AMERICAN JOURNAL OF BIOTECHNOLOGY AND MOLECULAR SCIENCES
ISSN Print: 2159-3698, ISSN Online: 2159-3701, doi:10.5251/ajbms.2013.3.2.29.32
© 2013, ScienceHuβ, http://www.scihub.org/AJBMS
Comparative Biochemical Characterization of alkaline phosphatase from
species of shrimps (Macrobrachium vollenhovenii and Penaeus notialis)
1
Raimi O.G., 2Hammed A.M., 1Adeola S.A., 2Jimoh, A.A. and 1Adeyemi, O.C.
Department of Biochemistry; 2Department of Fisheries Lagos State University, Ojo, Lagos,
Nigeria. PO Box 0001 LASU Post Office, Lagos
1
ABSTRACT
Alkaline phosphatase (ALP) EC 3.1.3.1) was isolated and partially characterized from two species
of decapod crustaceans. Highest activity of ALP enzyme was present in the head for both
Macrobachium vollenhovenii and Penaeus notialis 4881.52 ± 0.011 U/L and 4893.48 ± 0.006 U/L
respectively. ALP is more concentrated in the head of notialis sample compared to that of
vollenhovenii. For Macrobachium vollenhovenii the whole animal contained the highest
concentration of protein 12.29 ± 0.078 mg/ml followed by the head 11.452 ± 0.403 mg/ml and
also for Penaeus notialis sample, the whole animal contained the highest concentration of protein
17.01 ± 0.042 mg/ml followed by the head 12.95 ± 0.353 mg/ml. The K m and Vmax of the head
alkaline phosphatase were estimated to be 5.9mM and 5.1mM/min/ml respectively for the
Macrobrachium sample while the Km and Vmax of the head alkaline phosphatase were estimated
2+
to be 4.5 mM and 5.4 mM/min/ml for the Penaeus sample. Mg gradually increases the activity of
ALP in both species. EDTA gradually decreases the activity of the enzyme at concentration
above 10 mmol in both species. The ALP enzyme has pH optimum of 10 in both species and an
o
optimum temperature of 45 C.
Keywords: Shrimps, characterization, biochemical, alkaline phosphatase, comparative
INTRODUCTION
Alkaline phosphatase (ALP, EC 3.1.3.1) is a
hydrolase enzyme responsible for removing
phosphate
groups
from
many
types
of
phosphorylated molecules including nucleotide,
proteins and alkaloids (Martins et al., 2001). The
process of removing the phosphate group is called
dephosphorylation. As the name suggest, ALPs are
most effective in an alkaline environment. It is
sometimes
used
synonymously
as
basic
phosphatase (Tamas et al., 2002). The isolation and
characterization of ALP have been carried out from a
number of sources (Negrao et al., 2003, Raimi et al.,
2011; Raimi et al., 2012). ALPs are glycoproteins and
regarding their glycan moieties differ not only in the
way oligosaccharide chains are linked to the protein
but also the type of sugar chain present in the
molecule (Tamas et al., 2002).
ALP binds to the cellular membrane via a glycosylphosphatidy linositol anchor linkage and can be
differently released into intestinal lumen, plasma or
bile depending on both physiological and pathological
condition (Hoof et al., 1990).
The specific function of ALP remains unclear but
cellular location of ALP mainly in the plasma
membrane of exchange surface (for example liver,
kidney, bone, intestine and placenta) where
extensive transport take place is suggestive of a
biological role related to transport processes (Tamas
et al., 2002). ALP might also be involved in cellular
migration in intestinal lipid transport, in bone
mineralization and in regulation of a low conductance
chloride channel.
Macrobrachium vollenhovenii (African river prawn)
and Penaeus notialis (southern pink shrimp) are both
decapod crustaceans, which belong to the families
Palaemonidae and Penaeidae respectively. Both are
found in commercial quantity along the coast of West
Africa, and have been described by various authors
as the subject of important fisheries in West Africa
(Jimoh et al., 2012, Bello-Olusoji et al., 2006, LawalAre and Owolabi, 2012). The adults of M.
vollenhovenii are found in the freshwater
environment, while those of P. notialis inhabit the
marine coastal water. Generally, prawns/shrimps are
important food items both at the artisanal and
commercial trawl levels, hence studies have been
conducted on the acid and alkaline phosphatases
(ACP and ALP) in these decapod crustaceans
(Bhavan and Geraldine, 2004); however, there is a
dearth of information on the distribution of ALP in
Am. J. Biotechnol. Mol. Sci., 2013, 3(2): 29-32
Macrobrachium vollenhovenii and Penaeus notialis.
Effect of EDTA This was also carried out as
described for metal but a concentration range of 5.0
to 30.0 mmol was used differently.
This work is aimed at comparing the distribution and
characteristics of ALP from two species of decapod
crustaceans.
Optimum pH The reaction tube contain 2.0 ml of
PNPP and 0.1ml of tissues homogenates in a TrisHCl buffer of pH range between 7.0 and 11.0 as
described for enzyme activity and the absorbance
was read at 405nm against reagent blank.
MATERIALS AND METHODS
Tissue extraction Two different species of decapod
crustaceans (Macrobrachium vollenhovenii and
Penaeus notialis) with an average weight of 44.39
and 41.41g were used. The different parts of the
prawns were removed (head, skin, flesh, muscle, and
the whole animal).
Optimum temperature 0.1ml of sample homogenate
was added to 1ml of p-nitrophenol phosphate and the
reaction mixture was incubated for 10 min at different
o
o
o
o
o
o
temperature 20 C, 25 C, 30 C, 35 C, 40 C, and 50 C
after which 5.0 ml of 0.02M NaOH was added to
terminate the reaction. The absorbance was taken at
405nm against reagent blank.
