BLBS102-c14 BLBS102-Simpson March 21, 2012 13:17 Trim: 276mm X 219mm Printer Name: Yet to Come
268 Part 2: Biotechnology and Enzymology02040608010010 20 30 40 50 60 70 80 90
Temperature (°C)Relative activity (%)0204060801003456789101112
pHRelative activity (%)(A)(B)Figure 14.3.pH(A)and temperature(B)profiles of purified trypsin
from the pyloric ceca of pectoral rattail (Klomklao et al. 2009b).trypsins (Table 14.2). Moreover, the sequence of all trypsins
starts with IVGG after limited proteolysis of inactive trypsinogen
into the active trypsin (Cao et al. 2000, Klomklao et al. 2006b).
Chymotrypsin is another member of a large family of ser-
ine proteases functioning as a digestive enzyme. Chymotrypsins
have been isolated and characterized from marine species such
as anchovy (Heu et al. 1995), Atlantic cod (Asgeirsson and Bjar-
nason 1991), scallop (Chevalier et al. 1995), Monterey sardine
(Castillo-Yanez et al. 2006), and crucian carp (Yang et al. 2009).
In general, these enzymes are single-polypeptide molecules with
MWs between 25 kDa and 28 kDa. They are most active within
the pH range of 7.5–8.5 and are most stable at around pH 9.0
(Simpson 2000). Chymotrypsins have a much broader speci-
ficity than trypsins. They cleave peptide bonds involving aminoacids with bulky side chains and nonpolar amino acids such as
tyrosine (Tyr), phenylalanine, tryptophan, and leucine (Simpson
2000).Thiol/Cysteine ProteasesThiol or cysteine proteases are a group of endopeptidases that
have cysteine and histidine residues as the essential groups in
their catalytic sites. These enzymes require the thiol (-SH) group
furnished by the active site cysteine residue to be intact, hence,
this group is named “thiol” or “cysteine” proteases (Simpson
2000). The thiol proteases are inhibited by heavy metal ions and
their derivatives, as well as by alkylating agents and oxidizing
agents (Simpson 2000). The first three digits common to thiol
proteases are EC 3.4.22.
Digestive cysteine proteases have been found in the viscera
of fish and aquatic invertebrates. Digestive cysteine proteases
from marine animals are most active at acidic pH and inactive at
alkaline pH. Cathepsin B, cathepsin L, and cathepsin S are com-
mon examples of digestive thiol protease from marine animals
(Simpson 2000). Various researchers have described different
procedures for isolating marine cysteine or thiol proteases from
the digestive glands of marine animals.
Cathepsin B was isolated from a few aquatic animals including
the horse clam (Reid and Rauchert 1976), mussel (Zeef and
Dennison 1988), and carp (Aranishi et al. 1997a). Generally,
cathepsin B from marine animals is a single polypeptide chain
with molecular sizes ranging from 13.6 to 25 kDa. Cathepsins
from different species show maximum activity over a broad pH
range of 3.5–8.0. Cathepsin B is activated by Cl−ions, and
requires sulfhydryl-reducing agents or metal-chelating agents
for activity (Zeef and Dennison 1988).
Cathepsin L from carp hepatopancreas was purified by using
ammonium sulfate precipitation and a series of chromatogra-
phies, in which the enzyme had an affinity toward Concanavalin
A and Cibacron Blue F3GA (Aranishi et al. 1997b). Its homo-
geneity was established by a native-PAGE. Two protein bands
corresponding to MWs of 30 kDa and 24 kDa were found on
SDS-PAGE. The enzyme exhibited a maximal activity against
Z-Phe-Arg-MCA at pH 5.5–6.0 and 50◦C. All tested cysteine
protease inhibitors, TLCK and chymostatin, markedly inhib-
ited its activity, whereas the other serine protease inhibitors and
metalloprotease inhibitors showed no inhibitory effects on the
enzyme (Aranishi et al. 1997b). Recently, Cardenas-Lopez and
Haard (2009) isolated cathepsin L from jumbo squid hepatopan-
creas by a two-step procedure involving ammonium sulfate pre-
cipitation and gel filtration chromatography. The MW of the
enzyme was 24 kDa determined by SDS-PAGE and 23.7 kDa
by mass spectrometry. The activity had an optimum pH of 4.5
and optimum temperature of 55◦C using benzyloxycarbonyl-
Phe-Arg-7-amino-4-trifluoro methyl coumarin as substrate.
Cathepsin S from hepatopancreas of carp (Cyprinus car-
pio) was purified by ammonium sulfate fractionation, fol-
lowed by SP-Sepharose, Sephacryl S-200, and Q-Sepharose,
respectively, (Pangkey et al. 2000). The MW of purified pro-
tease was 37 kDa estimated by SDS-PAGE. It hydrolyzed
Z-Phe-Arg-MCA but not Z-Arg-MCA. The optimal pH and