Characterization of a Chitosanase Isolated from a Commercial Ficin Preparation

Abstract

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).

Characterisation of general proteolytic, milk clotting and antifungal activity of Ficus carica latex during fruit ripening

“…Such a result is not surprising since it is in accord with our result of chitinolytic activity. In fact, the great majority of recently described antifungal proteins of genus Ficus are chitinases, or chitinase‐like enzymes …”

“…Ficin from fig latex is known as an enzyme with milk clotting activity (ancient writings such as the Iliad indicate that fig latex can be used for cheese‐making), but it also has high proteolytic activity over a broad temperature and pH range . Apart from ficin, fig latex also contains the newly described collagenolytic serine protease, as well as a few recently described chitinolytic enzymes …”

Cysteine Proteases

“…Therefore the depolymerisation of chitosan could facilitate the application of chitosan-related materials in a variety of fields. Commercial crude papain, bromelain and ficin are widely used for chitosan depolymerisation (Li et al, 2005;Chang et al, 2005). However, the hydrolysis of chitin and chitosan by means of stem bromelain was the result of chitinase and chitosanase activities present in the crude enzyme and not bromelain itself (Hung et al, 2002).…”

Structural characterization of two papaya chitinases, a family GH19 of glycosyl hydrolases

“…Several commercial crude sources of proteases have been shown to be able to depolymerize chitosan because they contain chitinases. This is not only the case for the already mentioned papain, but also for, for instance, ficin [42], bromelain [43] and pepsin [44]. Another indication was provided by the rye seed chitinase-a (RSC-a) constituted by a N-terminal cysteine-rich chitin-binding domain, called the hevein domain, and a C-terminal catalytic domain; these two domains are linked by a flexible segment known as the hinge region.…”