Proteases can hydrolyse soluble as well as insoluble substrates. The effect will be depndent on particle size, mecanical action during the reaticon , temperature , pH, actual substrate and substrate concentrations, amino acid and pretreatment Do you have specific sustrates in mind? Kind regards Hans

The protease must be able to bind the substrate in its active site. An insoluble or immobilized protein might still have an exposed terminus or loop that can be bound and cleaved by the protease. However, proteolysis will be much more efficient if the protein is in solution because the entire protein will be exposed.

Hello Hans ( Hans Sejr Olsen ), Thanks for your reply. Cereal proteins normally contain quite a lot of alochol-soluble proteins, such as Zein from maize, which has quite poor solubility in aqueous. Then if this portein molecule is insoluble, how can it contact with the active site of enzyme. In my mind, enzymatic hydrolysis will happen in one system, except lipase that can work on the interface between oil and aqueous, right?

Best, Yuhong

Hey Adam ( Adam B Shapiro ) Thanks for your reply. So you think for such aqueous-insoluble proteins, enzyme will probably hydrolyze their surface (loops exposured in solution) firstly and then more and more exposured, by which to increase the solubility?

The enzyme attach to the surface of the substrate, and thereby open the it more and more. That is why it can solubilise proteins attached to surfaces of textiles or hard surfaces. That is maybe a too simple explanation ?

You may find that some of the protein is resistant to proteolysis because it is too inaccessible, even after exposed surface loops are cleaved. It depends on how large and densely packed the insoluble protein is. After an initial proteolytic treatment, you could try boiling the remaining protein to unfold it, then add more protease.

Some proteases are active in the presence of detergents and mild denaturants, which you could add to help solubilize the insoluble protein.