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Future prospects of enzyme engineering and enzyme technology

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        <h2><strong>Future prospects of enzyme engineering</strong></h2>
<p>Enzyme engineering is the recent technology growing rapidly due to its
higher application in a lot of fields and due to having bright and clear
future vision. A most exciting development over the last few years is the
application of genetic engineering techniques to enzyme technology. There
are a number of properties which may be improved or altered by genetic
engineering including the yield and kinetics of the enzyme, the ease of
downstream processing and various safety aspects. Enzymes from dangerous
or unapproved microorganisms and from slow-growing or limited plant or
animal tissue may be cloned into safe high-production microorganisms. The
amount of enzyme produced by a microorganism may be increased by
increasing the number of gene copies that code for it. For example; The
engineered cells, aided by the plasmid amplification at around 50 copies
per cell, produce penicillin – G – Amidase constitutively and in
considerably higher quantities than does the fully induced parental
strain. Such increased yields are economically relevant not just for the
increased volumetric productivity but also because of reduced downstream
processing costs, the resulting crude enzyme being that much purer. New
enzyme structures may be designed and produced in order to improve on
existing enzymes or create new activities. Much protein engineering has
been directed at Subtilisin (from Bacillus amyloliquefaciens), the
principal enzyme in the detergent enzyme preparation, Alcalase. This has
been aimed at the improvement of its activity in detergents by
stabilizing it at even higher temperatures, pH and oxidant strength. A
number of possibilities now exist for the construction of artificial
enzymes. These are generally synthetic polymers or oligomers with enzyme-
like activities, often called synzymes. Enzymes can be immobilized i.e.,
an enzyme can be linked to an inert support material without loss of
activity which facilitates reuse and recycling of the enzyme.Use of
engineered enzyme to form biosensor for the analytical use is also recent
activity among the developed countries. Some enzymes make use in diseases
diagnosis so they can be genetically engineered to make the task easier.
Thus it is obvious that there is huge scope of the enzyme technology in
the future as well as in present.</p>
<p><strong>Introduction</strong></p> <p>Enzymes are Organic compounds,
produced in the living cells to speed up chemical reaction in the
biological systems so that they can take place at relatively lower
temperature, but themselves remain apparently unchanged during the
process. Therefore enzymes are termed as biocatalysts. Biocatalysts are
either proteins (<em>enzymes</em>) or, in a few cases, they may be
nucleic acids (<em>ribozymes</em>; some RNA molecules can catalyze the
hydrolysis of RNA. Today, we know that enzymes are necessary in all
living systems, to catalyze all chemical reactions required for their
survival and reproduction – rapidly, selectively and efficiently.
Isolated enzymes can also catalyze these reactions. In the case of
enzymes however, the question whether they can also act as catalysts
outside living systems had been a point of controversy among biochemists
in the beginning of the twentieth century. It was shown at an early stage
however that enzymes could indeed be used as catalysts outside living
cells, and several processes in which they were applied as biocatalysts
have been patented These excellent properties of enzymes are utilized in
enzyme technology. For example, they can be used as biocatalysts to
catalyze chemical reactions on an industrial scale in a sustainable
manner. Their application covers the production of desired products for
all human material needs (e.g., food, animal feed, pharmaceuticals, fine
and bulk chemicals, fibers, hygiene, and environmental technology), as
well as in a wide range of analytical purposes, especially in
diagnostics. In fact, during the past 50 years the rapid increase in our
knowledge of enzymes – as well as their biosynthesis and molecular
biology – now allows their rational use as biocatalysts in many
processes, and in addition their modification and optimization for new
synthetic schemes and the solution of analytical problems</p> <p>Enzymes
have become big business. They are used in many industrial processes to
catalyze biological reactions. Enzymes are exploited in a variety of
manufacturing processes such as food processing and for the synthesis of
medicines such as antibiotics like artificial penicillin. They are also
used to clean up factory effluents and pollution in water and soil. Many
processes can be made faster and cheaper by using the right enzyme and
conditions.</p> <p>Optimum conditions are maintained during factory
production by use of bioreactors. These are vessels which are designed to
provide the ideal environment for reactions involving enzymes or living
organisms. Source of enzymes used commercial production is plant, animal
and microbial cells. Animal enzymes used currently are lipases, tripsin,
rennets etc. Most prevalent plant enzymes are papain, proteases, amylases
and soybean lipoxygenase. These enzymes are used in food industries, for
example, papain extracted from papaya fruit is used as meat tenderizer
and pancreatic protease in leather softening and manufacture of
detergents. In addition microbial enzymes have gained much popularity.
Production of primary and secondary metabolites by microorganism is
possible only due to involvement of various enzymes. They are of two
types: the extracellular and the intracellular enzymes. There is a wide
range of extracellular enzymes produced by pathogenic and saprophytic
microorganisms such as cellulose, polymethylegalactouronase,
pectinmethylesterase etc. These enzyme helps in establishment in host
tissues or decomposition of organic substrates. The intracellular enzyme
like invertase, uricoxidase, asparaginase are of high economic value and
difficult to extract as they produced inside the cell. They can be
extracted by breaking the cells by means of a homogenizer or a ball mill
and extracted them through the biochemical process.</p> <p>Biotechnology
offers an increasing potential for the production of goods to meet
various human needs. In enzyme technology – a sub-field of
biotechnology – new processes have been and are being developed to
manufacture both bulk and high added- value products utilizing enzymes as
biocatalysts, in order to meet needs such as food (e.g., bread, cheese,
beer, vinegar), fine chemicals (e.g., amino acids, vitamins), and
pharmaceuticals. Enzymes are also used to provide services, as in washing
and environmental processes, or for analytical and diagnostic purposes.
