Extraction and purification of hyaluronoglucosidase (EC 3.2.1.35) from Norway lobster (Nephrops norvegicus)

Abstract The waste of Norway lobster ( Nephrops norvegicus also known as Dublin Bay prawn and scampi) constitutes 75–80% of the whole animal by weight. This includes the hepatopancreas which is a good source of hyaluronoglucosidase (EC 3.2.1.35), commonly referred to as hyaluronidase. This enzyme was partially purified by acetone fractionation, ion-exchange column chromatography on a Type-I polystyrene-based anion-exchange resin, Amberlite™ IRA 420 and subsequently by gel filtration on Sephadex™ G-200. The anion-exchange step of the purification was optimised by including a wash with 10 mM sodium acetate buffer containing 12.5 mM NaCl after loading the column with the enzyme preparation. This was followed by gradient elution with 12.5–500 mM NaCl in 500 ml of the same buffer. The gel filtration step was optimised using Sephacryl™ S-200-HR gel filtration medium. As a result of these modifications to the purification process a 763-fold purification was achieved, with 32% of the enzyme from the original crude extract in 0.25 M sucrose solution being recovered. A loss of 41% of the enzyme activity was recorded during acetone fractionation and 34% during gel filtration. However, recovery from the anion-exchange step using Amberlite™ IRA 420 was as high as 81%. A sample of the purified extract was subjected to native polyacrylamide gel electrophoresis with PhastGel™ Gradient 10-15 using PhastGel Native Buffer Strips which indicated the presence of three proteins.