FISHERIES SCIENCE 2000; 66: 182–183
Short Paper
Comparison of protease activity in liver among several species
of squid and cuttlefish
Hideo HATATE,1* Ryusuke TANAKA,1 Nobutaka SUZUKI1 AND Yoichiro HAMA2
1Food Chemistry and Technology, National Fisheries University, Shimonoseki, Yamaguchi 759-6595, and
2Applied Biological Science, Saga University, Saga 849-8502, Japan
KEY WORDS: cuttlefish, liver, protease, squid.
Squid are widely distributed in the sea areas of the world,
and their muscles are used for a variety of foodstuffs. On
the other hand, squid livers are mostly discarded without
use, although they are known to contain various and
large amounts of enzymes.1–4 Enzyme preparations origi-
nating from micro-organisms are frequently used in the
pharmaceutical, chemical, and food industries. However,
interest in utilization of natural materials has increased
because of their high safety. Therefore, enzymes from
squid liver, a natural product, will be preferable to those
from micro-organisms at least for applications in the food
industry. Recently, it was reported that angiotensin I-
converting enzyme inhibitors (antihypertensive pep-
tides) were produced during autolysis of squid liver and
mantle muscle homogenates.5 This report also indicates
the effectiveness of squid proteases2–4 as well as their
muscle proteins in food processing. In order to utilize
squid liver as a useful enzyme resource, the present study
compared the potential of protease production in the
livers among several species of squid and cuttlefish.
Table 1 shows samples used in this study. Sample nos.
1–5 were caught in sea areas far from Japan and were
obtained in the frozen state at -20°C, while sample nos.
6–9, caught in Japan, were fresh enough to be obtained
in iced storage without freezing. Although the freshness
of each sample was different, this difference is not a crit-
ical problem for comparing the potential of proteolytic
activity between these livers, as previously described.6
Each liver was excised and subsequently homogenized.
The homogenate was mixed with 15 parts (v/w) of
cooled acetone (-20°C) and filtered with suction for
removal of lipids. The precipitate collected on the filter
was again washed with cooled acetone and then with
cooled ether. The washed precipitate was dried com-
pletely under reduced pressure. The dried precipitate
(100 mg) was mixed with 10 mL of 0.1 M sodium acetate
buffer (pH 4.0) and the crude enzyme extract in the
supernatant was recovered by centrifugation (10 000 g,
30 min, 4°C). After transporting the supernatant, the
precipitate was again extracted with the same procedure.
Both supernatants were combined and used as the crude
enzyme extract from liver.6
Proteolytic activity of the crude enzyme extract was
measured using each of 2% hemoglobin in 0.1 M sodium
acetate buffer (pH 4.0) and 2% casein in 0.1 M phos-
phate buffer (pH 7.0) as the substrates.6 An aliquot
(0.2 mL) of the crude enzyme extract was added to
2 mL of 2% substrate solution and incubated at 37°C for
10–60 min, according to the potency of proteolytic activ-
ity. The enzyme reaction was stopped by adding 5 mL
of 5% trichloroacetic acid. After standing for 30 min,
the mixture was filtered. The digestion degree of the sub-
strate in the filtrate was estimated by the method of
Lowry et al.7 using tyrosine as the standard. One unit of
enzyme activity was defined as the amount of enzyme
that liberated 1 mM of tyrosine per min.8
Table 2 shows the proteolytic activity in the crude
enzyme extracts from the samples shown in Table 1. All
the samples showed stronger activity at pH 4.0 than at
pH 7.0, while significant differences among them were
also observed in the potency of proteolytic activity, body-
and liver weights, and liver percentages. It was reported
that squid livers contain various types of cathepsin-like
proteases.1–4 These cathepsin analogs will participate as
the major acid proteases in the crude enzyme extracts,
although their enzymatic characterizations have not
been carried out yet. The recovery of enzymes from the
liver is dependent not only on specific enzyme activity
but also on liver weight. Considering these factors,
Antarctic flying squid (sample no. 1), jumbo flying squid
(no. 3), purpleback squid (no. 4), and Japanese common
squid (no. 8) will be more applicable to enzyme
resources, at least for the preparation of proteases.
Nevertheless, because all livers contained different but
*Corresponding author: Tel: 0832 865111. Fax: 0832 867434.
Received 26 April 1999.
Hatcher
