BLBS102-c14 BLBS102-Simpson March 21, 2012 13:17 Trim: 276mm X 219mm Printer Name: Yet to Come
274 Part 2: Biotechnology and EnzymologyEndogenous TGases from the muscle tissues of different fish
species vary in properties such as different MWs, pH, and tem-
perature optima. TGase activity has been found in muscle of
red sea bream (Pagrus major), rainbow trout (Oncorhynchus
mykiss), atka mackerel (Pleurogrammus azonus) (Muruyama
et al. 1995), walleye pollock liver (Theragra chalcogramma)
(Kumazawa et al. 1996), muscle of scallop (Patinopecten
yessoensis), botan shrimp (Pandalus nipponensis), and squid
(Todarodes pacificus) (Nozawa et al. 1997). Worratao and
Yongsawatdigul (2005) demonstrated that tropical tilapia (Ore-
ochromis niloticus) muscle contained high TGase activity. Gen-
erally, the optimum temperature of TGase ranged from 25◦C
to 55◦C, depending on fish species. Nozawa et al. (1997) de-
scribed TGase in fish muscle as calcium dependent. Worratao
and Youngsawatdigul (2005) also reported that purified tilapia
TGase had an absolute requirement for calcium ions. TGase ac-
tivity increased with Ca^2 +concentration and reached the maxi-
mum at 1.25 mM. Optimal Ca^2 +concentrations for TGase from
red sea bream liver, Japanese oyster, scallop, and pollock liver
were at 0.5 mM, 25 mM, 10 mM, and 3 mM, respectively
(Yasueda et al. 1994, Kumazawa et al. 1996,, Nozawa et al.
2001). It was postulated that the calcium ions induced confor-
mational changes of the enzyme that consequently exposed the
cysteine located at the active site to a substrate (Ashie and Lanier
2000). Noguchi et al. (2001) reported that the calcium ions attach
to a binding site of red sea bream TGase molecule, resulting in
conformational changes. Subsequently, Tyr covering the cataly-
sis cystine (Cys) was removed. Then, the acyl donor binds with
the Cys at the active site, forming an acyl-enzyme intermediate.POLYPHENOLOXIDASE
Polyphenoloxidase (PPO, EC 1.14.18.1) is a copper enzyme
(Goulart et al. 2003), whose active site contains two copper
atoms, each of which is liganded to three histidine residues (Fig.
14.5) (Whitaker 1995, Kim et al. 2000). This copper pair is
the site of interaction of PPO with both molecular oxygen and
its phenolic substrates (Kim et al. 2000). PPO is also known
as phenoloxidase (PO), polyphenolase, phenolase, tyrosinase,
and cresolase (Whitaker 1995). This enzyme catalyzes the hy-
droxylation of monophenols too-diphenols and the oxidation of
o-diphenols too-quinones. Quinones are highly reactive prod-His 187His 281His 193 His 306His 104His 95Cu2+ Cu2+OOFigure 14.5.Coordination of copper to six histidine residues in
active site of polyphenoloxidase.ucts and can polymerize spontaneously to form brown pigments
(melanin), or react with amino acids and proteins that enhance
the brown color produced (Fig. 14.6) (Goulart et al. 2003,
Martinez-Alvarez et al. 2008). Enzyme isolated from various
sources exhibited different activities depending on the substrates
used. Tyr has been reported to be a natural substrate for PO ac-
tivity in crustacea. Hydroxylation of Tyr leads to the formation
of dihydroxyphenylalanine (DOPA) (Kim et al. 2000), which
is then converted to dopaquinone and water and can be read-
ily converted to the orange-red pigment, dopachrome (Simpson
et al. 1987).
PO has been shown to play a central role in the innate immune
system of crustacea (Williams et al. 2003). This enzyme is ex-
pressed from their hemocytes as its precursor, prophenoloxidase
(proPO) and functions in various phases of the live organism
such as sclerotization, pigmentation, wound healing on cuticles,
and defense reaction (Adachi et al. 2001). PO has been detected
in the hemocyte, hemolymph, cuticle, accessory gland, egg case,
salivary gland, and midgut in insects (Adachi et al. 1999). In
crustaceans, PO is localized in different parts. It is found on the
exoskeleton, chiefly on the region of the pleopods’ connection
(Montero et al. 2001).
The activation of proPO in hemocyte, or hemolymph, is initi-
ated by minute amounts of bacterial or fungal cell wall compo-
nents (β-1,3-glucans, lipopolysaccharides, and peptidoglycans)
and mediated by several factors including protease cascade re-
action (Adachi et al. 1999, Williams et al. 2003). The serine
protease that exhibits trypsin-like activity cleaves the proPO be-
tween an arginine residue and a Thr residue to yield active PO
(Williams et al. 2003). The proPO of kuruma prawn homocyte
was activated by SDS and methanol but not by trypsin (Adachi
et al. 1999).
PPO has been found in crustaceans such as shrimp (Chen et al.
1997), lobster (Chen et al. 1991), pink shrimp (Simpson et al.
1988), white shrimp (Simpson et al. 1987), black tiger shrimp
(Rolle et al. 1991), Florida prawn (Ali et al. 1994), kuruma prawn
(Benjakul et al. 2005), imperial tiger prawn (Montero et al.
2001), and deep water pink shrimp (Zamorano et al. 2009). PPO
is distributed in many parts of shrimps with different levels of
activity (Montero et al. 2001). PO is localized in the carapace of
the cephalothorax, in the caudal zone and in the cuticle of the ab-
domen, mainly where the cuticle segments are joined and where
the cuticle is connected to the pleopods (Ogawa et al. 1984).
Zamorano et al. (2009) studied the tissue distribution of PPO in
deepwater pink shrimp (Parapenaeus longirostris) postmortem.
PPO activity was highest in the carapace, followed by that in
the abdomen exoskeleton, cephalotorax, pleopods, and telson.
No PPO activity was found in the abdominal muscle and in the
pereopods and maxillipeds. Storage of whole shrimps and of the
different organs showed that melanosis required the presence of
the cephalotorax to be initiated, indicating that its development
depends on other factions in addition to the PPO levels.Factors Affecting PPO ActivityPPO differs in isoforms, latency, catalytic behavior, MW, speci-
ficity, and hydrophobicity. In addition, optimum pH, thermal