Microbial Enzyme Technology

By; Ms. Jawairia kiran

Introduction to enzymes
 The nature and classification of enzymes
• Enzymes are biological catalysts (also known as biocatalysts)

• speed up biochemical reactions in living organisms

• can also be extracted from cells and then used to catalyse a wide
range of commercially important processes

For example
• Production of sweetening agents
• Modification of antibiotics
• Used in washing powders and various cleaning products
• Play a key role in analytical devices and assays that have clinical,
forensic and environmental applications.
 The word ‘enzyme’ was first used by the
German physiologist Wilhelm Kühne in 1878
• He was describing the
ability of yeast to
produce alcohol from
sugars, and it is derived

• From the Greek words :

en (meaning ‘within’)
zume (meaning ‘yeast’).
• In the late nineteenth century and early twentieth
century, significant advances were made in the
extraction, characterization and commercial
exploitation of many enzymes

• 1920s that enzymes were crystallized, revealing that

catalytic activity is associated with protein molecules.

• For the next 60 years or so it was believed that all

enzymes were proteins

• In 1980s it was found that some ribonucleic acid (RNA)

molecules are also able to exert catalytic effects called
ribozymes, play an important role in gene expression.
• In the same decade, biochemists developed
the technology to generate antibodies that
possess catalytic properties.

• These so-called ‘abzymes’ have significant

potential both as novel industrial catalysts and
in therapeutics.
• As catalysts, enzymes are required in very low
concentrations

• They speed up reactions without themselves

being consumed during the reaction

• Enzymes are describe as being capable of

catalysing the conversion of substrate molecules
into product molecules as follows:
Substrate ⇌ Enzyme Product
 Enzymes are potent catalysts
• The enormous catalytic activity of enzymes
can be expressed by a constant, kcat referred as
the turnover rate, turnover frequency or
turnover number.

• This constant represents the number of

substrate molecules that can be converted to
product by a single enzyme molecule per unit
time (usually per minute or per second).
Examples of turnover rate values are:
 For example

Single molecule of carbonic anhydrase can

catalyse the conversion of over half a million
molecules of its substrates, carbon dioxide
(CO2) and water (H2O), into the product,
bicarbonate (HCO3−), every second—a truly
remarkable achievement.
 Enzymes are specific catalysts
• They generally catalyse the conversion of only
one type (or at most a range of similar types)
of substrate molecule into product molecules.
• Some enzymes demonstrate group specificity.

• For example, alkaline phosphatase can remove

a phosphate group from a variety of
substrates.
• Other enzymes demonstrate much higher specificity,
which is described as absolute specificity.

• For example, glucose oxidase shows almost total

specificity for its substrate, β-D-glucose, and virtually
no activity with any other monosaccharides.

• This specificity is of paramount importance in many

analytical assays and devices (biosensors) that measure
a specific substrate (e.g. glucose) in a complex mixture
(e.g. a blood or urine sample).
 Enzyme names and classification
• the enzyme committee (EC) of the International
Union of Biochemistry and Molecular Biology
(IUBMB) recommended the classification of
enzymes into six groups.

• The IUBMB committee also defines subclasses

and sub-subclasses.

• Each enzyme is assigned an EC (Enzyme

Commission) number.
 For example
The EC number of catalase is EC 1.11.1.6.
The first digit indicates that the enzyme belongs to
oxidoreductase (class1).
 Subsequent digits represent subclasses and sub-
subclasses.
• The enzyme rennet used in cheese
manufacture and also known as chymosin, has
the number of EC 5.3.1.5.
The six major EC groups are as follows:
1. Oxidoreductases :
Catalyze a variety of oxidation-reduction reactions
Common names include:
dehydrogenase, oxidase, reductase and catalase.

2. Transferases:
Catalyze transfers of groups (acetyl, methyl, phosphate,
etc.).
Common names include acetyltransferase, methylase,
protein kinase, and polymerase.
 The first three subclasses play major roles in the regulation
of cellular processes.
3. Hydrolases:
Catalyze hydrolysis reactions where a molecule
is split into two or more smaller molecules by
the addition of water.
Some examples are:
i. Proteases: Proteases split protein molecules.