The driving force in the development of enzyme technology, both in
academia and industry, has been and will continue to be:</p> <ul> <li>
<p>The development of new and better products, processes and services to
meet these needs; and/or</p> </li> <li> <p>The improvement of processes
to produce existing products from new raw materials as   biomass.</p>
</li> </ul> <p>The goal of these approaches is to design innovative
products and processes that are not only competitive but also meet
criteria of sustainability. A positive effect in all these three fields
is required for a sustainable process. Criteria for the quantitative
evaluation of the economic and environmental impact are in contrast with
the criteria for the social impact, easy to formulate. In order to be
economically and environmentally more sustainable than an existing
processes, a new process must be designed to reduce not only the
consumption of resources (e.g., raw materials, energy, air, water), waste
production and environmental impact, but also to increase the recycling
of waste per kilogram of product.</p> <p><strong>Sources of
enzymes:</strong> Biologically active enzymes may be extracted from any
living organism. A very wide range of sources are used for commercial
enzyme production from <em>Actinoplanes</em> to <em>Zymomonas</em>, from
spinach to snake venom. Of the hundred or so enzymes being used
industrially, over a half are from fungi and yeast and over a third are
from bacteria with the remainder divided between animal (8%) and plant
(4%) sources. A very much larger number of enzymes find use in chemical
analysis and clinical diagnosis. Non-microbial sources provide a larger
proportion of these, at the present time. Microbes are preferred to
plants and animals as sources of enzymes because:</p> <ol> <li> <p>they
are generally cheaper to produce.</p> </li> <li> <p>their enzyme contents
are more predictable and controllable,</p> </li> <li> <p>reliable
supplies of raw material of constant composition are more easily
arranged, and</p> </li> <li> <p>plant and animal tissues contain more
potentially harmful materials than microbes, including phenolic compounds
(from plants), endogenous enzyme inhibitors and proteases.</p> </li>
</ol>   <p><strong>Table 1 </strong>. Some important industrial enzymes
and their sources.</p> <p>Enzyme</p> <p>EC number</p> <p>Source</p>
<p>Intra/extra<br> -cellular</p> <p>Scale of production</p>
<p>Industrial use</p> <p><strong>Animal enzymes</strong></p> <br> <br>
<br> <br> <p>Catalase</p> <p>1.11.1.6</p> <p>Liver</p> <p>I</p> <p>-</p>
<p>Food</p> <p>Chymotrypsin</p> <p>3.4.21.1</p> <p>Pancreas</p> <p>E</p>
<p>-</p> <p>Leather</p> <p>Lipase</p> <p>3.1.1.3</p> <p>Pancreas</p>
<p>E</p> <p>-</p> <p>Food</p> <p>Rennet</p> <p>3.4.23.4</p>
<p>Abomasum</p> <p>E</p> <p>+</p> <p>Cheese</p> <p>Trypsin</p>
<p>3.4.21.4</p> <p>Pancreas</p> <p>E</p> <p>-</p> <p>Leather</p>
<p><strong>Plant enzymes</strong></p> <br> <br> <br> <br>
<p>Actinidin</p> <p>3.4.22.14</p> <p>Kiwi fruit</p> <p>E</p> <p>-</p>
<p>Food</p> <p>a-Amylase</p> <p>3.2.1.1</p> <p>Malted barley</p> <p>E</p>
<p>+++</p> <p>Brewing</p> <p>b-Amylase</p> <p>3.2.1.2</p> <p>Malted
barley</p> <p>E</p> <p>+++</p> <p>Brewing</p> <p>Bromelain</p>
<p>3.4.22.4</p> <p>Pineapple latex</p> <p>E</p> <p>-</p> <p>Brewing</p>
<p>b-Glucanase</p> <p>3.2.1.6</p> <p>Malted barley</p> <p>E</p> <p>++</p>
<p>Brewing</p> <p>Ficin</p> <p>3.4.22.3</p> <p>Fig latex</p> <p>E</p>
<p>-</p> <p>Food</p> <p>Lipoxygenase</p> <p>1.13.11.12</p>
<p>Soybeans</p> <p>I</p> <p>-</p> <p>Food</p> <p>Papain</p>
<p>3.4.22.2</p> <p>Pawpaw latex</p> <p>E</p> <p>++</p> <p>Meat</p>
<p><strong>Bacterial enzymes</strong></p> <br> <br> <br> <br> <p>a-
Amylase</p> <p>3.2.1.1</p> <p><em>Bacillus</em></p> <p>E</p> <p>+++</p>
<p>Starch</p> <p>b-Amylase</p> <p>3.2.1.2</p> <p><em>Bacillus</em></p>
<p>E</p> <p>+</p> <p>Starch</p> <p>Asparaginase</p> <p>3.5.1.1</p>
<p><em>Escherichia coli</em></p> <p>I</p> <p>-</p> <p>Health</p>
<p>Glucose isomerase</p> <p>5.3.1.5</p> <p><em>Bacillus</em></p> <p>I</p>
<p>++</p> <p>Fructose syrup</p> <p>Penicillin amidase</p> <p>3.5.1.11</p>
<p><em>Bacillus</em></p> <p>I</p> <p>-</p> <p>Pharmaceutical</p>
<p>Protease</p> <p>3.4.21.14</p> <p><em>Bacillus</em></p> <p>E</p>
<p>+++</p> <p>Detergent</p> <p>Pullulanase</p> <p>3.2.1.41</p>
<p><em>Klebsiella</em></p> <p>E</p> <p>-</p> <p>Starch</p>
<p><strong>Fungal enzymes</strong></p> <br> <br> <br> <br> <p>a-
Amylase</p> <p>3.2.1.1</p> <p><em>Aspergillus</em></p> <p>E</p> <p>++</p>
<p>Baking</p> <p>Aminoacylase</p> <p>3.5.1.14</p>
<p><em>Aspergillus</em></p> <p>I</p> <p>-</p> <p>Pharmaceutical</p>
<p>Glucoamylase</p> <p>3.2.1.3</p> <p><em>Aspergillus</em></p> <p>E</p>
<p>+++</p> <p>Starch</p> <p>Catalase</p> <p>1.11.1.6</p>
<p><em>Aspergillus</em></p> <p>I</p> <p>-</p> <p>Food</p>
<p>Cellulase</p> <p>3.2.1.4</p> <p><em>Trichoderma</em></p> <p>E</p> <p>-
</p> <p>Waste</p> <p>Dextranase</p> <p>3.2.1.11</p>
<p><em>Penicillium</em></p> <p>E</p> <p>-</p> <p>Food</p> <p>Glucose
oxidase</p> <p>1.1.3.4</p> <p><em>Aspergillus</em></p> <p>I</p> <p>-</p>
<p>Food</p> <p>Lactase</p> <p>3.2.1.23</p> <p><em>Aspergillus</em></p>
<p>E</p> <p>-</p> <p>Dairy</p> <p>Lipase</p> <p>3.1.1.3</p>
<p><em>Rhizopus</em></p> <p>E</p> <p>-</p> <p>Food</p> <p>Rennet</p>
<p>3.4.23.6</p> <p><em>Mucor miehei</em></p> <p>E</p> <p>++</p>
<p>Cheese</p> <p>Pectinase</p> <p>3.2.1.15</p>
<p><em>Aspergillus</em></p> <p>E</p> <p>++</p> <p>Drinks</p> <p>Pectin
lyase</p> <p>4.2.2.10</p> <p><em>Aspergillus</em></p> <p>E</p> <p>-</p>
<p>Drinks</p> <p>Protease</p> <p>3.4.23.6</p> <p><em>Aspergillus</em></p>
<p>E</p> <p>+</p> <p>Baking</p> <p>Raffinase</p> <p>3.2.1.22</p>
<p><em>Mortierella</em></p> <p>I</p> <p>-</p> <p>Food</p>
<p><strong>Yeast enzymes</strong></p> <br> <br> <br> <br>
<p>Invertase</p> <p>3.2.1.26</p> <p><em>Saccharomyces</em></p> <p>I/E</p>
<p>-</p> <p>Confectionery</p> <p>Lactase</p> <p>3.2.1.23</p>
<p><em>Kluyveromyces</em></p> <p>I/E</p> <p>-</p> <p>Dairy</p>
<p>Lipase</p> <p>3.1.1.3</p> <p><em>Candida</em></p> <p>E</p> <p>-</p>
<p>Food</p> <p>Raffinase</p> <p>3.2.1.22</p>
<p><em>Saccharomyces</em></p> <p>I</p> <p>-</p> <p>Food</p> <br> <br>
<br> <br> <br> <br> <br> <br> <br> <br> <br> <br> <br> <br> <br> <br>
<p>Once the enzyme has been purified to the desired extent and
concentrated, the manufacturer's main objective is to retain the
activity. Enzymes for industrial use are sold on the basis of overall
activity. To achieve stability, the manufacturer should follow the recent
advanced technology even genetic engineering thechniques.Most industrial
enzymes contain relatively little active enzyme (&lt; 10% w/w, including
isoenzymes and associated enzyme activities), the rest being due to
inactive protein, stabilisers, preservatives, salts and the diluent which
allows standardisation between production batches of different specific
activities.The key to maintaining enzyme activity is maintenance of
conformation, so preventing unfolding, aggregation and changes in the
covalent structure. Three approaches are possible: use of additives, the
controlled use of covalent modification, and enzyme immobilization. So if
the genetic engineering along with the advanced technique for enzyme
engineering are employed there might be the great possibility of
increasing the half life of active protein and their stability as well as
specificity which will certainly reduce conventional methods for
stabilizing the enzymes.</p> <p><strong>Screening for novel
enzymes:</strong> One of the major skills of enzyme companies and
suitably funded academic laboratories is the rapid and cost-effective
screening of microbial cultures for enzyme activities. Natural samples,
usually soil or compost material found near high concentrations of likely
substrates, are used as sources of cultures.</p> <p><strong>Preparation
of enzymes:</strong> After the screening of the novel enzyme having great
commercial as well as industrial use, enzyme is prepared by optimizing
the condition of higher production with available resources. Purification
of enzyme after preparation depends upon its future use. Often the enzyme
may be purified several hundred-fold but the yield of the enzyme may be
very poor, frequently below 10% of the activity of the original material.
