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Method for extracting crystals of urease in canavalia ensiformis

A technology of urease and extraction method in soybeans, applied in the direction of hydrolytic enzymes, etc., can solve the problems of insufficient purification factor, low purity of urease, complex process flow, etc., to solve the problem of operation time and production, improve purity and extraction rate, without affecting The effect of protein activity

Active Publication Date: 2019-05-10
CHINA UNIV OF PETROLEUM (EAST CHINA)
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Problems solved by technology

[0004] my country is rich in soybean resources. At present, domestic research reports on urease extraction mainly focus on the extraction and purification of urease from soybeans, soybeans and other plants. There are research reports on the extraction of soybean urease, which uses dry soybeans as raw materials and adopts the method of fractional precipitation. Separation of urease, and finally Sephadex G~150 gel chromatography filtration, purification of the protein solution to obtain a urease product with high enzyme activity and high purity, but the process is complicated, the loss of urease is high, and it also involves coagulation Gel chromatography filtration technology, the project scale-up cost is relatively high; there are also reports that soybean seed coats are used as raw materials to obtain refined urease through solvent extraction, ammonium sulfate fractional precipitation and acetone precipitation. This technology is simple to operate, and the extraction rate of urease can be achieved. Reach 50-60%, but the purity of the product is not ideal
[0005] Compared with soybean urease, the activity of canavalia urease is higher, but the stability is worse. At present, the extraction technology research using canavalia as raw material is not mature enough. The conventional salting-out precipitation method is adopted, although the extraction rate of urease is higher. , but the purity of the product is very unsatisfactory
Chromatography, ultrafiltration membrane and other technologies can meet the extraction rate and purity requirements at the same time, but the process is complicated, the operation time is long, and the production cost is high.
Chinese patent CN85104238A proposes a method for extracting urease. First, the beans are soaked and wet-milled, and then the urease is extracted in batches in a low-concentration phosphate weakly acidic buffer solution. not high enough
Chinese patent CN104480092A discloses a method for extracting urease from sword bean, through extraction, sedimentation, neutralization, and a series of processes such as ceramic membrane microfiltration, dialysis, and organic membrane ultrafiltration, but the process is cumbersome and complicated. The operation cycle is as long as 45 hours, the cost is high, and it is difficult to scale up industrial production

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  • Method for extracting crystals of urease in canavalia ensiformis
  • Method for extracting crystals of urease in canavalia ensiformis

Examples

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Embodiment 1

[0031] Take by weighing 2 kg of pineapple powder, sieve to remove large particles of impurities, add 2L of ethanol solution containing 35% mass fraction, wherein the solvent is a sodium citrate buffer solution with an ionic strength of 0.025mol / L at pH=6.0, at room temperature at 700rpm Stir for 10 minutes to obtain a urease extract, and the stirring paddle is made of glass to prevent metals from affecting the urease activity. The resulting extract was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. After the supernatant was refrigerated at 6° C. for 4 h, a suspension containing urease precipitate was obtained. The suspension was centrifuged at 4000 rpm to obtain a crude precipitate of urease. Add 2L of sodium citrate buffer solution with an ionic strength of 0.025mol / L pH=6.0 to the crude precipitate of urease obtained, fully stir and dissolve at room temperature, centrifuge at 2000rpm for 20min to discard insoluble impurities, collect the supernatant,...

Embodiment 2

[0036] Take 2 kg of jack bean powder, sieve to remove large particles of impurities, add 2 L of ethanol solution containing 25% mass fraction, wherein the solvent is a sodium citrate buffer solution with an ionic strength of 0.025 mol / L and pH=6.5, and stir for 15 min at room temperature at 700 rpm , to obtain the leaching solution of urease, and the stirring blade is made of glass. The resulting extract was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. After the supernatant was refrigerated at 5° C. for 4 h, a suspension containing urease precipitate was obtained. The suspension was centrifuged at 4000 rpm to obtain a crude precipitate of urease. Add 2L of sodium citrate buffer solution with an ionic strength of 0.025mol / L pH=6.5 to the crude precipitate of urease obtained, fully stir and dissolve at room temperature, centrifuge at 2000rpm for 20min to discard insoluble impurities, collect the supernatant, and obtain a concentration of urease of Add...

Embodiment 3

[0041] Take 2 kg of jack bean powder, sieve to remove large particles of impurities, add 2 L of acetone solution containing 25% mass fraction, wherein the solvent is a sodium citrate buffer solution with an ionic strength of 0.025 mol / L and pH=7.0, stir at room temperature for 20 min at 700 rpm , to obtain the leaching solution of urease, and the stirring blade is made of glass. The resulting suspension was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. After the supernatant was refrigerated at 4° C. for 4 h, a suspension containing urease precipitate was obtained. The suspension was centrifuged at 4000 rpm to obtain a crude precipitate of urease. Add 2L of sodium citrate buffer solution with an ionic strength of 0.025mol / L pH=7.0 to the crude precipitate of urease obtained, fully stir and dissolve at room temperature, centrifuge at 2000rpm for 20min to discard insoluble impurities, collect the supernatant, and obtain a concentration of urease of Add ...

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Abstract

The invention discloses a method for extracting crystals of urease in canavalia ensiformis. The method comprises the following steps of 1, extraction, wherein canavalia ensiformis is ground into powder, an extracting solution is added after screening is carried out, full stirring is carried out, urease in the canavalia ensiformis powder is all digested, the leaching liquor is centrifugalized at room temperature, and the supernate is collected; 2, rough precipitating, wherein the supernate stands and refrigerated, centrifugalization is carried out, precipitate is collected, and rough urease precipitate is obtained; 3, cooling crystallization, wherein the rough urease precipitate is dissolved in a sodium citrate solution and stirred to be fully dissolved, centrifugalization is carried out, supernate is collected, a protein solution is obtained, a precipitator solution is slowly added to the protein solution, crystallization starts to be carried out after the materials are mixed to be uniform, a crystal solution is obtained, the crystal solution is subjected to centrifugal separation, urease crystal precipitate is collected and freeze-dried, and a urease product with the purity exceeding 96% is obtained. The method is easy to operate, short in technological process and low in energy consumption, a conventional solid-liquid separation means is adopted in the preparation process, and the urease gross activity recovery rate reaches 60% or above.

Description

technical field [0001] The invention relates to the technical field of crystallization, in particular to a method for crystallization and extraction of urease in jack bean. Background technique [0002] As an important biological agent, urease is widely distributed in the seeds of plants, and the content is most abundant in soybean and canavalia. Urease is an oligomerase that can specifically catalyze the hydrolysis of urea to release ammonia and carbon dioxide. Its catalytic reaction rate is more than 1000 times higher than the non-catalytic reaction rate without urease. It is widely used in agriculture, pharmaceutical industry and other fields . In animal husbandry, urease plays a key role in the urea nitrogen cycle of ruminants such as cattle and sheep; in the pharmaceutical industry, urease is an important component in the kit for clinical diagnosis of urea content in serum; Urease can decompose urea to detect the content of 0.1% soybean powder in milk powder; in addit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80
Inventor 于筱溪杨宇平黄方吴一宁刘敬平孙永鹏
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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