Enzyme Catalysis

For centuries enzymes have been used in fermentation reactions to

produce bread, alcoholic beverages, yogurt, cheese, and vinegar long
before their properties and structures were understood.

Historically, enzymes were defined as large macromolecular polypeptides

synthesized by living organisms with molecular weights of 104-106;
recently, this definition has been expanded to include ribozymes
(composed of RNA).
Enzyme Catalysis
Enzymes are proteins that consist of many amino acids coupled to each
other by peptide bonds. The rather small enzyme insulin, for example,
consists of 51 amino acids.

The chain of amino acids folds into a defined 3D conformation, which is

known in detail for many enzymes. Somewhere in this body is a
functional group, e.g. a carboxyl or an amine, which acts as the active
site.
Enzyme Catalysis
Amino acids contain two active groups, namely a carboxylic (COOH) and
an amino (NH2) group.
Enzyme Catalysis
Twenty different amino acids are known. They combine to give proteins
by forming an amide or peptide bond between the carbon from COOH
and nitrogen from NH2.
Enzyme Catalysis
Each enzyme has a unique three-dimensional structure with a binding
site or pocket that is chemically and geometrically compatible with a
single reactant molecule (substrate) or group of chemically related
reactants-in other words, enzymes have molecular-recognition
capability.
Enzyme Catalysis
Enzymes are unique in their ability to catalyze biochemical reactions with
high selectivity (essentially 100%) at extraordinarily high turnover rates
(TOFs), i.e. 10-10,000 molecules/enzyme-s compared to typical TOFs of
1-10 s-1 or less for conventional heterogeneous catalysts.

These activities enable enzymes to be effective catalysts at extremely

low concentrations, e.g. 10-5 to 10-10 mol/L. Typical substrate (reactant)
concentrations are greater than 10-6 mol/L.
Enzyme Catalysis
Enzymes are unique in their ability to catalyze biochemical reactions with
high selectivity (essentially 100%) at extraordinarily high turnover rates
(TOFs), i.e. 10-10,000 molecules/enzyme-s compared to typical TOFs of
1-10 s-1 or less for conventional heterogeneous catalysts.

These activities enable enzymes to be effective catalysts at extremely

low concentrations, e.g. 10-5 to 10-10 mol/L. Typical substrate (reactant)
concentrations are greater than 10-6 mol/L.
Enzyme Catalysis
Enzymes are typically named according to the reactions, which they
catalyze, with the suffix ase denoting enzymatic action. For example,
the enzyme urease catalyzes the hydrolysis of urea:
Enzyme Catalysis
Enzymes are classified into according to six different groups based on the
type of reaction they catalyze. General enzyme classes include
oxidoreductases, transferases, hydrolases, lyases, isomerases, and
ligases for oxidation-reduction, functional group transfer, hydrolysis,
addition to or formation of double bonds, isomerization and bond
formation, respectively.

Each class is subdivided to the point that individual enzymes are

identified by a six-figure code.
Enzyme Catalysis
Enzyme Catalysis
Enzyme Catalysis
The high activity of enzymes is illustrated by the following data for
urease:

The activation energy for acid-catalyzed hydrolysis of urea is 104 kJ/mol,

while the activation energy for the urease-catalyzed reaction is 29
kJ/mol.

At equal concentrations, urease is about 1012 times more active than acid
catalysts.
Enzyme Catalysis
In the decomposition of hydrogen peroxide:

The activation energies for the uncatalyzed, Pt-catalyzed, and catalase-

catalyzed reactions are 75.4, 50.2, and 8.4 kJ/mol, while relative reaction
rates are 1, 2 x 104, and 3 x 1011, respectively.
Enzyme Catalysis
The stereochemical specificity of
enzymes is unmatched and
absolute, i.e. most enzymes are
only active for a single reaction to
produce a single stereoisomer.

Essentially, their sites can

distinguish between optical and
geometrical isomers, almost always
catalyzing only the reaction of one
isomer of an enantiomeric pair.
Enzyme Catalysis
Some enzymes catalyze reactions of chemically unrelated species; for
example, nitrogenase reduces N2 to NH3 as well as hydrogenating
acetylene to ethylene.
Enzyme Catalysis
In 1976 there were 1800 known enzymes, and new enzymes were
being discovered at the rate of about 60 per year.

