In vitro stability of natural and synthetic proteins for potential application in animal feeding

Milk Bundle-1 is a de novo designed protein with 100 amino acids, having a molecular weight of 11.4 kilodaltons. MB-1 is enriched with 57% of selected essential amino acids (methionine, threonine, lysine and leucine) and is intended as a feed additive for dairy cows. In order to predict the behaviour of MB-1 in rumen environment, in vitro experiments were conducted to investigate its structural as well as its proteolytic stability.

Optimization of fluorescence technique was conducted by studying the structural stability of jack bean urease. The results indicated the hexameric urease structure dissociates into folded dimers which then aggregate or dissociate into unfolded monomers.

Fluorescence spectroscopy was used to study the thermal stability of MB-1. Denaturation studies of MB-1 were conducted in the presence of physiological levels of volatile fatty acids in a buffer with ionic strength and pH similar to rumen fluid. Under these conditions, the melting temperature was obtained to be around 50$\sp\circ$C. This suggested that MB-1 will have a folded structure at the nrmen temperature of 39$\sp\circ$C. This feature is important in that it indicates that MB-1's structure and folding will contribute to a higher resistance to in vivo degradation.

Proteolytic stability was investigated using two proteases of microbial origin. Pronase E (from Strepromyces griseus) and Neutrase (from Bacillus subtilis) were used according to a procedure currently used as a standard methodology in feed evaluation laboratories. The results of these studies revealed that MB-1 is more degradable than cytochrome c, ribonuclease A and urease. (Abstract shortened by UMI.).