ISOLATION OF ALFALFA SEED TRYPSIN INHIBITOR USING AFFINITY CHROMATOGRAPHY

Authors

  • Galyna V. Krusir Одесская национальная академия пищевых технологий http://orcid.org/0000-0001-6464-5754
  • Valentina G. Zakharchuk Odessa National Economic University
  • Elena V. Sevastyanova Odessa National Academy of Food Technologies
  • Liudmyla N. Pylypenko Odessa National Academy of Food Technologies
  • Sergey О. Moshtakov Odessa National Academy of Food Technologies

DOI:

https://doi.org/10.15421/082029

Keywords:

alfalfa seeds, extraction, affinity chromatography, trypsin inhibitor, amino acid composition, molecular weight

Abstract

A protein inhibitor of trypsin of the cultivar Eva alfalfa seeds was isolated and characterized. It has significant antiproteolytic activity and is promising for the creation of compositions that are designed to correct nutrition in various conditions, accompanied by increased activation of proteolytic enzymes. The stages of purification of the trypsin inhibitor from alfalfa seeds included: extraction of 0.05 M borate buffer (pH 7.6), fractionation of the protein component of the extract with ammonium sulfate, followed by dialysis of the fraction between 75% and 100% saturation and affinity chromatography on a biospecific sorbent trypsin - sepharose 4B. Calculations showed that from 100 g of alfalfa seeds of the cultivar Eva, 67.14 mg of trypsin inhibitor was obtained, the inhibitory activity of which is 27.6 IU / mg protein. Experimental data show that the obtained inhibitor reduces trypsin activity by 63.05% with a weight ratio of inhibitor: enzyme of 1: 1. It is known from literature that a soybean trypsin inhibitor reduces trypsin activity by 30%. The isolated trypsin inhibitor has significant inhibitory activity, the value of which is higher than the inhibitory activity of the plant trypsin inhibitor from soybeans. Affinity chromatography allowed us to obtain a pure, homogeneous trypsin inhibitor, which has significant antiproteolytic activity, which can be compared with the antiproteolytic activity of known plant trypsin inhibitors. From the obtained experimental data, it follows that both, the obtained inhibitor and the inhibitor, which is part of a commercial preparation, effectively reduce trypsin activity, slightly inhibit chymotrypsin activity, and do not affect α-amylase and protease activity. The above data indicate that the trypsin inhibitor of alfalfa seeds of the cultivar Eva is not a bifunctional inhibitor, since it does not affect the activity of amilolytic enzymes. The isolated inhibitor belongs to proteins with a high degree of hydrophobicity. The composition of amino acids, the side chains of which can take part in the formation of hydrogen bonds, is 25% of the total number of amino acid residues of the alfalfa seed trypsin inhibitor. For STI, this value is 23%.

Author Biography

Galyna V. Krusir, Одесская национальная академия пищевых технологий

кафедра экологии и природоохранных технологий, д.т.н., профессор

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Published

2021-01-10