Hydrolysis of α-lactalbumin by cardosin A immobilized on highly activated supports

• Author(s): Barros, Rui M. ; Extremina, Clara I. ; Gonçalves, Inês C. ; Braga, Beatriz O. ; Balcão, Victor M. ; Malcata, F. Xavier
• Date: 2003
• Persistent ID: http://hdl.handle.net/10400.14/6826
• Origin: Veritati - Repositório Institucional da Universidade Católica Portuguesa
• Subject(s): Enzyme; Protease; Agarose; Attachment; Structural stabilization; Dairy foods; α-Lactalbumin; Cardosin
• Document Type: Article
• Language: English

Description

In the present research effort, production of derivatives of cardosin A (a plant protease) encompassing full stabilization of its dimeric structure has been achieved, via covalent, multi-subunit immobilization onto highly activated agarose-glutaraldehyde supports. Boiling such enzyme derivatives in the presence of sodium dodecyl sulfate and β-mercaptoethanol did not lead to leaching of enzyme, thus providing evidence for the effectiveness of the attachment procedure. Furthermore, the cardosin A derivatives prepared under optimal conditions presented ca. half the specific activity of the enzyme in soluble form, and were successfully employed at laboratory-scale trials to perform (selective) hydrolysis of α-lactalbumin (α-La), one of the major proteins in bovine whey. Hydrolysates of α-La were assayed for by the OPA method, as well as by FPLC, SDS–PAGE and HPLC. Thermal inactivation of the immobilized cardosin A was also assessed at 40, 50 and 55 °C; at these temperatures, no thermal denaturation took place during incubation for 48 h. The highest degree of hydrolysis was attained by 5 h reaction, at 55 °C and pH 5.2. SDS–PAGE of α-La hydrolysates displayed bands corresponding to low molecular weight peptides. Our results suggest that cardosin A in immobilized form is a good candidate to bring about proteolysis in the dairy industry, namely in whey processing.