Method for extracting CEL I nuclease in celery

Abstract

The invention relates to a method for extracting CEL I nuclease in celery and discards measures adopting a plurality of high-speed or ultra-speed centrifugation and a plurality of gel filtration and ion exchange chromatography in the prior art. The method comprises the following steps: adding a small amount of sodium sulfite to extract so as to reduce and clear colored substances in the extract; adopting a thermal denaturation physical method to rapidly clear a great amount of foreign protein with poor temperature toleration; utilizing the characteristic that CEL I is the combination of glycoprotein and activated concanavalin to combine CEL I nuclease and concanavalin so as to remove the foreign protein and further purify the CEL I; and finally, selecting DEAE-Sepharose FF as a suitable medium for the CEL I by once chromatography purification. Accordingly, the extraction method replaces the time-consuming and expensive measures adopting a plurality of high-speed even ultra-speed refrigerated centrifugation, a plurality of gel filtration and ion exchange chromatography, and the like and achieves the aims that the CEL I nuclease is rapidly extracted from the celery and purified.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to the preparation of enzymes. Background technique [0002] CEL I nuclease is a single-strand specific nuclease with an isoelectric point of 6.0-6.5 and a molecular weight of 43kDa. Its catalytic activity requires Mg 2+ and Zn2 + Participation, belonging to one of the S1 endonuclease family. CEL I nuclease is the only enzyme found in the biological world that can accurately cut the mismatched sites in heteroduplex DNA. It plays an important role in genetic research and application fields and has broad application prospects. [0003] At present, the acquisition route of CEL I nuclease is mainly extracted from celery. People such as Oleykowski reported a kind of method extracting CEL I nuclease from celery at first in 1998, and this method method is as follows: first by continuous ammonium sulfate precipitation, then through concanavalin A-agarose column extraction (with [ α]-d+-manno...

AI Technical Summary

Problems solved by technology

This method requires multiple precipitation extractions, ion exchange chromatography, gel filtration, and repeated ultrahigh-speed centrifugation, resulting in a very low output per unit time

In addition, the total yield of this method is also very low, about 7kg of celery stems containing 350g protein can only be extracted to obtain 0.1 μg / μL of CEL I nuclease 3ml (NucleicAcides Res, 26, 4597-4602, 1998)

[0004] Subsequently, Yang et al. improved the above-mentioned method (PCT / US01 / 05502), adopted α-methylmannoside to overcome the problem of aggregation reaction between CEL I nuclease and endogenous selectin, and obtained crude from 105kg celery 0.5mL of pure enzyme was extracted from the enzyme solution, and the specific enzyme activity was changed from the original ×10 2 U / mg increased to 3.1×10 7 U / mg, but the total enzyme activity was changed from the original 1.9×10 7 U reduced to 3.1×10 5 U, the recovery rate is only 1.63%

However, this method cannot avoid multiple precipitation extractions, ion exchange chromatography, gel filtration, and repeated ultrahigh-speed centrifugation, and the extraction rate is not high.

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