Food Biochemistry and Food Processing (2 edition)

BLBS102-c14 BLBS102-Simpson March 21, 2012 13:17 Trim: 276mm X 219mm Printer Name: Yet to Come

14 Seafood Enzymes: Biochemical Properties and Their Impact on Quality 269

Table 14.2.N-Terminal Amino Acid Sequence of Fish and Mammalian Trypsins

Source of Trypsin N-Terminal Sequence Reference

Vertebrate

Pectoral rattail IVGGYECQEHSQ Klomklao et al. (2009a)

Skipjack tuna IVGGYECQAHSQPPQVSLNA Klomklao et al. (2009b)

Sardine IVGGYECQKYSQ Bougatef et al. (2007)

Yellowfin tuna IVGGYECQAHSQPHQVSLNA Klomklao et al. (2006b)

Tongol tuna IVGGYECQAHSQPHQVSLNA Klomklao et al. (2006a)

True sardine IVGGYECKAYSQPWQVSLNS Kishimura et al. (2006c)

Japanese anchovy IVGGYECQAHSQPHTVSLNS Kishimura et al. (2005)

Arabaesque greenling IVGGYECTPHTQAHQVSLDS Kishimura et al. (2006c)

Walleye pollok IVGGYECTKHSQAHQVSLNS Kishimura et al. (2008)

Spotted mackerel IVGGYECTAHSQPHQVSLNS Kishimura et al. (2006b)

Bluefish IVGGYECKPKSAPVQVSLNL Klomklao et al. (2007c)

Atlantic bonito IVGGYECQAHSQPWQPVLNS Klomklao et al. (2007b)

Cod IVGGYECTKHSQAHQVSLNS Gudmundsdottir et al. (1993)

Salmon IVGGYECKAYSQTHQVSLNS Male et al. (1995)

Invertebrate

Cuttlefish IVGGJESSPYNQ Balti et al. (2009)

Starfish IVGGKESSPHSR Kishimura et al. (2002)

Mammal

Porcine IVGGYTCAANSVPYQVSLNS Hermodson et al. (1973)

Bovine IVGGYTCGANTVPYQVSLNS Walsh (1970)

temperature for the hydrolysis of Z-Phe-Arg-MCA were 7.0

and 37◦C, respectively. This protease activity was inhibited by

E-64, leupeptin, 5–5’-dithiobis (2-nitro-benzoic acid), andp-

tosyl-lys-chloromethylketone.

Metalloproteases

The metalloproteases are hydrolytic enzymes whose activity de-

pends on the presence of bound divalent cations (Simpson 2000).

There may be at least one tyrosyl residue and one imidazole

residue associated with the catalytic sites of metalloproteases

(Whitaker 1994). The metalloproteases are inhibited by chelat-

ing agents such as 1, 10-phenanthroline, EDTA, and sometimes

by the simple process of dialysis. The metalloproteases have

been characterized from marine animals (e.g., rockfish, carp, and

squid mantle) but have not been found in the digestive glands

except in the muscle tissue (Simpson 2000). Metalloproteases

do not seem to be common in marine animals (Simpson 2000).

However, Sivakumar et al. (1999) purified collagenolytic metal-

loprotease with gelatinase activity from carp hepatopancreas by

ammonium sulfate fractionation and gel filtration chromatogra-

phy. The enzyme had a MW of 55 kDa and was active against

native type I collagen. Optimum temperature and pH were 25◦C

and 7–7.5, respectively. The enzyme was strongly inactivated by

10 mM EDTA.

Muscle Proteases from Marine Animals

Muscles of marine animals contain a variety of proteases. These

enzymes perform different metabolic functions in the living or-

ganisms. Their activity depends on the species, life cycle, and the

feeding status of the animal (Kolodziejska and Sikorski 1996).

Endogenous proteases in fish muscle are found in the sarcoplas-

mic (or soluble) component of muscle tissues, in association

with cellular organelles, connective tissues, and myofibrils and

in the interfiber space (Kolodziejska and Sikorski 1996). A num-

ber of endogenous proteases have been investigated as enzymes

contributing to postmortem softening of fish flesh, which also

participate to a different extent in the degradation of myofib-

rillar protein (Visessanguan et al. 2001). The action of these

enzymes exerts detrimental effects on the sensory quality and

functional properties of muscle food (Visessanguan et al. 2001).

The muscles of marine fish and invertebrates contain lysosomal

cathepsins, as well as different proteases active in neutral and

alkaline range of pH (Table 14.3).

Cathepsins

Lysosomal proteases such as cathepsin A (carboxypeptidase A),

cathepsin B (carboxypeptidase B), cathepsin D, cathepsin H,

and L have been recovered and identified from various fish

and aquatic invertebrates (Shahidi and Kamil 2001). Except for

cathepsin D, all other lysosomal proteases belong to serine or

cysteine (thiol). However, cathepsin D, which is an aspartic pro-

tease, has its pH optimum within the acidic range (Simpson

2000).

Cathepsin A (EC 3.4.16.1) is now called carboxypeptidase

A. The enzyme has a pH optimum of 5–6 and is readily inacti-

vated by heat and alkaline, and has a molecular mass of about

35 kDa. Cathepsin A has been purified about 1700-fold to homo-

geneity from carp muscle (Toyohara et al. 1982). The molecular

mass was estimated to be 36 kDa and the pH optimum was 5.0.