ASSIGNMENT

ON
ENZYMOLOGY
Topic:

Enzyme Production

SUBMITTED TO:

SUBMITTED

MAM. AAFIA RABBANI

BY:
SIDRA ASGHAR

Roll No. 27

BS (Botany)
7th Semester

DEPARTMENT OF BOTANY

THE WOMEN UNIVERSITY, MULTAN

TABLE OF CONTENTS

Abstract

02

1.

Enzymology

03

1.1

03

2.

Definition:

Enzyme Production

03

2.1

Definition:

03

2.2

Uses of Enzymes

03

2.3

Manufacturing

04

3.

Production of Microbial Enzymes

06

4.

Enzyme Technology

07

4.1

Chemical Process

08

4.2

Recombinant DNA Technology

08

4.3

Structure

08

5.

Involvement in disease

10

6.

Method and References

11

7.

Objective

12

ABSTRACT

The whole summary is about measuring the different aspects of enzyme

production. This assignment outlines a number of methods of production, their way of
construction and use of enzyme. There is a complete explanation of manufacturing of
enzyme production based on the technology. In this assignment also gave a summary of
microbial enzyme based on enzyme production. And last topic is about involvement in
disease.

1.

ENZYMOLOGY

1.1

Definition:

The study of enzymes is called enzymology.

2.

ENZYME PRODUCTION

2.1

Definition:

The process in which to obtain enzymes from synthetic process such as fermentation
microorganisms and minipreps is called enzyme production.
The production of enzymes is central to the modern biotechnology industry. The
traditional industrial enzymes continue to have expanding markets, and the recognition of
potential to use biocatalysis in various industrial sectors for new applications generates
demand for enzymes with novel activities and/or improved stability.

2.2

Uses of Enzymes
Man has utilized enzymes throughout the ages either in the form of:

vegetables rich in enzymes,

microorganisms used for a variety of purposes

for instance in brewing

baking, and

in cheese production.

This was initiated by the isolation of an enzyme complex from malt by Payen and Persoz
in 1833, termed diastase that converts gelatinized starch into sugars, primarily maltose.
The history of modern enzyme production really began in 1874 when the Danish chemist
Christian Hansen first produced rennet by extracting it from dried calves' stomachs with

saline solution. During the early part of the last century, in the Far East, an age-old
tradition involving the use of mould fungi called koji in the production of certain
foodstuffs and flavour additives based on soya protein and fermented beverages, formed
the basis on which the Japanese scientist Takamine developed a fermentation process for
the industrial production of fungal amylase. The process included the culture of
Aspergillus oryzae on moist rice or wheat bran, and the product was called 'Takadiastase'
which is still used as a digestive aid.

2.3

Manufacturing

Enzymes have been used for thousands of years to produce food and beverages, such as
cheese, yoghurt, beer and wine. They were used in cheese manufacturing using yeasts and
bacteria in food manufacturing. Enzymes which were isolated were first used in
detergents earlier. The protein nature was discovered later and slowly the industrial
applications also increased.
Enzyme production process can be divided into following phases:
Selection of an enzyme
Selection of a production strain
Construction of an overproducing strain by genetic engineering
Optimization of culture medium and production conditions
Optimization of recovery process (and purification if needed)
Formulation of a stable enzyme product

Criteria used in the selection of an industrial enzyme include specificity, reaction rate, pH
and temperature optima and stability, effect of inhibitors and affinity to substrates.
Enzymes used in paper industry should not contain cellulose-degrading activity as a side
activity because this activity would damage the cellulose fibres. Enzymes used in animal
feed industry must be thermo tolerant to survive in the hot extrusion process used in
animal feed manufacturing. The same enzymes must have maximal activity at the body
temperature of the animal. Enzymes used in industrial applications must usually be

tolerant against various heavy metals and have no need for cofactors. They should be
maximally active already in the presence of low substrate concentration so that the
desired reaction proceeds to completion in a realistic time frame.

3.

Production of Microbial Enzymes

Enzymes occur in every living cell, hence in all microorganisms. Each single strain of
organism produces a large number of enzymes, hydrolyzing, oxidizing or reducing, and
metabolic in nature. Hence, it is customary to select strains for the commercial production
of specific enzymes which have the capacity for producing highest amounts of the
particular enzymes desired. Commercial enzymes are produced from strains of molds,
bacteria, and yeasts as shown in table 1.

