Food Biochemistry and Food Processing (2 edition)

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14 Seafood Enzymes: Biochemical Properties and Their Impact on Quality 271

et al. (2008) reported that the predominant enzyme responsible

for autolysis in true sardine was a cathepsin D, which exhib-

ited the maximal activity at 55◦C, pH 3.5 and was effectively

inhibited by pepstatin A.

Cathepsin L (EC 3.4.22.15), is a typical cysteine protease

found in lysosomes and is considered to have major biological

roles in proteolysis. It is a 24-kDa molecule, existing in multiple

forms with pH between 5.9 and 6.1 and it exhibits the activ-

ity in a wide range of pH (3.0–6.5). It is strongly inhibited by

iodoacetate, leupeptin, and antipain (Kang and Lanier 2000).

Visessanguan et al. (2003) purified cathepsin L from arrowtooth

flounder muscle by heat treatment, followed by a series of chro-

matographic separations. The apparent molecular mass of the

purified enzyme was 27 kDa by size exclusion chromatogra-

phy and SDS-PAGE. The enzyme had high affinity and activity

toward Z-Phe-Arg-NMec. Activity was inhibited by sulfhydryl

reagents and activated by reducing agents. The purified enzyme

displayed optimal activity at pH 5.0–5.5 and 60◦C, respectively.

Cathepsin L from the muscle of anchovy was purified and char-

acterized (Heu et al. 1997). The enzyme was activated by thiol

reagents and inhibited by thiol-blocking reagents. The MW was

estimated to be 25.8 kDa by SDS-PAGE. The enzyme exhibited

its maximal activity at pH 6.0 and 50◦C. Cathepsin L is a major

protease, which degrades myofibrillar proteins in antemortem

or postmortem muscle of chum salmon (Yamashita and Kon-

agaya 1990), spotted mackerel (Lee et al. 1993), and Pacific

whiting (Visessanguan et al. 2001). Cathepsin L is capable of

hydrolyzing a broad range of proteins including myosin, actin,

nebulin, cytosolic proteins, collagen, and elastin (Visessanguan

et al. 2001).

Alkaline Proteases

Alkaline proteases are located in the muscle sarcoplasm, micro-

somal fraction or are bound to myofibrils (Makinodan and Ikeda

1971). Alkaline proteases are active at neutral to slightly alka-

line pH and are unstable under acidic conditions (Simpson 2000).

They are sometimes characterized as trypsin-like serine enzyme,

which show great capacity of degrading intact myofibrils in vitro

(Busconi et al. 1987). Generally, alkaline proteases are heat sta-

ble and also become active at the neutral pH of fish meat paste.

Properties of alkaline proteases vary with sources. Alkaline pro-

teases are oligomeric protease with high MWs ranging from

560 to 920 kDa (Kolodziejska and Sikorski 1996). However,

an alkaline protease isolated from Atlantic croaker was much

smaller with MW of 80–84 kDa (Lin and Lanier 1980). These

enzymes normally exhibit little or no catalytic activity unless

assayed at a nonphysiologically high temperature (60–65◦C) or

activated by protein denaturing agents such as urea, fatty acids,

or detergents (Visessanguan et al. 2001). Two alkaline proteases

(A and B) were purified from Atlantic menhaden muscle (Choi

et al. 1999). The MWs of purified enzymes were 707 kDa and

450 kDa, respectively. Both are probably serine proteases, and

optimum caseinolytic activity was observed at pH 8 and 55◦C.

Purification of a novel myofibril-bound serine protease from the

ordinary muscle of the carp was carried out by solubilizing it

from the myofibril fraction with acid treatment, followed by

Ultrogel AcA 54 and Arginine-Sepharose 4B chromatography

(Osatomi et al. 1997). The MW of purified enzyme was 30 kDa

by SDS-PAGE and gel filtration. The optimum pH and temper-

ature of the enzyme were 8.0 and 55◦C, respectively. Benjakul

et al. (2003a) reported that heat stable alkaline proteases puri-

fied from bigeye snapper had the optimum pH and temperature

for casein hydrolysis at 8.5 and 60◦C, respectively. The MW of

purified enzyme was estimated to be 72 kDa by gel filtration.

Neutral Ca^2 +-Activated Proteases

Neutral proteases in skeletal muscle have been classified with

the Ca^2 +-activated, neutral endopeptidases known as CANP, and

recently as calpains (EC 3.4.33.17) (Kolodziejska and Sikorski

1996). These enzymes are further subclassified intoμ-calpain

andm-calpain, which differ in sensitivity to calcium ions. Both

are heterodimers: the large subunit and the small subunit for

μ- andm-calpain, which have MWs near 80 kDa and 28 kDa,

respectively (Cheret et al. 2007). The calcium requirement of

μ- andm-calpain was 50–70μM and 1–5 mM, respectively.

Calpastatin is known to be the endogenous specific inhibitor of

calpain (Kolodziejska and Sikorski 1996). Calpains have been

isolated and characterized from the muscles of seafood species

like carp, American lobster, and scallop (Toyohara et al. 1982,

Kolodziejska and Sikorski 1996). Wang et al. (1993) purified

m-calpains from tilapia muscle by Phenyl-Sepharose CL-4B,

Sephacryl S-200, Q-Sepharose, and Superose 12 HR column

chromatography. The enzyme had a MW of 110,000 containing

two subunits of 80 kDa and 28 kDa. It has optimal temperature

and pH of 30◦C and 7.5, respectively. The protease was activated

by dithiothreitol, glutathione, andβ-mercaptoethanol and was

inhibited by calpain inhibitor I, calpain inhibitor II, leupeptin,

antipain, iodoacetic acid, but not affected by pepstatin A and

N-ethylmaleimide. Calpain was purified to homogeneity from

the arm muscle ofOctopus vulgaris(Hatzizisis et al. 1996).

The enzyme consists of a 65-kDa protein when separated by

SDS-PAGE. The Ca^2 +requirements for its half maximal and

maximal activities are 1.5 mM and 7 mM, respectively. It is

strongly inhibited by thiol protease inhibitors such as leupeptin,

E-64, and antipain and by alkylating agents such as iodoacetic

acid and iodoacetamide. The enzyme displayed maximal activity

at 30◦C and showed broad pH optimum between 6.5 and 7.5.

Fish Protease Applications

Proteases are by far the most studied enzymes for industrial

bioprocessing. Almost half of all industrial enzymes are pro-

teases, mostly used in the detergent, leather, and food industries

(Klomklao et al. 2005). The food industry uses proteases as

processing aids for many products including baked goods, beer,

wine, cereals, milk, fish products, and legumes. Production of

protein hydrolysates and flavor extracts has been known to be

successful by using selected proteases (Table 14.4) (Klomklao

2008).

Commercial supplies of food processing enzymes are

presently derived from various plants, animal, and microbial

sources. Compared with these enzymes used in food processing,