Proteolytic Enzymes and their Protein Inhibitors from Triticale Grain

It is known, that proteolytic enzymes play a significant role in various physiological processes: the degradation of reserve proteins, the post-translational process of proteins, the activation of inactive precursors of physiologically active proteins and peptides, participate in the processes of secretion, transport, adaptation of cellular metabolism to changes in the environment. Along with the reasons for the physiological character, interest in proteolytic enzymes is also due to the fact that they are a convenient object for studying the structure of proteins, active enzyme centers, mechanisms for the regulation of enzymatic activity, other important issues of enzymology and protein chemistry. From a practical point of view, interest in proteolytic enzymes is associated with their direct participation in processes that take place during the storage and processing of plant raw materials, as well as the use of protease preparations in various sectors: agriculture, food industry, medicine. At the present stage, the priority of any food technology is the preservation of the native properties of biologically valuable components of raw materials that possess therapeutic and prophylactic properties and the modification of food raw materials, including vegetable raw materials, in order to obtain components with certain technological and functional properties. Currently, the most widely used methods of enzymatic modification of grain raw materials using microbial enzyme preparations of proteolytic and cellulolytic action. At the same time, the study of their own proteolytic enzymes, their isolation and purification, with the aim of using them to modify the endogenous proteins to which they show the greatest affinity, seems to be a very interesting task, in terms of using the entire grain biopotential and ecofriendly products. Such a study is inextricably linked with the study of protein protease inhibitors, which have traditionally been considered only as antialimentary components of raw materials and food products, but are of exceptional interest as a factor in the regulation of the proteolysis process, only in vivo, but also in vitro. The article presents experimental data on the isolation, partial purification and characterization of proteolytic enzymes of triticale grain acting in acidic neutral and alkaline pH regions. It is shown that acid and neutral proteinases of triticale grain are thiol type enzymes, since they are activated by cysteine and is inhibited p-chloromercuriumbenzoate, a specific inhibitor of thiol enzymes. Alkaline proteinases belong to serine proteinases because they are inhibited by specific inhibitors of serine enzymes: diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The molecular mass of triticale grain proteinases was determined by gel chromatography: acidic - 75000 Da, neutral - 250000 Da, alkaline - 50000 Da. A scheme is proposed for the isolation and purification of protein inhibitors of endogenous proteinases from triticale grain. Their ability to inhibit the activity of their own proteinases and trypsin is shown. The separation of inhibitors of trypsin and alkaline proteinases with triticale (with a molecular weight of 30000 ÷ 35000 Da) and inhibitors of acidic and neutral proteinases of triticale (molecular weight 15000 ÷ 20000 Da) was carried out by gel chromatography. Partially purified proteinase preparations and their protein inhibitors obtained by the proposed schemes can be used subsequently for enzymatic modification of plant proteins and regulation of proteolysis processes in various food technologies using grain raw materials.

triticale grain, proteolytic enzymes, protein inhibitors, isolation, purification, gel chromatography

1. Асатулоев И. А., Карпиленко Г. П., Витол И. С. Белковые ингибиторы протеаз из семян нута // Хлебопродукты. 2008. № 1. С. 58-59.

2. Браудо Е. Е., Даниленко А. И., Дианова В. Т., Кроха Н. Г. Альтернативные подходы к получению растительных белковых продуктов / в кн.: Растительный белок: новые перспективы (под ред. Е.Е. Браудо). М.: Пищепромиздат, 2000. С. 6-23.

3. Витол И. С., Кандроков Р. Х., Стариченков А. А., Коваль А. И., Жильцова Н. С. Белково-протеиназный комплекс зерна тритикале // Хранение и переработка сельхозсырья. 2015. № 8. С. 36-39.

4. Витол И. С., Мелешкина Е. П., Карпиленко Г. П. Биоконверсия тритикалевых отрубей с использованием ферментных препаратов целлюлолитического и протеолитического действия // Хранение и переработка сельхозсырья. 2016. № 10. С. 35-38.

5. Витол И. С., Мелешкина Е. П., Карпиленко Г. П. Функциональные свойства модифицированных продуктов переработки зерна тритикале // Хранение и переработка сельхозсырья. 2017. № 2. С. 27-29.

6. Исламов Р. А., Хакимжанов А. А., Фурсов О. В. Выделение ингибиторов сериновых протеаз и амилаз из зерна пшеницы // Биотехнология. Теория и практика. 2004. № 4. С.117-122.

7. Исламов Р. А., Хакимжанов А. А., Фурсов О. В. Ингибитор трипсина из зерна пшеницы, выделенный аффинной хроматографией на трипсин-сефарозе // Биотехнология. Теория и практика. 2005. № 2. С. 71-75.

8. Meleshkina E. P., Pankratov G. N., Vitol I. S., Kandrokov R. H., Tulyakov D. G. Innovative Trends in the Development of Advanced Triticale Grain Processing Technology // Foods and Raw Materials. 2017. Vol.5. № 2. P. 70-82. DOI: 10.21179/2308-4057-2017-2-70-82.

9. Мосолов В. В. Белковые ингибиторы как регуляторы процессов протеолиза. М.: Наука, 1983. 40 с.

10. Нечаев А. П., Траубенберг С. Е., Кочеткова А. А., Колпакова В. В., Витол И. С., Кобелева И. Б. Пищевая химия. Лабораторный практикум. СПб.: ГИОРД, 2006. 304 с.