TABLE 1
Some commercial enzymes and source microorganisms
Source

Enzyme

Microorganism

Fungal

Amylases

(Aspergillus oryzae

Glucosidasesf

I Aspergillus flavus

Prpteases

{.Aspergillus niger

Pectinases

Aspergillus niger

Glucose oxidase)

jPenicillium notatum

Catalase J

\Aspergillus niger

Bacterial

Amylases ]
Proteases ?

Bacillus subtilis

Penicillinasej
Yeast

Invert ase

Saccharomyces cerevisiae

Lactase

Saccharomyces fragilis

For fungal enzymes, modifications of Dr. Takamines original mold bran process have
usually been employed. In this process, the mold is cultivated on the surface of a solid
substrate. Takamine used wheat bran and this has come to be recognized as the most
satisfactory basic substrate although other fibrous materials can be employed. Other
ingredients may be added, such as nutrient salts, acid or buffer to regulate the pH, soy
bean meal or beet cosettes to stimulate enzyme production. In one modification of the
bran process, the bran is steamed for sterilization, cooled, inoculated with the mold
spores, and spread out on trays (Underkofler et at., 1947; Forbath, 1957). Incubation takes
place in chambers where the temperature and humidity are controlled within limits by
circulated air. It may be stated that instead of trays for incubation, Takamine, as well as
other producers, at one time used slowly rotating drums. Generally tray incubation gives
more rapid growth and enzyme production.
Bacterial enzymes have been and are also produced by the bran process. However, until
recently the process originally invented by Boidin and Effront (1917) was most
extensively employed (Wallerstein, 1939). In this process, the bacteria are cultivated in
special culture vessels as a pellicle on the surface of thin layers of liquid medium, the
composition of which is adjusted for maximum production of the desired enzyme. Different strains of Bacillus subtilis and different media are employed, depending on whether
bacterial amylase or protease is desired.

4.

Enzyme Technology

Enzyme-catalyzed processes are gradually replacing chemical processes in many areas of

industry. Enzymes have all the properties of true catalysts.

4.1

Chemical Process

In the presence of an appropriate enzyme, a chemical reaction occurs at a much higher

rate but the enzyme is not consumed by the reaction. Their ability to perform very
specific chemical transformations (biotransformation) has made them increasingly
popular in industries where less specific chemical processes produce unwanted byproducts. Purity and predicability are of particular importance in food manufacture where
by-products may be harmful or affect flavour, and because of their specificity,
pharmaceutical companies favour biotransformations in the development of novel
therapeutic agents.

4.2

Recombinant DNA Technology

The best example is a DNA polymerase isolated from Thermus aquaticus which is used
in the Polymerase Chain Reaction (PCR). The half-life of this enzyme is 40 minutes at
95C and so Taq DNA polymerase can be repeatedly heated during the DNA denaturation
steps of the reaction. PCR has so revolutionised molecular genetics that its inventor, Kary
Mullis, shared the Nobel Prize for Chemistry in 1993.
To the rescue came recombinant DNA technology or .Genetic Engineering.

4.3

STRUCTURE

Enzymes are generally globular proteins, acting alone or in larger complexes. Like all
proteins, enzymes are linear chains of amino acids that fold to produce a threedimensional structure. The sequence of the amino acids specifies the structure which in
turn determines the catalytic activity of the enzyme. Although structure determines
function, a novel enzyme's activity cannot yet be predicted from its structure
alone. Enzyme structures unfold (denature) when heated or exposed to chemical
denaturants and this disruption to the structure typically causes a loss of activity.
Enzyme are usually much larger than their substrates. Sizes range from just 62 amino acid
residues, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the
animal fatty acid synthase. Only a small portion of their structure (around 24 amino
acids) is directly involved in catalysis: the catalytic site. This catalytic site is located next
to one or more binding sites where residues orient the substrates. The catalytic site and
binding site together comprise the enzyme's active site. The remaining majority of the
enzyme structure serves to maintain the precise orientation and dynamics of the active
site.
In some enzymes, no amino acids are directly involved in catalysis; instead, the enzyme
contains sites to bind and orient catalytic cofactors. Enzyme structures may also
contain allosteric sites where the binding of a small molecule causes a conformational
change that increases or decreases activity.
A small number of RNA-based biological catalysts called ribozymes exist, which again
can act alone or in complex with proteins. The most common of these is
the ribosome which is a complex of protein and catalytic RNA components.