In contrast, industrial enzymes will be purified as little as possible,
only other enzymes and material likely to interfere with the process
which the enzyme is to catalyze, will be removed.
                        <strong>Fig.1</strong>
Flow diagram for the preparation of enzymes.</p> <p><strong>Genetic
Protein Engineering of Enzymes</strong></p> <p><strong><br> </strong>A
most exciting development over the last few years is the application of
genetic engineering techniques to enzyme technology. Recombinant DNA
technology has allowed the transfer of useful enzyme genes from one
organism to another. Thus, when an enzyme has been identified as a good
candidate enzyme for industrial use, the relevant gene can be cloned into
a more suitable production host microorganism and an industrial
fermentation carried out. In this way, it becomes possible to produce
industrial enzymes of very high quality and purity. A recent example of
this technology is the detergent enzyme Lipolase produced by Novo Nordisk
A/S, which has improved removal of fat stains in fabrics. The enzyme was
first identified in the fungus Humicola languinosa at levels
inappropriate for commercial production. The gene DNA fragment for the
enzyme was cloned into the fungus Aspergillus oryzae and commercial
levels of enzyme achieved. The enzyme has proved to be efficient under
many wash conditions. The enzyme is also very stable at a variety of
temperature and pH conditions relevant to washing.</p> <p>There are a
number of properties which may be improved or altered by genetic
engineering including the yield and kinetics of the enzyme, the ease of
downstream processing and various safety aspects. Enzymes from dangerous
or unapproved microorganisms and from slow-growing or limited plant or
animal tissue may be cloned into safe high-production microorganisms.</p>
<p>All proteins, including enzymes, are based on the same 20 different
amino acid building blocks arranged in different sequences. Enzyme
proteins typically comprise sequences of several</p> <p>hundred amino
acids folded in a unique three-dimensional structure. Only the sequence
of these 20 building blocks determines the three-dimensional structure,
which in turn determines all properties such as catalytic activity,
specificity and stability. Nature has been performing ‘protein
engineering’ for billions of years since the very start of evolution.
Natural spontaneous mutations in the DNA coding for a given protein
result in changes of the protein structure and hence its properties. This
natural variation is part of the adaptive evolutionary process
continuously taking place in all living organisms, allowing them to
survive in continuously changing environments. Natural variants of enzyme
proteins are adapted to perform efficiently in different environments and
conditions. This explains why in nature enzymes belonging to the same
enzyme family but isolated from different organisms and environments
often show a variation in amino acid sequence of more than 50%. The
properties of enzymes used for industrial purposes sometimes also require
some adaptations in order to function more effectively in applications
for which they were not designed by nature. Traditionally, such enzyme
optimization is performed by screening naturally occurring
microorganisms, followed by classical mutation and selection. The
disadvantage of this method is, however, that it may take a very long
time until the enzyme with the desired properties is found. This is why
protein engineering was developed.<strong> </strong></p>
<p><strong>Assumptions for Protein Engineering</strong></p> <p>While
attempting protein engineering, one should recognize the following
properties of enzymes: <br> <br> (i) many amino acid substitutions,
deletions or additions lead to no change in enzyme activity, so that they
are silent mutations; <br> <br> (ii) proteins have a limited number of
basic structures and only minor changes are superimposed on them leading
to variation;<br> <br> (iii) similar patterns of chain folding and domain
structure can arise from different amino acid sequences, which show
little or no homology (although same amino acid sequence never gives
different folding and domain structures).</p> <p>The above properties
suggest that while many major changes sometimes may lead to no alteration
in function, some of the minor changes at specific positions may lead to
the desired favourable change.<br> <br> For example, a single amino acid
replacement (glycine to aspartic acid) in E. coli asparate
transcarbamylase leads to <br> <br> (i) loss of activity and to <br> <br>
(ii) an alteration in the binding of catalytic and regulatory subunits.
Another example involved the engineering of a single chain biosynthetic
antibody binding site (BARS), which is though only one sixth of the size
of the complete antibody, but retains its antigen binding
specificity.</p> <p>This synthetic fragment has heavy and light chain
variable regions (V H and V J connected by a 15 - amino acid linker. A
synthetic gene has also been prepared for the fragment, which expressed
in E. coli. This fragment binds to digoxin, a cradiac glycoside. Single
amino acid replacements in BABS fragment have sometimes led to major
changes in its binding affinity.<br> <br> In view of the above, it is
necessary to examine not only the crystal structure but also the active
sites therein, so that the gene may be modified or artificially
synthesized for protein engineering to meet the desired needs.</p>
<p><strong>Methods for Protein Engineering</strong> –</p> <p>A variety
of methods have been used and proposed for future use in protein
engineering. In this connection mutagenesis, selection, and recombinant
DNA are being used and will be increasingly utilized in future.</p>
<p><strong>1. Mutagenesis and Selection for Protein Engineering</strong>
- Mutagenesis and selection can be effectively utilized for improving a
specific property of an enzyme. Following are some of the examples of
selection of mutant enzymes:<br> <br> (i) E. coli anthranilate synthetase
enzyme is normally sensitive to tryptophan inhibition due to feedback
inhibition. An MTR 2 mutation of E. coli was found to possess an altered
form of enzyme anthranilate synthetase that is insensitive to tryptophan
inhibition. They may help in continuous synthesis of tryptophan without
any inhibition by tryptophan accumulated as a product.<br> <br> (ii)
Xanthine dehydrogenase enzyme oxidizes 2 hydroxy-purine at position 8,
but a mutant has been inolated which oxidizes 2 hydroxy-purine at
position 6.</p> <p>(iii) Lactate dehydrogenase (LDU) from a bacterial
system was modified to malate dehydrogenase able a natural mutation
leading to a single amino acid substitution (Gln 02... Arg; see later m
thIS chapter).<br> <br> In the above and other cases of naturally
occurring mutant enzymes, single amino acid modification or
addition/deletion has been observed.<br> <br> However, if improvement
requires changes in several amino acids, such a mutant will be rare or
nonexistent and modifications of this type will be possible only through
gene modification techniques discussed in the following section.</p>
<p><strong>2. Production of Artificial Semi Synthetic Oxido Reductases -
Flavo Enzymes</strong> - Artificial oxido reductases can be prepared by
covalently attaching redoxactive prosthetic groups to existing sites.
Linking of 10-methyilsoalloxazine derivatives (as redox-active groups) to
specific sites of several proteins has been achieved. The efficiency of
these semisynthetic enzymes (e.g. flavopapain) compares favourably with
that of naturally occurring flavoenzymes.</p> <p><strong>3. Modification
of Proteases into Peptide Ligases</strong> -Peptide ligation to native
enzymes may lead to high specificity and stereoselecitivity, and may
suppress side reactions. Therefore, synthesis of any enzyme that may
catalyze peptide ligation will be most welcome. <br> <br> Protease
'subtilisin' has been modified (by converting a serine into cysteine or
seleno-cysteine) into thiol-and selenolsubtilisin, the two semi synthetic
enzymes (they are damaged proteases), which can catalyse peptide
ligation. Both these damaged proteases are efficient peptide ligases.
