Historical background
Most of the
reactions in living organisms are catalyzed by protein molecules called
enzymes. Enzymes can rightly be called the catalytic machinery of
living systems. The real break through of enzymes occurred with the
introduction of microbial proteases into washing powders. The first
commercial bacterial Bacillus protease was marketed in 1959 and major
detergent manufactures started to use it around 1965.
The
industrial enzyme producers sell enzymes for a wide variety of
applications. The estimated value of world market is presently about
US$ 2 billion. Detergents (37%), textiles (12%), starch (11%), baking
(8%) and animal feed (6%) are the main industries, which use about 75%
of industrially produced enzymes.
Enzyme classification
Presently
more than 3000 different enzymes have been isolated and classified. The
enzymes are classified into six major categories based on the nature of
the chemical reaction they catalyze:
1. Oxidoreductases catalyze oxidation or reduction of their substrates.
2. Transferases catalyze group transfer.
3. Hydrolases catalyze bond breakage with the addition of water.
4. Lyases remove groups from their substrates.
5. Isomerases catalyze intramolecular rearrangements.
6. Ligases catalyze the joining of two molecules at the expense of chemical energy.
Only
a limited number of all the known enzymes are commercially available .
More than 75 % of industrial enzymes are hydrolases. Protein-degrading
enzymes constitute about 40 % of all enzyme sales. More than fifty
commercial industrial enzymes are available and their number is
increasing steadily.
Enzyme production
Some enzymes
still extracted from animal and plant tissues. Enzymes such as papain,
bromelain and ficin and other speciallity enzymes like lipoxygenase are
derived from plants and enzymes pepsin and rennin are derived from
animal. Most of the enzymes are produced by microorganisms in submerged
cultures in large reactors called fermentors. The enzyme production
process can be divided into following phases:
1. Selection of an enzyme.
2. Selection of production strain.
3. Construction of an overproducing stain by genetic engineering.
4. Optimization of culture medium and production condition.
5. Optimization of recovery process.
6. 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 the industrial
applications must usually tolerant against various heavy metals and
have no need for cofactors.
Microbial production strains
In
choosing the production strain several aspects have to be considered.
Ideally the enzyme is secreted from the cell. Secondly, the production
host should have a GRAS-status. Thirdly, the organism should be able to
produce high amount of the desired enzyme in a reasonable life time
frame. Most of the industrially used microorganism have been
genetically modified to overproduce the desired activity and not to
produce undesired side activities.
Enzyme production by microbial fermentation
Once
the biological production organism has been genetically engineered to
overproduce the desired products, a production process has to be
developed. The optimization of a fermentation process includes media
composition, cultivation type and process conditions. The large volume
industrial enzymes are produced in 50 -500 m3 fermentors. The
extracellular enzymes are often recovered after cell removal (by vacuum
drum filtration, separators or microfiltration) by ultrafiltration.
Protein engineering
Often
enzymes do not have the desired properties for an industrial
application. One option is find a better enzyme from nature. Another
option is to engineer a commercially available enzyme to be a better
industrial catalyst. Another option is to engineer a commercially
available enzyme to be a better industrial catalyst. Two different
methods are presently available: a random method called directed
evaluation and a protein engineering method called rational design.
Enzyme technology
This
field deals with how are the enzymes used and applied in practical
processes. The simplest way is to use enzymes is to add them into a
process stream where they catalyze the desired reaction and are
gradually inactivated during the process. This happens in many bulk
enzyme applications and the price of the enzymes must be low to take
their use economical.
An alternative way to use enzymes is to
immobilize them so that they can be reused. Enzyme can be immobilized
by using ultra filtration membranes in the reactor system. The large
enzyme molecule cannot pass through the membrane but the small
molecular reaction products can. Many different laboratory methods for
enzyme immobilization based on chemical reaction, entrapment, specific
binding or absorption have been developed.
Large scale Enzyme applications
1] Detergents
Bacterial
proteinases are still the most important detergent enzymes. Lipases
decompose fats into more water-soluble compounds. Amylases are used in
detergents to remove starch based stains.
2] Starch hydrolysis and fructose production
The
use of starch degrading enzymes was the first large scale application
of microbial enzymes in food industry. Mainly two enzymes carry out
conversion of starch to glucose: alpha-amylase and fungal enzymes.
Fructose produced from sucrose as a starting material. Sucrose is split
by invertase into glucose and fructose, fructose separated and
crystallized.
3] Drinks
Enzymes have many applications in
drink industry. Lactase splits milk-sugar lactose into glucose and
galactose. This process is used for milk products that are consumed by
lactose intolerant consumers. Addition of pectinase, xylanase and
cellulase improve the liberation of the juice from pulp. Similarly
enzymes are widely used in wine production.
4] Textiles
The
use of enzymes in textile industry is one of the most rapidly growing
fields in industrial enzymology. The enzymes used in the textile field
are amylases, catalase, and lactases which are used to remove the
starch, degrade excess hydrogen peroxide, bleach textiles and degrade
lignin.