5.

Involvement in disease

In phenylalanine hydroxylase over 300 different mutations throughout the structure

cause phenylketonuria. Phenylalanine substrate and tetra hydro biopterin coenzyme in
black, and Fe2+ cofactor in yellow.
Since the tight control of enzyme activity is essential for homeostasis, any malfunction
(mutation, overproduction, underproduction or deletion) of a single critical enzyme can
lead to a genetic disease. The malfunction of just one type of enzyme out of the thousands
of types present in the human body can be fatal. An example of a fatal genetic disease due
to enzyme insufficiency is Tay-Sachs disease, in which patients lack the enzyme
hexosaminidase.
One example of enzyme deficiency is the most common type of phenylketonuria. Many
different single amino acid mutations in the enzyme phenylalanine, which catalyzes the
first step in the degradation of phenylalanine, result in build-up of phenylalanine and
related products. Some mutations are in the active site, directly disrupting binding and
catalysis, but many are far from the active site and reduce activity by destabilizing the
protein structure, or affecting correct oligomerisation. This can lead to intellectual
disability if the disease is untreated. Another example is pseudo cholinesterase deficiency,
in which the body's ability to break down choline ester drugs is impaired. Oral
administration of enzymes can be used to treat some functional enzyme deficiencies, such
as pancreatic insufficiency and lactose intolerance.
Another way enzyme malfunctions can cause disease comes from gremlins in genes
coding for DNA repair enzymes. Defects in these enzymes cause cancer because cells are
less able to repair mutations in their genomes. This causes a slow accumulation of
mutations and results in the development of cancers. An example of such a

10

hereditary cancer syndrome is xeroderma pigmentosum, which causes the development of

skin in response to even minimal exposure to ultraviolet light.

6.

Method and References

Aunstrup K., Andresen O., Falch E.A., and Nielsen T.K. (1979) Production of microbial
enzymes. In Microbial Technology. 2nd edition, vol. 1, Academic Press, pp. 281-309.
[This article reviews all aspects of microbial enzyme production including applications of
some selected enzymes].
Babu K.R. and Satyanarayana T. (1996) Production of bacterial enzymes by solid state
fermentation. J. Sci. Indust. Res. 55, 464-467. [This paper provides consolidated
information on the production of extracellular enzymes by bacteria in solid state
fermentation].
Brewer J.W. (1987) The production and purification of fine enzymes. In: Basic
Biotechnology (Bu'lock, J. and Kristiansen, B., eds.) Academic Press, London, pp. 407424. [This review deals with the purification of speciality enzymes from microbial,
animal and plant sources, activity and purity analysis, storage and distribution].
Frost G.M. and Moss D.A. (1987) Production of enzymes by fermentation. In
Biotechnology Vol. 7a, (Rehm, H.-J.,

11

7.

OBJECTIVE

1.

MCQs
1.

The history of enzyme production was began:

(a) 1874

2.

(b) 1876

(b) yeast

(b) 6

(d) all of these

(c) 7

(d) 8

The best example of recombinant DNA technology is:

(a) DNA helicase

MCQs Answers: 1. (a)

2.

(c) fungi

Enzyme production process can be divided into phases:

(a) 5

4.

(d) 1880

The source of enzyme for amylase is

(a) Bacteria

3.

(c) 1878

(b) DNA polymerase

2. (c)

3. (b)

(c) Gyrase

(d) None

4. (b)

Fill in the Blanks

1.

_______________ are rich in enzyme.

2.

Enzyme which were isolated first used in _______________earlier.

3.

_______________ disease in which patient lack the enzyme.

Blanks Answers: 1. vegetable

3.

2. detergents

3. Tay-sachs

True / False
1.

Defects in the gremlins enzyme cause cancer.

2.

A small number of RNA based upon chemical catalysts called ribozymes.

3.

In PCR technique Kary Mullis shared the noble prize for chemistry in 1993.

T/F Answers: 1. T

2. F

3. T

12