22 - Cloning/Biotech Part 2 Flashcards
Isolated enzymes efficiency
Less wasteful than whole enzymes as whole microorganisms use up more substrate + they work at higher concentration than possible in a cell
Isolated enzymes artificial
Maximise efficiency - isolated enzymes can be given ideal conditions for maximum product formation
Isolated enzymes waste
More specific as no unwanted enzymes are present so no wasteful side reactions
What is downstream processing?
Series of steps to purify a sample/extract products - whole microorganisms gives a variety of products making isolation harder!!!
Extracellular > Intracellular
When secreted, extracellular enzymes are easier to isolate and cells produce a few of extracellular enzymes but a range of intracellular enzymes (processing)
Robust IE OR EE
EE as conditions outside cell less controlled so adapted to greater variations in temp and ph than ie
When are IE used?
Bigger range of IE so in some cases its ideal
Disadvantage of EE
Expensive extraction/isolation process and need for tightly controlled conditions
Example of IE used in industry
Penicillin acylase for converting penicillin into drugs which are more effective
Why are enzymes immobilised?
As free enzymes are often very wasteful - because they are now held in place they can be recovered from the reaction mixture and reused time after time - enzymes do not contaminate thus less downstream processing = economical
Advantage of immobilised enzymes
Reused hence cheaper
Less downstream processing can easily separate/no contaminate
More reliable = more control over environment
Greater temperature tolerance - less easily denatured
Immobilised enzymes Ease of manipulation??
Catalytic properties can be altered to fit a particular process - immobilised glucose isomerase can be continuously used for over 1000 hours ; keeps costs low
Disadvantage of immobilised enzymes
Reduced efficiency - may reduce its activity rate
HIGHER INITIAL COSTS OF BIOREACTOR BUT ENZYMES DO NOT NEED REPLACING
More technical issues
4 methods of immobilised enzymes
Adsorption to inorganic carriers
Covalent/ionic bonding
Entrapment in matrix
Membrane entrapment
Adsorption to inorganic carriers
Very simple and cheap/enzymes accessible to substrate BUT enzymes can be lost from matrix
Surface immobilisation covalent/ionic
Cost varies/enzymes strongly bound - ph has little effect on activity BUT active site may be modified in process so less effective
Entrapment in gelatin/polysaccharides
Widely applicable to different processes BUT may be expensive/difficult to entrap/diffusion of substrate can be slow and hold up activity
Membrane entrapment
Simple and small effect on enzyme activity BUT relatively expensive and can hold up rate of reaction with slow substrate diffusion
Microorganisms need
Food/oxygen and a carefully controlled environment
Penicillin acylase
Semi synthetic penicillins from naturally producing penicillins - many bacteria still vulnerable even with antibiotic resustance
Immobilised glucose isomerase
Fructose made from glucose - much sweeter than sucrose or glucose and is widely used as a sweetener
Lactase
Produces lactose-free milk ; hydrolyses lactose to glucose and galactose for some people/cats are intolerant of lactose
Aminoacylase
Produces L-amino acids for pharmaceuticals
Glucoamylase
Breakdown of starch to glucose syrup (dextrins to glucose)
Nitrile hydratase
Allows hydration of acrylonitrile to acrylamide - without using suphuric acid/reduced copper catalyst but conditions needed were extreme (yield is poor and unwanted by products were formed)
