Biotechnological Applications of Enzymes - New Age International

CHAPTER 

5 

5.1 ENZYMES IN FOOD PROCESSING 

Biotechnological Applications 

of Enzymes 

(A) Introduction 

One knows that, enzymes catalyze chemical changes with a speed and precision that turn a 

laboratory worker green with envy. One of the really exciting practical research aim today is 

the application of enzymes to industries such as food processing. In one sense one has been 

doing this at least since the start of recorded history without understanding the biochemical 

basis. Conversion of carbohydrates to alcohol by fermentation certainly takes advantage of 

yeast enzymes. In this case, however, the organism providing the enzyme remains intact and 

alive. The yeast cells are providing a life support system for the enzymes of fermentation. 

However, what is really needed is individual specific enzymes to do just what is wanted and 

nothing more. 

(B) Use of Some Specific Enzymes in Manufacture of Food Products 

(I) Cheese making 

The first step in cheese production is the precipitation (coagulation) of casein, the chief milk 

protein. This is done with the enzyme chymosin (rennin) obtained either from calf stomach or 

more recently from a microorganism. Chymosin is an aspartic acid protease which causes the 

coagulation of milk, a process which involves cleavage of a single peptide bond in k-casein 

between Phe105 and Met 106. This process releases the acidic C-terminal peptide. The high 

specificity of chymosin however, does not entirely depend on the recognition of residues 105 

and 106 ; all the residues from 98 to 111 appear to be involved in the recognition process. The 

release of the C-terminal peptide is followed by Ca2+ -induced aggregation of the modified micelles 

to form a gel. Significantly chymosin cleaves the Phe105–Met106 peptide bond in k-casein 

about 100 times faster than any other peptide bonds in it. 

Other proteolytic enzymes such as trypsin also bring about the clotting of milk, but 

suffer from the disadvantage of further degrading casein which leads to undesirable flavours. 

The flavour of foods is not dependent on proteins. The peptides and amino acids are largely 

responsible for sweet, sour, bitter and salty flavours. Peptides having chain lengths of 3–15 

amino-acid residues and rich in hydrophobic amino acids are the major components which 

give bitter taste. In the search for a substitute for chymosin, enzymes that cause clotting but 

only limited proteolysis so as to preserve a desirable flavour are desirable. This is particulary 

important since now there is a reduced availability of chymosin which is obtained from calf 

stomachs. As said above the fungal aspartate proteases are now largely substituting for 

chymosin. However, these donot display a more desireable cloting/proteolysis ratio like chymosin 

obtained from calf stomachs. 

189

190 Bioorganic, Bioinorganic and Supramolecular Chemistry 

The chymosin is now made by recombinant DNA technology whereby the chymosin 

gene has been cloned in systems like Aspergillus oryzae and is in use since 1994 

for the manufacture of cheese. 

(II) Meat tenderizing enzymes 

Ordinarily meat is tough and this toughness is due to the presence of collagen, elastin and 

actomyosin. It is thus required to make the meat soft/tender. Moreover, the favourable 

flavour of meat depends largely on the concentration of peptides and amino acids in the meat. 

For the purpose of tenderizing, marination and flavour proteases are used. Papain is the traditional 

enzyme used, but others such as bromelain, trypsin, chymotrypsin and microbial 

proteases from Aspergillus are also used. 

(III) Sweeteners 

A very important use of enzymes is the generation of sweet syrups from cornstarch. D-glucose 

is not as sweet as sucrose, but D-fructose is sweeter. A mixture of D-glucose and D-fructose is 

sweeter than glucose alone and is used in place of sucrose in canned fruits, fruit drinks, and 

carbonated beverages. This mixture is prepared by use of two enzymes and these are exo-1, 4α-D-glucosidase 

obtained from Aspergillus niger and xylose isomerase also known as glucose 

isomerase mainly from Streptomyces spp. 

Starch glucose fructose 

Another example of the use of an enzyme to cause an increase in sweetness is the use of 

β-D-galactosidase in the manufacture of ice-cream. The mixture of glucose and galactose is 

sweeter than lactose, this enzyme can be used to catalyze an increase in sweetness of a product. 

(C) Immobilized Enzymes 

(I) The merit of using immobilized enzymes 

Enzymes are generally rather difficult to isolate in pure form. However, after purification, 

they spontaneously denature. Finally to be used, they need to be mixed with the substrate, 

after which it may be very difficult to get the enzymes back, even though they are perfectly 

reusable. 

A variety of techniques are solving these problems. This is done by adding cross-links to 

the enzyme molecule to hold the enzyme more strongly in the correct molecular pattern (I, 

Scheme 5.1). This prevents denaturation, extending the useful life of the enzyme. In another 

method the enzymes are attached to a large, insoluble support (II, Scheme 5.1). The enzymes 

can be added to substrate, then easily centrifuged away from the product later. It would also 

be possible to pack the solid supports to which enzymes were attached into a column, then 

pour the substrate through (III, Scheme 5.1) 

E 

— 

E 

— 

E 

— 

— 

— 

— 

E 

E 

E 

E 

E 

(I) 

— 

— 

— 

— 

— 

— 

— 

— 

— 

— 

— 

E 

— 

E 

Intermolecular crosslinkage 

generated by the use of reagents 

e.g., bifunctional reagents like 

1, 5,-difluoro-2, 4-dinitrobenzene 

SCHEME 5.1 Continued... 

E 

E 

E E 

(II) 

Support is e.g., silica glass beads

Biotechnological Applications of Enzymes 191 

SCHEME 5.1 

(II) Immobilized enzymes—In the production of syrups, from corn starch 

The use of enzymes in the production of glucose syrups has been mentioned above. From 1974 

onwards several million tonnes of high-fructose corn syrup is produced annually involving the 

use of immobilized enzymes. This advancement has largely displaced sucrose in traditional 

applications. Moreover, one knows that the role of fructose as a sweetening agent is much 

more satisfactory since it is more sweeter than glucose and also it does not crystallize readily 

from a concentrated solution. The two main enzyme catalyzed steps are (Scheme 5.2). The 

enzyme exo-1, 4-α-D-glucosidase (from Aspergillus niger) is immobilized on porous silica while 

the second enzyme is immobilized via cross linking with glutaraldehyde. 

exo-1, 4-a-D glucose 

Cornstarch glucose fructose 

glucosidase 

isomerase 

SCHEME 5.2 

(III) Role of immobilized enzymes in the synthesis of semi synthetic penicillins 

Several derivatives of naturally occuring penicillin are far more effective as antibiotics. These 

semi synthetic penicillins are made from 6-aminopenicillanic acid which is obtained from 

naturally occuring penicillins (Scheme 5.3). The semisynthetic penicillins have largely replaced 

natural penicillins and about 85% of penicillins marketed for medicinal use are semi synthetic. 

6-Aminopenicillanic acid is obtained by the hydrolysis of the amide bond of the naturally occuring 

penicillin with the enzyme penicillin amidase, which unlike chemical hydrolysis does not open 

the β-lactam ring. 

S CH3 Penicillin + 

RCONH — CH 

amidase NH3 — CH 

O 

C N 

— 

— 

CH 3 

COO 

O 

C N 

Penicillin 6-Aminopenicillanic acid 

SCHEME 5.3 

S 

— 

— 

CH3 +RCOO 

CH3 

COO


(IV) Resolution of racemic amino acid mixtures—Production of L-amino acids 

Synthetic methods are used for the production of huge amounts of (±)-amino acids (about 5 × 

107 kg of DL-Amino acids per year). The production of chiral chemicals is necessary for biological 

purposes and also for use in pharmaceutical industry. Optically active compounds are needed 

as starting materials for asymmetric synthesis and only one of the enantiomers of a recemic 

mixture may have the desireable pharmacological action. These preconditions thus require 

the resolution of racemic mixture of amino acids by an efficient technique. The classical method 

of resolution employing optically active bases is both expenssive and time consuming. 

