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Brief review on alkaline phosphatase-an overview

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INTERNATIONAL JOURNAL OF MICROBIOLOGY AND BIOINFORMATICS

ISSN: 2277-789X REVIEW ARTICLE

International Journal of Microbiology and Bioinformatics - Vol.2, Issue, 1, pp.1- 4, January, 2012

BRIEF REVIEW ON ALKALINE PHOSPHATASES-AN OVERVIEW

*Kirti Rani, Sanchi Datt and Rachita Rana
Amity Institute of Biotechnology, Amity University, Noida, 201303 (U.P.), India

ART IC LE INF O ABSTR ACT

Article History:
Received, 10th ,Novemer,2011
Received in revised form,
10th,December,2011
Accepted, 30th,Dcember,2011
Published online, 30th,January ,2012
Key words:
Alkaline phosphatise (ALP, ALKP) (EC 3.1.3.1) is a hydrolase enzyme responsible
for removing phosphate groups from many types of molecules, including
nucleotides, proteins, and alkaloids. The process of removing the phosphate group
is called dephosphorylation. As the name suggests, alkaline phosphatases are most
effective in an alkaline environment. This enzyme serves many important roles in
human beings and also in various industries and therefore has a wide range of
applications. This review aims to study this enzyme in detail laying special emphasis
on it’s mechanism, sources and industrial applications.

Calf intestinal Alkaline phosphatase,

shrimp alkaline phosphatase placental
© Copy Right, IJMB, 2011, Bret Research Journals. All rights reserved.
alkaline phosphatise

INTRODUCTION for other Bacillus species.However, the extracellular

Alkaline Phosphatase (EC 3.1.3.1) enzyme hydrolyzes the
phosphomonoesters from number of organic molecules
like ribonucleotides, deoxy-ribonucleotides, proteins,
alkaloids, phosphate esters and anhydrides of phosphoric
acid . Alkaline Phosphatase is a metallodependent enzyme
which shows its catalytic activity optima at alkaline pH.
Alkaline Phosphatase can be isolated from variety of
microorganisms including E.Coli, Pseudomonas,
Aerobactor and Bacillus species. In all bacteria, ALPase
found in the periplasmic membrane which is external to
the cell membrane of bacteria. Usually the ALPase is
produced at commercial level from E.coli or Calf intestine.
production of ALPase have been studied in Micrococcus
sodonensis, Pseudomonas spp, Alkalophilic bacterium,
Haloarcula marismortui and Arthobacter.
Intracellular production of ALPase is quite tedius and
expensive process in comparison to extracellular. This
statement is supported by the study of Hulett et al, (1986)
which explained that extracellular ALPase gave higher
specific activity than intracellular ALPase is because of
short and simple steps of purification (Mahesh et al,
2010).
Mechanism

Studies have been carried out for production,
purification and characterization of ALPase from genus
Bacillus. Like B.subtilis, B.subtilis JH646MS strain, they
purified the two ALPase producing proteins. B.cereus,
B.subtilis KIBGE-HAS strain newly synthesized .All these
studies based on intracellular production of ALPase.
Alkaline phosphatase removes 5' phosphate groups from
DNA and RNA. It will also remove phosphates from
nucleotides and proteins. These enzymes are most active at
alkaline pH.

Very little work has been done with respect to

extracellular production of ALPase in genus Bacillus.The
extracellular production of ALPase in Bacillus
licheniformis was investigated which shows that it
synthesizes 10 times more ALPase activity than is reported

