Restricted enzymatic hydrolysis of legumin of broad beans (Vicia faba L.) by trypsin in concentrated solutions. Control of hydrolysis process at the expense of change of enzyme‐substrate ratio

Abstract

SummaryKinetics and mechanism of proteolysis of broad bean (Viciu ,fubu L.) legumin by trypsin in concentrated solutions at different enzyme-substrate ratios ( E / S = I /40, 1 /loo, 1/200, lj1000) were studied. By the method of HPLC it was established that during proteolysis process ( E / S = 1/40) take place both protein content dccrease in hydrolysate and change of its molecular mass (from 360 to 280 kD), which evidences for the mixed type of proteolysis, including cooperative and non-cooperative mechanisms. Decrease of E / S from 1/40 up to IjlOO-l/lOOO leads not only to unproportional drop in proteolysis rate, but also to decrease of the relative contribution of cooperative proteolysis which is, possibly, connected with presence in protein molecule of at least two groups of attack sites, differentiating by their affinity with enzyme. Change of enzyme-substrate ratio is accompanied also by change of subunit composition of product of non-cooperative proteolysis ('modified protein') and also or temperature and enthalpy of its denaturation. ZusammenfassungBegrenzte enzymatische Hydrolyse von Legumin aus Ackerbohnen (Viciu fubu L.) durch Trypsin in konzentrierten Losungen. Kontrolle des Hydrolyseprozesses in Abhangigkeit von der Veranderung des Enzym-Substrat-Vcrhaltnisses Es wurden die Kinetik und der Mechanismus der Proteolyse des Legumins von Ackerbohnen (Viciu juhu L.) durch Trypsin in konzentrierten Losungen bei unterschiedlichen Enzym-Substrat-VerhHltnissen ( E / S = 1/40, 1/100, 1/200, I/lOOO) untersucht. Mittels HPLC wurde festgestellt, dal3 wahrend des Proteolyseprozesses ( E / S = 1/40) der Proteingehalt im Hydrolysat abnahm und eine Veranderung der Molmasse (von 360 auf 280 kD) vonstatten ging; dies ist ein Beweis fur eine gemischte Hydrolyse, die kooperative und nicht-kooperative Mechanismen einschlieBt. Eine Abnahme von E / S von 1/40 auf 1/100 bis 1/1000 fiihrt nicht nur zu einer unproportionalen Abnahme der Proteolyserate, sondern auch zu einer Abnahme des relativen Beitrages der kooperativen Proteolyse, die moglicherweise in Zusammenhang steht mit wenigstens zwei Angriffsstellen im Proteinmolekiil, die in ihrer Affinitat zum Enzym differieren. Eine Veranderung des Enzym-Substrat-Verhaltnisses wird begleitet von einer Veranderung der Zusammensetzung der Subeinheiten des Produktes der nicht-kooperativen Proteolyse (,,modifiziertes Protein") und auch der Temperatur und Enthalpie dessen Denaturierung.

Comparative effects of limited tryptic hydrolysis on physicochemical and structural features of seed 11S globulins

“…Similar results were later obtained when the tryptic hydrolysis of vetch [2] and broad been [3] legumins, two other 11S globulins, was studied. The faster process is of limited non-cooperative ("zipper") type, while the much slower one is of cooperative ("one by one") type [4].…”

Controlled in vitro proteolysis of casein using immobilized trypsin. Influence of variation of the enzyme‐substrate ratio and the re‐use of the immobilized enzyme on the preparation of phosphopeptide‐rich fractions

“…This result suggests that the mainly hydrophilic areas of the casein are, with excess substrate or lack of enzyme, preferentially hydrolysed or rather that these areas with a tryptic proteolysis in hydrous systems are primarily accessible. DANILENKO et al [9] describe also that with low trypsin concentrations preferentially peptide bonds are broken down for which the enzyme exhibits the higher affinity. This finding is indicative of the limits existing, also from the economic point of view, as regards a minimization of the use of enzyme, because lack of enzyme may lead to a qualitatively differing composition of the resulting proteolysates.…”