Similarly histidine residue can also be modified to yield peptide
ligases.</p> <p><strong>4. Enzyme PEG Conjugates</strong> - An enzyme L-
asparaginase (isolated from microbes) has antitumour properties, but is
toxic with a life time of less then 18hr thus reducing its utility. It
has been shown that E. coli LÂ--asparaginase can be modified by
polyethylene glycol derivatives to produce PEG-asparaginase conjugates ,
which differ from the native enzyme in following features:<br> (i) it
retains only 52% of the catalytic activity of native enzyme; <br> (ii) it
becomes resistant to proteolytic degradation; (Hi) it does not cause
allergy. In view of this, PEG-asparaginase has been used to treat
malignant murine (mouse), canine (cats, etc.) and human tumours. PEG
conjugates of a large number of enzymes (adenosine deaminase, uricase,
catalase, etc.) have been prepared and will be utilized in industry
also.</p> <p><strong>5. Production of Site Specific Nucleases -
Restriction Enzymes</strong> - The DNA recognition and binding properties
of proteins can be combined using chemical cleavage agents. Cys178 of E.
coli CAP protein; has been modified using 'S-iodoacetamide -1, 10-
phenanthroline' yielding a DNA cleaving agent that recognized and cleaved
DNA at the centre of the recognition site (22 bp) for CAP. <br> <br> This
may give restriction enzymes recognizing upto 20 bases instead of 6 or 8
bases and may, therefore, be useful for isolating long DNA fragments
needed for sequencing and mapping. Nucleases may also be produced by
fusion of non-specific phosphodiesterases to oligonucleotides of defined
sequence. <br> <br> For a nuclease from Staphylococcus modified by this
approach, it was shown that oligonucleotide component of fused product
pairs with its complementary sequence and the hybrid enzyme hydrolyses
single stranded DNA or RNA adjacent to the oligonucleotide binding site.
This approach thus can also be used for developing artificial restriction
enzymes.</p> <p><strong>Protein engineering and how it is applied to
enzymes</strong></p> <p>A most exciting development over the last few
years is the application genetic engineering techniques to enzyme
technology. Protein engineering of enzymes is a faster, more controlled,
more targeted and more accurate method to optimize the properties of
enzymes for a specific industrial application than the traditional method
described above. It makes it possible to sidestep the high number of
natural isolate screenings that would otherwise be necessary to find the
enzyme with the desired properties, and increases the likelihood that a
suitable enzyme will be found. The protein engineering technique involves
genetic modification by means of recombinant DNA technology of the enzyme
producing microorganism, in particular the enzyme encoding gene,
resulting in substitution of one or more amino acids in the amino acid
sequence of the enzyme protein. Strategies for making such amino acid
substitutions and developing protein engineered enzymes are based on the
knowledge of the structure/function relationships of enzymes, computer
modeling and techniques for creating and testing enzyme variants.</p>
<p>Enzyme technology is the application of modifying an enzyme's
structure (and thus its function) or modifying the catalytic activity of
isolated enzymes to produce new metabolites, to allow new (catalyzed)
pathways for reactions to occur, or to convert from some certain
compounds into others (biotransformation). These products will be useful
as chemicals, pharmaceuticals, fuel, food or agricultural additives. An
<em>enzyme reactor</em> consists of a vessel containing a reactional
medium that is used to perform a desired conversion by enzymatic means.
Enzymes used in this process are free in the solution or immobilized in
particulate, membranous or fibrous support. There are many directions in
which enzyme technologists are currently applying their art and which are
at the forefront of biotechnological research and development. Some of
these have already been examined in some detail earlier. At present,
relatively few enzymes are available on a large scale (i.e. &gt; kg) and
are suitable for industrial applications. These shortcomings are being
addressed in a number of ways:</p> <ol> <li> <p>New enzymes are being
sought in the natural environment and by strain selection</p> </li> <li>
<p>Novel enzymes are being designed and produce by genetic
engineering;</p> </li> <li> <p>New organic catalysts are being designed
and synthesized using the 'knowhow' established from enzymology; and</p>
</li> <li> <p>More complex enzyme systems are being utilized.</p> </li>
</ol> <p>Each of these areas has a extensive and rapidly expanding
literature. Some advances possibly belong more properly to other areas of
science. Thus, the development of genetically improved enzymes is
generally undertaken by molecular biologists and the design and synthesis
of novel enzyme-like catalysts is in the provenance of the organic
chemists. Both groups of workers will, however, base their science on
data provided by the enzyme technologist.</p> <p>There are a number of
properties which may be improved or altered by genetic engineering
including the yield and kinetics of the enzyme, the ease of downstream
processing and various safety aspects. Enzymes from dangerous or
unapproved microorganisms and from slow growing or limited plant or
animal tissue may be cloned into safe high-production microorganisms. In
the future, enzymes may be redesigned to fit more appropriately into
industrial processes; for example, making glucose isomerase less
susceptible to inhibition by the Ca2+ present in the starch
saccharification processing stream.</p> <p>The amount of enzyme produced
by a microorganism may be increased by increasing the number of gene
copies that code for it. This principle has been used to increase the
activity of penicillin-G-amidase in <em>Escherichia coli</em>. The
cellular DNA from a producing strain is selectively cleaved by the
restriction endonuclease HindIII. This hydrolyses the DNA at relatively
rare sites containing the 5'-AAGCTT-3' base sequence to give identical
'staggered' ends.</p> <p>[Fig2]<br> intact DNA cleaved DNA</p> <p>The
total DNA is cleaved into about 10000 fragments, only one of which
contains the required genetic information. These fragments are individual
cloned into a cosmid vector and thereby returned to <em>E. coli</em>.
These colonies containing the active gene are identified by their
inhibition of a 6-amino-penicillanic acid-sensitive organism. Such
colonies are isolated and the penicillin-G-amidase gene transferred on to
pBR322 plasmids and recloned back into <em>E. coli</em>. The engineered
cells, aided by the plasmid amplification at around 50 copies per cell,
produce penicillin-G-amidase constitutively and in considerably higher
quantities than does the fully induced parental strain. Such increased
yields are economically relevant not just for the increased volumetric
productivity but also because of reduced downstream processing costs, the
resulting crude enzyme being that much purer.</p> <p>The process starts
with the isolation and characterisation of the required enzyme. This
information is analysed together with the database of known and putative
structural effects of amino acid substitutions to produce a possible
improved structure. This factitious enzyme is constructed by site-
directed mutagenesis, isolated and characterised. The results, successful
or unsuccessful, are added to the database, and the process repeated
until the required result is obtained.</p> <p>Another extremely
promising area of genetic engineering is protein engineering. New enzyme
structures may be designed and produced in order to improve on existing
enzymes or create new activities. An outline of the process of protein
engineering is shown in Figure 2. Such factitious enzymes are produced by
site-directed mutagenesis (Figure 3). Unfortunately from a practical
point of view, much of the research effort in protein engineering has
gone into studies concerning the structure and activity of enzymes chosen
for their theoretical importance or ease of preparation rather than
industrial relevance. This emphasis is likely to change in the
future.<strong> Figure 2</strong>. The protein engineering cycle.</p>
<p>As indicated by the method used for site-directed mutagenesis (Figure
3), the preferred pathway for creating new enzymes is by the stepwise
substitution of only one or two amino acid residues out of the total
protein structure. Although a large database of sequence-structure
correlations is available, and growing rapidly together with the
necessary software, it is presently insufficient accurately to predict
three-dimensional changes as a result of such substitutions. The main
problem is assessing the long-range effects, including solvent
interactions, on the new structure. As the many reported results would
attest, the science is at a stage where it can explain the structural
consequences of amino acid substitutions after they have been determined
but cannot accurately predict them. Protein engineering, therefore, is
presently rather a hit or miss process which may be used with only little
realistic likelihood of immediate success. Apparently quite small
sequence changes may give rise to large conformational alterations and
even affect the rate-determining step in the enzymic catalysis. However
it is reasonable to suppose that, given a sufficiently detailed database
plus suitable software, the relative probability of success will increase
over the coming years and the products of protein engineering will make a
major impact on enzyme technology.</p> <p>Much protein engineering has
been directed at subtilisin (from <em>Bacillus amyloliquefaciens</em>),
the principal enzyme in the detergent enzyme preparation, Alcalase. This
has been aimed at the improvement of its activity in detergents by
stabilising it at even higher temperatures, pH and oxidant strength. Most
of the attempted improvements have concerned alterations to:</p> <ol>
<li> <p>the P1 cleft, which holds the amino acid on the carbonyl side of
the targeted peptide bond;</p> </li> <li> <p>the oxyanion hole
(principally Asn155), which stabilises the tetrahedral intermediate;</p>
</li> <li> <p>the neighbourhood of the catalytic histidyl residue
(His64), which has a general base role; and</p> </li> <li> <p>the
methionine residue (Met222) which causes subtilisin's lability to
oxidation.</p> </li> </ol> <p>It has been found that the effect of a
substitution in the P1 cleft on the relative specific activity between
substrates may be fairly accurately predicted even though predictions of
the absolute effects of such changes are less successful. Many
substitutions, particularly for the glycine residue at the bottom of the
P1 cleft (Gly166), have been found to increase the specificity of the
enzyme for particular peptide links whilst reducing it for others. These
effects are achieved mainly by corresponding changes in the Km rather
than the Vmax. Increases in relative specificity may be useful for some
applications. They should not be thought of as the usual result of
engineering enzymes, however, as native subtilisin is unusual in being
fairly non-specific in its actions, possessing a large hydrophobic
binding site which may be made more specific relatively easily (e.g. by
reducing its size). The inactivation of subtilisin in bleaching solutions
coincides with the conversion of Met222 to its sulfoxide, the
consequential increase in volume occluding the oxyanion hole.