5] Animal feed
Addition of xylanase to wheat-based
broiler feed has increased the available metabolizable energy 7-10% in
various studies. Enzyme addition reduces viscosity, which increases
absorption of nutrients, liberates nutrients either by hydrolysis of
non-degradable fibers or by liberating nutrients blocked by these
fibers, and reduces the amount of faeces.
6] Baking
Alpha-amylases
have been most widely studied in connection with improved bread quality
and increased shelf life. Use of xylanases decreases the water
absorption and thus reduces the amount of added water needed in baking.
This leads to more stable dough. Proteinases can be added to improve
dough-handling properties; glucose oxidase has been used to replace
chemical oxidants and lipases to strengthen gluten, which leads to more
stable dough and better bread quality.
7] Pulp and Paper
The
major application is the use of xylanases in pulp bleaching. This
reduces considerably the need for chlorine based bleaching chemicals.
In paper making amylase enzymes are used especially in modification of
starch. Pitch is a sticky substance present mainly in softwoods. Pitch
causes problems in paper machines and can be removed by lipases.
8] Leather
Leather
industry uses proteolytic and lipolytic enzymes in leather processing.
Enzymes are used to remove unwanted parts. In dehairing and dewooling
phases bacterial proteases enzymes are used to assist the alkaline
chemical process. This results in a more environmentally friendly
process and improves the quality of the leather . Bacterial and fungal
enzymes are used to make the leather soft and easier to dye.
9] Speciality enzymes
There
are a large number of specialty applications for enzymes. These include
use of enzymes in analytical applications, flavour production, protein
modification, and personal care products, DNA-technology and in fine
chemical production.
10] Enzymes in analytics
Enzymes are
widely used in the clinical analytical methodology. Contrary to bulk
industrial enzymes these enzymes need to be free from side activities.
This means that elaborate purification processes are needed.
An
important development in analytical chemistry is biosensors. The most
widely used application is a glucose biosensor involving glucose
oxidase catalysed reaction.
Several commercial instruments are
available which apply this principle for measurement of molecules like
glucose, lactate, lactose, sucrose, ethanol, methanol, cholesterol and
some amino acids.
11] Enzymes in personal care products
Personal
care products are a relatively new area for enzymes. Proteinase and
lipase containing enzyme solutions are used for contact lens cleaning.
Hydrogen peroxide is used in disinfections of contact lenses. The
residual hydrogen peroxide after disinfections can be removed by
catalase enzyme. Some toothpaste contains glucoamylase and glucose
oxidase. Enzymes are also studied for applications in skin and hair
care products.
12] Enzymes in DNA-technology
DNA-technology
is an important tool in enzyme industry. Most traditional enzymes are
produced by organisms, which have been genetically modified to
overproduce the desired enzyme. The specific order of the organic bases
in the chain of DNA constitutes the genetic language. Genetic
engineering means reading and modifying this language. Enzymes are
crucial tools in this process.
13] Enzymes in fine chemical production
In
spite of some successes, commercial production of chemicals by living
cells using pathway engineering is still in many cases the best
alternative to apply biocatalysis. Isolated enzymes have, however, been
successfully used in fine chemical synthesis. Some of the most
important examples are represented here.
13 A] Chirally pure amino acids and aspartame
Natural
amino acids are usually produced by microbial fermentation. Novel
enzymatic resolution methods have been developed for the production of
L- as well as for D-amino acids. Aspartame, the intensive non-calorie
sweetener, is synthesized in non-aqueous conditions by thermolysin, a
proteolytic enzyme.
13 B] Rare sugars
Recently enzymatic
methods have been developed to manufacture practically all D- and
L-forms of simple sugars. Glucose isomerase is one of the important
industrial enzymes used in fructose manufacturing.
13 C] Semisynthetic penicillins
Penicillin
is produced by genetically modified strains of Penicillium strains.
Most of the penicillin is converted by immobilised acylase enzyme to
6-aminopenicillanic acid, which serves as a backbone for many
semisynthetic penicillins.
13 D] Lipase based reactions
In
addition to detergent applications lipases can be used in versatile
chemical reactions since they are active in organic solvents. Lipases
used in transesterification and also used for enantiomeric separation
of alcohols and separate racemic amine mixtures. Lipases have also been
used to form aromatic and aliphatic polymers.
13 E] Enzymatic oligosaccharide synthesis
The
chemical synthesis of oligosaccharides is a complicated multi-step
effort. Biocatalytic syntheses with isolated enzymes like
glycosyltransferases and glycosidases or engineered whole cells are
powerful alternatives to chemical methods. Oligosaccharides have found
applications in cosmetics, medicines and as functional foods.
Future trends in industrial enzymology
Industrial
enzyme market is growing steadily. The reason for this lies in improved
production efficiency resulting in cheaper enzymes, in new application
fields. Tailoring enzymes for specific applications will be a future
trend with continuously improving tools and understanding of
structure-function relationships and increased search for enzymes from
exotic environments.
New technical tools to use enzymes as
crystalline catalysts, ability to recycle cofactors, and engineering
enzymes to function in various solvents with multiple activities are
important technological developments, which will steadily create new
applications.