BLBS102-c14 BLBS102-Simpson March 21, 2012 13:17 Trim: 276mm X 219mm Printer Name: Yet to Come
266 Part 2: Biotechnology and Enzymology45,000-24,000-18,400-14,300-M1 2Figure 14.2.Protein pattern of purified pepsins A and B from
pectoral rattail stomach determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. M, MW standard; lane 1,
pepsin A; lane 2, pepsin B (Klomklao et al. 2007a).as estimated by SDS-PAGE (Fig. 14.2) and gel filtration on
Sephacryl S-200.
Pepsins and pepsin-like enzymes can be extracted from the
stomach of marine animals such as capelin (Mallotus villo-
sus) (Gildberg and Raa 1983), polar cod (Boreogadus saida)
(Arunchalam and Haard 1985) sardine (Sardinops melanostica)
(Noda and Murakami 1981), Monterey sardine (Sardinops sagax
caerulea) (Castillo-Yanez et al. 2004), sea bream (Sparus latus
Houttuyn) (Zhou et al. 2007), pectoral rattail (Coryphaenoides
pectoralis) (Klomklao et al. 2007a), smooth hound (Mustelus
mustelus) (Bougatef et al. 2008), and European eel (Anguilla
anguilla) (Wu et al. 2009).
Several methods have been described in the literature for the
isolation and purification of pepsins from marine animals. Gild-
berg et al. (1990) purified pepsin from stomach of Atlantic cod
(Gadus morha) by ammonium sulfate fractionation (20–70%
saturation), followed by ion-exchange chromatography using S-
Sepharose column. Klomklao et al. (2007a) purified two pepsins,
A and B, from the stomach of pectoral rattail by acidification,
ammonium sulfate precipitation (30–70% saturation), followed
by a series of column chromatographies including Sephacryl
S-200, diethylaminoethyl (DEAE)–cellulose and Sephadex G-- Pepsin from the stomach of smooth hound was purified by
20–70% ammonium sulfate precipitation, Sephadex G-100 gel
filtration, and DEAE–cellulose anion exchange chromatogra-
phy (Bougatef et al. 2008). Recently, Wu et al. (2009) isolated
pepsins from the stomach of freshwater fish European eel by am-
monium sulfate fractionation, column chromatography on anion
exchange (DEAE–Sephacel) and gel filtration (Sephacryl S-200
and Superdex G-75).
Pepsin activity is very dependent on pH values, temperatures,
and the type of substrate. Hemoglobin is the substrate most
commonly used for determination of pepsin activity (De-Vecchi
and Coppes 1996, Klomklao et al. 2004). Pepsin from polar
cod stomach exhibited a maximal activity against hemoglobin
at pH 2.0 and 37◦C (Arunchalam and Haard 1985). Gildberg
et al. (1990) reported that the optimal pH of Atlantic cod pepsin
for hemoglobin hydrolysis was 3.0. Fish pepsins were shown
to hydrolyze hemoglobin much faster than casein (Gildberg
and Raa 1983). Most fish species contain two or three ma-
jor pepsins with an optimum hemoglobin digestion at pH be-
tween 2 and 4 (Gildberg and Raa 1983). Gildberg et al. (1990)
found that the affinity of cod pepsin, especially pepsin I toward
hemoglobin, was lower at pH 2 than at pH 3.5. Klomklao et al.
(2007a) found that pepsins A and B from pectoral rattail stom-
ach had maximal activity at pH 3.0 and 3.5, respectively, and
had the same optimal temperature at 45◦C using hemoglobin
as the substrate. Pepsins I, II, and III purified from the stom-
ach of European eel showed maximal activity at pH 3.5, 2.5,
and 2.5, respectively, with hemoglobin as substrate (Wu et al.
2009). Bougatef et al. (2008) found that the optimal pH and
temperature for the pepsin activity of smooth hound stomach
were pH 2.0 and 40◦C, respectively, using hemoglobin as a
substrate.
Pepsins from marine species are quite stable from pH 2 to
about 6, but rapidly lose activity at pH 6 and above due to the
denaturation (Simpson 2000). Pepsin from sardine stomach was
stable between pH 2 and 6 and showed drastic loss of activity at
pH 7.0 (Noda and Murakami 1981). Castillo-Yanez et al. (2004)
found that Monterey sardine acidic pepsin-like enzymes were
stable at pH ranging from 3.0 to 6.0. Smooth hound pepsin was
stable within the pH range of 1.0–4.0 (Bougatef et al. 2008).
Klomklao et al. (2007a) also reported that both pepsins A and B
from the stomach of pectoral rattail were stable in the pH range
of 2.0–6.0. Pepsin from the stomach of albacore tuna was stable
in the pH range of 2–5 (Nalinanon et al. 2010).
Chymosins (EC 3.4.23.4), commonly known as rennins, have
also been described as acid proteases with some characteristics
distinct from other acid proteases (Shahidi and Kamil 2001). For
example, these enzymes are most active and stable around pH 7.0
unlike other acid proteases. They also have relatively narrower
substrate specificity, compared with other acid proteases such
as pepsin (Simpson 2000). Digestive proteases with chymosin-
like activity were purified as zymogens from the gastric mucosa
of young and adult seals (Pagophilus groenlandicus) (Sham-
suzzaman and Haard 1984) by a series of chromatographies
including DEAE–Sephadex A-50, Sephadex G-100, and Z-D-
Phe-T-Sepharose gel. The enzyme had optimal pH of 2.2–3.5 for
hemoglobin hydrolysis. The chymosins from marine animals did
not hydrolyze the specific synthetic substrate for pepsin (i.e.,
N-acetyl-l-phenylalanine diiodotyrosine) and were also more
susceptible to inactivation by urea (Simpson 2000).