Download Enzymology - Lectures For UG-5

Enzymology
Lecture 7
Introduction






Enzymes found in nature have been used since ancient times in the
production of food products, such as cheese, sourdough, beer, wine and
vinegar, and in the manufacture of commodities such as leather, indigo and
linen.
All of these processes relied on either enzymes produced by spontaneously
growing microorganisms or enzymes present in added preparations such as
calves’ rumen or papaya fruit.
The development of fermentation processes during the later part of the last
century, aimed specifically at the production of enzymes by use of selected
production strains, made it possible to manufacture enzymes as purified, wellcharacterized preparations even on a large scale.
This development allowed the introduction of enzymes into true industrial
products and processes, for example, within the detergent, textile and starch
industries.
The use of recombinant gene technology has further improved manufacturing
processes and enabled the commercialization of enzymes that could
previously not be produced.
Furthermore, the latest developments within modern biotechnology,
introducing protein engineering and directed evolution, have further
revolutionized the development of industrial enzyme.
The development of enzymes
Usage in the industry






The majority of currently used industrial enzymes are
hydrolytic in action, being used for the degradation of various
natural substances.
Proteases remain the dominant enzyme type, because of
their extensive use in the detergent and dairy industries.
Various carbohydrases, primarily amylases and cellulases,
used in industries such as the starch, textile, detergent and
baking industries, represent the second largest group.
The technical industries, dominated by the detergent, starch,
textile and fuel alcohol industries, account for the major
consumption of industrial enzymes.
Overall, the estimated value of the worldwide use of industrial
enzymes has grown from $1 billion in 1995 to $1.5 billion in
2000.
The fastest growth over the past decade has been seen in the
baking and animal feed industries.
Application of Enzymes in Various
industries

Excel sheet
Use in the detergent Industry
Their use as detergent additives still represents the
largest application of industrial enzymes, both in
terms of volume and value.
 The major component is proteases, but other and
very different hydrolases are introduced to provide
various benefits, such as the efficient removal of
specific stains .
 To save energy, the temperature used in household
laundering and automated dishwashers has been
reduced in recent years.
 This often results in problems with efficient
cleaning and stain removal that enzyme technology
can help overcome.







Recent examples of second-generation detergent enzymes include
the development of novel amylases that have enhanced activity at
lower temperatures and alkaline pH, while maintaining the necessary
stability under detergent conditions.
These enzymes were developed by the combined use of microbial
screening and rational protein engineering.
Proteases displaying activity at low temperatures have been isolated
from nature, but have also been evolved in the laboratory.
Furthermore, from a starting material of 26 subtilisin proteases
Ness and coworkers utilized one round of DNA shuffling to isolate
new proteases with various improved properties. The improvements
included characteristics very relevant for detergent proteases
(i.e. improved activity and stability at alkaline pH).
The most recent introduction of a new enzyme class into a
detergent has been the addition of a mannanase — the result of a
joint development between Procter and Gamble and Novozymes.
This enzyme helps remove various food stains containing guar gum,
a commonly used stabilizer and thickening agent in food products.
Enzymes for starch conversion





The enzymatic conversion of starch to high fructose corn syrup is a
well-established process and provides a beautiful example of a
bioprocess in which the consecutive use of several enzymes is
necessary.
The first step in the process is the conversion of starch to
oligomaltodextrins by the action of α-amylase. The concomitant
injection of steam puts extreme demands on the thermostability of
the enzyme.
Using traditional α-amylases, the pH has to be adjusted to an
undesirable high level and calcium must be added to stabilize the
enzyme.
New α-amylases with optimized properties, such as enhanced thermal
stability, acid tolerance, and ability to function without the addition of
calcium, have recently been developed and offering obvious benefits to
the industry.
Engineering efforts have also been undertaken to develop improved
versions of the enzymes used later in the process (i.e. glucoamylase
and glucose isomerase).
Fuel alcohol production
In the alcohol industry, the use of enzymes for the production
of fermentable sugars from starch is also well established.
 Over the past decade, there has been an increasing interest in
fuel alcohol as a result of increased environmental concern,
higher crude oil prices.
 Therefore, intense efforts are currently being undertaken to
develop improved enzymes that can enable the utilization of
cheaper and partially utilized substrates such as lignocellulose,
to make bio-ethanol more competitive with fossil fuels.
 The cost of enzymes needed to turn lignocellulose into a
suitable fermentation feed-stock is a major issue, and current
work focuses both on the development of enzymes with
increased activity and stability as well as on their efficient
production.

Application in textile industry
Application in the feed industry
The use of enzymes as feed additives is also well
established.
 For example, xylanases and β-glucanases have been
used throughout the past decade in cereal-based feed
for monogastric animals which, contrary to ruminants,
are unable to fully degrade and utilize plant-based feeds
containing high amounts of cellulose and hemicellulose.
 During recent years focus has been on the utilization of
natural phosphorus bound in phytic acid in cereal-based
feed for monogastrics. Better utilization of total plant
phosphorus, of which 85–90% is bound in phytic acid, is
only obtained by adding the enzyme phytase to the feed.

Application in the food industry
Much work has been carried out on the
application of transglutaminase as a texturing
agent in the processing of, for example,
sausages, noodles and yoghurt.
 At present only the transglutaminase from
Streptoverticillium sp. is commercially available at
a reasonable scale, and work is ongoing to
increase the availability of the enzyme by
recombinant production in Escherichia coli.






Within the baking industry there is an increasing focus on lipolytic
enzymes.
Recent findings suggest that (phospho)lipases can be used to
substitute or supplement traditional emulsifiers, as the enzymes
degrade polar wheat lipids to produce emulsifying lipids in situ.
Studies have confirmed previous findings showing that water-binding
capacity and retention in the starch and hemicellulose fractions of
the bread, being the substrates of α-amylases and xylanases,
respectively, to be critical for maintaining softness and elasticity.
The recently determined three-dimensional structure of the widely
applied amylase for antistaling (NovamylTM) provided further insight
into the mechanism of enzyme action .
This amylase is probably capable of degrading amylopectin to a
degree that prevents re-crystallization after gelatinization, without
completely degrading the amylopectin network which provides the
bread with elasticity.

The use of laccase for clarification of juice
(laccases catalyze the cross-linking of
polyphenols, resulting in an easy removal
of polyphenols by filtration) and for flavor
enhancement in beer are recently
established applications within the
beverage industry.
Enzymes for organic synthesis





Chemical synthesis is an area where the use of enzyme catalysis has
long been seen as having great promise.
At present, however, we are seeing very significant growth in this area
and enzyme-based processes are now, finally, being widely introduced
for the production of a diversity of different chemicals;
one key example is in the production of single-enantiomer
intermediates used in the manufacture of drugs and agrochemicals.
This market is characterized by a very high degree of fragmentation,
as very few enzymes have applicability in a broad range of different
processes.
Recently introduced enzyme-based processes include the use of
lipases for the production of enantiopure alcohols and amides,
nitrilases for the production of enantiopure carboxylic acids, and
acylases for the production of new semisynthetic penicillins. As many
companies are currently at an early stage in the exploitation of
enzyme-based catalysis, many new developments are expected in this
area over the next few years.