Module 210
Energy in Cells
In conjunction with
INTRODUCTION TO THIS RESOURCE ............................................................ ERROR! BOOKMARK NOT DEFINED.
ENZYMES ................................................................................................................................................................................. 4
MEDICAL APPLICATIONS OF ENZYMES .................................................................................................................................... 4
PROTEIN ENGINEERING ............................................................................................................................................................ 5
Subtilisin............................................................................................................................................................................. 5
BIOSENSORS ............................................................................................................................................................................ 6
Bacterial Diagnostics ......................................................................................................................................................... 6
THE BACTERIAL AND BIOSENSOR DIAGNOSTIC MARKET COVERS A WIDE RANGE OF APPLICATIONS INCLUDING CLINICAL
DIAGNOSTICS, FOOD TESTING, VETERINARY MEDICINE AND BIOTERRORISM. THE MARKET FOR GENERAL DIAGNOSTIC KITS
COVERING ALL THESE AREAS WAS $6 BILLION IN 2000. OF THIS THE CLINICAL DIAGNOSTIC AND RESEARCH SEGMENTS ARE
WORTH ABOUT $2 BILLION AND SEEM TO BE EXPERIENCING GROWTH RATE OF ALMOST 25% PER YEAR. ................................ 6
SOME EXAMPLES OF SOME DIAGNOSTIC MACHINERY ARE:....................................................................................................... 6
IMMOBILISED ENZYMES ........................................................................................................................................................... 6
Introduction to this resource
This resource is designed with one aim in mind, that being to help the students taking module 520
appreciate the diverse ways in which biological knowledge has been harnessed to create wealth and
resolve problems.
The facts and examples are by no means exhaustive, but they should help illustrate the biological and
economic importance of the topics covered within the module. This is also written for the students rather
than academics, and all of this material, including that for other modules is on the York BioEnterprise
website.
http://www.york.ac.uk/depts/biol/bioenterprise/
Enzymes
A huge number of commercial and industrial enzymes exist, and the market figure for the commercial
worth of industrially produced enzymes as of 2000 was in the region of $1.5bn.
Most enzymes mentioned in the course are available to buy commercially from companies such as
United States Biochemical (USB) or Invitrogen, Merck and Fisher Scientific.
http://www.usbweb.com/category.asp?cat=bio&id=16965 - Hexokinase
Medical Applications of Enzymes
Since the 19th century, proteolytic enzymes have been used to treat gastrointestinal disorders, eg, pepsin
for dyspepsia, but, other than as digestion aids, enzymes were largely ignored as drugs until Emmerich
observed in 1902 that an extracellular secretion of Bacillus pyocyaneus was capable of killing anthrax
bacilli and could protect mice from otherwise lethal innocula of the bacterium. Nowadays, development
of enzymes for medical uses is as extensive as that for industrial uses. The most successful applications
are still extracellular such as the removal of toxic substances from the blood. Some examples include
collagenase, which is used to treat skin ulcers, hyaluronidase which is used to treat heart attacks (as are
the anti-coagulants streptokinase and urokinase), lysozyme which hydrolses chitins and mucopeptides of
bacterial cell walls and is used as an antibacterial agent and -lactamase which is used to treat penicillin
allergy.
Therapeutic enzymes have to have very high degree of purity as they are used in relatively tiny amounts.
A low Km and a high Vmax are also desirable so that they are active at low enzyme and substrate
concentrations. The costs of such enzymes may be quite high but still comparable to those of competing
therapeutic agents or treatments. As an example, urokinase is prepared from human urine (some
genetically engineered preparations are being developed) and used to dissolve blood clots. The cost of
the enzyme is about £100 per mg, with the cost of treatment in a case of lung embolism being about
£10,000 for the enzyme alone. In spite of this, the market for the enzyme is worth about £70m.
However there are problems with using enzymes. Enzymes from sources foreign to the body are
antigenic and can cause adverse immune responses. It has proved possible to circumvent this problem,
in some cases, by disguising the enzyme using covalent modification. For example asparaginase,
modified by covalent attachment of polyethylene glycol, has been shown to retain
its anti cancer effect whilst having no immunogenicity. This technique was
developed by Dr Abraham Abuchowski who started the work at Rutgers
University, New Jersey, USA, before starting his own company called Enzon.
Enzon have since gone on to develop this “PEG technology” launching
ADAGEN®, ONCASPAR®, DEPOCYT® and PEG-INTRON® onto the world
markets. Combined product sales of Enzon products were $146 million in 2004.
http://www.enzon.com/products.html - Enzon’s portfolio of products.