Recently the use of an immobilized enzyme to resolve the racemic mixture of amino 

acids has been developed on commercial basis. The enzyme is aminoacylase which is immobilized 

by binding to DEAE-Sephadex Columns (Scheme 5.4). The enzyme is specific for the 

hydrolysis of only L-N-acetylated amino acids. Moreover, advantage is also taken of the fact 

that the solubilities of free amino acids, largely differ from their N-acetylated derivatives. 

Following this procedure (Scheme 5.4) free L-amino acid crystallizes out from the system. 

DL-Amino acid 

racemic mixture 

Chemical 

acetylation 

DL-N -Acetylamino acid 

Chemical 

racemization 

SCHEME 5.4 

Aminoacylase 

D-N -Acetylamino acid 

D-N -Acetylamino acid 

+L-Amino acid 

L-Amino acid 

Crystallization 

(V) Other common uses of immobilized enzymes 

Immobilized galactosidase generally from Aspergillus oryzae is largely used in food industry. 

The unwanted trisaccharide raffinose interferes with crystallization of sucrose from beet sugar 

syrups. Raffinose increases in concentration on storage of the beets in cold weather. An 

α-galactosidase is used to convert the raffinose to sucrose and galactose in the syrups. Lactose 

malabsorption is quite common throughout the world population and this has led to a demand 

for lactose-free milk, which can be prepared by hydrolysing the lactose to glucose and galactose, 

using the immobilized enzyme β-galactosidase. 

The carticosteriod cortisol is a useful medicine for the treatment of arthritis and it can 

be made from the cheap precursor 11-deoxycortisol. Two immobilized steroid enzymes 

11β-monooxygenase and a ∆′-dehydrogenase are used to produce prednisolone, which is a 

superior drug compared to cortisone (Scheme 5.5).


O 

CH 3 

11-Deoxycortisol 

CH 3 

CO OH 

2 

CO 

OH 

Steroid 11 

monooxygenase 

O 

HO 

CH 3 

SCHEME 5.5 

Cortisol 

CH 3 

CO OH 

2 

C — O 

OH 

' Dehydrogenase 

O 

HO 

CH 3 

CH 3 

Prednisolone 

CO OH 

2 

CO 

OH 

One future potential for such processing looks impressive. For example if a stable cellulose 

preparation can be researched, the cellulose of plants could be converted to glucose to be 

used as food. The enormous quantities of discarded cellulose in the form of paper, cotton, 

wood, garden debris, agricultural wastes, and food processing wastes could be converted to 

glucose. This glucose could then be fermented to produce ethanol. Every 12.8 lb of glucose 

produces a gallon of alcohol. Such ethanol could be produced at a low price enough to be considered 

as a supplement to or replacement for petroleum fuels in automobiles. This process 

would help solve both an energy problem and a garbage disposal problem. 

Immobilized enzymes are made by the attachment of an enzyme to an insoluble 

support which allows its reuse and continuous use and thus eliminating the tedious 

recovery process. Immobilization stabilizes the enzyme, moreover two or more 

enzymes catalyzing a series of reactions may be placed in close proximity to one 

another. Adsorption, covalent linkage, cross linking, matrix entrapment or 

encapsulation are different methods for making immobilized enzymes. Production 

of glucose syrups from starch by the use of immobilized enzymes is one of the most 

important processes of food industry. 

5.2 CLINICAL USES OF ENZYMES 

(A) Introduction 

It was first discovered in 1954 that the activity of enzyme aspartate aminotransferase in serum 

increased shortly after myocardial infraction [myocardial infraction is defined as necrosis of 

myocardium (heat muscle) due to cessation of blood flow i.e., a process of formation of an area 

of dead heart muscle]. This observation has led to widen the scope of measurement of enzyme 

activity in such body fluids as plasma or serum and has become a valueable tool in medical


diagnosis (clinical enzymology). Certain enzymes that function in the plasma, such as the 

enzymes involved in blood clotting, are continually secreted into the blood by the liver. Most 

other enzymes, however, are normally present in plasma in very low concentrations. They are 

derived from the routine destruction of erythrocytes, leukocytes, and other cells. When cells 

die, their soluble enzymes leak out of the cells and enter the bloodstream. However, all cells 

donot contain the same complement of enzymes, those that are specific to a particular organ 

can be important in aiding diagnosis. Therefore, an abnormally high level of a particular enzyme 

in the blood often indicates specific tissue damage, as in hepatitis and myocardial infarction 

(myo, muscle ; cardi, heart ; an infarct is an area of dead tissue). For example, elevated blood 

levels of creatine kinase (CK) and glutamic-oxaloacetic transaminase (GOT) accompany some 

forms of severe heart disease. A blood analysis that show high levels of CK may indicate that 

the heart muscle has suffered serious damage. 

(B) Clinical enzymology of heart disease 

Several enzymes are present in the heart muscle (myocardium). When the blood flow to the 

myocardium is interrupted, myocardial necrosis (heart attack) occurs. The enzymes present 

in the myocardium leak into the circulating blood. Measurement of these enzymes is useful in 

detecting myocardial infarction. 

The following enzymes are present in myocardium : creatine kinase (CK), lactate 

dehydrogenase (LDH) and Glutamic-oxaloacetic transaminase (GOT). Creatine kinase is the 

earliest to be detectable rising 4-6 hours after the chest pain, reaching a peak at 24-36 hour 

and then rapidly declining (Scheme 5.6). Creatine kinase is also present in skeletal muscle and 

brain. In skeletal muscle this enzyme has almost eight times the concentration on a gram wet 

weight basis compared to that of cardiac muscle. Generally an increase in CK activity in serum 

is mostly associated with damage to the cardiac or skeletal muscle and less frequently to brain 

damage. Moreover since some of these enzymes are also present in other organs such as liver, 

lungs and red blood cells, hence they are not specific to the heart. To further refine the detection 

of infarction various sub-types of these enzymes have been identified. For example CK 

has three isoforms i.e., BB, MB and MM. BB isoform is present mostly in the brain, MM mostly 

in the skeletal muscle and MB mostly in the heart. Measurement of MB isoform is, therefore, 

most useful in making the diagnosis of infarction. 

Extent of enzyme increase 

(multiple of normal) 

5X 

4X 

3X 

2X 

1 

(Normal) 

Chest pain 

Creatine kinase 

Glutamic oxaloacetic 

transaminase 

Lactic dehydrogenase 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

Days after episode 

SCHEME 5.6


Modern medical practices have automated and computerized the assay procedures for 

most of these serum enzymes. It is important to note that the precise patterns of enzyme 

changes in certain tissue diseases are characteristic. For example, in a myocardial infarction, 

the GOT/GPT ratio is usually high ; the reverse is true in liver disease. 

(C) Clinical enzymology of liver disease 

The aminotransferases (formerly called transaminases), alkaline phosphatase and GGT are 

present in liver cells. With liver cell injury or death, these enzymes leak into the blood stream. 

Measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is 

particularly helpful in detecting liver disease. 

(D) Clinical enzymology of cancer 

Certain enzymes such as L-aasparaginase have been found to be useful in treating cancer. 

L-aasparagine is a non-essential amino acid that is required by cancer cells for their growth. 

L-aasparaginase occurs in plants, animals and bacteria. Most common mammals lack this 

enzyme. L-aasparaginase is mostly derived from the bacterium Escherichia coli. Laasparaginase 

by lowering the concentration of asparagine retards the growth of cancer cells. 

It has proven particularly useful in treating lymphoblastic leukemia and certain forms of 

lymphomas. 

(E) Clinical enzymology of pancreatitis 

α-Amylase is largely present in the slivary glands and in the pancreas. This enzyme is an 

endoamylase which brings about the hydrolysis of the 1, 4-α linkage in amylose and amylopectin 

(Scheme 5.7). Low emylase activity is detected in the serum and urine of normal subjects. In 

the case of acute pancreatitis the amylase activity increases 20-30 times the normal levels. 

