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

Rui M. Barros, Clara I. Extremina, Inês C. Gonçalves, Beatriz O. Braga, Victor M. Balcão, F. Xavier Malcata*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Citations (Scopus)
57 Downloads

Abstract

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 48h. The highest degree of hydrolysis was attained by 5h 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.
Original languageEnglish
Pages (from-to)908-916
Number of pages9
JournalEnzyme and Microbial Technology
Volume33
Issue number7
DOIs
Publication statusPublished - 2 Dec 2003

Keywords

  • Agarose
  • Attachment
  • Cardosin
  • Dairy foods
  • Enzyme
  • Protease
  • Structural stabilization
  • α-Lactalbumin

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