Substitution of this methionine by serine or alanine produces mutants
that are relatively stable, although possessing somewhat reduced
activity.</p>   <p><strong>Figure 3.</strong> An outline of the process
of site-directed mutagenesis, using a hypothetical example. (a) The
primary structure of the enzyme is derived from the DNA sequence. A
putative enzyme primary structure is proposed with an asparagine residue
replacing the serine present in the native enzyme. A short piece of DNA
(the primer), complementary to a section of the gene apart from the base
mismatch, is synthesised. (b) The oligonucleotide primer is annealed to a
single-stranded copy of the gene and is extended with enzymes and
nucleotide triphosphates to give a double-stranded gene. On reproduction,
the gene gives rise to both mutant and wild-type clones. The mutant DNA
may be identified by hybridisation with radioactively labelled
oligonucleotides of complementary structure.</p> <p>An example of the
unpredictable nature of protein engineering is given by trypsin, which
has an active site closely related to that of subtilisin. Substitution of
the negatively charged aspartic acid residue at the bottom of its P1
cleft (Asp189), which is used for binding the basic side-chains of lysine
or arginine, by positively charged lysine gives the predictable result of
abolishing the activity against its normal substrates but unpredictably
also gives no activity against substrates where these basic residues are
replaced by aspartic acid or glutamic acid.</p> <p>Considerable effort
has been spent on engineering more thermophilic enzymes. It has been
found that thermophilic enzymes are generally only 20-30 kJ more stable
than their mesophilic counterparts. This may be achieved by the addition
of just a few extra hydrogen bonds, an internal salt link or extra
internal hydrophobic residues, giving a slightly more hydrophobic core.
All of these changes are small enough to be achieved by protein
engineering. To ensure a more predictable outcome, the secondary
structure of the enzyme must be conserved and this generally restricts
changes in the exterior surface of the enzyme. Suitable for exterior
substitutions for increasing thermostability have been found to be
aspartate , glutamate, lysine , glutamine, valine , threonine, serine ,
asparagine, isoleucine , threonine, asparagine , aspartate and lysine ,
arginine. Such substitutions have a fair probability of success. Where
allowable, small increases in the interior hydrophobicity for example by
substituting interior glycine or serine residues by alanine may also
increase the thermostability. It should be recognised that making an
enzyme more thermostable reduces its overall flexibility and, hence, it
is probable that the factitious enzyme produced will have reduced
catalytic efficiency.</p> <p><strong>Artificial enzymes: </strong></p>
<p>A number of possibilities now exist for the construction of artificial
enzymes. These are generally synthetic polymers or oligomers with enzyme-
like activities, often called synzymes. They must possess two structural
entities, a substrate-binding site and a catalytically effective site. It
has been found that producing the facility for substrate binding is
relatively straightforward but catalytic sites are somewhat more
difficult. Both sites may be designed separately but it appears that, if
the synzyme has a binding site for the reaction transition state, this
often achieves both functions. Synzymes generally obey the saturation
Michaelis-Menten kinetics . For a one-substrate reaction the reaction
sequence is given by</p> <p>synzyme + S (synzyme-S complex) synzyme +
P</p> <p>Some synzymes are simply derivatised proteins, although
covalently immobilised enzymes are not considered here. An example is the
derivatisation of myoglobin, the oxygen carrier in muscle, by attaching
(Ru(NH3)5)3+ to three surface histidine residues. This converts it from
an oxygen carrier to an oxidase, oxidising ascorbic acid whilst reducing
molecular oxygen. The synzyme is almost as effective as natural ascorbate
oxidases.</p> <p>It is impossible to design protein synzymes from scratch
with any probability of success, as their conformations are not presently
predictable from their primary structure. Such proteins will also show
the drawbacks of natural enzymes, being sensitive to denaturation,
oxidation and hydrolysis. For example, polylysine binds anionic dyes but
only 10% as strongly as the natural binding protein, serum albumin, in
spite of the many charges and apolar side-chains. Polyglutamic acid,
however, shows synzymic properties. It acts as an esterase in much the
same fashion as the acid proteases, showing a bell-shaped pH-activity
relationship, with optimum activity at about pH 5.3, and Michaelis-Menten
kinetics with a Km of 2 mm and Vmax of 10-4 to 10-5 s-1 for the
hydrolysis of 4-nitrophenyl acetate. Cyclodextrins (Schardinger dextrins)
are naturally occurring toroidal molecules consisting of six, seven,
eight, nine or ten a-1, 4-linked D-glucose units joined head-to-tail in a
ring (a-, b-, g-, d- and e-cyclodextrins, respectively: they may be
synthesised from starch by the cyclomaltodextrin glucanotransferase (EC
2.4.1.19) from <em>Bacillus macerans</em>). They differ in the diameter
of their cavities (about 0.5-1 nm) but all are about 0.7 nm deep. These
form hydrophobic pockets due to the glycosidic oxygen atoms andÂ
inwards-facing C-H groups. All the C-6 hydroxyl groups project to one end
and all the C-2 and C-3 hydroxyl groups to the other. Their overall
characteristic is hydrophilic, being water soluble, but the presence of
their hydrophobic pocket enables them to bind hydrophobic molecules of
the appropriate size. Synzymic cyclodextrins are usually derivatised in
order to introduce catalytically relevant groups. Many such derivatives
have been examined. For example, a C-6 hydroxyl group of b-cyclodextrin
was covalently derivatised by an activated pyridoxal coenzyme. The
resulting synzyme not only acted a transaminase but also showed
stereoselectivity for the L-amino acids. It was not as active as natural
transaminases, however. Polyethyleneimine is formed by polymerising
ethyleneimine to give a highly branched hydrophilic three-dimensional
matrix. About 25% of the resultant amines are primary, 50% secondary and
25% tertiary:Ethyleneimine                    Â
polyethyleneimine</p> <p>The primary amines may be alkylated to form a
number of derivatives. If 40% of them are alkylated with 1-iodododecane
to give hydrophobic binding sites and the remainder alkylated with 4(5)-
chloromethylimidazole to give general acid-base catalytic sites, the
resultant synzyme has 27% of the activity of a-chymotrypsin against 4-
nitrophenyl esters. As might be expected from its apparently random
structure, it has very low esterase specificity. Other synzymes may be
created in a similar manner.</p> <p>Antibodies to transition state
analogues of the required reaction may act as synzymes. For example,
phosphonate esters of general formula (R-PO2-OR')- are stable analogues
of the transition state occurring in carboxylic ester hydrolysis.
