanical stirring for 60 minutes. The extracting solution consisted of 32％acetone, 65％phosphate, 0.05％DTT, 0.3％EDTA and water and was with a pH of 7.4. The mixture was mechanically separated to obtain a supernatant liquid, i.e. the extracted urease solution. The extraction process can be repeated for twice for a higher yield.
The pH of the extracted urease solution was adjusted to 8.2. And the extracted urease solution was heated at a temperature of 40 degree for 45 minutes and then was centrifuged to obtain a purified urease liquid. Then the pH of the extracted urease liquid was adjusted to 5.5. The acid urease liquid was aged for 16 hours and was centrifuged to obtain the urease precipitates which were dried by lyophilization to obtain purified urease with an activity of 934 IU/mg and a yield of 75％.
Parameters adopted in Examples 1-62 are listed in Table. 1.
Table 1: Parameters adopted in Examples 1-62
Figure PCTCN2017108319-appb-000001
Figure PCTCN2017108319-appb-000002
Figure PCTCN2017108319-appb-000003
Figure PCTCN2017108319-appb-000004
Figure PCTCN2017108319-appb-000005
Figure PCTCN2017108319-appb-000006
Figure PCTCN2017108319-appb-000007
Figure PCTCN2017108319-appb-000008
Figure PCTCN2017108319-appb-000009
Comparing examples 14, 15 and 16, it can be seen that the ratio between JBM/Extraction has influence on the final product performance including activity and recovery. As shown in example 14, the higher performance product can be obtained under the ration of 1/5.
Comparing examples 3 and 5, it can be seen that the DTT introduction can improve the final product performance dramatically.
Comparing examples 1, 7 and 14, it can be seen that the acetone concentration has great influence on the final product performance and the higher performance can be obtained under the 32％acetone concentration.
Comparing examples 8, 9, and 14, it can be seen that the different organic reagent has influence on the final product performance. As shown in example 14, the better performance can be obtained by using the acetone reagent.
Comparing examples 10, 11, and 14, it can be seen that the type of buffer does not have obvious influence on the final product performance. The comparable final product performance can be obtained by using phosphate buffer, tris buffer or BTP buffer.
Comparing examples 45, 47 and 48, it can be seen that the buffer concentration has great influence on the final product performance. As shown in example 45, the higher performance can be obtained under the 65％buffer concentration.
Comparing examples 14 and 17, it can be seen that the pH adjustment followed by extraction step has great influence on the final product performance. As shown in example 14, the better performance can be obtained by adjusting the pH value to 8.5 after extraction step.
Comparing examples 14, 18, 21 and 22, it can be seen that the heat treatment temperature followed by pH adjustment has influence on the final product performance. As shown in example 14, the better performance can be obtained by controlling the heat treatment temperature at around 40℃.
Comparing examples 18, 19, and 20, it can be seen that the aging time has great influence on the final product performance. As shown in examples 18 and 20, the better performance can be obtained by extending the aging time more than 16 hours.
Comparing examples 14, 23, and 24, it can be seen that the pH adjustment followed by heat treatment has great influence on the final product performance. As shown in example 14, the better performance can be obtained by adjusting the pH to 5.4 followed by heat treatment.
Comparing examples 25, 28, 29, 30 and 31, it can be seen that the different extraction solution formulation has great influence on the final product performance. As shown in example 25, the better performance can be obtained by using 32％acetone with0.05％DTT, 0.3％EDTA, 65％buffer solution and others for water.

Claims (59)

