Enzymes Flashcards
What are proteins?
Macromolecules composed of amino acids
Peptide: molecule of 2 or more amino acids (conventional limit 50 a-a)
What are the structures seen in protein?
Primary protein structure
Secondary protein structure
Tertiary protein structure
Quaternary protein structure
What are enzymes?
Proteins that catalyse biochemical reactions
Increase rate of reaction
How are enzymes specific to the substrate?
Group specificity
Absolute specificity
What is the mechanism of enzyme activity?
Shape and charge of the active site enable binding to the type of substrate molecule
What are some examples of enzyme uses in human society history?
Cheese making rennin
Baking, brewing and distilling- yeast
Detergents- hydrolases
Leather tanning- proteases in dog and pigeon faeces
What are the advantages of using isolated enzymes?
Very substrate specific and product specific
Fewer by products
No biomass to dispose of
Energy and resource efficient
Reactions proceed at low temperatures and pressures
Higher enzyme concentrations can be used than would be available in cell
What are the disadvantages of enzymes in cell culture?
Majority of the nutrients get converted into biomass (more cells)
Cells perform many biochemical reactions, not all of which are wanted
Conditions for product stability may not be compatible with growth
Product may ‘stick’ to cells
What are the main types of enzymes?
Oxido-reductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
When do many enzymes only perform their catalytic role?
When they are associated with a coenzyme
These are relatively small, non protein molecules, which form a complex with the enzyme
What are the main sources of enzymes?
May come from any organism
Frequently produced by microorganisms- 50% from fungi e.g. yeasts, Aspergillus spp, -35% bacteria. Plants/animals account for the rest
What was one of the first uses of modern industrial enzyme?
takadiastase, a mix of hydrolytic enzymes from Aspergillus oryzae
Species have been used for thousands of years of Asia
Why are microbial sources of enzymes regarded as preferable?
Easy and cheap to grow
Easier to control enzyme/protein production
What 2 groups can enzymes be classified into?
Enzymes can be intracellular or extracellular
Extracellular enzymes: Enzymes that are secreted into the medium, tend to be more robust
Extracellular present in a less complex mixture, thus purification easier
Intracellular enzymes- have to purify desired enzyme away from others
How can enzymes be used during industrial processes?
Can be added directly to solutions of the substrate, but this can be expensive and is single use, as the enzyme is present with the final product
Preferable to immobilise enzymes, as the enzyme can then be used many times and is not present in the final product
What methods exist in order to use enzymes in industrial processes?
Methods:
Caging/entrapment
- Enzymes trapped inside a porous bead of silica, agarose etc
- Substrate/product able to diffuse in and out of the beads
Surface absorption
- Relies on weak attraction between enzyme and surface, can result in enzyme loss
Cross-linking
- Enzymes are covalently joined to bead surfaces
- Need to be careful not to inhibit activity
What are extremophiles?
Extremophiles are defined as organisms that live in extreme environments under high pressure and temperature, many of which are bacteria and archeans
What are extremozymes?
Enzymes of extremophiles are referred to as extremozymes
What are the characteristics of thermophiles?
Thermophiles >45 degrees
Heat resistant proteins:
- Increased resistance to folding
- Increase in protein compactness
- Increase in the number of hydrogen bonds
- Increased in the number of charged amino acids
What are the characteristics of Mesophiles?
24 to 40 degrees
Proteins denature at high temperatures (ca. 40 degrees)
Proteins unfolds- deactivated
What are the characteristics of the Psychrophiles?
-20 to 20 degrees
Cold resistant proteins:
- Flexibility
- Reduction in core hydrophobicity
- Decreased ionic/electrostatic interactions
- Additional surface loops
What are the Tardigrads?
Water bears, moss piglets
Small (<1mm) eight legged invertebrates in their own phylum
Why are the Tardigrads significant?
Can endure up to 30 years without food or water
Survive at absolute zero or boiling
Endure pressures 6 times greater than the deepest ocean trench
Endure extreme conditions by entering dormant state, compared with true extremophiles that are adapted to extreme environments and thrive in them
Possess unique protein, Damage suppressor protein (Dsup), that associates with nuclear DNA, believed to protect it from strand breaks
What are the areas of extremophile application?
Dairy industry
Biofuel industry
Bioremediation
Detergent industry
Starch liquification
Water treatment
What are some examples of extremozymes?
Acidophillic extremozymes
Heat-adapted extremozymes
Cold-adapted extremozymes
Halophillic extremozymes
Alkaliphillic extremozymes
What is an example of enzyme application in food and drink industry?
High fructose corn syrup (HFCS)
Corn starch = infinite lengths of glucose
Immobilised glucose isomerase coverts glucose to fructose
What is another example of enzyme application in the food and drink industry?
Bread textures
Bacterial xylenases from marine Antarctic bacteria used to improve bread quality
What is an example of enzyme application in the dairy industry?
Digestive enzymes
Interest in marine proteolytic enzymes for application in dairy industry
Investigated enzymes of Munida
Purified 6 forms of trypsin
Fragment associated with bitter flavours in cheese, thought that novel trypsins could accelerate maturation process
Why are enzymes used in animal feed?
Many feed ingredients not fully digestible/absorbed cannot digest phytic acid from legumes
Feeds supplemented with phytase maximise phosphorous availability in food, reduces amount released to environment
Improves digestibility for livestock
Extends range of raw materials
What enzymes do animal feed contain?
Xylanase- non starch polysaccharides
Glucanase and alpha amylase starch
Subtilisin protease
Polygalacturonase breaks down pectin
When were enzymes first used in detergents?
Started in 1913, with addition of trypsin from pig pancreas
Low activity/stability made enzymes unpopular additions until the exploitation of bacterial proteases which were more tolerant to pH
What were the uses of enzymes in detergents limited to for many years?
Use of enzyme based detergent initially limited to removing blood stains from hospital and abattoir uniforms
Proteases dominant for many years until amylases, lipases, and cellulases started to be added
More recent enzyme groups used include mannanases and pectate lyases
What does each enzyme used in detergents do?
Cellulases: improve cleaning and fabric of cotton-based garments
Lipases: act as alternatives to current surfactants, acting on lipids thus targeting grease
Mannanases and pectinases: Improve removal of plant/fruit based stains such as salad dressing, ketchup, frozen desserts
Proteases- effective in removing egg stains, minced meat and oatmeal
Amylases- remove starch containing material
What are some molecular applications of enzymes?
Mesophile polymerases destroyed by high temp
Taq DNA polymerase (operate at 72 degrees) isolated from Thermus aquaticus
Found in marine sand surrounding sulfurous volcanoes
Anaerobic (lives without oxygen)
Capable of growing at 70-103 degrees
Maintains chromosomal integrity up to 103 degrees
Highly resistant to radiation
What types of enzymes are desirable in molecular biology?
Psychrophiles
Used in molecular biology for de-phosphorylation of DNA vectors prior to cloning to avoid interference with the subsequent steps
Psychrophillic (heat-labile) enzymes inactivated by moderate heat, allowing performance of subsequent steps in the same reaction vessel