• They are further classified by their optimum

pH as acid, alkaline or neutral.

• They may also be classified on the basis of

their active centers into the following:
a. Serine proteases:
These have a residue in their active center and are
specifically inhibited by diisopropyl
phosphofluoridate and other organophosphorus
derivates.

b. Thiol proteases:
• The activity of these depends on the presence of
an intact-SH group in their active center.

• They are specifically inhibited by thiol reagents

such as heavy metal ions and their derivatives, as
well as alkylating and oxidizing agents.
c. Metal proteases: These depend on the
presence of more of less tightly bound divalent
cations for their activity.

d. Acid proteases: Acid proteases contain one or

more side chain carboxyl groups in their active
center.
ii. Nucleases split nucleic acids (DNA and
RNA).

• Based on the substrate type, they are divided

into RNase and DNase.
– RNase catalyzes the hydrolysis of RNA
– DNase acts on DNA.
• They may also be divided into Exonuclease
and Endonuclease.
– The exonuclease progressively splits off single
nucleotides from one end of DNA or RNA.
– The endonuclease splits DNA or RNA at internal
sites.
iii. Phosphatase catalyzes dephosphorylation
(removal of phosphate groups).
4. Lyases
Catalyze the cleavage of C-C, C-O, C-S and C-N
bonds by means other than hydrolysis or
oxidation.
Common names include decarboxylase and
aldolase.
5. Isomerases catalyze atomic rearrangements
within a molecule.
Examples include
• rotamase, protein disulfide isomerase (PDI), epimerase
and racemase.

6. Ligases catalyze the reaction which joins two

molecules.
Examples include peptide
• synthase, aminoacyl-tRNA synthetase, DNA ligase and
RNA ligase.
 Enzyme structure and substrate
binding
• Amino acid-based enzymes are globular proteins

• Range in size from less than 100 to more than 2000 amino
acid residues.

• These amino acids can be arranged as one or more

polypeptide chains

• that are folded and bent to form a specific three-

dimensional structure, incorporating a small area known as
the active site where the substrate actually binds
• The active site may
well involve only a
small number (less
than 10) of the
constituent amino
acids
It is the shape and charge properties of the
active site that enable it to bind to a single
type of substrate molecule, so that the
enzyme is able to demonstrate considerable
specificity in its catalytic activity.
• German chemist Emil Fischer in 1894, first
proposed “Enzyme specificity results from the
complementary nature of the substrate and its
active site”

Became known as Fischer's ‘lock and key

hypothesis’
• Through techniques such as X-ray
crystallography, it became clear that enzymes
are not rigid structures, but are in fact quite
flexible in shape.

• In 1958 Daniel Koshland extended Fischer's

ideas and presented the ‘induced-fit model’ of
substrate and enzyme binding
• The enzyme molecule changes its shape
slightly to accommodate the binding of the
substrate
• Large number of enzymes consist solely of protein, many
also contain a non-protein component, known as a cofactor
that is necessary for the enzyme's catalytic activity.

• A cofactor may be another organic molecule called a

coenzyme

• or it may be an inorganic molecule, typically a metal ion

such as iron, manganese, cobalt, copper or zinc.

• A coenzyme that binds tightly and permanently to the

protein is generally referred to as the prosthetic group of
the enzyme.
• When an enzyme requires a cofactor for its
activity, the inactive protein component is
generally referred to as an apoenzyme

• the apoenzyme plus the cofactor (i.e. the

active enzyme) is called a holoenzyme
• The need for minerals and vitamins in the human diet is
partly attributable to their roles within metabolism as
cofactors and coenzymes.
• Suggested books:
1. Ray, R. C., 2005. Microbial Biotechnology in
Agriculture and Aquaculture. NBN
International.
2. Kreuzer, H. and Massey, A., 2005. Biology and
Biotechnology Science, Applications, and
Issues American Society for Microbiology.
Thank you