Эффективность протеаз панкреатина и трипсина при ферментативном гидролизе коллагена

Abstract

The analysis of molecular-mass characteristics (MMC) / molecular mass (MM) and polydispersity coefficient (PC) of high molecular weight fish collagen (FC) hydrolysate has been carried out in the process of enzymatic hydrolysis by two proteolytic enzymes: pancreatin and trypsin at room temperature. High efficiency of enzymes has been shown: the main part of FC is hydrolyzed within the first minute in the case of both pancreatin and trypsin. As a result, several fractions of FC hydrolysate are formed. A small oligomeric fraction is observed in the case of trypsin. The values of the polydispersity coefficient M w / M n for fractions in the hydrolysis process are typically no more than 1.2. This indicates the homogeneity of collagen fractions in MM. The original native FC with MM ~ 300 kDa is absent in the hydrolysate fractions. These data indicate a high hydrolysis rate in the presence of these enzymes. Formation of two low-molecular fractions of hydrolysate with close values of MM ~ 17 kDa and ~ 9 kDa has been observed for both enzymes, and the share of the latter fraction in the solution is more than 80 %. Further control of MMC has shown that there is a slow decrease in the proportion of all fractions, to almost complete disappearance in three days, except for the low-molecular fraction with MM ~ 9 kDa, the content of which gradually increases. The type of molecular weight distribution curves for different enzymes is almost the same. However, there is a small difference in the ratio of hydrolysate fractions at different stages of hydrolysis with the use of these enzymes, due to a minor difference in the efficiency of the enzymes used, which may affect the structure of the scaffold during its formation. Differences in the MM values and the ratio of fractions with different MM indicate the influence of the enzyme nature on hydrolysis of high-molecular collagen. The presented results are in good agreement with the known literature data on the nature of pancreatin enzymes (it contains the proteolytic enzymes: trypsin, pepsin and chymotrypsin). These three proteases catalyze the hydrolysis of peptide bonds formed by residues of the amino acids arginine and lysine. The strongly pronounced limited substrate specificity of enzymes leads to practically quantitative formation of a narrowly dispersed oligomer with MM ~ 9 kDa in the process of hydrolysis. In combination with the results of research on the effect of enzymes upon other components of the scaffold design, as well as biomimetic tests, these will model the composition, structure, and major characteristics of scaffolds with the optimized properties. This is especially important if the enzymatic hydrolysis reactions are used in formation of scaffolds and biomedical cell products, because the resulting cell matrix is formed not from the native molecules, but from their hydrolysates.