A major potential therapeutic application of enzymes is in the treatment of cancer. It was found that
when cells become cancerous their biochemistry often changes. Certain tumor cells are deficient in their
ability to synthesize the nonessential amino acid L-asparagine, and are forced to extract it from body
fluids; by contrast, most normal cells can produce their own L-asparagine. Almost a 60% incidence of
complete remission has been reported in a study of almost 6000 cases of acute lymphocytic leukaemia
after intravenous administration of asparaginase. Various products are on the market including
Leucoginase and Leunase.
http://www.vhbgroup.com/products_oncology.asp - Leucoginase and other products
http://www.kyowa.co.jp/eng - Leunase from Kyowa
http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Application_Index/The
rapeutic_Enzymes.html - List of available therapeutic enzymes from Sigma.
http://www.direvo.de/pharma.html - Screening and improvement of pharmaceutical proteins
Protein Engineering
Protein engineering is simply the design and mutation of proteins by site directed or random
mutagenesis to produce new enzyme structures that improve on or create new activities. Modern
techniques tend to be specific and site directed, even progressing as far as designing totally synthetic
enzymes (synzymes) though this is a relatively new field and it seems most products are still at the
research stage. Some companies however do offer custom peptide synthesis services, such as American
Peptides.http://www.americanpeptide.com/commerce/info/productservice.jsp
Subtilisin
Subtilisin from Bacillus amyloliquefaciens has been used in detergents for over 25 years, but the
Centre for Advanced Research in Biotechnology (CARB) used bisulphite mutagenesis of bacterial
plasmids to gain a 4-fold increase in stability at 65C. The discovery was licensed to Procter &
Gamble to be used in washing powders such as Ace, Bold and Ariel.
http://householdproducts.nlm.nih.gov/cgi-bin/household/brands?tbl=chem&id=2167
Subtilisin has been modified several times. The aims have been to improve its activity by stabilising
it at even higher temperatures, pH and oxidant strength. Most of the work has focused on the P1
cleft, which holds the amino acid on the carbonyl side of the targeted peptide bond; the oxyanion
hole (principally Asn155), which stabilises the tetrahedral intermediate; the region of the catalytic
histidyl residue (His64), which has a general base role; and the methionine residue (Met222) which
causes subtilisin's lability to oxidation.
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.
Biosensors
Biosensors are machines that utilise biochemical reactions to conduct a test, and
then transduce the signal into a suitable display (i.e. digital). They usually
consist of enzymes within a selectively permeable membrane, with a further
membrane for the product “test” molecule to cross before detection at the
transducer. They provide on the spot tests that previously would have been sent
to the lab, and as such have found applications in medical science, agriculture,
food and environmental monitoring. They are also being developed for use as
sensors for biological and chemical weapons such as Anthrax. The biosensor
market is currently predicted to be in the region of $1-2 billion
http://www.sensornetworks.net.au/biosens.html - Good overview of the technology
http://www.the-scientist.com/yr2002/mar/profile_020318.html - Review by the easy to read journal.
http://www.army.mod.uk/equipment/nbcds/nbcds_nai.htm - British army nerve agent detection using an
immobilised cholinesterase. (NIAD - Nerve Agent Immobilised Enzyme Alarm and Detector).
http://www.analox.com/ - Biosensors for molecules such as glucose, Lactate etc
http://www.ambri.com/ - Medical biosensors
http://biacore.com/products/ - Medical and general bioscience sensors
http://www.medisense.com/ - Market leaders with sales of $170m (mostly medical)
http://news.bbc.co.uk/2/hi/health/1857730.stm - Biosensors in the news
http://news.bbc.co.uk/2/hi/uk_news/wales/2779581.stm - Biosensors in the news
Bacterial Diagnostics
The bacterial and biosensor diagnostic market covers a wide range of applications including clinical
diagnostics, food testing, veterinary medicine and bioterrorism. The market for general diagnostic
kits covering all these areas was $6 billion in 2000. Of this the clinical diagnostic and research
segments are worth about $2 billion and seem to be experiencing growth rate of almost 25% per
year.
Some examples of some diagnostic machinery are:
http://www.accelr8.com/ - In development, advanced identification, counting and antibiotic
susceptibility screening in one package.
http://www.bacbarcodes.com/press_release_021203.htm - Bacterial biosensors linking bacterial
strain information to bioinformatics for quick identification.
Immobilised enzymes
The two main advantages of immobilising enzymes onto a substrate are:
 Easy separation of the enzymes from the reaction mixture, providing the ability to control reaction
times and minimise the enzymes lost in the product.
 Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme-mediated
reactions.
There are various methods of immobilising enzymes onto the substrate, but the main four are: physical
adsorption onto an inert carrier, such as a glass bead; inclusion into the lattices of a gel, cross-linking of
the protein with another reagent, and covalent binding to a reactive insoluble support. Most major
biological suppliers such as Invitrogen, Fisher Bioscience, sell various types of immobilised enzymes.
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/IMMOB/goel2nd.htm - Immobilisation methods
http://www.biocatalytics.com/immob.htm - Small company offering all types of enzymes
http://www.worthington-biochem.com/ - Similar company selling enzymes