Significantly, acute pancreatitis would be otherwise diffcult to diagnose in the absence of enzyme 

tests since the patient normally complains of intense upper abdominal (the area where 

pancreas are located) pain which could be due to several other ailments. Infact both α-amylase 

and lipase are elevated in acute pancreatitis. 

O 

HO 

CH OH 

2 

O 

1 

HO 

O 

HO 

CH2 4 

a-1,4-glycoside 

bond 

a-amylose 

O 

HO 

O 

O 

HO 

SCHEME 5.7 

CH OH 

2 

O 

HO 

b-1,4-glycoside 

bond 

HO 

O 

Cellulose 

CH OH 

2 

O 

OH 

Humans do not have enzymes which 

can hydrolyze -glycoside 

linkage and 

thereby utilize the component glucose 

O


5.3 MUTATIONS AND GENETIC DISEASES-FAILURE TO SYNTHESIZE A 

PARTICULAR ENZYME 

(A) Enzyme deficiencies and associated diseases-phenylketonuria 

One knows that DNA directs the synthesis of proteins and the sequence of bases in the DNA is 

critical and specific for the proper sequence of amino acids in proteins. Occassionally, however, 

the base sequence in DNA may get modified by exposure to heat, radiation or some 

chemicals leading to mutation. [Mutation is altering of genetic information in such a fashion 

that the message for sequence of amino acids of a specific protein is altered.] 

Thus bases in DNA can be altered or lost, the phosphodiester bonds in the backbone can 

be broken and strands can become covalently cross-linked. Thus e.g., some chemicals including 

acids and oxidising agents can modify DNA by alkylation, melhylation or deamination. DNA is 

also susceptible to spontaneous loss of heterocyclic bases (depurination or depyrimidization). 

[In replication of DNA alone, each time a human cell divides, a copy is made of 4 billion bases 

to generate a new strand of DNA. Probably there are 2000 errors each times replication occurs. 

Several of these errors are, however, not important, but many may lead to genetic diseases. 

Those diseases result from the inability to produce one of the enzymes of a metabolic pathway 

in an active form (a situation called genetic deficiency)]. 

In most genetic diseases, the resulting defective genes lead to the failure to synthesize a 

particular enzyme. One has already learnt that elevated enzyme activities in serum and urine 

form the basis of diagonsis of associated diseases. Often, on the other hand some subjects may 

suffer from enzyme deficiencies as a result of inborn errors in metabolism and about 400 such 

inborn errors (genetic diseases) in melabolism are now recognized. Phenylketonuria is one 

such inborn disorder (1 in 12,000 births in UK) and in most of these situations an enzyme 

deficiency has been recognized. In fact in many cases the presence of a metabolite is used for 

the diagnosis of the desease e.g., phenylketonuria is detected by estimating the metabolite 

phenylpyruvate in urine or phenylalanine in blood (Scheme 5.8). In phenylketonuria there is 

lack of the enzyme phenylalanine hydroxylase which is needed to convert phenylalanine to 

tyrosine (Scheme 5.8) which is the precursor of the neurotrans-mitters dopamine and 

norepinephrine as well as the skin pigment melanin. 

Phenylalanine is an essential amino acid which is formed regularly in a normal subject 

due to protein turnover and dietary in take. In a person suffering from phenylketonuria 

(a phenylketonuric subject) there is a high build up of phenylalanine or its metabolites. 

Phenylketonuria is associated with mental retardation and the desease can be controlled by 

CH CHCOOH 

2 

NH 2 

phenylalanine 

transamination 

O 

CH —C—COOH 

2 

phenylpyruvate 

Phenylalanine 

hydroxylase 

HO 

CH CHCOOH 

2 

tyrosine 

NH 2 

Tyrosine also serves as a precursor 

for the melanins, the pigments 

which color the skin, hair and eyes. 

This leads to albinism. 

SCHEME 5.8


using a diet with reduced phenylalanine content. On reducing the protein content in the normal 

diet would lead to deficiency in other essential amino acids. This has led to semisynthetic diets 

for phenylketonuric persons which contain protein hydrolysate from which only the 

phenylalanine contant is reduced. Thus preventing mental retardation, in a phenylketonuric 

person. 

A recent problem of significance is the use of the artificial 

sweetener arpartame [aspartame, a dipeptide was discovered in 

1969 is about 200 times sweeter than sugar and is made from 

amino acids phenylalanine and aspartate] which has significantly 

replaced other sweeteners (Scheme 5.9). Aspartame is convertad 

into L-phenylalanine, L-aspartic acid and methanol in the 

intestine. This has led to the importance of early detection of 

phenylketonuria and infact in some countries, screening is done 

right at birth. 

(B) SOME EXAMPLES OF DAMAGE TO DNA (GENETIC-DISEASES) AND 

THEIR ENZYMATIC REPAIR 

(I) Lesions in DNA-pyrimidine dimers formed by ultraviolet light and their repair-enzyme 

DNA photolyase 

On exposure to UV light two adjacent thymines of a DNA strand can become covalently linked 

to give a thymine dimer. It is an example of photodimerization. DNA replication cannot take 

place in the presence of such a dimer (Scheme 5.10) since a dimer distorts a template strand 

(I, Scheme 5.10). Thus the removal of the pyrimidine dimers is necessary for survival. The 

repair process involves an enzyme known as DNA photolyase. This enzyme recognizes and 

binds to the thymine dimer. In the presence of visible light the enzyme catalyzes the cleavage 

H H—N — N 

O 

O 

N 

— 

— 

CH 3 

CH 3 

P P 

A single strand of DNA 

with two adjacent pyrimidines 

(Thymines) 

— 

O 

N 

— 

N N—H — H 

O 

P 

UV 

SCHEME 5.10 

H H—N — N 

O 

3' 

5' 

O 

N 

— 

— 

CH 3 

S S 

P P 

— — 

— — 

— — 

H 3 C 

() 

H N 

O 

N 

— 

Formation of 

a thymine dimer 

— 

H 

O 

C 

NH2 H 

OCH3 

O 

aspartame 

SCHEME 5.9 

N N—H — H 

5' 

3' 

O 

P 

COOH


of the dimer and thus restores normal base pairing and thus DNA is repaired. The enzyme 

(photolyase) then dissociates from the repaired DNA leading to the normal A/T base pair reforming. 

The joined thymines called dimers prevent the replication of DNA. Several enzyme 

systems in normal cells recognize these dimers and either reverse the dimerization 

or cut out and replace the altered section of DNA. People suffering from 

(XP = Xeroderma pigmentosum–development of dry skin, skin and eye cancers) 

lack any one of the nine enzymes necessary for repairing this demage to the 

DNA. 

(II) Mutations caused by changes in the dase sequence of DNA 

One may consider a situation when one base pair is substituted by another. Some of the hydrogen 

atoms on each of the four bases may change their location to give a tautomer e.g., an amino 

group (—NH2 ) can tautomerise to an imino form (= NH) while a keto group (C = O) tautomerizes 

to an enol (= C—OH). These tautomers though transient can lead to unusual base pairs which 

can fit into a double helix. Thus the imino tautomer of adenine can pair with cytosine (Scheme 

5.12). This unusual pairing of A*—C (asterisk denotes the imino tautomer) then leads to C 

N 

H—C 

DNA-bases 

One may recall the structures of the four DNA bases and the Watson and Crick 

specificity of their pairing adenine (A) must pair with thymine (T) and guanine 

(G) with cytosine (C) i.e., A—T and G—C (Scheme 5.11) 

1 

2 

NH 2 

C C N 

6 

3 

N 

5 

4 

C 

Adenine 

(A) 

7 

8 

9 

N 

H 

C—H 

N 

HC 

1 

2 

HN 

H 2 N—C 

H 

C 

C 

N 

6 

3 

N 

1 

2 

5 

4 

C 

Purine 

O 

7 

8 

9 

N 

H 

CH 

C C N 

6 

3 

N 

5 

4 

C 

Guanine 

(A) 