*Corresponding author: +91 9990329492

Email: kirtisharma2k@rediffmail.com, krsharma@amity.edu
 International Journal of Microbiology and Bioinformatics - Vol.2, Issue, 1, pp.1- 4, January, 2012
Sources
Bacterial alkaline phosphatase (BAP) is the most active
of the enzymes, but also the most difficult to destroy at the
end of the dephosphorylation reaction.
In bacteria, alkaline phosphatase is located in the
periplasmic space, external to the cell membrane. Since
this space is much more subject to environmental variation
than the actual interior of the cell, bacterial alkaline
phosphatise is comparatively resistant to
inactivation,denaturation, and degradation, and also has a
higher rate of activity. Although the actual purpose of the
enzyme is still not fully understood, the simple hypothesis,
that it is a means for the bacteria to generate free
phosphate groups for uptake and use, is supported by the
fact that alkaline phosphatase is usually produced by the
bacteria only during phosphate starvation and not when
phosphate is plentiful.
However, other possibilities exist; for instance, the
presence of phosphate groups usually prevents organic
molecules from passing through the membrane, therefore
dephosphorylating they may be important for bacterial
uptake of organic compounds in the wild . Some
complexities of bacterial regulation and metabolism
suggest that other, more subtle, purposes for the enzyme
may also play a role for the cell. In thelaboratory,
however, mutant Escherichia coli lacking alkaline
phosphatase survive quite well, as do mutants unable to
shut off alkaline phosphatase production.
The optimal pH for the activity of the E. coli enzyme is
8.0 while the bovine enzyme optimum pH is slightly
higher at 8.5.
Characteristics of Alkaline Phosphatase from E. coli
 Molecular weight: 80,000
 Composition: E. coli alkaline phosphatase is a
dimeric, zinc and magnesium containing protein
.Though the subunits are believed to be coded by
the same gene, they develop molecular
heterogeneity after translation.
 There are two active sites, only one is functional at
a time. Two Zn2+ are needed for activity. A stable
apoenzyme can be reactivated with Zn2+ , Mn, Co,
Ni, Cu, Cd and Hg have been substituted for Zn but
only Co restores significant activity .Magnesium
does not activate the apoenzyme but enhances
activity of the enzyme containing 2g zinc
 Optimum pH: 8.0
 Isoelectric point: pH 4.5
 Activity: E. coli enzyme binds phosphate tightly
over a wide range of pH forming complexes that
may be intermediate in the hydrolytic action.
 Inibibitors: The enzyme is inhibited by chelating
agents and inorganic phosphates.
 Stability: Stable at 2 - 8°C for at least 6 - 12
months.[4]
Calf intestinal alkaline phosphatase (CIP) is purified
from bovine intestine. This is phosphatase most widely
used in molecular biology labs because, although less
active than BAP, it can be effectively destroyed by
protease digestion or heat (75C for 10 minutes in the
presence of 5 mM EDTA).
Calf Intestinal Alkaline Phosphatase (CIAP/CIP) is an
enzyme that catalyzes the 5' phosphate group's removal,
Dephosphorylation, from DNA. This enzyme is frequently
used in DNA sub-cloning, because DNA fragments that
lack the 5' phosphoryl termini cannot self-ligate. This is
used to prevent recircularization and religation of
linearized cloning vehicle DNA by removing phosphate
groups from both 5´-termini. Preventing self-ligation is
important both in improving the yield of properly ligated
product and in reducing the background of improperly
self-ligated contaminant.
Characteristics of Alkaline Phosphatase from Calf
Intestine
 Molecular Weight: 140,000.
 Optimum pH: 9.8.
 Isoelectric Point: 5.7.
 Activators: Zn, Mg++, Ca++.
Shrimp alkaline phosphatase (SAP) is derived from a
cold-water shrimp ,Pandalus borealis.It is a species of
shrimp found in cold parts of the Atlantic and Pacific
Oceans.and is promoted for being readily destroyed by
heat (65C for 15 minutes).
Placental alkaline phosphatase (PLAP) is an allosteric
enzyme that in humans is encoded by the ALPP gene.
Alkaline phosphatase, placental type is a membrane bound
glycosylated dimeric enzyme, also referred to as the heat
stable form that is expressed primarily in the placenta
although it is closely related to the intestinal form of the
enzyme as well as to the placental-like form.
General Properties of Alkaline Phosphatases: The
alkaline phosphates are salts of the purified phosphoric
acid; they are more or less polymerised and are acidic,
neutral or basic.
Depending on the number of P atoms, the usual name will
change as follow
 1 P atom: orthophosphates
 2 P atoms: pyrophosphates or diphosphates
 3 P atoms: tripolyphosphates
 4 P atoms: polyphosphates
The two main characteristics of the phosphates are their
chain length and their pH. These characteristics define
their four properties: buffer agent, sequestering power,
dispersing power and water holding capability. These four
properties will explain all uses of phosphates in food and
technical applications.
Buffer agent: Alkaline phosphates are able to buffer a
solution, which is to impose their pH to the solution. Short
chain phosphates such as orthophosphates and
pyrophosphates are the most efficient.
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 International Journal of Microbiology and Bioinformatics - Vol.2, Issue, 1, pp.1- 4, January, 2012
Sequestering power: Alkaline phosphates are able to
sequestrate polyvalent cations, this will isolate them from
the matrix. In general, the bigger the cations, the longer the
