RIBOZYME

RIBOZYME
• A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction.
• Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome.
• In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA could act as a catalyst
• RNase-P, which is responsible for conversion of a precursor tRNA into the active tRNA.
• RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme.
• Ribozymes often have divalent metal ions such as Mg2+ as cofactors.
Naturally occurring ribozymes include:
• Peptidyl transferase 23S rRNA
• RNase P
• Group I and Group II introns
• GIR1 branching ribozyme[9]
• Leadzyme - Although initially created in vitro, natural examples have been found
• Hairpin ribozyme
• Hammerhead ribozyme
Vancomycin
 Vancomycin is a glycopeptide antibiotic used in the prophylaxis and treatment of infections caused by Gram-positive bacteria.
 It has traditionally been reserved as a drug of "last resort", used only after treatment with other antibiotics had failed.
 The organism that produced it by the fermentation of the Actinobacteria species is Amycolatopsis orientalis (formerly designated Nocardia orientalis).
 The original indication for vancomycin was for the treatment of penicillin-resistant Staphylococcus aureus.
Vancomycin never became first line treatment for Staphylococcus aureus for several reasons:
1. The drug must be given intravenously, because it is not absorbed orally.
2. β-lactamase-resistant semi-synthetic penicillins such as methicillin (and its successors, nafcillin and cloxacillin) were subsequently developed.
 Vancomycin acts by inhibiting proper cell wall synthesis in Gram-positive bacteria. Specifically, vancomycin prevents incorporation of N-acetylmuramic acid (NAM)- and N-acetylglucosamine (NAG)-peptide subunits into the peptidoglycan matrix; which forms the major structural component of Gram-positive cell walls.
 In particular, vancomycin should not be used to treat methicillin-sensitive Staphylococcus aureus because it is inferior to penicillins such as nafcillin.
ANTIBIOTIC RESISTANCE
Intrinsic resistance
 There are a few gram-positive bacteria that are intrinsically resistant to vancomycin: these are Leuconostoc and Pediococcus species, but these organisms are rare causes of disease in humans.
 Most Lactobacillus species are also intrinsically resistant to vancomycin (the exception is the finding of a few strains (but not all) of L. acidophilus).
 Most gram-negative bacteria are intrinsically resistant to vancomycin because their outer membrane is impermeable to large glycopeptide molecules (with the exception of some non-gonococcal Neisseria species).
 There is some suspicion that agricultural use of avoparcin, another similar glycopeptide antibiotic, has contributed to the emergence of vancomycin-resistant organisms.
Red man syndrome
 Vancomycin must be administered in a dilute solution slowly, over at least 60 minutes (maximum rate of 10 mg/minute for doses >500 mg). This is due to the high incidence of pain and thrombophlebitis and to avoid an infusion reaction known as the red man syndrome or red neck syndrome. This syndrome, usually appearing within 4–10 minutes after the commencement or soon after the completion of an infusion, is characterized by flushing and/or an erythematous rash that affects the face, neck and upper torso.
TANDEM REPEAT
 Tandem repeats occur in DNA when a pattern of two or more nucleotides is repeated and the repetitions are directly adjacent to each other.
An example would be:
A-T-T-C-G-A-T-T-C-G-A-T-T-C-G
in which the sequence A-T-T-C-G is repeated three times
 When between 10 and 60 nucleotides are repeated, it is called a minisatellite.
 Those with fewer are known as microsatellites or short tandem repeats.
 When exactly two nucleotides are repeated, it is called a "dinucleotide repeat"; when three are repeated, it is called a "trinucleotide repeat" (as in trinucleotide repeat disorders.)
 When the number is not known, variable, or irrelevant, it is sometimes called a variable number tandem repeat (VNTR). MeSH classifies variable number tandem repeats under minisatellites.
 DNA is examined from microsatellites within the chromosomal DNA.
 Minisatellite is another way of saying special regions of the loci.
 Polymerase chain reaction (or PCR) is performed on the minisatellite areas.