7 

8 

9 

N 

H 

C—H 

SCHEME 5.11 

N 

HC 

3 

2 

H 

C 

4 CH 

1 

N 

5 

6 

Pyrimidine 

H—N 

3 

2 

CH 

O 

C 

4 C—CH3 

5 

6 

O—C 1 

N 

H 

C—H 

Thymine 

(T) 

O 

N 

3 

2 

NH 2 

C 

4 C—H 

5 

6 

C 1 

N 

H 

C—H 

Cytosine 

(C)


H 

C 

N 

C1 H 

C 

C 

N 

C H 

O 

Cytosine 

H 

N 

H 

H 

N 1 

C 

N 

C 

6 

N 

H 

C 

C 

Rare tautomer 

of adenine 

N 

N 

C—H 

C 1 

SCHEME 5.12 

The rare tautomer of adenine pairs 

with cytosine instead of thymine. This 

tautomer is formed by the shift of 

a proton from the 6-amino group 

to N-1. 

getting incorporated into a growing strand where istead normally it should have been T and 

this leads to mutation. Hydrolytic deamination of cytosine gives uracil which pair with adenine 

rather than guanine (Scheme 5.13)—another situation which damages DNA. 

H 

HC 

HC 

N 

C 

N 

Cytosine 

H 

N 

C 

O 

HO 

2 

Hydrolytic 

deamination 

NH 3 

SCHEME 5.13 

Most of these defects in DNA are repaired by general excision-repair pathway. In the 

first step of the repair pathway an enzyme–endonuclease recognizes the distorted, demaged 

DNA and both ends of the lesion are excised. This enzymatic cleavage releases an oligonucleotide 

with about 12 residues leading to a gap. This gap is filled by a DNA polymerase and the nick is 

sealed by DNA ligase (Scheme 5.14). 

(C) ROLE OF ENZYMES IN DRUG DESIGN 

(I) Lowering of Cholesterol Levels 

One has learnt the clinical situations where the enzyme overactivity is used to detect some 

diseases like heart disease. A deficiency of a particular enzyme may be the cause of a genetically 

inherited disease like e.g., phenylketonuria. It is now well established that many drugs function 

through their inhibitory effect on a critical enzyme in the cells of the invading organism. Another 

genetically inherited disease is hypercholesterolaemia in which there is a high circulating 

level of cholesterol/cholesterol esters in the blood (generally 4-6 times the normal circulating 

levels of cholestrol). The disease infact involves a protein rather than an enzyme defect. The 

disease leads to the deposition of arterial plaques with a consequent symptoms of cornonary 

HC 

HC 

O 

C 

N 

Uracil 

NH 

C 

O


Damaged 

DNA 

Normal 

DNA 

Site of damage—a region e.g., containing a thymine dimer 

3' 

5' 

3' 

5' 

3' 

5' 

3' 

5' 

3' 

5' 

Excision of a 12 nucleotide 

fragment by endonuclease 

nicks the DNA backbone on 

both sides of the damage 

(see arrows) 

5' 

SCHEME 5.14 

A helicase removes the 

damaged DNA, leaving 

a gap. 

DNA synthesis by DNA 

polymerase fills the gap 

Joining by DNA ligase. 

disease in childhood or adolescence. The disease is due to deficiency in the low density lipoprotein 

receptors which are involved in the uptake of cholesterol/cholesterol esters by the tissues leading 

to an increase of levels of cholesterol/cholesterol esters. These high levels of circulating 

cholesterol/cholesterol esters can be brought down and thus also the tendency for the formation 

of arterial plaques by giving a drug–a competitive inhibitor of hydroxymethyl-glutaryl CoA 

reductase (HMG CoA reductases). HMG CoA reductase catalyses the rate-limiting step in the 

formation of cholesterol (Scheme 5.15). Three competitive inhibitors of HMG CoA reductase 

5' 

3' 

3' 

5' 

3' 

5' 

3' 

5' 

3'


CH 3 

HO 

C 

OH 

+ + 

HMG CoA + 2NADPH + 2H mevalonate + 2NADP 

O 

O - 

O 

Mevinolin 

O 

C 

CH 3 

H 

HO 

Compactin 

R=H 

O 

SCHEME 5.15 

O 

O 

O C 

R 

Monacol K 

R=CH 3 

CH 3 

HO 

O 

SCoA 

HMG CoA 

(Scheme 5.15) have been researched which inhibit the enzyme. All three are fungal products 

and have a structural resemblance to HMG CoA. Mevalonate is a precursor not only of steroids 

but also of a number of other important cell constituents. Sterol biosynthesis appears to be 

selectively inhibited at low concentrations of the enzyme (also see Scheme 4.46). 

(II) Role of sulfa drugs 

One of the best understood antimetabolites is the synthetic sulfa drug sulfanilamide which 

interferes with the normal metabolism of p-amino-benzoic acid, due to their close structural 

similarity (Scheme 5.16). The following points may be considered: 

HN— 

2 

HN 

2 

N 

HN 3 1 

2 

4 

O 

O 

H 

N 

8 

N 5 

H 

—C—OH 

p-aminobenzoic acid 

7 

6 

9 H 

CH 2 —N— 

10 

SCHEME 5.16 

CO H 

2 

CH 2 

O CH2 H 

—C—N—CH—CO2H HN— 

2 

—SO NH 

2 2 

p-aminosulfonamide, 

a sulfa drug 

• p-Aminobenzoic acid is vital for the growth of many pathogenic bacteria. 

• The vitamin folic acid serves as a coenzyme for several important biochemical 

processes. Folic acid is obtained by a human from its diets and from the bacteria in 

the digestive tracts.


• Bacteria can synthesize folic acid from available p-aminobanzoic acid, however, in 

the presence of structurally similar drug sulfanilamide, the bacterial enzyme easily 

incorporates instead the drug to produce a false nonfunctional folic acid. 

• This false folic acid cannot act as a proper coenzyme and it also acts as a competitive 

inhibitor for the enzyme. 

• Thus the bacteria are unable to biosynthesize vital compounds like certain amino 

acids and nucleotides for their survival and they die (also see Scheme 5.24). 

(III) Penicillin antibiotics 

Lastly mention may be made of several natually occuring 

penicillins which have structural similarity. All have 

O Cysteine 

the empirical formula C9H11O4SN2R, with a four 

membered ring fused to a five membered ring. A variety 

of structural variations in R groups are obtained by 

R—C 

N 

H 

C 

H 

C 

S 

CH3 adding an appropriate organic compound to the culture 

H C N CH3 medium. An antibiotic is a compound which is produced 

by one microorganism (bacterium, mold yeast) and is 

O 

COOH 

valine 

toxic to another. 

A penicillin (Scheme 5.17) is the most widely used 

a penicillin 

antibiotic in the world and functions by inhibiting an 

SCHEME 5.17 

enzyme (transpeptidase) which catalyzes the last step in bacterial cell wall biosynthesis leading 

to death of bacteria. 

(IV) Cancer drugs and biosynthesis of nucleotides 

Nucleotides are involved in the transfer of hereditary information and numerous other reactions 

and are thus crucial to life and all nucleotides can be made by the body and a dietary 

source is not needed. Again, each nucleotide has its own biosynthetic pathway. Deoxythymidine 

- 

O 

O - 

P 

O 

O 

CH 2 

H 

H 

O 

OH 

dUMP 

O 

HN 

C 

H 

H 

O O 

C 

N 

H 

CH 

CH 

Methylene 

+ 

THF 

Thymidylate 

synthase 

SCHEME 5.18 

- 

O 

O - 

P 

O 

O 

CH 2 

H 

H 

O 

OH 

dTMP 

O 

HN 

C 

H 

H 

C 

N 

H 

C—CH 3 

C—H 

+ DHF


monophosphate (dTMP) is formed from deoxyuridine monophosphate (dUMP) by the donation 

of a methyl group by a coenzyme derived from folic acid (methylene THF) one has already seen 

(Scheme 1.26) that uracil has the same shape and size as the drug 5-fluorouracil. This is the 

basis of discovery of 5-fluorocil as a cancer drug. Cancer cells divide more rapidly than most 

normal cells. Cell division requires DNA replication, which in turn requires the deoxynucleotides. 

