Abstract

Proteolytic activity in Norway lobster (Nephrops norvegicus) was studied. An improved separation and partial purification of the three proteases (designated as proteases I, II and III) was achieved from Norway lobster heads by a combination of acetone precipitation and DEAE-Sepharose CL-6B column chromatography.

The purification achieved was 63-, 25- and 217-fold at pH 8.2, and 40-, 25- and 160-fold at pH 6.4 for protease I, II and III, respectively.

With casein as substrate, protease III was most active at pH 8.2, whilst proteases I and II showed activity over a wide range of pH.

Protease III was characterized as an alkaline Zn-serine protease as it was strongly inhibited by PMSF, soybean trypsin inhibitor, Co2+, Mn2+ and 1–10 phenanthroline. Protease I was strongly inhibited by p-benzoquinone, iodo-acetamide, heavy metals (Ag+, Cu2+) and 1–10 phenanthroline and was thus characterized as a Zn-thiol protease. Protease II was also inhibited by the same inhibitors as protease I (but to a lesser extent) and was characterized as a thiol protease.

The molecular weights were determined to be 22.5, 45 and 42.5 kDa (with activity at 18 kDa) for proteases I, II and III, respectively.

It was found that protease III activates phenolase at pH 8.2, whilst proteases II and I can activate phenolase at both pH 6.7 and 8.2.