ENZYMES ................( Important tool in rDNA technology )

 What is enzyme .................? 

Enzymes are proteins that act as biological catalysts. Catalysts accelerate chemical reactions.

 The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products.

              Fig. 1. Enzyme. 

 Since enzymes are proteins, they are composed of monomers known as amino acids.

Type of enzymes :- 

There are main 5 categories of enzymes - 

1) lysing enzymes 

2) Cleaning enzymes 

3) Synthesizing enzymes 

4) Joining enzymes or sealing enzymes 

5) Alkaline phosphatase

In recombinant DNA technology enzymes plays very important role. 

                     Fig. 2. Enzymes 

A) Lysing enzymes :- 

What does lysing mean?

To lyse is to break apart a larger particle into smaller pieces. Lysis, or the process of lysing, can occur both inside and outside of the cell. 

 This causes destruction of the bacteria cell wall and results in the bacteria's death. 

Theses enzymes are used to open up the cell to get DNA for genetic experiment. 

Lysozyme is used to dissolve the bacterial cell wall in plant cell, cell wall is made up of cellulose and in fungi, it is made up of chitin. 

So the lysing enzymes are used to dissolve cellulose and chitin. 

B) Cleaving enzymes :- 

( Cleave - cut) 

A protein produced by bacteria that cleaves DNA at specific sites along the molecule.

 In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms.

These are used to break DNA molecule. They cut the internal phosphodiester bond of specific site. 

These are of three types :- 

1) Exonuclease :- 

They break or cut the DNA at 5' or 3' ends of DNA molecule. They remove nucleotide from terminal end of DNA in one strand of double helix. 

2) endonuclease :- 

They cut the DNA at specific site within DNA. These enzyme do not cleave the ends and involve only one strand of DNA  duplex .

( They may cut both strand DNA or sometime RNA also ).

3) Restriction endonuclease :-

They can break DNA helix at specific point in such a way that a short single stranded molecule with free end is formed .

These ends are called sticky ends .The can join similar complementary ends of DNA fragment from other sources .

The enzyme have the capacity to recognize specific sequences of DNA called restriction site .

The restriction site or recognition sequences are different and specific for different recognition endonuclease .

C) Synthesizing enzyme :- 

These enzyme are used for the synthesis of DNA strand on suitable template . They has two type -

a) DNA polymerase :- These enzyme are used to synthesize DNA strand on DNA template . It polymerase the DNA synthesis on DNA template .

                          ( A. Kornberg and coworkers discovered DNA polymerase in E.coli  in 1956.)

b) Reverse transcriptase :- These enzymes help to synthesis DNA or cDNA by using mRNA as template . These process called reverse transcription or teminism.

                           ( Temin and baltimore discovered this enzyme in 1970 .) 

D) Joining enzyme or sealing enzymes :- 

They help in joining the fragment by forming phosphodiester bond between them. It is called as ligation or splicing .

 Thus they help in sealing gaps in DNA fragment . Therefore they are also called as molecular glue or genetic gum .

T4 ligase is used most often in rDNA technology .( in 1969 Hargovind Khorana discovered DNA ligase in T4 bacteriophage .) 

E)  Alkaline phosphatases :- 

It is and enzyme needed to cut of phosphate group from 5' ends of double stranded or single stranded DNA or RNA . It prevent recircularisation of vector.

( Ligation i.e. joining of DNA fragment absolutely requires the presence of 5' phosphate group at DNA site to be ligated . If this phosphate is removed , the DNA cannot be ligated.)