Method for extracting crystals of urease in canavalia ensiformis

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...

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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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