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(TM) IRA 420 and subsequently by gel filtration on Sephadex(TM) 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(TM) 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(TM) IRA 420 was as high as 81. A sample of the purified extract was subjected to native polyacrylamide gel electrophoresis with PhastGel(TM) Gradient 10-15 using PhastGel Native Buffer Strips which indicated the presence of three proteins. Copyright (C) 1999 Elsevier Science Ltd