SHORT TANDEM REPEAT
 A short tandem repeat (STR) in DNA occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other.
 The pattern can range in length from 2 to 16 base pairs (bp) (for example (CATG)n in a genomic region) and is typically in the non-coding intron region.
 A short tandem repeat polymorphism (STRP) occurs when homologous STR loci differ in the number of repeats between individuals. By identifying repeats of a specific sequence at specific locations in the genome, it is possible to create a genetic profile of an individual.
 Shorter repeat sequences tend to suffer from artifacts such as PCR stutter and preferential amplification, as well as the fact that several genetic diseases are associated with tri-nucleotide repeats such as Huntington's disease. Longer repeat sequences will suffer more highly from environmental degradation and do not amplify by PCR as well as shorter sequences.
 Y-STRs (STRs on the Y chromosome) are often used in genealogical DNA testing.

SYNZYMES
 Synzymes are substances with catalytic capabilities. The name synzyme is derived from synthetic enzyme. Current synzymes consist mainly of organic molecules tailored in such a way that they catalyse certain kinds of reactions. Like enzymes, they bind a transition state of a substrate in an acitve site, and like enzymes they generally obey Michaelis-Menten kinetics.
 Derivatised proteins: If [Ru(NH3)5]3+ is attached to certain histidine residues in a myoglobin protein, myoglobin is no longer a passive oxygen carrier, but gains enzmatic activity of an oxidase. Ascorbic acid is oxidised with molecular oxygen.
 Antibodies can act as enzymes, then named abzymes, if they are selected against transition state analogues.
 Abzymes have a low KM, meaning that they readily bind a target molecule, but have low Vmax values, indicating a slow reaction rate.
 Synzymes from organic molecules: Cyclodextrins are cap structures with a hydrophilic exterior but a hydrophobic interior. If pyridoxal is anchored in the interior the cyclodextran shows transaminase activity.
 A primary metabolite is directly involved in normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes, but usually has an important ecological function. Examples include----------- antibiotics and pigments.
Antimetabolite
 An antimetabolite is a chemical that inhibits the use of a metabolite.
 Such substances are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid.
 The presence of antimetabolites can have toxic effects on cells, such as halting cell growth and cell division, so these compounds are used as chemotherapy for cancer
 Antimetabolites can be used in cancer treatment, as they interfere with DNA production and therefore cell division and the growth of tumors.
CANCER TREATMENT
 Anti-metabolites masquerade as a purine (azathioprine, mercaptopurine) or a pyrimidine - which become the building blocks of DNA. They prevent these substances becoming incorporated in to DNA during the S phase (of the cell cycle), stopping normal development and division.
 They also affect RNA synthesis. However, because thymidine is used in DNA but not in RNA (where uracil is used instead), inhibition of thymidine synthesis via thymidylate synthase selectively inhibits DNA synthesis over RNA synthesis.
 Antimetabolites may also be antibiotics, such as sulfanilamide drugs, which inhibit dihydrofolate synthesis in bacteria by competing with para-aminobenzoic acid.
Main representatives of these drugs are:
• purine analogues ,pyrimidine analogues, antifolates

