Phosphatases are enzymes that catalyze the hydrolysis of phosphomonoesters, removing a phosphate group from the substrate. This reaction is not reversible and is opposite to the activity of kinases and phosphotransferases that add phosphate groups. There are two main types of phosphatases - acid phosphatases that are most active at pH 3-6, and alkaline phosphatases that are most active at pH around 10. Alkaline phosphatases are found in many human tissues and are important for removing phosphate groups from phosphorylated proteins. Calf intestinal alkaline phosphatase is commonly used in molecular cloning to prevent self-ligation of DNA vectors.

1.  Phosphatases catalyze the hydrolysis of a
phosphomonoester, removing a phosphatemoiety from
the substrate. Water is split in the reaction, with the -OH
group attaching to the phosphate ion, and the H+
protonating the hydroxyl group of the other product
 This reaction is not reversible
 This shows totally opposite activity from enzyme like
kinases & phaspharylase that add a phosphate grope to
their substrate.
PHOSPHATASE

2. REACTION

3. TYPES OF PHASPHATASE
On the basis of their activity there are two types of
phosphatase
 ACID PHOSPHATASE
 ALKALINE PHOSPHATASE
 In both forms the alkaline phosphate are most
common special class of phophatase that remove a
phosphate group from protein called phasphoprotein
phosphatase

4. ACID PHOSPHATASE
It shows is optimal activity at Ph between 3 to 6 range
Acid phosphatase is a ubiquitous lysosomal enzyme
that hydrolyses organic phosphates at an acid pH.
Although the postpuberteral prostatic epithelial cell
contains a uniquely high concentration of acid
phosphatase, cellular components of bone, spleen,
kidney, liver, intestine, and blood also contain this
enzyme.

5. ALKALINE PHOSPHATASE
Alkaline phosphatase or basic phosphatase is a
homodimeric protein enzyme of 86 kilodaltons. Each
monomer contains five cysteine residues, two zinc
atoms, and one magnesium atom crucial to its catalytic
function, and it is optimally active at alkaline pH
environments.
They shows their optimal activity at pH of about 10
In humans, alkaline phosphatase is present in all
tissues throughout the entire body, but is particularly
concentrated in the liver, bile duct, kidney, bone,
intestinal mucosa and placenta.

6. Calf-intestinal alkaline
phosphatase (CIAP/CIP)
Calf-intestinal alkaline phosphatase (CIAP/CIP) is a type
of alkaline phosphatase that catalyzes the removal of
phosphate groups from the 5' end of DNA strands. This
enzyme is frequently used in DNA sub-cloning, as DNA
fragments that lack the 5' phosphate groups
cannot ligate. This prevents recircularization of
linearized vector DNA and improves the yield of vector
containing the appropriate insert.

7. Calf-intestinal alkaline
phosphatase (CIAP/CIP)

8. Transformation results in the genetic alteration of the
recipient cell. Exogenous DNA is taken up into the
recipient cell from its surroundings through the cell
membrane (s). Transformation occurs naturally in
some species of bacteria, but it can also be affected by
artificial means in other cells
GENETIC TRANSFORMATION

9. Transformation is the process by which an organism acquires
exogenous DNA. Transformation can occur in two ways:
natural transformation and artificial transformation. Natural
transformation describes the uptake and incorporation of
naked DNA from the cell’s natural environment. Artificial
transformation encompasses a wide array of methods for
inducing uptake of exogenous DNA. In cloning protocols,
artificial transformation is used to introduce recombinant DNA
into host bacteria (E. coli). The most common method of
artificial transformation of bacteria involves use of divalent
cations (e.g., calcium chloride) to increase the permeability of
the bacterium’s membrane, making them chemically
competent, thereby increasing the likelihood of DNA
acquisition. Another artificial method of transformation is
electroporation, in which cells are shocked with an electric
current, to create holes in the bacterial membrane. With a
newly-compromised cell membrane, the transforming DNA is
free to pass into the cytosol of the bacterium. Regardless of

10. ELECTROPORATION
Electroporation, or electropermeabilization, is
a microbiology technique in which an electrical field is
applied to cells in order to increase the permeability of
the cell membrane allowing chemicals, drugs, or DNA to
be introduced into the cell (also called electrotransfer)

11. HEAT SHOCK METHOD
Transformation of plasmid DNA into E. coli using the
heat shock method is a basic technique of molecular
biology. It consists of inserting a foreign plasmid or
ligation product into bacteria.
A short incubation in ice, a mixture of chemically
competent bacteria and DNA is placed at 42°C for 45
seconds (heat shock) and then placed back in ice.