There are currently over 3000 enzymes that have been functionally
characterized from the more than 7000 predicted to exist (Jaeger,
2004).
Enzyme Catalysis
The application of enzymes in industrial processes has rapidly increased in the
last 20 years, leading to:

Significant cost, materials and energy savings (up to 90% over traditional
processes)

More environmentally friendly processes

Significant simplification of difficult synthetic routes for pharmaceutical

processes and fine chemical synthesis where the production of
enantiomerically pure stereoisomers is required
(e.g. for amino acids, alcohols, organic acids and epoxides)
Catalytic characteristics of enzymes
The Catalytic characteristics include:

Their flexible structure, which facilitates an induced fit of the substrate, the
making and breaking of bonds, and the departure of products.

Their sensitivity to reaction effectors (inhibitors or activators), which

function similarly to the promoters added to heterogeneous catalysts.

Some enzymes require a cofactor, which combines with the enzyme to form a
catalytic site; metal ions are examples of cofactors.
Catalytic characteristics of enzymes

Enzymes generally function only under mild conditions of temperature

and pH observed in living organisms.

If exposed to severe conditions of temperature and pH, they undergo

denaturation, i.e. loss or modification of functional groups or amino acid
residues and/or changes in conformation, which can alter and
deactivate the active site.
Catalytic characteristics of enzymes
The activity of a typical enzyme increases exponentially with
temperature in accordance with the Arrhenius law up to about 50-60C,
where it passes through a maximum, and declines precipitously
between about 60-70C.

Thus catalyst life may be on the order of days to weeks at around 50C;
however, the deactivation rate is extremely high at only slightly higher
temperatures (for example, a 50% loss of activity in 5 min at 65-70C is
typical).
Catalytic characteristics of enzymes
Some enzymes are active and stable at temperatures exceeding 100C;
for example, -amylase catalyzes starch liquefaction at 105-115C.
Because their deactivation rates are highly temperature dependent,
enzymes are generally shipped and stored under refrigeration (0-4C);
at these low temperatures they are generally stable for months.
Catalytic characteristics of enzymes
Under typical commercial reaction conditions (40-60C, 1 atm) enzymes in
solution may deactivate rapidly; moreover, their separation from the product
is generally difficult and expensive or causes denaturation and loss of
catalytic activity.

Nevertheless, catalyst stability can be greatly improved and recovery

problems obviated by immobilizing (heterogenizing) enzymes on inert
supports.

Immobilization enables the catalytic process to be run continuously using a

reactor of substantially lower volume, thereby substantially reducing capital
and operating costs..
Enzymatic Processes and Biotechnology
Historically, biocatalysts have been used over many hundreds of years
in the manufacture of dairy products, bread and pastry products, and
alcoholic beverages, albeit on a limited scale.

However, in the past three decades they have begun to be widely

integrated into industrial practice accompanied by the emergence of a
substantial, growing biotechnology industry in just the past 15-20
years.
Enzymatic Processes and Biotechnology
This new industry is the result of several key scientific and engineering
developments:

The discovery and characterization of large numbers of enzymes that

catalyze industrially useful reactions has increased the number of
accessible reactions.

The development of techniques to engineer these enzymes in order

to optimize their performance and stability have significantly
increased the number of economically feasible industrial applications.
Enzymatic Processes and Biotechnology
Improved (cheaper) methods of enzyme production, isolation, and
immobilization have resulted in improved process economics.

The development of non-aqueous enzymatic processes has expanded

the number of products, reactants, and reactions that can be used.

Improvements in reactor technology have facilitated more precise

control of enzymatic processes
Enzymatic Processes and Biotechnology

Despite these improvements, limitations in enzyme availability,

substrate scope, and operational stability continue to hold back the
integration of enzymatic processes into industrial application
Some Industrially Important Enzymes, Sources and Applications
Some Industrially Important Enzymes, Sources and Applications
Sources and availability of enzymes
Enzymes can be. isolated from bacteria, molds, yeasts, plants and
animals. The pancreas, liver and blood of animals are particularly rich
sources and historically were the primary sources of enzymes.

However, significant, recent advancements in genetic engineering have

revolutionized enzyme production and isolation for industrial use. The
use of designer bugs, or genetically engineered (modified)
microorganisms to produce large amounts of the desired enzyme(s) is
practiced widely.
Sources and availability of enzymes
The purity required for a free enzyme depends on its application,
most large applications requiring only mildly pure enzymes, while
pharmaceutical, medical, and laboratory applications require high
purity.
In isolating and purifying the desired enzyme from its biological
sources, care must be taken to retain the native conformation of the
enzyme. Thus most isolation and purification procedures are
relatively mild in nature.
Techniques such as mechanical disruption are used to break open the
cells, and crystallization or chromatography are typically used to
isolate and purify the enzymes.
Enzyme engineering
Engineered enzymes presently account for roughly 90% of enzymes
used commercially.