Macromolecular crowding
 The phenomenon of macromolecular crowding alters the properties of molecules in a solution when high concentrations of macromolecules such as proteins are present.
 Such conditions occur routinely in living cells; for instance, the cytosol of Escherichia coli contains about 300-400 milligrammes per millilitre (mg/ml) of macromolecules. Crowding occurs since these high concentrations of macromolecules reduce the volume of solvent available for other molecules in the solution, which has the result of increasing their effective concentrations.
Abzyme
 An abzyme (from antibody and enzyme), also called catmab (from catalytic monoclonal antibody), is a monoclonal antibody with catalytic activity.
 Molecules which are modified to gain new catalytic activity are called synzymes.
 Abzymes are usually artificial constructs, but are also found in normal humans (anti-vasoactive intestinal peptide autoantibodies) and in patients with autoimmune diseases such as systemic lupus erythematosus, where they can bind to and hydrolyze DNA.
HIV treatment
In a June 2008 issue of the journal Autoimmunity Reviews, researchers S Planque, Sudhir Paul, Ph.D, and Yasuhiro Nishiyama, Ph.D of the University Of Texas Medical School at Houston announced that they have engineered an abzyme that degrades the superantigenic region of the gp120 CD4 binding site. This is the one part of the HIV virus outer coating that does not change, because it is the attachment point to T lymphocytes, the key cell in cell-mediated immunity. Once infected by HIV, patients produce antibodies to the more changeable parts of the viral coat. The antibodies are ineffective because of the virus' ability to change their coats rapidly. Because this protein gp120 is necessary for the HIV virus to attach, it does not change across different strains and is a point of vulnerability across the entire range of the HIV variant population.
The abzyme does more than bind to the site, it actually destroys the site, rendering the HIV virus inert, and then can attach to other viruses. A single abzyme can destroy thousands of HIV viruses. Human clinical trials will be the next step in producing treatment and perhaps even preventative vaccines and microbicide
CATALASE
 Catalase is a common enzyme found in nearly all living organisms which are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen.
 Catalase has one of the highest turnover numbers of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second.
 Catalase is a tetramer of four polypeptide chains.
 It contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide.
The reaction of catalase in the decomposition of hydrogen peroxide is:
2 H2O2 → 2 H2O + O2
• Any heavy metal ion (such as copper cations in copper(II) sulfate) will act as a noncompetitive inhibitor on catalase.
• Also, the poison cyanide is a competitive inhibitor of catalase, strongly binding to the heme of catalase and stopping the enzyme's action.
Cellular role
• Hydrogen peroxide is a harmful by-product of many normal metabolic processes: to prevent damage, it must be quickly converted into other, less dangerous substances. To this end, catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules
 Human catalase works at an optimum temperature of 37°C, which is approximately the temperature of the human body.
 In contrast, catalase isolated from the hyperthermophile archaea Pyrobaculum calidifontis has a temperature optimum of 90°C.
 Catalase is usually located in a cellular organelle called the peroxisome.
 Hydrogen peroxide is used as a potent antimicrobial agent when cells are infected with a pathogen.
 Pathogens that are catalase-positive, such as Mycobacterium tuberculosis, Legionella pneumophila, and Campylobacter jejuni, make catalase in order to deactivate the peroxide radicals, thus allowing them to survive unharmed within the host.
 Streptococcus species are an example of aerobic bacteria that do not possess catalase. Catalase has also been observed in some anaerobic microorganisms, such as Methanosarcina barkeri.
Catalase is used in the food industry for removing hydrogen peroxide from milk prior to cheese production. Another use is in food wrappers where it prevents food from oxidizing. A minor use is in contact lens hygiene.
CATALASE TEST
The presence of catalase enzyme in the test isolate is detected using hydrogen peroxide.
If the bacteria possess catalase (i.e. are catalase positive), when a small amount of bacterial isolate is added to hydrogen peroxide, bubbles of oxygen are observed.
In microbiology, the catalase test is used to differentiate between bacterial species in the lab.
The test is done by placing a drop of hydrogen peroxide on a microscope slide. Using an applicator stick, a scientist touches the colony and then smears a sample into the hydrogen peroxide drop.
• If bubbles or froth forms, the organism is said to be catalase-positive. Staphylococci] and Micrococci are catalase-positive.
• If not, the organism is catalase-negative. Streptococci and Enterococci are catalase-negative.
Pathology
The peroxisomal disorder acatalasia is due to a deficiency in the function of catalase.