Monoclonal antibodies raised to immunising protein conjugates covalently
attached to these phosphonate esters act as esterases. The specificities
of these catalytic antibodies (also called abzymes) depends on the
structure of the side-chains (i.e. R and R' in (R-PO2-OR')-) of the
antigens. The Km values may be quite low, often in the micromolar region,
whereas the Vmax values are low (below 1 s-1), although still 1000-fold
higher than hydrolysis by background hydroxyl ions. A similar strategy
may be used to produce synzymes by molecular 'imprinting' of polymers,
using the presence of transition state analogues to shape polymerising
resins or inactive non-enzymic protein during heat denaturation.</p>
<p>Coenzyme-regenerating systems</p> <p>Many oxidoreductases and all
ligases utilise coenzymes (e.g. NAD+, NADP+, NADH, NADPH, ATP), which
must be regenerated as each product molecule is formed. Although these
represent many of the most useful biological catalysts, their application
is presently severely limited by the high cost of the coenzymes and
difficulties with their regeneration. These two problems may both be
overcome at the same time if the coenzyme is immobilised, together with
the enzyme, and regenerated <em>in situ</em>.</p> <p>A simple way of
immobilising/regenerating coenzymes would be to use whole-cell systems
and these are, of course, in widespread use. However as outlined earlier,
these are of generally lower efficiency and flexibility than immobilised-
enzyme systems. Membrane reactors (may be used to immobilise the
coenzymes but the pore size must be smaller than the coenzyme diameter,
which is extremely restrictive. Coenzymes usually must be derivatised for
adequate immobilisation and regeneration. When successfully applied, this
process activates the coenzymes for attachment to the immobilisation
support but does not interfere with its biological function. The most
widely applied synthetic routes involve the alkylation of the exocyclic
N6-amino nitrogen of the adenine moiety present in the coenzymes NAD+,
NADP+, NADH, NADPH, ATP and coenzyme A.</p> <p>In some applications, such
as those using membrane reactors it is only necessary that the coenzyme
has sufficient size to be retained within the system. High molecular
weight water-soluble derivatives are most useful as they cause less
diffusional resistance than insoluble coenzyme matrices. Dextrans,
polyethyleneimine and polyethylene glycols are widely used. Relatively
low levels of coenzyme attachment are generally sought in order to allow
greater freedom of movement and avoid possible inhibitory effects. The
kinetic properties of the derived coenzymes vary, depending upon the
system, but generally the Michaelis constants are higher and the maximum
velocities are lower than with the native coenzymes. Coenzymes
immobilised to insoluble supports presently have somewhat less favourable
kinetics even when co-immobilised close to the active site of their
utilising enzymes. This situation is expected to improve as more
information on the protein conformation surrounding the enzymes' active
sites becomes available and immobilisation methods become more
sophisticated. However, the cost of such derivatives is always likely to
remain high and they will only be economically viable for the production
of very high value products.</p> <p>There are several systems available
for the regeneration of the derivatised coenzymes by chemical,
electrochemical or enzymic means. Enzymic regeneration is advantageous
because of its high specificity but electrochemical procedures for
regenerating the oxidoreductase dinucleotides are proving competitive. To
be useful in regenerating coenzymes, enzymic processes must utilise cheap
substrates and readily available enzymes and give non-interfering and
easily separated products. Formate dehydrogenase and acetate kinase
present useful examples of their use, although the presently available
commercial enzyme preparations are of low activity:</p>   <p>Genetically
Engineered Enzymes</p> <p>Enzymes are naturally occurring proteins that
speed up biochemical processes. They're used to produce everything from
wine and cheese to corn syrup and baked goods. Enzymes allow the
manufacturer to produce more of a particular product in a shorter amount
of time, thus increasing profit.</p> <p>Generally, the use of enzymes is
beneficial. In some cases, they can replace harmful chemicals and reduce
water and energy consumption in food production. However, enzymes
produced by genetically engineered organisms are cause for concern. Not
enough is known about the long-term effects of these enzymes on humans
and the ecosystem for them to be used across the board.</p> <p>FDA
regulations on enzyme use is a gray area. Enzymes used in the processing
of foods do not have to be listed on product labels because they are not
considered foods. Also, when enzymes are genetically engineered, the
manufacturer is not required to notify the FDA that the enzymes have been
modified. The lists of GE enzymes known by the FDA is, by their own
admission, "probably incomplete."</p> <p>Worldwide, the enzyme market is
a $1.3 billion industry. One of the largest enzyme manufacturers are Novo
Nordisk, which manufactures GE and non-GE enzymes. The FDA provided us
with this partial list of genetically engineered enzymes:</p> <ul> <li>
<p>Chymosin—used in the production of cheese</p> </li> <li>
<p>Novamyl(TM)—used in baked goods to help preserve freshness</p> </li>
<li> <p>Alpha amylase—used in the production of white sugar,
maltodextrins and nutritive carbohydrate          sweetenersÂ
(corn syrup)</p> </li> <li> <p>Aspartic (proteinase enzyme from R.