phosphate chain is. For example: Fe 2+ and Cu 2+ are
complex by pyrophosphates / Ca 2+ and Mg 2+ are
complex by tripolyphosphates or polyphosphates.
Dispersing power: Alkaline phosphates act as
polyelectrolytes are able to change the ionic charges
distributions. This effect increases with the phosphates
chain length. Polyphosphates are thus the most efficient.
Water holding capability: Alkaline phosphates are able
to increase the water holding capability of proteins and
find its application mainly in food such as meat processing
and processed cheese. This property results from the 3
previous ones. A maximum efficiency is obtained with
intermediate chain length phosphates.
Uses
There are two primary uses for alkaline phosphatase in
DNA manipulations:
 Removing 5' phosphates from plasmid and
bacteriophage vectors that have been cut with a
restriction enzyme. In subsequent ligation reactions,
this treatment prevents self-ligation of the vector and
thereby greatly facilitates ligation of other DNA
fragments into the vector (e.g. subcloning).
 Removing 5' phosphates from fragments of DNA
prior to labeling with radioactive phosphate.
Polynucleotide kinase is much more effective in
phosphorylating DNA if the 5' phosphate has
previously been removed.
It is usually recommended that dephosphorylation of
DNAs with blunt or 5'-recessed ends be conducted using a
higher concentration alkaline phosphatase or at higher
temperatures than for DNAs with 5' overhangs.
Alkaline phosphatase has become a useful tool in
molecular biology laboratories, since DNA normally
possesses phosphate groups on the 5' end. Removing these
phosphates prevents the DNA from ligating (the 5' end
attaching to the 3' end), thereby keeping DNA molecules
linear until the next step of the process for which they are
being prepared; also, removal of the phosphate groups
allows radiolabeling (replacement by radioactive
phosphate groups) in order to measure the presence of the
labeled DNA through further steps in the process or
experiment. For these purposes, the alkaline phosphatase
from shrimp is the most useful, as it is the easiest to
inactivate once it has done its job. Another important use
of alkaline phosphatase is as a label for enzyme
immunoassays.
Industrial Applications
Alkaline phosphatase have also been found in variety of
micro-organism including E.coli (Torriani, 1968)
Pseudomonas (Friedberg and Avigad, 1967), Aerobacter
(Wolfenden and Spence, 1967) and Bacillus species
(Takeda and Tsugita, 1967). In all these bacteria alkaline
phosphatase found in periplasmic space, external to the
cell membrane (Hulett-cowling, F.M. and Campbell, L.,
1971) and induces under low phosphate concentration that
indicates that bacterial alkaline phosphatase is also
involves in phosphate metabolism (Mc. Comb et al, 1979).
Alkaline phosphatase has become an important tool in
molecular cloning and DNA sequencing. It also used as an
important part of diagnostic kits component of different
ELISA base kits (Reid and Wilson, 1971).Bacterial
alkaline phosphatase is used more commonly in research
because it is comparatively resistant to inactivation,
denaturation, degradation and higher rate of activity (3).
Due to its so many industrial uses, it is necessary to purify
it on large scale for commercial and research purpose.
The main food applications are meat and seafood
processing, baking and processed cheese, but others such
as cereals, French fries, fruits and vegetables, beverages,
noodles and so on also may need the use of phosphates.
On the technical side, the main applications are the
detergent products, the water treatment and the metal
treatment. As for the food, many other applications require
phosphates such as ceramics, bone china, paper and paints.
In meat products, phosphates salts interact in a unique
way to bind water with proteins and improve the
tenderness in meats. Treated products will maintain their
juicy appearance as well as their natural nutritional
properties texture and colour. In fish and seafood products,
phosphates salts allow the retention of the natural juices of
frozen fish fillets, prawns, shrimps, scallops and other
seafood. Phosphates also help prevent the build-up of
struvite crystals in tinned tuna and crabmeat.
In processed cheese, phosphates are crucially important
in the production of processed cheese. These products
ensure a homogeneous and uniform melt of raw cheese
and product stability.
For bakery products, acid phosphates are well known
leavening agents. Their reaction with sodium bicarbonate
generates a controlled gas release that contributes to the
volume, appearance and taste of types of cakes and pastry.
(Hourant et al., 2004]
In detergent products, phosphates are essential
components of I&I and household detergent. The
formulations using phosphates have clear advantages
compared to alternative formulations. The main properties
of this “builder” are: the sequestering power (softener), the
dispersing power, emulsifier and buffer agent, the synergy
with tensio-actives and the alkaline content. The sodium
tripolyphosphate is proved to be the most efficient builder.
Phosphates are used for the metal surface phosphatizing.
This reduces the metal corrosion risks, to electrically
isolate them and to improve the painting of the metals. On
the other hand, phosphates are also used for the metal
cleaning and the manufacturing of magnetic metal sheets.
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 International Journal of Microbiology and Bioinformatics - Vol.2, Issue, 1, pp.1- 4, January, 2012