Chloramphenicol
Chloramphenicol was originally derived from the bacterium Streptomyces venezuelae,
Chloramphenicol (INN) is a bacteriostatic antimicrobial. It is considered a prototypical broad-spectrum antibiotic, alongside the tetracyclines.
Chloramphenicol is effective against a wide variety of Gram-positive and Gram-negative bacteria, including most anaerobic organisms it is sometimes used topically for eye infections.
The most serious adverse effect associated with chloramphenicol treatment is bone marrow toxicity, which may occur in two distinct forms: bone marrow suppression, which is a direct toxic effect of the drug and is usually reversible, and aplastic anemia, which is idiosyncratic (rare, unpredictable, and unrelated to dose) and generally fatal.
It is common for chloramphenicol to cause bone marrow suppression during treatment: this is a direct toxic effect of the drug on human mitochondria.
 Because it functions by inhibiting bacterial protein synthesisIt is not active against Pseudomonas aeruginosa, Chlamydiae, or Enterobacter species.
Gray baby syndrome
 Intravenous chloramphenicol use has been associated with the so called gray baby syndrome. This phenomenon occurs in newborn infants because they do not yet have fully functional liver enzymes (i.e. UDP-glucuronyl transferase), and so chloramphenicol remains unmetabolized in the body.
 Chloramphenicol increases the absorption of iron.
 Chloramphenicol is bacteriostatic (that is, it stops bacterial growth).
 It is a protein synthesis inhibitor, inhibiting peptidyl transferase activity of the bacterial ribosome, binding to A2451 and A2452 residues in the 23S rRNA of the 50S ribosomal subunit, preventing peptide bond formation
• Chloramphenicol passes into breast milk and should therefore be avoided during breastfeeding if possible.
 Chloramphenicol is a potent inhibitor of the cytochrome P450 isoforms CYP2C19 and CYP3A4 in the liver.

o In some countries, chloramphenicol is sold as chloramphenicol palmitate ester. Chloramphenicol palmitate ester is inactive, and is hydrolysed to active chloramphenicol in the small intestine. There is no difference in bioavailability between chloramphenicol and chloramphenicol palmitate.
o The intravenous (IV) preparation of chloramphenicol is the succinate ester, because pure chloramphenicol does not dissolve in water. This creates a problem: chloramphenicol succinate ester is an inactive prodrug and must first be hydrolysed to chloramphenicol; the hydrolysis process is incomplete and 30% of the dose is lost unchanged in the urine, therefore serum concentrations of chloramphenicol are only 70% of those achieved when chloramphenicol is given orally.
Ramoplanin
o Ramoplanin (INN) is a glycolipodepsipeptide antibiotic drug derived from strain ATCC 33076 of Actinoplanes.
 It exerts its bacteriocidal effect by inhibiting cell wall biosynthesis, acting by inhibiting the transglycosylation step of peptidoglycan synthesis. [Gate-2010]
 Used for treatment for multi-antibiotic resistant Clostridium difficile infection of the gastrointestinal tract,
DNA supercoiling
"Supercoiling" is an abstract mathematical property representing the sum of twist and writhe.
The twist is the number of helical turns in the DNA and the writhe is the number of times the double helix crosses over on itself (these are the supercoils).
The relationship of twist, writhe, and supercoiling is expressed as the equation:
S = T + W

Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling.
Many topoisomerase enzymes sense supercoiling and either generate or dissipate it as they change DNA topology. DNA of most organisms is negatively supercoiled.
 Supercoiled DNA forms two structures—
A plectoneme or a toroid, or a combination of both .
 A negatively supercoiled DNA molecule will produce either a one-start left-handed helix, the toroid, or a two-start right-handed helix with terminal loops, the plectoneme.
 Plectonemes are typically more common in nature, and this is the shape most bacterial plasmids will take.
 For larger molecules it is common for hybrid structures to form.
OCCURRENCE OF DNA SUPERCOILING
 DNA supercoiling is important for DNA packaging within all cells. Because the length of DNA can be thousands of times that of a cell, packaging this genetic material into the cell or nucleus (in eukaryotes) is a difficult feat.
 Supercoiling of DNA reduces the space and allows for a lot more DNA to be packaged.
 In prokaryotes, plectonemic supercoils are predominant, because of the circular chromosome and relatively small amount of genetic material.
 In eukaryotes, DNA supercoiling exists on many levels of both plectonemic and solenoidal supercoils, with the solenoidal supercoiling proving most effective in compacting the DNA.
 Solenoidal supercoiling is achieved with histones to form a 10nm fiber. This fiber is further coiled into a 30nm fiber, and further coiled upon itself numerous times more.
 DNA packaging is greatly increased during nuclear division events such as mitosis or meiosis, where DNA must be compacted and segregated to daughter cells.
 Condensins and cohesins are Structural Maintenance of Chromosome proteins that aid in the condensation of sister chromatids and the linkage of the centromere in sister chromatids.
 These SMC proteins induce positive supercoils.
 Supercoiling is also required for DNA/RNA synthesis. Because DNA must be unwound for DNA/RNA polymerase action, supercoils will result. The region ahead of the polymerase complex will be unwound; this stress is compensated with positive supercoils ahead of the complex. Behind the complex, DNA is rewound and there will be compensatory negative supercoils. It is important to note that topoisomerases such as DNA gyrase (Type II Topoisomerase) play a role in relieving some of the stress during DNA/RNA synthesis.
MODELING USING MATHEMATICS
 DNA supercoiling can be described numerically by changes in the 'linking number' Lk.
 Lko, the number of turns in the relaxed (B type) DNA plasmid/molecule, is determined by dividing the total base pairs of the molecule by the relaxed bp/turn which, depending on reference is 10.4-10.5.
Lko = bp / 10.4
 The topology of the DNA is described by the equation below in which the linking number is equivalent to the sum of TW, which is the number of twists or turns of the double helix, and Wr which is the number of coils or 'writhes'.
 If there is a closed DNA molecule, the sum of TW and Wr, or the linking number, does not change. However, there may be complementary changes in TW and Wr without changing their sum.
Lk = Tw + Wr
 The change in the linking number, ΔLk, is the actual number of turns in the plasmid/molecule, Lk, minus the number of turns in the relaxed plasmid/molecule Lko.
ΔLk = Lk − Lko
 If the DNA is negatively supercoiled ΔLk < 0. The negative supercoiling implies that the DNA is underwound.
 A standard expression independent of the molecule size is the "specific linking difference" or "superhelical density" denoted σ.
 σ represents the number of turns added or removed relative to the total number of turns in the relaxed molecule/plasmid, indicating the level of supercoiling.
σ = ΔLk / Lko
The Gibbs free energy associated with the coiling is given by the equation below-------------------
ΔG / N = 10RTσ2
Examples
Since the linking number L of supercoiled DNA is the number of times the two strands are intertwined (and both strands remain covalently intact), L cannot change.
The reference state (or parameter) L0 of a circular DNA duplex is its relaxed state. In this state, its writhe W = 0. Since L = T + W, in a relaxed state T = L. Thus, if we have a 400 bp relaxed circular DNA duplex, L ~ 40 (assuming ~10 bp per turn in B-DNA). Then T ~ 40.
• Positively supercoiling:
T = 0, W = 0, then L = 0
T = +3, W = 0, then L = +3
T = +2, W = +1, then L = +3
• Negatively supercoiling:
T = 0, W = 0, then L = 0
T = -3, W = 0, then L = -3
T = -2, W = -1, then L = -3
 Negative supercoils favor local unwinding of the DNA, allowing processes such as transcription, DNA replication, and recombination.
 Negative supercoiling is also thought to favour the transition between B-DNA and Z-DNA, and moderate the interactions of DNA binding proteins involved in gene regulation.


ENZYME ASSAY
Enzyme activity
• Enzyme activity = moles of substrate converted per unit time = rate × reaction volume.
• Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit.
• A more practical and commonly-used value is 1 enzyme unit (U) = 1 μmol min-1. 1 U corresponds to 16.67 nanokatals
Specific activity
• This is the activity of an enzyme per milligram of total protein (expressed in μmol min-1mg-1).
• Specific activity gives a measurement of the purity of the enzyme.
• It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of enzyme.
• Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of enzyme.
• The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min-1.
• Specific activity is a measure of enzyme processivity, usually constant for a pure enzyme.

 The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme).
 The impure sample has lower specific activity because some of the mass is not actually enzyme.
 If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated.