The tissues were weighed and then homogenized
using mortal and pestle differently in ice cold 50mM
Tris-HCl pH 7.5 and the homogenate were then
centrifuged at 5,000rpm for 10min. The supernatant
was collected and kept in the freezer until the time of
assay.
RESULTS AND DISCUSSION
Alkaline phosphatase was isolated from different
tissues of decapod crustaceans (Macrobrachium
vollenhovenii and Penaeus notialis). In both species
of prawn the whole prawn contains the highest
concentration of protein 12.29 ± 0.078 mg/ml and
17.01 ± 0.042 mg/ml for Macrobrachium and
Penaeus respectively followed by the head (Table 1).
The highest activity of ALP was observed in the
head for both Macrobrachum vollenhovenii and
Penaeus notialis 4881.52 ± 0.011 U/L and 4893.48 ±
0.006 U/L respectively (Table 2). This might be as a
result of the fact that the digestive system of this
invertebrate is contained in the head. At a constant
enzyme concentration, the kinetics of the enzyme
was defined by Michaelis-Menten equation. The Km
and Vmax for the head ALP (HALP) were estimated to
be 5.9 mM and 5.1 mM/min/ml respectively for
Macrobrachium vollenhovenii and 4.5mM and 5.4
mM/min/ml for Penaeus notialis respectively (Figure
1).
Protein determination Protein concentration of
tissue homogenates was determined using Biuret
method (Lyne et al., 1957). 1.0ml of the
homogenates was mixed with 3.0ml of Biuret reagent
which gives blue coloured complex and incubated for
10 min at 37C. Absorbance was taken at 540nm.
Enzyme activity Alkaline phosphatase activity in
tissue homogenates was determined by a modified
method of Bowers and MCcomb 1996. The method
involves the incubation of 0.1ml of diluted
heamolymph with 80mM Tris-HCl buffer (pH 10.5,
0.5mM P-nitrophenylphosphate (PNPP, 10mMol/l) at
37C for 30 minutes. The reaction was terminated by
adding 5.0ml of 2 mM NaOH after which absorbance
was taken at 405nm against reagent blank.
Determination of K m and Vmax: The Km and Vmax of
the tissue homogenate with the highest enzyme
activity was determined. The reaction mixtures
contain 0.1 ml of the tissue homogenate (head), 80
mM Tris-HCl buffer, (pH 10.5) and varying
concentration of p-nitrophenylphosphate (10 mmol/l).
The reaction mixtures were incubated for 10 min at
o
37 C after which 5.0 ml of 0.02 M NaOH was added
and the corresponding absorbance taken at 405 nm.
Table 1: Protein concentration in tissue homogenates
Effect of metal concentrations MgCl2 was used for
this assay. A range of 0.1 to 1.5 mol/l metal
concentrations were added to 0.1 ml of tissue
homogenate in the test tubes and 0.1 ml 80 mM TrisHCl buffer, (pH 10.5) and 2.0 ml of pnitrophenylphosphate was added. Other steps were
as described for enzyme assay.
Tissue
Macrobrachium
vollenhovenii
Penaeus notialis
Whole
12.29 ± 0.078
17.01 ± 0.042
Skin
10.58 ± 0.367
10.40 ± 0.113
Muscle
7.66 ± 0.240
10.10 ± 0.141
Head
11.452 ± 0.403
12.95 ± 0.353
Data represent Mean ± SD (n = 2) concentration expresses
total protein in mg/ml
30
Am. J. Biotechnol. Mol. Sci., 2013, 3(2): 29-32
Table 2: Alkaline phosphatase activity in tissue
homogenates
Macrobrachium
vollenhovenii
Penaeus
notialis
Whole
7420.68 ± 0.208
5533.89 ± 0.843
Skin
4005.68 ± 0.253
4796.88 ± 0.045
Muscle
1451.76 ± 0.113
903.43 ± 0.270
Head
4881.52 ± 0.011
4893.48 ± 0.006
Absorbance (405)
Tissue
inhibited the enzyme at concentration above 10mmol
(Figure 3).
Data represent Mean ± SD (n = 3) activity is expressed in
unit per liter U/L
A
B
Concentration (mmol)
2+
Absorbance (405)
Fig 2: Effect of Mg on alkaline phosphatase activity
(Macrobrachium and Penaeus)
B
Concentration, (mmol)
Fig 1: Reciprocal plot of absorbance against substrate
concentration ((A) Macrobrachium vollenhovenii and
(B) Penaeus notialis)
Figure Fig3: Effect of EDTA on alkaline phosphatase activity (
Macrobrachium and  Penaeus)
ALP is a divalent metalloenzyme that requires metal
2+,
such as Mg
for its activity, absence of these
divalent mental has been seen to lower the ALP
activity in serum and several cellular tissues (Tamas
2+
et al., 2002). Mg ion was observed to slightly
activate HALP activity (Figure 2) while EDTA
ALP in Macrobrachium vollenhovenii and Penaeus
notialis has an optimum pH of 10.0 (Figure 4) and
o
optimum temperature of 45 C (Figure 5).
31
Am. J. Biotechnol. Mol. Sci., 2013, 3(2): 29-32
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o
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CONCLUSION
The results of this study show that alkaline
phosphatase activity is highest in the whole animal
followed by the head of the Macrobrachium
vollenhovenii and Penaeus notialis. The activities of
ALP in prawns was activated by MgCl2 and gradually
inhibited by EDTA.
Tamas L, HuttovA J, Mistrk I, and Kogan G (2002): Effect of
Carbon Chitin-Glucan on the Activity of Some
Hydrolytic Enzymes in Maize plants” Chem. Pap. 56
(5): 326-329.
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