Inhibition of dTMP synthesis kills rapidly dividing cells. The drug 5-fluorouracil is 

metabolized to an inhibitor of thymidylate synthase, a key enzyme in the synthesis of 

deoxytymidylate. This nucleotide is needed for the synthesis of DNA, which must be synthesized 

before cell division can occur. If there is little or no DNA synthesis, there will be little or no cell 

division (Scheme 5.18). Of the side effects of cancer chemotherapy can be traced to the loss of 

normal rapidly dividing cells. Hair cells die, and cells lining the gastrointestinal tract are 

affected as are the cells that divide to form blood cells. Hair loss, gastrointestinal problems, 

and suppressed immunity are all side effects of chemotherapy (also see Problem 2.13). 

5.4 RECOMBINANT DNA TECHNOLOGY (GENETIC ENGINEERING OR 

CLONING) 

(A) Introduction-Understanding recombinant DNA technology 

DNA, the genetic material is a long, unbranched polymer. The gene may be regarded as a 

segment of the DNA molecule which directs the synthesis of all the protein molecules made by 

the cell. DNA molecules are among the longest molecules known. The DNA found in one human 

cell has about 5.5 billion nucleotide base pairs which probably make up 1 million genes. 

Recombinant DNA technology is the transplantation of genes from one organism into 

another. It depends first on having enzymes that can cleave DNA chains into specific fragments 

which can be manipulated. A new piece of DNA is then inserted into a gap created by cleavage 

and resealing the chains (ligation of DNA fragments with DNA ligases) and its introduction 

into host cells. When the recombination is successful, the gene which is transplanted will 

express (synthesize) its normal protein product thus bacteria which receive human genes can 

be induced to express human proteins of value to treat a particular disease. In short recombinant 

DNA technology is based on nucleic acid enzymology, which involves alteration of DNA of 

some organism with a goal of having that organism produce a desired protein. 

Recombinant DNA technology has produced useful proteins for human therapy. One 

example is the use of insulin. The insulin obtained from hogs has a similarity in amino acid 

composition to human insulin, however, insulin from this source often causes allergic response. 

Human insulin produced by bacteria via recombinant DNA technology is on the market for use 

by diabetic patients. The recombinant DNA technology can thus be studied via the study of the 

following overall aspects : 

• The structural outline and properties of DNA 

• DNA replication—use of DNA polymerases 

• Cleavage of DNA duplex at specific sequences—use of enzymes—restriction endonucleases


• Sealing of gap in DNA—use of DNA ligases. 

• Introduction of modified DNA into host cells and the study of replication and expression 

of recombinant DNA in host cells. 

(B) Structure of DNA 

This may be studied by looking to the following points : 

• The molecular mass of a DNA molecule is very high as several billion, on the other 

hand RNA molecules are much smaller–generally falling in the range 20,000 to 40,000. 

• DNA is very long thread like macromolecule which is composed of a large number of 

deoxyribonucleotides and each of these is composed of a nitrogen base, a sugar and a 

phosphate group. The sugar is deoxyribose (see Scheme 1.27) while four different 

nitrogen bases (heterocyclic amines) are found in DNA. Two of these bases adenine 

(A) and guanine (G) are derivatives of purine, the other two thymine (T) and cytosine 

(C) are derivatives of pyrimidine (see Schemes 1.26 and 1.27). 

• The sequence of nucleotide bases of DNA molecules carry the genetic information 

(Majority of plant and animal genes occur in pieces spread out along the DNA) while 

their sugar and phosphate groups plays the structural role. [using DNA as a pattern 

or template, the genetic information is transferred to RNA. The RNA enters the 

cytoplasm and controls the order in which the amino acids are assembled into new 

protein] 

• The structures of four deoxynucleoside monophosphates (or nucleotides) are in 

(Scheme 5.19) and represent deoxyadenosine monophosphate (dAMP), deoxythymidine 

monophosphate (dTMP), deoxyguanosine monophosphate (dGMP), and deoxycytidine 

monophosphate (dCMP). These are linked in the polymer by an ester bond between 

the 5′-phosphate of the nucleotide and the 3′-hydroxyl of the sugar of the next as 

shown (Scheme 1.27). One end of this linear polynucleotide is said to be 5′ (as no 

residue is attached to its 5′ carbon) and the other is said to be 3′ (since no residue is 

attached to its 3′-carbon). 

- 

O 

O – 

P 

O 

5'CH2 

H 

H 

O 

OH 

O 

H 

H 

2' 

N 

N 

H 

NH 2 

2'-Deoxyadenosine 5'-monophosphate (Deoxyadenylate, dAMP) 

(I) 

N 

N 

- 

O 

SCHEME 5.19 Continued... 

O - 

P 

O 

CH 2 

H 

H 

O 

HC 

3 

O 

OH H 

2'-Deoxythymidine 5'-monophosphate (Thymidylate, dTMP) 

(II) 

H 

O 

N 

H 

NH 

O


 

O 

O 

P 

O 

CH 2 

H 

H 

O 

O 

OH H 

2'-Deoxyguanosine 5'-monophosphate (Deoxyguanylate, dGMP) 

(III) 

H 

N 

N 

H 

O 

N 

NH 

NH 2 

SCHEME 5.19 

 

O 

O 

P 

O 

CH 2 

H 

H 

O 

O 

OH H 

2'-Deoxycytidine 5'-monophosphate ( Deoxycytidylate, 

dCMP) 

(IV) 

• The sugar units and phosphate groups are same in all the nucleotides thus the structural 

abbreviations repersent only the sequence of the nitrogen bases. By convention 

one starts at the left working in the 5′ —→ 3′ direction. Thus a tetranucloetide can be 

referred to as A—G— T—C when it is clear that the reference is to DNA. On may 

5′ ⎯⎯⎯⎯→ 3′ 

often write these abbreviations for the base sequence in DNA by putting a lower case 

d in front of the base sequence i.e., dA— G— T— C. 

This shows that all the sugar 

5′ ⎯⎯⎯⎯→ 3′ 

units of the sugar-phosphodiester backbone of the molecule are deoxyribose in DNA 

(since the phosphate ester bridges holding the nucleotides together each contain two 

phosphate ester linkages, these bridges are called phosphodiesters). 

• These polymeric molecules, consist of two strands of polynucleotide and each base of 

one strands forms hydrogen bonds with a base of the opposite strand to give a base 

pair (I, Scheme 5.20), commonly between the lactam and amino tautomers of the 

bases. 

• Because A in one strand pairs with T in the other strand while G pairs with C, the 

strands are complementary. In any DNA molecule, A = T and G = C. 

• The strands of DNA run in opposite directions i.e., these are antiparallel. Each end of 

double-stranded DNA is made up of the 5′ end of one strand and the 3′ end of another. 

This base pairing, thus enables two complementary strands of DNA to form a duplex. 

Whose shorthand structure may be represented as (I, Scheme 5.20). 

• The duplex is in fact a right-handed double helix (II) (Watson and Crick, the Noble 

prize 1962) where the two helical polynucleotide chains wrap around around each 

other (two stranded helical structure around a common axis). Thus the DNA molecule 

can be imagined as a “ladder” which has been twisted into a helix. 

H 

NH 2 

N 

H 

N 

O


SCHEME 5.20 

• The purine and pyrimidine bases are on the inside of the helix while the sugarphosphate 

units on the nucleotide are on the outside of the DNA molecule. The planes 

of the bases are perpendicular to the helix axis, while the plane of the sugars are 

almost at right angles to those of the bases. 

