Professional Documents
Culture Documents
ON
ENZYMOLOGY
Topic:
Enzyme Production
SUBMITTED TO:
SUBMITTED
BY:
SIDRA ASGHAR
Roll No. 27
BS (Botany)
7th Semester
DEPARTMENT OF BOTANY
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.
06
4.
Enzyme Technology
07
4.1
Chemical Process
08
4.2
08
4.3
Structure
08
5.
Involvement in disease
10
6.
11
7.
Objective
12
ABSTRACT
1.
ENZYMOLOGY
1.1
Definition:
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:
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.
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
4.1
Chemical Process
4.2
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
10
6.
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.
2.
(b) 1876
(b) yeast
(b) 6
(c) 7
(d) 8
(c) fungi
4.
(d) 1880
3.
(c) 1878
3. (b)
(c) Gyrase
(d) None
4. (b)
2.
3.
2. detergents
3. Tay-sachs
True / False
1.
2.
3.
In PCR technique Kary Mullis shared the noble prize for chemistry in 1993.
T/F Answers: 1. T
2. F
3. T
12