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Intracellular and extra cellular industrial enzymes Presentation Transcript:
1.Intracellular and extra cellular industrial enzymes
2.Enzymes are being used more and more for industrial bioconversion i.e. making a chemical product using purified enzymes rather by pure chemical methods (e.g. citric acid production) or using whole cells (e.g. yeast in brewing).
3.Extracellular enzyme
The enzymes that function in our digestive systems are manufactured in cells - but work extracellularly.eg
Spiders and flies are two examples of animals that have taken extracellular digestion.
They secrete an enzyme soup into or on their food. In spiders, this is injected into the prey's body. The enzyme soup digests the prey's body contents (specific enzymes breaking down proteins to AAs, lipids into FAs and glycerol and polysaccharides into monosaccharides) and the spider simply sucks up the resulting already digested food.
Saprophytic fungi also secrete enzymes through their hyphal tips in order to digest their food.
4.Intracellular enzyme
Enzymes that act inside cells are responsible for catalysing the millions of reactions that occur in metabolic pathways such as glycolysis in the mitochondria and in the photosynthetic pathway in the chloroplast.
The lysosome contains many enzymes that are mainly responsible for destroying old cells.
5.Considerations when selecting a strain:
Does it do what is required?
Is it safe?
Is it cost effective?
Enzymes may be intracellular or extracellular. What is the advantage of extracellular production?
Already outside cell
Limited number secreted so easier to isolate
More robust so less likely to be broken down by heat of chemicals
6.Why are intracellular enzymes more difficult to isolate than extracellular ones?
Because they are inside the cell, first the cell has to be broken open then the enzyme separated from the mixture of all the cellular contents
Why is it more efficient to use isolated enzymes than whole cells?
Isolated enzymes are usually more efficient in biotechnology than whole cells because enzyme concentration is higher and no unwanted enzymes are present
7.Enzymes and Sources
Proteases
Overproducing strains of Bacillus, Aspergillus, Rhizopus, and Mucor.
Pectinases
Aspergillus niger.
Lactases
Yeast and Aspergillus.
Lipases
Certain strains of yeast and fungi.
Glucose isomerase
Flavobecterium arborescens or Bacillus coagulans
8.Production of Enzymes
Cultivate the organisms producing the desired enzymes.
Production can be regulated
Fermentation conditions ca be optimized for overproduction.
9.Enzyme production
Surface and submerged techniques:
Surface = enzyme produced on the surface of a solid medium
Submerged = the mould or bacterium producing the enzyme is grown throughout a liquid medium
Advantages and disadvantages?
Submerged – more yield as growth throughout but aeration necessary
10.The maximum enzyme production is usually in stationary phase of microbe growth, so a batch or fed-batch process are usually used.
The medium must be chosen to stimulate the microbe into synthesizing the correct enzyme.
For example to stimulate a microbe to synthesize amylase enzymes, a medium with starch but no sugars is used.
11.Production of Enzymes
What type of medium would you use to stimulate a microbe to synthesize a protease?
A medium with proteins but no amino acids is used.
12.Microbes are encouraged into the log phase initially with a medium with a lot of protein
This encourages rapid increase in the number of cells, but not much protease is produced.
Cells are then introduced into the fermentation vessel and allowed to grow for a further 1-8 days.
The medium now has very little protein in it. Why?
13.The microbe must produce a lot of protease because as the enzyme leaves the cell it doesn’t immediately come into contact with protein that it can break down – more protease produced to maximise the amount of amino acids from the small amount of protein.
14.Down stream processing
The remaining mixture contains enzymes, waste materials, nutrients and cells
The enzyme is extracted by downstream processing
15.Down stream processing
Cell separated from the media usually by filtration or something by centrfugation.
Depending on intra/extracellular nature of the enzyme, the cell or fermentation broth is further processed.
Recovery of intracellular enzymes is more complicated and involves the disruption of cells and removal of debris and nucleic acids.
Increasing permeability of cell membrane (CaCl2 (salt) or dimethylsulfoxide (DMSO) or change in pH
Last resort is cell disruption.