• The precise sequence of bases carries the genetic information while the Watson-Crick 

pairing rules for bases that adenine (A) must pair with thymine (T) ; and guanine (G) 

with Cytosine (C), (specificity for the pairing of bases) reflect on the double helical 

structure regarding steric and hydrogen bonding factors. There is just the right amount 

of space in the center of the helix for one purine and one pyrimidine to fit across from 

each other (III Scheme 5.20). In contrast the room is insufficient for two purines, 

there is more than enough space for two pyrimidines and in that case these would be 

too far apart to form effective hydrogen bonding. 

Thus out of the base pair in a DNA helix one must always be a purine and other 

pyrimidine (steric factors). The base pairing in further restricted by hydrogen bonding 

requirements. When thymine is across from adenine, the hydrogen bond can form at 

two places, however, if thymine were across from guanine only one bond would be


possible. Thus adenine and thymine always pair. In the case of guanine and cytosine, 

three hydrogen bond can form, adenine would be unable to hydrogen bond to cytosine 

under the same conditions. Guanine and cytosine are thus also uniquely suited to pair. 

• The DNA molecules from many sources are circular. 

(C) DNA replication by DNA polymerases that take instructions from temptates 

The DNA can be replicated by an enzyme DNA polymerase and replication proceeds exclusively 

in the 5′ ——→ 3′ direction. DNA polymerase catalyses the step by step addition of 

deoxyribonucleotide units to a DNA chain (Scheme 5.21). The synthesis of DNA uses as starting 

materials the nucleoside 5′ triphosphates of the bases found in DNA (the abbreviation dNTP 

represents any deoxyribonucleoside triphosphate and PPi denotes the pyrophosphate group). 

The specific nucleotide unit to be added under the DNA polymerase catalysis is determined by 

Watson–Crick base pairing to the template strand, adenine (A) pairs with thymine (T) and 

guanine (G) pairs with cytosine (C) as shown (Scheme 5.22). 

 

(DNA) n residues + dNTP (DNA) n+1 + PP O—P—O—P—O—P— OCH O 

i 2 

SCHEME 5.21 

O 

O 

O 

O 

O 

O 

dNTP 

H H 

H H 

HO H 

DNA polymerase catalyzes the formation of a phosphodiester bond (see arows) 

only if the base on the incoming nucleotide is complementary to the base on 

the template strand. Thus e.g., the base of dNTP must be thymine (T) so as to 

match with adenine (A) on template strand. 

Chain-elongation reaction catalyzed by DNA polymerase 

SCHEME 5.22 

Thus for DNA replication using DNA polymerase as a catalyst one must have a template 

strand of DNA, (template–a polymer molecule whose sequence is used for charting a sequence 

for another molecule. DNA serves as a template for DNA synthesis during replication) a DNA 

Base


O O O 

5' DNA 

O 

CH 2 

H 

H 

3' 

O—P—O—P—O—P— 

O 

O O O 

5' DNA 

O 

CH 2 

H 

H 

3' 

O 

dNTP 

O 

O— 

P O 

O 

5'CH2 

H 

H 

OH 

H 

H 

2' 

H 

O 

O 

5'CH2 

H 

New strand 

New strand 

N 

N 

H 

H 

H 

N 

N 

O 

H 

H 

OH 

O 

N 

H 

H 

H 

2' 

H 

O 

H 

N 

N 

N 

N 

N 

H 

H 

H 

N H 

H 

H 

H 

N 

H 

N 

N 

N 

O 

N 

(G) 

H 

N 

N 

(A) 

H 

N 

N H 

H 

H H 

N 

N N 

O 

H 

O 

N 

CH 3 

N N 

O 

SCHEME 5.23 

H 

H 

N N 

O 

O (C) 

CH 3 

H 

N N 

O (T) 

Hydrogen bonds 

have formed 

H 

3' DNA 

O 

O 

O 

H H 

H 

O 

H 

H 

3' DNA 

O 

O 

O 

H H H 

O 

H 

CH 2 

O 

P O - 

O 

H 

H H 

Template strand 

H 

CH 2 

O 

P O - 

O 

H 

H H 

H 

CH 2 

O 

5' DNA 

Template strand 

H 

CH 2 

O 

5' DNA


or RNA primer which is annealed to the tamplate and which has a 3′ hydroxyl group on the 

deoxyribose and an appropriate dNTP as a source of monomers (Scheme 5.22). 

The shorthand representation shown (Scheme 5.22) for elongation of the DNA chain is 

elaborated in (Scheme 5.23). The incoming dNTP forms a base pair with the residue on the 

tamplate strand and after this correct base pair is formed, the 3′–OH group of the primer 

carries out a nucleophilic attack on the α-phosphorus atom (innermost phosphorus atom) of 

the incoming dNTP. This leads to the addition of a nucleoside monophosphate residue with 

displacement of pyrophosphate. The subsequent hydrolysis of the pyrophosphate (PPi not shown) 

makes the polymerization reaction essentially irreverssible. As true in the case of other 

nucleotide reactions which release pyrophosphate, the presence of Mg2+ is a must, Mg2+ complexes 

with the β and γ-phosphate groups to make nucleoophilic attack feasible. Thus one may 

simplify the information (Scheme 5.23 to Scheme 5.24). 

In summary the DNA is replicated by DNA polymerase by taking instruction 

(presence of a suitable base) from template. The dNTP forms a phosphodiester 

bond provided it has a complimentary base to the base on the template strand. 

The shorthand representation is in (Scheme 5.22) which is elaborated for 

understanding in (Scheme 5.23) and may be represented as in (Scheme 5.24). The 

DNA polymerase is a template–directed enzyme. The enzyme takes instructions 

from the template leading to a synthesis of a feature with a base sequence 

complimentary to that present in the template. 

(D) Restriction enzymes split DNA in to specific fragments 

Restriction enzymes (restriction endonucleases) recognise specific base sequences in double 

helical DNA and bring out cleavage of both strands of the duplex in regions of defined sequence. 

One of their important uses is in recombinant DNA technology. Restriction enzymes cleave foreign 

DNA molecules. The term restriction endonuclease comes from the observation that certain 

bacteria can block virus infections by specifically destroying the incoming viral DNA. Such 

bacteria are known as restricting hosts, since they restrict the expression of foreign DNA. 

Restricting hosts synthesize nucleases which digest foreign DNA. The cell’s own DNA is 

not degraded by restriction enzymes since it is methylated at critical base sites at the sites 

recognized by endonucleases. In fact many bacteria have at least one highly specific restriction 

endonuclease as well as a restriction methylase of identical specificity (this protects the host 

DNA by methylating critical bases). Many restriction enzymes recognize specific sequences of 

four to eight base pairs and bring about the hydrolysis of a phosphodiester bond in each strand 

in this region and nearly in all cases the recognition sites have a two fold axis of symmetry (C2 axis) i.e., the 5′ —→ 3′ sequence of residues is the same in both the strands of the DNA molecule. 

Thus the paired sequences “read” the same in either direction (one says that the recognized 

sequence is polindromic, these sequences are termed palindromes–palindromes in English 

include BIB, DEED, RADAR and MADAMI’ MADAM) and the cleavage sites are symmetrically 

positioned. EcoRI was one of the first restriction enzymes to be discovered. It is 

present in many strains of Escherichia coli this enzyme has a palindromic recognition sequence 

of six base pairs (the 5′ → 3′ sequence is GAATTC on each strand). This endonuclease 

catalyzes the hydrolysis of the phosphodiesters which link G to A in each strand and thus DNA 

is cleaved (Scheme 5.25).