miehei)—used in the production of cheese</p> </li> <li>
<p>Pullulanase—used in the production of high fructose corn syrup</p>
</li> </ul> <p>If you want to absolutely avoid genetically engineered
enzymes you will have two choices: avoid foods in the following
categories, or call the food manufacturers directly and ask them if their
enzymes are genetically engineered. They will probably have no idea. Ask
them to check and call them back again. Let us know if you get written
confirmation.</p> <ul> <li> <p>Beers, wines and fruit juices—(Enzymes
used: Cereflo, Ceremix, Neutrase, Ultraflo, Termamyl, Fungamyl, AMG,
Promozyme, Viscozyme, Finizym, Maturex, Pectinex, Pectinex Ultra SP-L,
Pectinex BE-3L, Pectinex AR, Ultrazym, Vinozym, Citrozym, Novoclairzym,
Movoferm 12, Glucanex, Bio-Cip Membrane, Peelzym, Olivex/Zietex)</p>
</li> <li> <p>Sugar—Enzymes used: Termamyl, Dextranase, Invertase,
Alpha Amylase</p> </li> <li> <p>Oils—Enzymes used: Lipozyme IM, Novozym
435, Lecitase, Lipozyme, Novozym 398, Olivex, Zeitex</p> </li> <li>
<p>Dairy products—Enzymes used: Lactozym, Palatase, Alcalase,
Pancreatic Trypsin Novo (PTN), Flavourzyme, Catazyme, Chymosin</p> </li>
<li> <p>Baked goods—Enzymes used: Fungamyl, AMG, Pentopan, Novomyl,
Glutenase, Gluzyme</p> </li> </ul> <p>In many cases the enzymes named
above are brand names. They may appear under other names as well. Enzymes
are usually found in minuscule quantities in the final food product. The
toxin found in genetically engineered tryptophan was less than 0.1
percent of the total weight of the product, yet it was enough to kill
people. The use of enzymes is pervasive in the food industry. Nothing is
known about the long-term effects of genetically engineered enzymes. We
include this information so you can make an informed choice about whether
you want to eat them or not.</p> <p>Enzymes produced by genetically
modified microorganisms</p> <p><strong>Novozymes’ enzymes produced by
genetically modified microorganisms</strong></p> <p>Novozymes A/S markets
a range of enzymes for various industrial purposes. Many of these enzymes
are produced by fermentation of genetically modified microorganisms
(GMMs).</p> <p><strong>There are several advantages of using GMMs for the
production of enzymes, including:</strong></p> <ul> <li> <p>It is
possible to produce enzymes with a higher specificity and purity</p>
</li> <li> <p>It is possible to obtain enzymes which would otherwise not
be available for economical, occupational health or environmental
reasons</p> </li> <li> <p>Due to higher production efficiency there is an
additional environmental benefit through reducing energy consumption and
waste from the production plants</p> </li> <li> <p>For enzymes used in
the food industry particular benefits are for example a better use of raw
materials (juice industry), better keeping quality of a final food and
thereby less wastage of food (baking industry) and a reduced use of
chemicals in the production process (starch industry)</p> </li> <li>
<p>For enzymes used in the feed industry particular benefits include a
significant reduction in the amount of phosphorus released to the
environment from farming</p> </li> </ul> <p>Due to an efficient
separation process the final enzyme product does not contain any
GMMs.</p> <p>The enzymes are produced by fermentation of the genetically
modified micro organisms (the production strain) which then produces the
desired enzyme. The process takes place under well-controlled conditions
in closed fermentation tank installations.</p> <p>After fermentation the
enzyme is separated from the production strain, purified and mixed with
inert diluents for stabilisation.</p> <p>The following is a list of
Novozymes' enzymes produced by genetically modified organisms.<br> <br>
<strong>Food Applications:</strong></p> <p><strong>Brand
name</strong></p> <p><strong>Type of enzymes</strong></p> <p><strong>Main
Application</strong></p> <p>Amylase® AG XXL</p> <p>Glucoamylase</p>
<p>Juice Industry</p> <p>Dextrozyme®</p> <p>Pullulanase /
Amyloglucosidase</p> <p>Starch industry</p> <p>Finizym® W</p>
<p>Phospholipase</p> <p>Starch industry</p> <p>Gluzyme® Mono</p>
<p>Glucose oxidase</p> <p>Baking industry</p> <p>Lecitase® Novo</p>
<p>Lipase</p> <p>Oils and fats industry</p> <p>Maltogenase®</p>
<p>Maltogenic amylase</p> <p>Starch industry</p> <p>Maturex®</p>
<p>Alpha-acetodecarboxylase</p> <p>Brewing industry</p> <p>NovoCarne®
Tender</p> <p>Protease</p> <p>Meat industry</p> <p>Novoshape®</p>
<p>Pectinesterase</p> <p>Fruit processing</p> <p>Novozym® 27080</p>
<p>Carbohydrase / Lipase</p> <p>Baking industry</p> <p>NOVOZYM®
27122</p> <p>Xylanase</p> <p>Protein Hydrolysis</p> <p>Novozym®
33081</p> <p>Polygalacturonase</p> <p>Juice Industry</p> <p>Novozym®
46016</p> <p>Phospholipase</p> <p>Dairy industry</p> <p>Novozym®
46019</p> <p>Cellobiose oxidase</p> <p>Dairy Industry</p> <p>Pectinex®
XXL</p> <p>Pectin lyase / Polygalacturonase</p> <p>Juice Industry</p>
<p>Promozyme® D2</p> <p>Pullulanase</p> <p>Starch industry</p>
<p>Saczyme®</p> <p>Glucoamylase</p> <p>Alcohol Industry</p>
<p>Toruzyme®</p> <p>Transferase</p> <p>Starch industry</p> <p>
<strong>Feed Applications:</strong></p> <p><strong>Brand
name</strong></p> <p><strong>Type of enzymes</strong></p> <p><strong>Main
Application</strong></p> <p>Bio-Feed® Wheat</p> <p>Xylanase</p>
<p>Animal feed industry</p> <p>Bio-feed® Phytase</p> <p>Phytase</p>
<p>Animal feed industry</p> <p> <strong>Other Applications:</strong></p>
<p><strong>Brand name</strong></p> <p><strong>Type of
enzymes</strong></p> <p><strong>Main Application</strong></p>
<p>Alcalase®</p> <p>Subtillisin</p> <p>Detergent industry</p>
<p>Aquazym® LT-L</p> <p>Alpha-amylase</p> <p>Textile industry</p>
<p>BioPrep®</p> <p>Pectate lyase</p> <p>Textile industry</p>
<p>Carezyme®</p> <p>Cellulase</p> <p>Detergent industry</p> <p>Clear-
Lens® LIPO</p> <p>Lipase</p> <p>Personal care industry</p>
<p>DeniLite®</p> <p>Laccase</p> <p>Textile industry</p> <p>DeniMax®
601</p> <p>Cellulase</p> <p>Textile Industry</p> <p>Duramyl®</p>
<p>Alpha-amylase</p> <p>Detergent industry</p> <p>Everlase®</p>
<p>Subtillisin</p> <p>Detergent industry</p> <p>Extruzyme® Pro</p>
<p>Alpha-amylase</p> <p>Pet food industry</p> <p>Greasex®</p>
<p>Lipase</p> <p>Leather industry</p> <p>Kannase®</p> <p>Subtillisin</p>
<p>Detergent industry</p> <p>Lipex®</p> <p>Lipase</p> <p>Detergent
industry</p> <p>Lipolase®</p> <p>Lipase</p> <p>Detergent industry</p>
<p>Liquanase®</p> <p>Subtilisin</p> <p>Detergent industry</p>
<p>Liquozyme®</p> <p>Alpha-amylase</p> <p>Starch and Ethanol
industry</p> <p>Mannaway®</p> <p>Mannanase</p> <p>Detergent industry</p>
<p>NovoBate® 100</p> <p>Trypsin</p> <p>Leather Industry</p>
<p><strong>Chemical Modification of Enzymes</strong> –</p> <p>We know
that the proteins synthesized under the control of gene sequences in a
cell undergo post translational modification. This leads to stability,
structural integrity, altered solubility and viscosity of individual
proteins. This may also alter the chemical reactivity.<br> <br> These
alterations can be achieved in vitro and may .sometimes even create
entirely new enzyme, by creating new active sites or modifying the old
ones. Some of the examples will be described in this section.</p>
<p><strong>Protein Modelling</strong></p> <p>Utilizing the data generated
through X-ray diffraction and NMR studies, models can be constructed with
the help of computer graphics. There are computer programmes available
(interactive colour graphics programmes) with the help of which a protein
structure can be fitted to the electron density map (obtained from X-ray
diffraction) by simultaneous display on the screen of computer monitor.