  1. A method for extracting and purifying urease from beans comprising the steps of:
    extracting urease from the beans with an extracting solution to obtain an extracted urease solution;
    adjusting the pH of the extracted urease solution to greater than 7;
    obtaining a purified urease liquid by separation;
    adjusting the pH of the purified urease liquid to less than 7; and
    obtaining purified urease by drying urease precipitates obtained from the purified urease liquid.
  2. The method of claim 1, wherein the hulls of the beans are removed prior to the step of extracting urease from the beans.
  3. The method of claim 1, wherein the beans are ground to make bean meal prior to the step of extracting urease from the beans.
  4. The method of claim l, wherein the pH of extracting solution is 7～8.
  5. The method of claim l, wherein the extracting solution is an aqueous solution.
  6. The method of claim 1, wherein the extracting solution consists of water and acetone.
  7. The method of claim 6, wherein the mass percent of acetone in the extracting solution is 10％～50％.
  8. The method of claim 1, wherein the extracting solution consists of water, acetone, EDTA and DTT.
  9. The method of claim 8, wherein the acetone can be replaced by methanol, ethanol, or petroleum ether.
  10. The method of claim 1, wherein the extracting solution consists of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
  11. The method of claim 5, wherein the extracting solution consists of water, EDTA and DTT.
  12. The method of claim 1, wherein the extracting solution consists of water, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
  13. The method of claim 1, wherein the extracting solution consists of water, acetone, EDTA, DTT and buffer solution.
  14. The method of claim 1, wherein the extracting solution consists of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution.
  15. The method of claim 1, wherein the extracting solution consists of water, EDTA, DTT and buffer solution.
  16. The method of claim 1, wherein the extracting solution consists of water, 0.01wt. ％～0.5wt. ％％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution.
  17. The method of claim 1, wherein the buffer solution is at least any one of phosphate buffer solution, Tris buffer solution or Bis-tris propane buffer solution.
  18. The method of claim 1, wherein the step of extracting urease from the beans comprises mixing the beans with the extracting solution to create a mixture.
  19. The method of claim 1, wherein the mass ratio between the beans and the extracting solution is 1/20 to 1/1.
  20. The method of claim 18, wherein the step of mixing the beans with the extracting solution comprises agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic.
  21. The method of claim 18, wherein the step of extracting urease from the beans comprises mechanically separating the mixture to obtain the extracted urease solution.
  22. The method of claim 1, wherein the pH of the extracted urease solution is adjusted to greater than 7 and not greater than 9.
  23. The method of claim 1, wherein the step of adjusting the pH of the extracted urease solution to greater than 7 is followed by a step of heat-treatment.
  24. The method of claim 23, wherein the heat-treatment comprises heating the extracted urease solution at a temperature of 30～50 degree centigrade.
  25. The method of claim 1, wherein the pH of the purified urease liquid is adjusted to 3～6.
  26. The method of claim 1, wherein the step of adjusting the pH of the purified urease liquid to less than 7 is followedby a step of aging the urease liquid for at least 12 hours.
  27. The method of claim 26, wherein the step of aging the urease liquid for at least 12 hours is followed by a step of separation for obtaining the urease precipitates.
  28. The method of claim 27, wherein the step of separation is followed by the step of drying the urease precipitates by spry drying, lyophilization or supercritical drying.
  29. The method of claim 1, wherein the purified urease with activity of no less than 300 IU/mg and/or yield of no less than 50％is obtained.
  30. A method for extracting and purifying urease from beans comprising the steps of: extracting urease from the beans with an extracting solution to obtain an extracted urease solution, wherein the extracting solution is an aqueous solution or an extracting solution consisting of water and at least any one of acetone, EDTA, DTT and buffer solution; and purifying the extracted urease solution to obtain purified urease.
  31. The method of claim 30, wherein the hulls of the beans are removed prior to the step of extracting urease from the beans.
  32. The method of claim 30, wherein the beans are ground to make bean meal prior to the step of extracting urease from the beans.
  33. The method of claim 30, wherein the pH of extracting solution is 7～8.
  34. The method of claim 30, wherein the extracting solution is an aqueous solution.
  35. The method of claim 30, wherein the extracting solution consists of water and acetone.
  36. The method of claim 35, wherein the mass percent of acetone in the extracting solution is 10％～50％.
  37. The method of claim 30, wherein the extracting solution consists of water, acetone, EDTA and DTT.
  38. The method of claim 37, wherein the acetone can be replaced by methanol, ethanol, petroleum ether.
  39. The method of claim 30, wherein the extracting solution consists of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
  40. The method of claim 30, wherein the extracting solution consists of water, EDTA and DTT.
  41. The method of claim 30, wherein the extracting solution consists of water, 0.01wt. ％～0.5wt. ％of EDTA and 0～0.1wt. ％of DTT.
  42. The method of claim 30, wherein the extracting solution consists of water, acetone, EDTA, DTT and buffer solution.
  43. The method of claim 30, wherein the extracting solution consists of water, 10wt. ％～50wt. ％of acetone, 0.01wt. ％～0.5wt. ％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution.
  44. The method of claim 30, wherein the extracting solution consists of water, EDTA, DTT and buffer solution.
  45. The method of claim 30, wherein the extracting solution consists of water, 0.01wt. ％～0.5wt. ％of EDTA, 0～0.1wt. ％of DTT and 45wt. ％～90wt. ％of buffer solution.
  46. The method of claim 30, wherein the buffer solution is at least any one of phosphate buffer solution, Tris buffer solution or Bis-tris propane buffer solution.
  47. The method of claim 30, wherein the step of extracting urease from the beans comprises mixing the beans with the extracting solution to create a mixture.
  48. The method of claim 30, wherein the mass ratio between the beans and the extracting solution is 1/20 to 1/1.
  49. The method of claim 30, wherein the step of mixing the beans with the extracting solution comprises agitating the mixture by mechanical stirring or by magnetic stirring or by ultrasonic.
  50. The method of claims 47, wherein the step of extracting urease from the beans comprises mechanically separating the mixture to obtain the extracted urease solution.
  51. The method of claim 30, wherein the step of purifying the extracted urease solution comprises:
    adjusting the pH of the extracted urease solution to greater than 7;
    obtaining a purified urease liquid by separation;
    adjusting the pH of the purified urease liquid to less than 7; and
    obtaining purified urease by drying urease precipitates obtained from the purified urease liquid.
  52. The method of claim 51, wherein the pH of the extracted urease solution is adjusted to greater than 7 and not greater than 9.
  53. The method of claim51, wherein the step of adjusting the pH of the extracted urease solution to greater than 7 is followed by a step of heat-treatment.
  54. The method of claim 53, wherein the heat-treatment comprises heating the extracted urease solution at a temperature of 30～50 degree centigrade.
  55. The method of claim 51, wherein the pH of the purified urease liquid is adjusted to 3～6.
  56. The method of claim 51, wherein the step of adjusting the pH of the extracted urease liquid to less than 7 is followed by a step of aging the urease liquid for at least 12 hours.
  57. The method of claim 56, wherein the step of aging the urease liquid for at least 12 hours is followed by a step of separation for obtaining the urease precipitates.
  58. The method of claim 57, wherein the step of separation is followed by the step of drying the urease precipitates by spry drying, lyophilization or supercritical drying.
  59. The method of claim30, wherein the purified urease is urease with activity of no less than 300 IU/mg and/or yield of no less than 50％.
PCT/CN2017/108319 2017-10-30 2017-10-30 Urease purification from beans WO2019084713A1 (en)

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CN113817713A (en) * 2021-11-10 2021-12-21 江苏海洋大学 Method for extracting urease from red sword beans

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