Water treatment is the last major application.The use of 4.
Phosphates also prevents scale formation, controls the
black and red water phenomenon and reduces pipe
corrosion risks.Moreover, some phosphates are used in the
biological purification of industrial and used water. 5.
CONCLUSION
6.
Phosphates salts are very important helpers in everyday
life. They are used in food processing but also in many
technical applications. Phosphorus is in all living
organisms and we could not live without. A minimum
intake per day is required for the proper nutrition of plants,
animals and human beings. They participate to the
biological energy transfer through a natural phosphate 7.
called adenosine triphosphate (ATP). On the other hand,
many industries would be under serious troubles and
would have a major challenge should they reformulate 8.
without using phosphate salts and for sure some currently
available products would completely disappear from the
market or would become anecdotal.
9.
References
1. Fermley, N.H., 1971. The Enzyme. 3rd edition, 10.
vol.4: 417-474.
2. Friedberg, I. and Avigad, G., 1967. Some
properties of alkaline phosphatase of Ps.
flourescens. Eur. J. Biochem. 1: 193-198. 11.
3. Hourant P., 2004. General Properties of the
Alkaline Phosphates: Major Food and 12.
Technical Applications. Phosphorus Research
Bulleti.15, 85-94. 13.
Hulett-cowling, F.M. and Campbell, L., 1971.
Purification and properties of an alkaline
phosphate of B. licheniformis. J. Biochem. 10:
1364-1371.
Mc. Comb, R.B., Bowers, G.N. and Posen, S.,
1979. Alkaline phosphatase. Plenum. Press, New
York.
Mahesh.M, Guleria Neha, Rajesh.T.S,
Somashekhar.R, Puttaiah.E.T, Jnana Sahyadri.,
2010. Isolation And Characterization of
Extracellular Thermostable Alkaline Phosphatase
Enzyme From Bacillus Spp. International Journal
of apllied Biology and Pharmaceutical
Technology. 1 (1), 21-33.
Reid, T. and Wilson, I., 1971. E.coli Alkaline
Phosphatase, The Enzyme, P. Boyer, Academic
press, New York. 373, 3rd edition, vol.4.
Takeda, K. and Tsugita, A., 1967.
Phosphoesterase of B. subtilis. II crystallization
and properties of alkaline phosphatase. J.
Biochem. 61: 231-241.
Torriani, A., 1968. Alkaline phosphatase subunit
and their dimerization in vivo, J. Bacteriol. 96:
1200-1207.
Wolfenden, R. and Spence, G., 1967. Depression
of phosphomonoesterase and phosphodiesterase
activities in Aerobacter aerogenes. J. Biochim.
Biophys. Acta, 46: 296-298.
http://en.wikipedia.org/wiki/Alkaline_phosphatas
e.
http://www.vivo.colostate.edu/hbooks/genetics/b
otech/enzymes/phosphatase.html
http://www.worthingtonbiochem.com/bap/default
.html

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