Immobilized trypsin as well as preparation method and application thereof

Abstract

The invention discloses immobilized trypsin as well as a preparation method and application thereof. The preparation method comprises the steps of: with a large-aperture polymeric microsphere as an immobilized enzyme carrier, completely cleaning, activating functional groups of the microsphere, mixing the microsphere with a trypsin solution, carrying out vibration reaction so as to generate covalent binding by virtue of enzyme molecules and the functional groups on the surface of the carrier, standing, adjusting the pH value to be 9 or over, and carrying out maintenance for a certain period oftime, so as to obtain the immobilized trypsin. The preparation method is stable in process and mild in reaction conditions; the protein immobilization rate of the prepared immobilized trypsin can reach 92.7%; the activity recovery rate is high, and the fluctuation is small; the stability of the immobilized trypsin in an organic solvent medium is substantially improved; and meanwhile, the pH valueapplication range is relatively wide, the acid and alkali resistances are relatively good, and the immobilized trypsin can still preserve very good activity in an environment with a pH value of 2-12and has relatively wide application range.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to an immobilized trypsin and its preparation method and application. Background technique [0002] Trypsin is a kind of protease and an important proteolytic enzyme. It is also an endolytic tool enzyme commonly used in the industry. It can cut off the carboxyl side of lysine and arginine residues in the polypeptide chain, It can digest and dissolve denatured proteins, and its applications in various fields of life sciences such as food, chemical industry, animal husbandry, textiles, cosmetics, biological experiments, pharmaceuticals, medical care, and proteomics are becoming more and more popular. Its sources are extracted from the pancreas tissue of cattle, sheep, pigs and other animals, or obtained through microbial expression. The enzyme is easily soluble in water, becomes a free enzyme after being dissolved in water, and has good catalytic activity. However, free enzymes are ver...

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Problems solved by technology

[0004] Although the use of soluble carriers and carrier-free immobilization methods has advantages, compared with insoluble carriers, its disadvantages are more obvious. The technology is not mature enough, and there is still a long way to go before industrial application.

For example, the patent document with the publication number CN 1869213A discloses a preparation method for immobilizing trypsin with a soluble carrier, which is prepared by using a dextran derivative to make a carrier, and then linking the enzyme and the carrier through a cross-linking agent. The advantage of this method is that the prepared immobilized enzyme is soluble under the condition of pH 6-10, and the catalytic specific surface area is high, but the disadvantage is that the dextran derivative material is relatively expensive, which is not conducive to reducing costs. The activity recovery rate is low, and the unstable preparation process leads to large fluctuations in the activity recovery rate, which fluctuates in the range of 60% to 85%. The third is that the applicable range of the pH value is narrow, and it is only suitable for the environment of pH 6 to 10. The fourth is the recovery process Compared with the insoluble carrier, it needs to adjust the pH<4 to make it precipitate, which also requires the substrate to have better stability under the condition of pH<4, which further reduces its application range

For example, the advantages of the adsorption method and the embedding method are that the preparation methods are very simple and the loss of enzyme activity is small, but the disadvantage of the adsorption method is that the combination is not firm, and it is easy to fall off during use, resulting in insufficient stability. The disadvantage of the embedding method is the substrate diffusion resistance The advantage of the cross-linking method is that it is more firmly combined than the adsorption method, and it is not easy to fall off, but the disadvantage is that the reaction conditions are more intense and the enzyme activity is lost. The disadvantage is that 100% covalent binding rate cannot be guaranteed, and the remaining enzyme molecules that have not undergone covalent binding in the carrier pores are the root cause of its insufficient stability

Before the breakthrough of the present invention, the researchers of the present invention tried a lot of preparation methods such as adsorption method, cross-linking method, embedding method and covalent bonding method according to the preparation methods of immobilized trypsin reported in relevant literature and patents, and obtained The stability of the immobilized trypsin in the organic solvent medium is not good, and the ideal results have not been obtained after repeated optimization.

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