Enzyme Biotechnology Flashcards
(18 cards)
What are enzymes
Biological catalysts that convert substrate into product
Nature of enzymes
Catalytic effect-single molecule per unit time can convert numberous substrate molecule to product molecule
Specific for example glucose oxidase specific to beta- D -glucose
Enzyme structure and substrate binding
Amino acids are arranged into polypeptide chains that are folded into 3D structure . The 3D structure contains small area known as active site where substrate bind . Shape and charge properties of active site allow binding to occur
Protein molecule act to stabilize active sites
A cofactors may be present to help with catalytic effect
Cofactor, coennzyme , apoenzyme
When an enzyme requires a cofactor for its activity the inactive protein component referred to as the apoenzyme and apoenzyme plus cofactor is called holoenzyme
Coenzyme that binds tightly and permanently to protein is referred to as prosthetic group of enzyme
Cofactor may be organic molecule (coenzyme) but may be inorganic typically a metal ion such as iron
Enzyme kinetics
Study of factors that determine speed of enzyme catalyses reaction
Continuous vs discontinuous assay
Continuous-mix the enzyme with substrate and continuously measure appearance of product over time
Discontinue assay- involve mixing substrate and enzyme together and measuring the product formed after a set of period time
Chromagen
Artificial substrate that yields bright color making reaction easy to follow using spectrophotometer
1st enzyme kinetics
Mixing chromagen with buffer solution and then add enzyme . Place mixture in spectrophotometer and appearance of colored product will be measured
Shows a rapid reaction, then after few minutes will slow down due to ph changing ,unstable enzyme, substrate being used up
Describe the graph
Initially when substrate conc increases the rate of rxn increased , but as you increase substrate conc further the effects of reaction rate start to decrease till stage is reached where no effect on rate even if increase conc. It is said the enzyme has come close to saturation with substrate demonstrating its maximal velocity (Vmax)
Give formula for initial rate of reaction
Vmax *substrate conc/ substrate con + Km
Factors influence enzyme kinetics
pH- as ph changes the ionization of groups both at enzymes active site and on the substrate can alter influencing rate of binding
Temperature-increase temp increase molecular movement but can also lead to protein denature
Inhibitors- ie DFP inhibit acetycholinesterase activity by reacting with serine residue in active site of enzyme
Enzyme immobilization
During production of commercially important products via enzymatic catalysis removal of enzymes involves a costly downstream processing star and generally render the enzyme inactive. So immobilization fix the enzyme so that it can be reused many times making it commercially favored
Techniques of immobilization
Adsorption: adsorption of yeast invertase into activated charcoal and the subsequent use of this preparation for sucrose hydrolysis
Covalent bonding : immobilization of enzymes by covalent bonding to activated polymers
Entrapment: involves 3 methods
Inclusion within matrix of highly cross-linked polymer
Separation from bulk phase by semi-permeable microcapsule
Dissolution in a distinct non-aq phase
Ie gel immobilized living yeast cell and has been used for successf manufacture of champagne
Give application of enzyme biotechnology for
1.industrial catalyst ie alpha amylase for starch hydrolysis for conversion of starch to glucose or dextrans in food industry
2. Therapeutic agents -urokinase for plasminogen activation found in humans fkr removal of fibrin clots from bloodstream
3. Analytic agents - glucose oxidation found in asperigillus niger fkr detection of glucose in blood
4. Manipulative tools - Nucleases fro hydrolysis of phosphodiester bonds from various bacteria used to restrict enzymes used in genetic manipulation to cut DNA
Give examples of industrial catalyst, therapeutic agents, manipulative tools
Catalyst- aplha amylase, aminoacylase for production of L -amino acids
Therapeutic agents - urokinase, L.asparaginase
Manipulative tools- DNA polymerase for dna amplification used in polymerase chain reaction
Biosensors
Device that measures biological or chemical reactions by generating signals proportional to the concentration of an analyte in the reaction.
Components of biosensors
- Analyte-substance of interest that needs detection ie glucose
- Bioreceptor - molecule that recognize analyte for example enzymes . Bio-recognition takes place upon interaction of bioreceptor with analyte and signal is generated (form of light, heat, pH)
- Transducer- element that converts one form of energy into another ,it converts bio-recognition into measurable signal
- Electronics- process transduced signal and prepares it for display , signals conveyed from analogue into digital form.
Application of biosensors
Pollution monitoring
Bio markers so clinical tools to detect protein cancer bio markers
Used in artificial implantable devices such as pacemakers and other prosthetic device
Used to detect number of chemical and biological agents considered to be toxic materials of defense interest