Types of assay
1. Continuous assays
Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There are many different types of continuous assays.
1. Spectrophotometric
 In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are called colorimetric assays.
 The MTT assay, a redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay.
 UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidized forms.
 An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme.
Direct versus coupled assays
 Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by using a coupled assay.
 Here, the product of one reaction is used as the substrate of another, easily-detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-phosphate dehydrogenase.


Coupled assay for hexokinase using glucose-6-phosphate dehydrogenase.
2. Fluorometric
 Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength.
 Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction.
 These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light.
 An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH.
 Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase.
 Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-galactoside for assaying β-galactosidase.
3. Calorimetric
 Calorimetry is the measurement of the heat released or absorbed by chemical reactions.
 These assays can be used to measure reactions that are impossible to assay in any other way.

4. Chemiluminescent
 Chemiluminescence is the emission of light by a chemical reaction.
 Some enzyme reactions produce light and this can be measured to detect product formation.
 These types of assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be detected.
 The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in western blotting.
 Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.
5. Light Scattering
 Static light scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution.
 Light scattering assays of protein kinetics is a very general technique that does not require an enzyme.
2. Discontinuous assays
Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples.
1. Radiometric
 Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates.
 The radioactive isotopes most frequently used in these assays are 14C, 32P, 35S and 125I.
 Since radioactive isotopes can allow the specific labelling of a single atom of a substrate, these assays are both extremely sensitive and specific.
 They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).
 Radioactivity is usually measured in these procedures using a scintillation counter.
2. Chromatographic
 Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography.
 This is usually done by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography.
 Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag.
FACTORS TO CONTROL IN ASSAYS
• Salt Concentration: Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. As usual there are exceptions such as the halophilic (salt loving) algae and bacteria.
• Effects of Temperature----The idea of an "optimum" rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the reaction rate and the denaturation rate. If you were to use an assay measuring activity for one second, it would give high activity at high temperatures, however if you were to use an assay measuring product formation over an hour, it would give you low activity at these temperatures.
• Effects of pH: The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds(Like temp). Most enzymes function between a pH of 6 and 8; however pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.
• Level of crowding, large amounts of macromolecules in a solution will alter the rates and equilibrium constants of enzyme reactions, through an effect called macromolecular crowding.



CHEMOSTAT
 A chemostat is a bioreactor to which fresh medium is continuously added, while culture liquid is continuously removed to keep the culture volume constant.
 By changing the rate with which medium is added to the bioreactor the growth rate of the microorganism can be easily controlled.
Steady State
 One of the most important features of chemostats is that micro-organisms can be grown in a physiological steady state. In steady state, growth occurs at a constant rate and all culture parameters remain constant.
 Micro-organisms grown in chemostats naturally strive to steady state: if a low amount of cells are present in the bioreactor, the cells can grow at growth rates higher than the dilution rate, as growth isn't limited by the addition of the limiting nutrient.
 However, if the cell concentration becomes too high, the amount of cells that are removed from the reactor cannot be replenished by growth as the addition of the limiting nutrient is insufficient. This results in an equilibrium situation (steady state), where the rate of cell growth is equal to the rate of cell removal.
 Because obtaining a steady state requires at least 5 volume changes,chemostats require large nutrient and waste reservoirs
Dilution Rate
At steady state the specific growth rate (μ) of the micro-organism is equal to the dilution rate (D). The dilution rate is defined as the rate of flow of medium over the volume of culture in the bioreactor:

Maximal growth rate
Each microorganism growing on a particular substrate has a maximum specific growth rate (μmax) (the rate of growth observed if none of the nutrients are limiting). If a dilution rate is chosen that is higher than μmax, the culture will not be able to sustain itself in the bioreactor, and will wash out.
Variations
 Fermentation setups closely related to the chemostats are the turbidostat, the auxostat and the retentostat.
 In retentostats culture liquid is also removed from the bioreactor, but a filter retains the biomass. In this case, the biomass concentration increases until the nutrient requirement for biomass maintenance has become equal to the amount of limiting nutrient that can be consumed.
 Chemostats can also be used to enrich for specific types of bacterial mutants in culture such as auxotrophs or those that are resistant to antibiotics or bacteriophages for further scientific study