Controlled Protein Hydrolysis with Immobilized Alkaline Endo-Protease

Abstract

Enzymatic hydrolysis is used to enhance the functional properties of proteins; the modified proteins exhibit improved solubility, lower hydrophobicity and viscosity; but high cost and lower stability of proteases pose problems in their commercial use. In current work, different endo-proteases were screened for proteolytic activity and 89L (alkaline protease) was selected based on higher specific activity. Glutaraldehyde coupling was used for immobilization with CR20 resin as support matrix; various parameters such as glutaraldehyde concentration (1%), activation time (16hrs), enzyme loading (4mg/ml of resin) and coupling time (2hrs) were optimized for maximum immobilization efficiency. Immobilized preparation exhibited high catalytic activity i.e. 9260 Casein Digesting Units per ml with improved operational and storage stability. Soy proteins, whey proteins, bovine serum albumin (BSA) were subjected to controlled protein hydrolysis using immobilized 89L. Hydrolysis reaction with BSA revealed substrate concentration is inversely proportional to degree of hydrolysis. Gel permeation chromatography analysis of BSA and soy protein hydrolysates depicted formation of peptides in the range of 1-10kDa and <1kDa. MS analysis confirmed presence of smaller peptides with molecular weight around 1kDa. Optimum hydrolysis of soy proteins was obtained in batch mode with 1:10 enzyme to substrate ratio at 50°C in 200 min with 16% degree of hydrolysis. Packed bed column hydrolysis produced homogeneous hydrolysates for 15 cycles without any loss of enzymatic activity. Thus, protein hydrolysis reaction can be customised using immobilized 89L to obtain peptides with desired molecular weight distribution.