Nitrile hydratase recombinant from E.coli

Melting point / freezing point

Link to relevant study record(s)

Reference

Endpoint:

melting point/freezing point

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Description of key information

In accordance with REACH Annex XI, section 2, a melting point study is not required as testing is technically not possible as a consequence of the properties of the substance.

The substance, nitrile hydratase, is a substance that is handled in a state where a mixture of the enzyme and the dead bacteria that produced it is dispersed in water.

A fundamental requirement for testing the physicochemical properties of a substance, is that the substance is tested as such, excluding all solvents. However, since the higher order structure of the constituent molecules of the proposed substance (nitrile hydratase) is damaged by the removal of water, it cannot be said that the proposed substance would be correctly evaluated by performing this test after the removal of water. It should therefore be avoided.

In order to avoid deterioration it would be possible to conduct the test by leaving the substance as a suspension (ie dispersed in water). However, to do so would render the test results meaningless as the physical properties of water would greatly influence the results of such a test. As such, it would not be possible to consider the results of any such test as indicative of the physicochemical properties of the substance as such.

Based on the above, it is not considered that the physicochemical properties of the proposed substance would be correctly evaluated in a melting point test conducted after removing water, and therefore it is not considered necessary to conduct the test.

Additionally, even if water were to be removed from the test material suspension a classic melting point test would still not be relevant. Indeed, enzymes are polymers of 20 different naturally occurring amino acids in various length and order. The amino acids sequence determines the structure of the enzyme since at biological conditions the structure of this polymer is defined by the energy minimum fold. The enzyme fold and structure is held together primarily by hydrogen bonds, hydrophobic interactions, ion bonds and van der Waals forces between the different amino acids and cofactors. When heating the enzymes above the biological conditions, typically 80°C and higher, the attracting forces within the enzymes are broken, the fold is disrupted and the enzyme denatures. Denatured the enzymes lose their activity and typically coagulate. As such it is technically impossible to obtain a meaningful result.