Sepabeads-EP (a new epoxy support) has been utilized to immobilize-stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 micromol/mL). These features should permit a very intense enzyme-support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three-step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds-fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 degrees C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).

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Chickpea protein isolates and the protease alcalase were used for the production of protein hydrolysates that inhibit angiotensin I-converting enzyme (ACE). The highest degree of inhibition was found in a hydrolysate obtained by 30 min of treatment with alcalase. This hydrolysate was used as starting material for the puri®cation of ACE-inhibitory peptides. After Biogel P2 gel ®ltration chromatography and HPLC C 18 reverse phase chromatography, four peptides with ACE-inhibitory activity were puri®ed. Two of them were competitive inhibitors of ACE, while the other two were uncompetitive inhibitors. These results show that chickpea proteins are a good source of ACEinhibitory peptides when hydrolysed with the protease alcalase.

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