Intracellular and extra cellular industrial enzymes Presentation Transcript:
1.Intracellular and extra cellular industrial enzymes
2.Enzymes are being used more and more for industrial bioconversion i.e. making a chemical product using purified enzymes rather by pure chemical methods (e.g. citric acid production) or using whole cells (e.g. yeast in brewing).
3.Extracellular enzyme
The enzymes that function in our digestive systems are manufactured in cells - but work extracellularly.eg
Spiders and flies are two examples of animals that have taken extracellular digestion.
They secrete an enzyme soup into or on their food. In spiders, this is injected into the prey's body. The enzyme soup digests the prey's body contents (specific enzymes breaking down proteins to AAs, lipids into FAs and glycerol and polysaccharides into monosaccharides) and the spider simply sucks up the resulting already digested food.
Saprophytic fungi also secrete enzymes through their hyphal tips in order to digest their food.
4.Intracellular enzyme
Enzymes that act inside cells are responsible for catalysing the millions of reactions that occur in metabolic pathways such as glycolysis in the mitochondria and in the photosynthetic pathway in the chloroplast.
The lysosome contains many enzymes that are mainly responsible for destroying old cells.
5.Considerations when selecting a strain:
Does it do what is required?
Is it safe?
Is it cost effective?
Enzymes may be intracellular or extracellular. What is the advantage of extracellular production?
Already outside cell
Limited number secreted so easier to isolate
More robust so less likely to be broken down by heat of chemicals
6.Why are intracellular enzymes more difficult to isolate than extracellular ones?
Because they are inside the cell, first the cell has to be broken open then the enzyme separated from the mixture of all the cellular contents
Why is it more efficient to use isolated enzymes than whole cells?
Isolated enzymes are usually more efficient in biotechnology than whole cells because enzyme concentration is higher and no unwanted enzymes are present
7.Enzymes and Sources
Proteases
Overproducing strains of Bacillus, Aspergillus, Rhizopus, and Mucor.
Pectinases
Aspergillus niger.
Lactases
Yeast and Aspergillus.
Lipases
Certain strains of yeast and fungi.
Glucose isomerase
Flavobecterium arborescens or Bacillus coagulans
8.Production of Enzymes
Cultivate the organisms producing the desired enzymes.
Production can be regulated
Fermentation conditions ca be optimized for overproduction.
9.Enzyme production
Surface and submerged techniques:
Surface = enzyme produced on the surface of a solid medium
Submerged = the mould or bacterium producing the enzyme is grown throughout a liquid medium
Advantages and disadvantages?
Submerged – more yield as growth throughout but aeration necessary
10.The maximum enzyme production is usually in stationary phase of microbe growth, so a batch or fed-batch process are usually used.
The medium must be chosen to stimulate the microbe into synthesizing the correct enzyme.
For example to stimulate a microbe to synthesize amylase enzymes, a medium with starch but no sugars is used.
11.Production of Enzymes
What type of medium would you use to stimulate a microbe to synthesize a protease?
A medium with proteins but no amino acids is used.
12.Microbes are encouraged into the log phase initially with a medium with a lot of protein
This encourages rapid increase in the number of cells, but not much protease is produced.
Cells are then introduced into the fermentation vessel and allowed to grow for a further 1-8 days.
The medium now has very little protein in it. Why?
13.The microbe must produce a lot of protease because as the enzyme leaves the cell it doesn’t immediately come into contact with protein that it can break down – more protease produced to maximise the amount of amino acids from the small amount of protein.
14.Down stream processing
The remaining mixture contains enzymes, waste materials, nutrients and cells
The enzyme is extracted by downstream processing
15.Down stream processing
Cell separated from the media usually by filtration or something by centrfugation.
Depending on intra/extracellular nature of the enzyme, the cell or fermentation broth is further processed.
Recovery of intracellular enzymes is more complicated and involves the disruption of cells and removal of debris and nucleic acids.
Increasing permeability of cell membrane (CaCl2 (salt) or dimethylsulfoxide (DMSO) or change in pH
Last resort is cell disruption.
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