Primer strand 

O 

H 2 C 

Primer strand 

O 

O 

H 2 C 

H H 

H H 

O H 

O—P O 

O 

H 2 C 

O 

O 

H H 

H H 

3 

HO H 

H H 

H H 

HO H 

elongation of 

DNA chain to 

occur here 

G C— D 

N 

A 

A 

G C—D 

N 

A 

t 

e 

m 

p 

l 

a 

t 

e 

T— s 

t 

r 

a 

n 

d 

t 

e 

m plate 

s tr 

T— a 

n 

d 

O 

O 

O—P—O—P—O—P 

O O 

O 

H 2 C 

O 

O 

appropriate dNTP 

Polymerase 

O 

O 

H H 

H H 

H 2 C 

HO H 

Primer strand 

O 

O 

O —P—O—P—O—P 

O O 

O 

A 

H H 

H H 

H 

H 2 C 

O H 

O 

O 

O 

O 

H H 

H H 

HO H 

DNA polymerase brings about the synthesis of DNA by adding one nucleotide, 

unit at a time to the 3′ end. The nucleotide substrate is a suitable 

deoxyribonucleoside 5′-triphosphate (dNTP). The specificity of the dNTP 

whether dATP, dGTP, dTTP and dCTP is determined by Watson-Crick base 

pairing to the template strand ; adenine (A) pairs with thymine (T) and guinine 

(G) pairs with cytosine (C). 

SCHEME 5.24 

D 

G C— N 

A 

A 

T— 

t 

e 

m 

p 

l 

a 

t 

e 

s 

t 

r 

a 

n 

d


Cleavage 

site 

5'—G—A—A—T—T—C— 

3' 

3'—C—T—T—A—A—G— 

5' 

Symmetry 

axis 

Cleavage 

site 

Eco Rl 

5'—G 

3'—C—T—T—A—A 

+ A—A—T—T—C— 3' 

G— 5' 

The sequences which are recognized by these enzymes contain a two fold 

axis of symmetry the two strands in these regions are related by C2 axis-a 

180° rotation around the axis (shown by a dot). 

GAATTC sequence is recognized by the enzyme and both strands of foreign 

DNA are cleaved to give staggered ends. 

SCHEME 5.25 

A piece of DNA formed by the action of one restriction enzyme can be further 

specifically cleaved into smaller fragments by another restriction enzyme. The 

pattern of these fragments serves as a fingerprint of a DNA molecule. 

(E) Gaps in DNA are sealed by DNA ligases 

Certain nicks in duplex DNA can be sealed by an enzyme-DNA ligase which generates a 

phosphodiester bond between a 5′-phosphoryl group and a directly adjacent 3′-hydroxyl, using 

either ATP or NAD + as an external energy source. The overall process is represented (eq I, 

Scheme 5.26) 

5' 

3' 

+ 

DNA (nicked) + NAD 

O 

OH P 

O - 

nick to be sealed 

in DNA 

O 

O - 

3' 

5' 

+ NAD + 

DNA ligase 

DNA 

ligase 

SCHEME 5.26 

+ 

DNA (sealed) + NMN + AMP 

O 

O—P—O 

sealed DNA 

O - 

(I) 

+ AMP + NMN + 

The mechanism of action of DNA ligase to seal the nick is represented (Scheme 5.27) 

which shows the formation of a phosphodiester linkage at the nick in DNA. In the first step the 

lysine side chain on the enzyme attacks (nucleophilic) NAD + to form an AMP-DNA ligase 

intermediate with a phosphoamide bond generating NMN + (nicotinamide mononucleotide). In 

the second step an oxygen atom of the free 5′-phosphate group of the DNA attacks the phosphate 

group of the AMP-enzyme complex to give ADP-DNA intermediate. In the last step 3 the 

nucleophilic 3′-hydroxyl group on the terminal residue of the adjacent DNA strand attacks the 

activated 5′ phosphate group of ADP-DNA complex to release AMP and to form a phosphodiester


Adenine 

enzyme 

DNA ligase 

Lys 

enzyme 

liberated 

-O 

H H 

H H 

OH 

Lys 

O 

O 

(CH ) —NH 

24 2 

P—O—P 

O 

O 

CH 2 

O- 

O 

H 2 C 

O 

H H 

H H 

OH OH OH 

NAD + 

O 

+ 

N 

Ad—O—P—O—P—O—R—N + 

(CH ) —NH 

24 2 

O - 

Nick 

is 

sealed 

5' DNA 

H 2 C 

-O 

+ 

O 

O - 

O 

H 2 C 

O 

H H 

H H 

O 

—P O 

O 

H H 

H H 

O 

3' DNA 

H 

B 

H 

B 

Sealed DNA strand 

O 

– 

O—P—O—AD 

O – 

AMP 

O 

NH 2 

NMN + 

Step 1 

step 

3 

SCHEME 5.27 

Lys 

+ 

(CH 24 ) —NH2 

- 

O P—O 

Adenine 

H H 

H H 

OH 

AMP-DNA-ligase 

intermediate 

(a) 

Lys 

Adenine 

O 

O 

OH 

O 

CH 2 

H H 

H H 

OH 

OH 

Step 2 

O 

CH 2 

-O 

5' DNA 

H 2 C 

O- 

—P O 

O 

H 2 C 

O 

H2C 5' 

O 

H H 

H H 

O 

3' DNA 

O 

H 

H 

3' 

H 

H 

OH H 

5' DNA 

H 2 C 

O 

Nick 

to be 

sealed 

H 

H H 

H 

+ 

(CH 24 ) —NH2 

3' 

O H 

O O- 

O P—O—P 

O 

Nick 

getting sealed 

- 

H 

H 

H H 

H H 

O 

3' DNA 

ADP- DNA intermediate 

O 

H 

B 

B 

B 

B


linkage which seals the nick in the DNA strand. This overall detailed process may be represented 

in a short hand way (Scheme 5.28). 

E—(Lys)—NH 2 

lysine 

residue an 

enzyme 

H 

O 

E—(Lys)—N + —P—O—Ad + 

Ad 

O - 

O 

H O - 

(a) 

O 

OH P 

P 

O 

O 

O 

Ad—O—P—O—P—O—R—N + 

O 

O - 

O - 

+ 

O 

O - 

NAD (coenzyme) 

(simplified form) 

Step 

3 

O 

OH P 

O - 

O 

O - 

nicked DNA 

Step 

1 

O 

Step 

2 

O—P—O 

O - 

sealed DNA 

SCHEME 5.28 

H 

O 

E—(Lys)—N + —P—O—Ad+NMN + 

+H + 

H O - 

(a) 

AMP-DNA-ligase 

(intermediate) 

O O 

OH P 

O - 

O O 

P 

O O—Ad 

- 

ADP-DNA 

(intermediate) 

+ 

O 

- 

O— P—O—Ad 

- 

O AMP 

+ E(Lys)—NH2 

(enzyme) 

In summary role of restriction enzymes and DNA ligase to form recombinant DNA 

molecules is central. The DNA molecule is split at a unique site by using a restriction 

enzyme (e.g., Eco RI). The DNA fragment thus produced can be annealed/joined 

using DNA ligase. The DNA ligase requires a free 3′ OH group and a 5′-phosphate 

group. This leads to new combinations of unrelated genes, which are introduced 

into suitable cells. Thus a recombinant DNA molecule contains unrelated genes. 

(F) The Recombinant DNA Technique 

In an example of recombinant DNA technology, one begins with certains circular DNA molecules 

found in the cells of the bacteria Escherichia coli. These molecules, called plasmids 

(I, Scheme 5.29), consist of double-stranded DNA arranged in a ring. Restriction enzymes cleave 

DNA molecules at specifric locations (a different location for each enzyme). For example, one 

of these enzymes may split a double-stranded DNA as shown (Scheme 5.29).


(I) 

H—GAATTC—H 

H—CTTAAG—H 

B—GAATTC—B 

B—CTTAAG—B 

H—G 

+ 

H—CTTAA 

AATTC—B 

G—B 

enzyme H—G 

+ 

H—CTTAA 

AATTC—H 

G—H 

enzyme B—G 

+ 

B—CTTAA 

AATTC—B 

G—B 

enzyme 

H—GAATTC—B 

H—CTTAAG—B 

Recombinant DNA technology. 

