Preparation And Application Of Buckwheat Trypsin Inhibitor Affinity Materials

Affinity chromatography has the advantages of high efficiency and rapidity,and sometimes purification can be achieved in only one step.Therefore,affinity chromatography is the preferred method for the purification of biological enzymes.Buckwheat trypsin inhibitor?BTI?is a competitive inhibitor of trypsin.BTI has good thermal stability,acid-base stability and strong inhibitory effect on trypsin,which is suitable for immobilized reaction.It can be used in the preparation of affinity chromatography medium to achieve the purpose of one-step chromatography to obtain high purity trypsin,providing important theoretical basis for large-scale preparation of trypsin and development of new trypsin in the future.Part One: Based on the specific affinity between the inhibitor and the enzyme,the BTI with good stability was immobilized to prepare an affinity adsorbent of trypsin,and the efficient purification of trypsin was realized.Electrophoretic pure BTI was obtained by prokaryotic expression,Ni²⁺-NTA affinity chromatography and Superdex G-25 gel filtration.With Sepharose as the carrier,three activation carrier methods?hydrogen bromide,epichlorohydrin,carbonyl diimidazole?and BTI as the ligand were selected to prepare BTI-sephorose affinity material.The results showed that the heat resistance of BTI,the specificity of inhibition of trypsin and the good acidbase stability of BTI can be used in the preparation of affinity chromatography medium to achieve the purpose of one-step chromatography to obtain high purity trypsin.Carbonyl diimidazole is an ideal activator and can be used for immobilizing other enzymes by affinity chromatography.Part Two: the specific adsorption of trypsin by BTI-sepharose was detected with bovine trypsin as the material,and the adsorption and desorption conditions of trypsin by BTI-sepharose were further studied.The results showed that the prepared BTI-sepharose had specific adsorbability to trypsin and could be used for the affinity purification of trypsin.The p H value of buffer had an important influence on the adsorption and desorption of BTI-sepharose and trypsin,and the optimal p H value of the adsorption of both was 7-8.When the p H was 3.5,they could be completely separated without affecting the biological activity of trypsin.The BTI-sepharose prepared in this experiment can be used to prepare trypsin with high purity by one-step chromatography,which provides a theoretical basis for the efficient purification of trypsin and the research and development of new trypsin.Part Three: Trypsin was extracted from the hepatopancreas of grass carps in this study.After homogenization and ammonium sulfate precipitation,crude enzyme solution was prepared by dialysis.Then,trypsin of electrophoretic purity was obtained by only one step of BTI-sepharose affinity chromatography.The results showed that the molecular weight of trypsin in grass carp was about 27 k Da,and the K_m was 3.4×10^-5mol/L.The optimum reaction temperature of the enzyme was 60? and it maintained stability under the condition of lower than 60?.Its optimal p H was 9.5,and the p H tolerance range was 6.0 12.0.Ba²⁺,Mg²⁺ and Fe²⁺ all could activate the enzyme in the concentration range of 0-10 mmol/L.K⁺ and EDTA inhibited the enzyme in the concentration range of 0-10 mmol/L,and EDTA inhibited the enzyme more obviously than K⁺.