Solubilization of cheese whey protein by trypsin and a process to recover the active enzyme from the digest

Porcine trypsin (EC 3.4.4.4) converted, within approximately 2 hr at 50°C, its 1000‐fold weight of water‐insoluble, heat‐denaturated cheese whey protein into a water‐soluble product. In the course of this digestion, the enzyme increased the α‐amino nitrogen of the protein by a factor of >20, from 0.40 to 9.40%. After digesting the water‐insoluble whey protein, fully active trypsin could be recovered from the soluble digest with the aid of a cellulose‐based affinity adsorbent. The enzyme which was eluted from a column of p‐aminobenzamidine, bound to succinylated aminododecylcellulose, was fully active and showed essentially unchanged kinetic properties with a synthetic substrate, L‐benzoyl‐arginine p‐nitroanilide. It was possible to perform, with the same amount of trypsin, three subsequent and equally effective solubilizations of whey protein, followed by a fourth digestion which still yielded a soluble product, but was considerably slower and incomplete. During each digestion, an estimated 30% of the trypsin was lost. The was not due to a decreased efficiency of the affinity adsorbent, as its trypsin‐binding capacity was essentially unaffected after over 10 cycles of use.