Similarly, Van der Waals surfaces for the protein can be displayed and
interaction between several molecules simulated. <br> <br> There are also
other interactive molecular graphics which can be used (with the help of
programmes) to find out the perturbations (disturbances) in protein
structure that will result from specific modifications of amino acid
sequences. We know that to some extent the three dimensional structure of
a protein can be predicted from the amino acid sequence, but we still
have to depend partly on X-ray diffraction patterns for determining the
three dimensional structure.<br> <br> In future when the three
dimensional structure can be accurately predicted from amino acid
sequence data, this will lead to long term success in protein
engineering. The models of proteins, made on the basis of amino acid
alterations, can then be tested for the predictions about structure
function relationships.</p> <p><strong>Multienzyme Systems by Gene Fusion
( Bi and Polyfunctional Enzymes)</strong> –</p> <p>Multienzyme systems
have been artificially synthesized, which can catalyze sequential
reactions in many biotechnological production processes. Although,
proximity of more than one enzymes can also be achieved by co-
immobilization and chemical cross linking, gene fusion appears to have
the highest potential in enzyme technology. The gene fusion technology,
for preparation of bi-and polyfunctional enzymes, involves joining of
structural genes of two or more enzymes. The translational stop singal at
the 3' end of the first gene is removed and ligated in frame to the A TG
start codon of the second gene. Alternatively, short linkers (2-10 amino
acids) are used. The novel chimaeric gene gives a single polypeptide
chain carrying active sites of both genes. This fusion may involve</p>
<p>(i)Â Â Â Â two monomeric enzymes</p> <p>(ii)Â Â a monomeric and a
dimeric enzyme or</p> <p>(iii) two dimeric enzymes.</p>
<p><strong>Rationale of Protein Enzyme Engineering</strong> - Although
thousands of proteins have been characterized in prokaryotes and
eukaryotes, only few became commercially important. This is due to the
high cost of isolating and purifying enzymes in sufficient quantities.
<br> <br> Although the cost aspect has been overcome by producing an
enzyme in large quantities in bacteria, for its industrial application,
an enzyme (outside the cell) should also have some characteristics in
addition to those of enzymes in the cells. These characteristics may
include the following: <br> (i) enzyme should be robust with a long
life;</p> <p>(ii) enzyme should be able to use the substrate supplied in
the industry even if it differs slightly from that in the cell; <br>
(iii) enzyme should be able to work under conditions (e.g. extremes of
pH, temperature and concentration) of the industry even if they differ
from those in the cell.<br> </p> <p>In view of the above, enzyme should
be engineered to meet the altered needs. Therefore, efforts have been
made to alter the properties of the enzymes. Following is the list of
properties that one needs to alter in a predictable manner for protein or
enzyme engineering.</p> <p>(1)Â Â Â Â Â Kinetic properties of enzyme
turnover and Michaelis Constant, Km. <br> (2) Theremostability and the
optimum temperature for the enzyme.<br> (3) Stability and activity of
enzyme in nonaqueous solvents.<br> (4) Substrate and reaction
specificity.<br> (5) Cofactor requirements.<br> (6) Optimun pH.<br> (7)
Protease resistance.<br> (8) Allosteric regulation.<br> (9) Molecular
weight and subunit structure.<br> <br> For a particular class of enzymes,
variation in nature may occur for each of the above properties, so that
one may like to combine the optimum properties to get the most efficient
form of the enzyme.</p> <p>This aspect of protein engineering will be
illustrated using the example of glucose isomerases, which convert
glucose into other isomers like fructose and are used to make high
fructose corn syrup vital for soft drink industry. It exhibits wide
variation in its properties.<br> Sometimes, it may not be possible to get
a combination of optimum properties. For instance, an enzyme with highest
activity may not be the most stable. Therefore, a compromise in
properties may have to be made, if we have to select an enzyme from the
available variability or even if we create variability by mutagenesis.
<br> However, if structure and function relationship of an enzyme is
known, the structural features for desirable function may be combined and
protein engineering techniques may then be used to create a novel enzyme
exhibiting a combination of all desirable functional properties.</p>
<p>Glucose isomerase belongs to a TIM barrel family of enzymes which
resemble each other in having a highly characteristic domain called TIM
barrel, with active site for catalytic action at one end. This TIM barrel
may be found in enzymes that may differ in sequences and may catalyze
different reactions. <br> As earlier discussed, since similarities of
structure of protein meant similarities in function, TIM barrel presents
a challenge to this concept. However, it is curious tbat some enzymes in
this family appear in pairs in their metabolic pathways so that they
catalyse two consecutive steps thus showing coupling of their
functions.<br> As an example of two enzymes of TIM barrel family, while
'triose phosphate isomerase' is one of the most efficient catalysts,
'glucose isomerase' is relatively very inefficient. <br> Therefore, if
'glucose isomerase' enzyme is redesigned to use the highly efficient
domain of TIM barrel family, it will be a remarkable achievement for soft
drink industry. Efforts in this direction are being made (see later for
methods of protein engineering).</p> <p><strong>Acheivements of Protein
Engineering</strong></p> <p>A number of proteins are known, now, where
efforts have been made to know the effects of site specific mutagenesis
involving substitution of one or more amino acids. Efforts have also been
made to study in detail the function of different regions of a protein.
Following are some results of such efforts.<br> <br> <strong>?-
lactamase</strong>. This enzyme functions in the periplasmic space of
bacterial cells. The enzyme hydrolyses and inactivates the beta- lactam
ring of penicillin derivatives and helps in transport across the inner
membrane. During transport a polypeptide (signal sequence peptide of 23
amino acids) is cleaved off.<br> <br> Detailed analysis suggested that,
transport and processing does not depend on this polypeptide of 23 amino
acids alone. An active site involving amino acid serine has also been
identified, since its replacement by cysteine leads to reduction in the
activity of this enzyme.</p> <p><strong>Dihydrofolate reductase</strong>.
In this enzyme, replacement of a single amino acid, aspartic add (ASP) by
asparagine (ASN), led to a decrease in specific activity by a thousand
fold, suggesting that aspartic acid is very important.(or the active
site. Other similar modifications were also examined. <br> <br>
<strong>Insulin</strong>. It consists of A and B chains linked by C-
peptide of 35 amino acids. It was shown that a sequence of 6 amino acids
for C-peptide was adequate for the, linking function.</p>
<p><strong>Lactose permease (product of, gene of 'lac' operon).</strong>
This enzyme is involved in transport of lactose and a cysteine to glycine
substitution showed that this amino acid was not essential for transport.
Further, out of four histidine residues, two at positious 35 and '39 do'
not play any essential role in transport, while the mutation in any of
the other two histidines at positions 208 and 322, lead to loss of
transport function.<br> <br> <strong>T4 lysozyme</strong>. A mutation of
isoleucine to cystine in this enzyme leading to formation of a disulphide
bridge led to thermal stability and a 200 fold increase in enzyme
activity even at 6T'C. <br> <br> <strong>Human beta interferon</strong>.
Removal of one of the three cysteine residues' I led to an improvement in
stability of the enzyme.<br> <br> <strong>? repressor.</strong> This
protein could be engineered to develop a specific site for cro protein,
since the alteration led to development of a cro recognition site.I</p>
<p><strong>Acetylcholine receptor</strong>. This protein is involved in
transport, of acetylcholine through. the membrane. Specific regions of
this protein involved in acetylcholine binding and channel formation have
been, identified.<br> <br> <strong>Cytochrome C</strong>. A phenylalanine
residue has been identified to be non-essential for electron transfer but
is involved in determining the reduction potential of the protein.<br>
<br> <strong>Trypsin</strong>. It could be redesigned to have altered
substrate specificity.<br> <br> <strong>Subtilisin</strong>. Another
successful alteration of substrate specificity involved the enzyme
subtilisin reported in 1986-87.</p> <p><strong>Lactate
dehydrogenase</strong>. A lactate dehydrogenase (LDH) from Bacillus
stearothermophilus was modified separately by each of the three
substitutiens of amino acids (resulting from mutations; Asp197... Asn;
Thr246"'Gly; Gln102...Arg). The substitution, Gln102"'Arg, led to change
in specificity from lactate to malate, with high efficiency, comparable
to that which the native LDH had for lactate.<br> <br> <strong>Lactic
protease</strong>. Substrate specificity of lactic protease (in E. coli),
has been shown to be dramatically modified by replacing active site
methionine by alanine (Met19        <!--INFOLINKS_OFF-->
        </div>

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