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14
Seafood Enzymes: Biochemical Properties
and Their Impact on Quality
Sappasith Klomklao, Soottawat Benjakul, and Benjamin K. Simpson

Introduction
Proteases
Digestive Proteases from Marine Animals
Acid/Aspartic Proteases
Serine Proteases
Thiol/Cysteine Proteases
Metalloproteases
Muscle Proteases from Marine Animals
Cathepsins

Alkaline Proteases
Neutral Ca2 -Activated Proteases
Fish Protease Applications
Transglutaminase
Polyphenoloxidase
Factors Affecting PPO Activity
Temperature
pH
Activators/Inhibitors
Melanosis
Trimethylamine-N-Oxide Demethylase
Distribution of TMAOase
Purification and Characterization of TMAOase
Prevention of TMAOase Activity
Lipase
Fish Lipase Applications
References

Abstract: Recovery and characterization of enzymes from seafood
has taken place and this has led to the emergence of some interesting
new applications of these enzymes in food processing. The major
enzymes isolated from marine organism and used for industrial application are protease, transglutaminase, and lipase. However, some
enzymes found in the fish and aquatic invertebrates have exhibited
an adverse effect on seafood quality such as polyphenoloxidase and
TMAOase. Therefore, appropriate approach to tackle the problem
associated with undesirable enzyme has become the technical concern for processors to maintain the prime quality of seafood and
seafood products. On the other hand, the maximization of desir-

able enzyme activities is the means to come across the fulfillment
of seafood processing. In addition, the cost of enzyme from other

sources could be lowered, leading to the sustainability of seafood
industry, in which all resources could be fully utilized.

INTRODUCTION
The marine environment contains a wide variety of genetic material, offering enormous potential as a source of enzymes. Because of the biological diversity of marine species, a wide array of enzymes with unique properties can be recovered and
effectively utilized. Most enzymes from fish and aquatic invertebrates are present in terrestrial organisms. However, the
enzymes from different marine species, as well as their organs
or environment habitat display differences in molecular weight
(MW), amino acid composition, pH and temperature optimum,
stability, and inhibition characteristics and kinetic properties.
Recovery and characterization of enzymes from seafood has
been studied extensively, and this has led to the emergence of
some interesting new applications of these enzymes in food processing. The major enzymes isolated from marine organisms
and used for industrial application are proteases, transglutaminases, and lipases. However, some enzymes found in fish and
aquatic invertebrates exhibit adverse effects on seafood quality
such as polyphenoloxidases (PPO) and trimethylamine-N-oxide
demethylase (TMAOases). Therefore, appropriate approaches
to tackle the problem associated with undesirable enzymes in
seafood products have become a technical concern for processors to maintain the prime quality of seafood and seafood products. Conversely, the activities of desirable enzyme should be
maximized to fully exploit the marine resources. Furthermore,
the need of commercial enzymes can be reduced, thereby lowering the operation cost associated with those enzymes.

Food Biochemistry and Food Processing, Second Edition. Edited by Benjamin K. Simpson, Leo M.L. Nollet, Fidel Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H. Hui.
C 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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PROTEASES
Proteases play an important role in the growth and survival of all
living organisms. Protease catalyzes the hydrolysis of peptide
bonds in polypeptides and protein molecules (Garcia-Carreno
and Hernandez-Cortes 2000). On the basis of their in vitro properties, proteases have been classified in a number of ways such as
the pH range over which they are active (acid, neutral, or alkaline
proteases), their ability to hydrolyze specific proteins, and their
mechanism of catalysis. Proteases may be classified based on
their similarities to well-characterized proteases such as trypsinlike, chymotrypsin-like, chymosin-like or cathepsin-like, and so
on (Haard 1990). In addition, proteases are classified according
to their catalytic action (endopeptidase or exopeptidase) and the
nature of the catalytic site. In Enzyme Commission (EC) system
for enzyme nomenclature, all proteases (peptide hydrolases) belong to subclass 3.4, which is further divided into 3.4.11–19,
the exopeptidases and 3.4.21–24, the endopeptidases or proteinases (Nissen 1993). Endopeptidases cleave the polypeptide

chain at particularly susceptible peptide bonds distributed along
the chain, whereas exopeptidases hydrolyze one amino acid unit
sequentially from the N-terminus (aminopeptidases) or from Cterminus (carboxypeptidases) (Fig. 14.1). Exopeptidases, especially aminopeptidases, are ubiquitous, but less readily available
as commercial products, since many of them are intracellular or
membrane bound (Simpson 2000). For endopeptidases or proteinases, the four major classes of endopeptidases can be distinguished according to the chemical group of their active site,
including serine proteinases (EC 3.4.21), thiol or cysteine proteinases (EC 3.4.22), acid or aspartic proteinases (EC 3.4.23),
and metalloproteinases (EC 3.4.24) (Simpson 2000). The enzymes in the different classes are differentiated by various criteria, such as the nature of the groups in their catalytic sites,
their substrate specificity, and their response to inhibitors or by
their activity/stability under acid or alkaline conditions (Nissen
1993).
For marine animals, proteases are mainly produced by the
digestive glands. Like the proteases from plants, animals, and
microorganisms, digestive proteases from marine animals are
polyfunctional enzymes catalyzing the hydrolytic degradation
of proteins (Garcia-Carreno and Hernandez-Cortes 2000). For

some fish species, proteases are present at high levels in the muscle (Kolodziejska and Sikorski 1996), and are associated with
the induced changes of proteins during postmortem storage or
processing. Marine animals have adapted to different environmental conditions, and these adaptations, together with interand intraspecies genetic variations, are associated with certain
unique properties of their proteinases, compared with their counterpart from land animals, plants, and microorganisms (Simpson
2000). Some of these distinctive properties include higher catalytic efficiency at low temperature and lower thermal stability
(Klomklao et al. 2009a).

Digestive Proteases from Marine Animals
Digestive proteases have been studied in several species of
fish and decapods (De-Vecchi and Coppes 1996). Proteases
found in the digestive organs of fish include pepsin, gastricsin,
trypsin, chymotrypsin, collagenase, elastase, carboxypeptidase,
and carboxyl esterase (Simpson 2000). Pepsin, chymotrypsin,
and trypsin are three main groups of proteases found in fish

viscera. Pepsin is localized in fish stomach (Klomklao et al.
2007a), while chymotrypsin and trypsin are concentrated in tissues such as the pancreas, pyloric ceca, and intestine (Klomklao
et al. 2004). The distribution and properties of protease vary
depending on species and organs (Table 14.1). According to
the International Union of Applied Biochemists classification,
digestive proteases from fish and aquatic invertebrates may be
classified into four major groups such as acidic/aspartic protease,
serine protease, thiol or cysteine protease, and metalloprotease
(Simpson 2000).

Acid/Aspartic Proteases
The acid or aspartic proteases are a group of endopeptidases
characterized by high activity and stability at acidic pH. They
are referred to as “aspartic” proteases (or carboxyl proteases)
because their catalytic sites are composed of the carboxyl group
of two aspartic acid residues (Whitaker 1994). On the basis
of the EC system, all the acid/aspartic proteases from marine
animals have the first three digits in common as EC 3.4.23.
Three common aspartic proteases that have been isolated and

Endopeptidase

Exopeptidase

Figure 14.1. Cleavage of proteins by endopeptidases and exopeptidases.

+

+