SCHEME 5.29 

H is human gene 

which makes e.g., 

insulin 

B is a plasmid 

from bacteria (shown 

in linear form for clarity) 

A modified plasmid 

(recombinant DNA 

molecule with a desired gene 

A plasmid (B) is cut by the specific restriction enzyme at the restriction site. In this cut 

one can fit e.g., a human gene (H) which is responsible for making insulin using the enzyme 

DNA ligase to give a modified (recombinant) DNA molecule (Scheme 5.29). In summary the 

following steps are involved to produce insulin by recombinant technology : 

• A plasmid (a circular DNA molecule) from a bacterium e.g., E coli is cut using a 

specific restriction enzyme to produce a double stranded chain (Scheme 5.29/5.30) 

with sticky ends since each of the strands has several free bases which are ready to 

pair up with a complementary strip (section) of gene (i.e., this gene must be a strip of 

double stranded DNA that has necessary base sequence. This e.g., can be a human 

gene which makes insulin. 

• The gene strip which is to be inserted into the cut DNA (Scheme 5.30) can be made in 

two ways : 

1. In a laboratory by chemical synthesis ; that is, chamists can combine the nucleotides 

in the proper sequence to make the gene. 

2. One may cut a human chromosome with the same restriction enzyme. As it is the 

same enzyme, it cuts the human gene so as to leave the same sticky ends : 

• The gene strip (cut gene for insulin) and the cut DNA are mixed in the presence of a 

DNA ligase which joins the sticky ends and the cut DNA gets converted into a circle 

(plasmid) once again which now contains the desired gene. 

The modified plasmid (which does all things that DNA does) is then introduced into a 

bacterial cell, where it replicates. All these cells now generate human insulin thus one can use 

bacteria as a factory to manufacture specific proteins. This new technology has tremendous 

potential for lowering the price of drugs that are now manufactured by isolation from human 

or animal tissues. Not only bacteria but also plant cells can be used. 

Provided recombinant DNA techniques can be applied to humans and not just to bacteria, 

genetic diseases can be cured by this powerful technology. For instance, an infant or fetus 

who is missing a gene might be given that gene. Once in the cells, the gene would reproduce 

itself and perform for the individual’s lifetime.


T 

A 

A 

T 

A 

T 

G 

C 

T 

A 

G 

C 

G 

CTTAA 

CT TAA 

G 

GG A A A A T T T T C C 

CCT 

TTTAAAAG G 

GGAAAA 

CC 

TTTT 

CC TT T T A A A A G G 

restriction 

enzyme 

Cut DNA 

modified 

DNA with 

insulin gene 

inserted 

Recombinant DNA 

PROBLEMS AND EXERCISES 

Plasmid 

Sticky ends 

Cut 

gene 

for 

insulin 

Bacterium 

DNA ligase 

plasmid 

enters new 

bacterium 

SCHEME 5.30 

AATTC AATTC 

GG 

Foreign DNA to 

be cloned [restriction 

enzyme ( EcoRI)] 

GG 

CTTAA CTTAA 

Synthesis of 

desired protein 

5.1. Write a short note on the role of enzymes in food processing taking an example from 

cheese making. 

5.2. How enzymes are immobilized? Give their role in the production of syrups from corn 

starch? 

5.3. How L-amino acids are prepared from their racemic mixture using immobilized enzymes? 

5.4. How the level of cholesterol is controlled in humans? Explain briefly the pathway for 

biosynthesis of cholesterol and one typical enzyme involved. 

5.5. How enzymes are used to detect heart problem? 

5.6. What are genetic diseases? Give one example with the enzyme deficiency to cause it.


5.7. How a sulfa drug is incorporated into an enzyme to produce nonfunctional folic acid? 

Explain. 

5.8. What bases are required to repair the demaged portion of the DNA molecule. 

—A T— 

—G 

—T A— 

—T A— 

C— 

—A T— 

—A T— 

—G C— 

5.9. Compare hydrogen bonding in the α-helix of proteins to that in double helix of DNA. 

5.10. Write a short note on the role of restriction enzymes (endonucleases). What are stickly 

ends? 

5.11. EcoRI restriction endonuclease recognizes the sequence GAATTC and cuts it between G 

and A. What will be the stickly ends of the following double-helical sequence when EcoRI 

acts on it? 

CAAAGAATTCG 

GTTTCTTAAGC 

5.12. Recombinant DNA technology is based on isolation of DNA, its cleavage at particular 

sequences, ligation of DNA fragments and their incorporation into host cells. Give the 

mechanism of any of these techniques. 

5.13. Two different restriction endonucleases act on the following sequence of a double-stranded 

DNA : 

The endonuclease EcoRI is specific for the sequenc GAATTC and cuts the sequence 

between G and A. The other endonuclease, TaqI, recognizes the sequence TCGA and 

cuts the sequence between T and C. What stickly ends will be created by these 

endonucleases? 

5.14. In forming recombinant DNA molecules it may be necessary to control the enzymatic 

replication of DNA. How it can be achieved? 

SELECTED ANSWERS TO PROBLEMS AND EXERCISES 

5.8. C pairing with G and G pairing with C. 

5.9. In the α-helix the hydrogen bonds form between carbonyl oxygen of one residue and 

amide hydrogen four residues away. These hydrogen bonds are roughly parallel to the 

axis of the helix and involve the atoms in the backbone. The amino acid side chains 

which point away from the backbone are not involved in intrahelical hydrogen bonding.


5.13. 

In the case of double stranded DNA. The sugar-phosphate backbone is not involved in 

hydrogen bonding. However, two or three hydrogen bonds which are roughly 

perpendicular to the axis of the helix involve complimentary bases in opposite strands. 

In the α-helix the cumulative effect of all the hydrogen bonds tends to stabilize the 

helical structure particularly within the hydrophobic interior of the protein where water 

does not compete for hydrogen bonding. On the other hand in DNA the major role of 

hydrogen bonding is to allow one strand to be complementary to the other. The helix in 

DNA no doubt is stabilized by hydrogen bonds between complimentary bases, however, 

the helix gets, major setability from stacking interactions between base pairs in the 

hydrophobic interior. 

EcoRI 

Taql 

AATG 

TTACTTAA 

AATGAATT 

TTACTTAAGC 

AATTCGAGGC 

GCTCCG 

CGAGGC 

TCCG 

5.14. The synthesis of a new strand of DNA is achieved by the successive addition of nucleotides 

to the end of the growing chain. DNA polymerase synthesizes DNA by the addition of 

one nucleotide at a time to the 3′-end of the newly synthesized DNA. The nucleotide 

substrate is a deoxyribonucleoside 5′-triphosphate (dNTP). The specific nucleotide is 

determined by Watson-Crick base pairing to the template strand i.e. adenine (A) pairs 

with thymine (T) and guanine (G) pairs with cytosine (C). 

The DNA synthesis can be terminated by using Sanger’s method by employing 2′, 

3′-dideoxynucleoside triphosphates (dd NTP) which differ from dNTP by lacking a 3′-OH 

group. The ddNTP serves as a substrate for DNA polymerase. The addition of this analog 

blocks further growth of the new chain because it lacks the 3′-hydroxyl terminus needed 

to form the next phosphodiester bond. Hence, fragments of various lengths are formed 

in which the dideoxy analog is at the 3′ end (In addition to the four dNTP’s the incubation 

mixture contains 2′, 3′-dideoxy analog of one of them 

P—P—POCH 2 

ddNTP 

O 

H H 

H H 

H 

3 2 

H 

Base 

DNA to be sequenced 

3 ——GAATTCGCTAATGC———— 

5 ——CTTAA 

Primer 

DNA polymerase I 

Labeled dATP, dTTP, 

dCTP, dGTP 

Dideoxy analog of dATP A 


5 ——CTTAAGCGATT 

A 

+ 


5 ——CTTAAGCG 

A 

New DNA strands are separated 

and electrophoresed

Biotechnological Applications of Enzymes - New Age International

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