Most generally, changes are made in the amino acids at active sites,
leading to improved activity and/or selectivity.

This tailoring of the enzyme can be done in many ways, including

random gene mutation, rational protein engineering, and directed
enzyme evolution.
Limitations to industrial applications of
enzymatic processes
Enzyme availability is an issue, given the small quantities of enzymes
commercially available and their high cost per unit weight, which
limits economic feasibility for many potential reactions.

The scope of available enzyme reactions and substrates is limited,

even with the discovery of many new enzymes and the development
of non-aqueous processes; this is especially a problem for reactions
involving the formation of carbon-carbon bonds.
Limitations to industrial applications of
enzymatic processes

Efficient reactor technology and operational stability continue to

limit the use of biocatalysts. That is, product quality control, enzyme
lifetime, and downstream product separation, which require complex
reactors and separation schemes, depend on highly complex variables
that are difficult to design for and control.

While the use of designer bugs has improved enzyme availability

and should continue to do so, there is still room for improvement in
enzyme isolation and purification
Limitations to industrial applications of
enzymatic processes

Lack of experience in the biocatalysis industry in the application of

reactor technology and process control may be the most critical
limiting factor in the development of economical enzymatic
processes.While improvements in enzyme engineering, biocatalyst
immobilization techniques, and enzyme chemistry are on the
increase, improvements in reactor and separation technologies for
biocatalytic applications have lagged
Limitations to industrial applications of
enzymatic processes

Further substantial improvements are needed in process modeling

and systems engineering of biocatalyst, reactor, and process to
facilitate greater stability and lifetime under industrially relevant
conditions
The Future of Enzyme Catalysis
It is evident that enzymes have substantial advantages relative to
conventional catalysts in catalytic processes involving reactions of
complex organic molecules requiring high specificity or stereospecificity
for reactants.

Because of their high selectivities (often 99.5+%), biocatalytic

processes are the essence of green chemistry.
The Future of Enzyme Catalysis
Green Chemistry:

Conventional catalysts can be replaced with enzymes of higher

selectivity in order to avoid costly product/side-product separations
and disposal of undesirable waste products.

Enzymatic processes are also less energy intensive and often

sustainable.
The Future of Enzyme Catalysis
Production of fine chemicals:
Enzymes are beginning to find application in the production of fine
chemicals, i.e., in unusual environments to catalyze reactions of
unnatural substrates.

Biocatalytic processes are being developed or have already been

developed for production of alcohols, aldehydes, amines, amino acids
(, , and racemic mixtures), epoxides, organic acids, peroxides, and
sugars, including glucose.
The Future of Enzyme Catalysis
Enzyme engineering: to improve the activity, selectivity, and thermal
stability of enzymes.

Aplication of enzymes in two-phase separation systems: Examples are

the application of enzymes in organic solvents and membrane systems
to processes in which traditional separation techniques are ineffective
and where the unique selectivity of enzymes is required, such as low-
cost separations of organic acids and alcohols.
The Future of Enzyme Catalysis
Development of new enzyme systems that enable breakthrough
technologies: Research is ongoing to utilize biocatalysts for the
transformation of many types of biomass to liquid fuels such as
bioethanol and biodiesel.

Development of artificial enzymes: Recent developments have shown

that it is possible to design and synthesize new catalysts based on the
active site of a specific enzyme. (e.g. an artificially synthesized inorganic
iron-sulfur complex, similar to the catalytic center of iron-only
hydrogenase enzyme)
The Future of Enzyme Catalysis
Using of new approaches to stabilize or immobilize biocatalysts:
Stabilization by use of additives (metals, salts, substrates, sugars, and
antioxidants).
Chemical modifications such as amino acid modification, cross-
linking, and immobilization.
Genetic manipulation.
Isolation of enzymes from thermophiles (high-temperature
microorganisms)
Immobilization of plant cells, mammalian cells and organelles.
Coimmobilization of enzymes and cells, coupling of enzymes to living
cells, or combinations ofimmobilization with other biocatalytic
technologies
The Future of Enzyme Catalysis

Improvements in process technology and control:

Improved reactor and process equipment technologies are needed to
improve catalyst life, throughput, and process economics.