Cloning & Biotech

Name some immobilised enzymes in industry

• Glucose isomerase

• Penicillin acylase

• Lactase

• Aminoacylase

• Glucoamylase

Suggest why using immobilised enzymes rather than free enzymes may be cheaper in industrial processes.

- Bioreactors can be run continuously for long periods, so less emptying and cleaning is needed

- The product will not be contaminated with enzyme, therefore the extraction of the enzyme from the product will not be needed

- The immobilised enzyme can be reused

- The process can run over a wider temperature range

What is glucose isomerase used for?

The conversion of glucose to fructose

What is penicillin acylase used for?

The formation of semisynthetic penicillins (to which some penicillin resistant organisms are not resistant)

What is lactase used for?

The hydrolysis of lactose to glucose and galactose

What is Aminoacylase used for?

The production of pure samples of L-amino acids

What is glucoamylase used for?

The conversion of dextrins to glucose.

Glucose Isomerase

- Produces fructose from glucose

- Fructose is sweeter than sucrose & glucose, therefore used as a sweetener in food industries

- Glucose is produced from cheap, starch rich plant material, and glucose isomerase is then used to turn cheap glucose into marketable fructose

Penicillin acylase

- It makes semi synthetic penicillins from naturally produced penicillins

- Many bacteria are resistant to naturally occurring penicillins, but are still vulnerable to semi synthetic penicillins

- Therefore this immobilised enzyme is important in treating infections caused by bacteria resistant to naturally occurring penicillins

Lactase

- Immobilised lactase hydrolyses lactose to glucose and galactose, giving lactose free milk

- Many people are lactose intolerant

Aminoacylase

- Produces pure samples of L-amino acids

- L-amino acids are used in the production of pharmaceuticals, organic chemicals, cosmetics, and food

Glucoamylase

- Amylase enzymes break starch down into short chain polymers called dextrins

- The final breakdown of dextrins to glucose is catalysed by immobilised glucoamylase

What are immobilised enzymes?

The enzymes are attached to an insoluble material to prevent mixing with the product

4 Methods of Immobilisation

• Encapsulation

• Entrapment

• Cross-linked

• Carrier-bound

Encapsulation

Enzyme is trapped in a semi-permeable membrane

Entrapment

Enzyme is trapped in silica gel matrix

Cross-linked

Covalent or ionic bonds between the amino acids of the enzymes, sometimes using glutaldehyde

Carrier-bound

The enzyme is attached to a carrier, such as activated carbon or clay

What are the advantages of immobilised enzymes?

1. Enzyme can be removed to be used again

2. There is less downstream processing / leads to a purer product

3. More stable than free enzymes.

What does less downstream processing mean?

After the reaction is complete, there is less work needed to purify the final product.

There will be less downstream processing because in a typical reaction with free enzymes, the enzymes would have to be removed, but if the enzymes are immobilised, then they wouldn’t be mixed in the solution.

The enzyme doesn’t need to be extracted from it

Why is being more stable than free enzymes good?

They are less likely to be denatured by pH changes or high temperatures

What are the disadvantages of immobilised enzymes?

1. The process of immobilising an enzyme can reduce its activity as it cannot mix freely with its substrate

2. Higher initial cost of equipment (bioreactor) to set up the reaction

3. Higher cost of using immobilised enzymes vs free enzymes

4. More complex procedure and therefore higher possibility of technical issues

Why do we use a fermenter / bioreactor?

To culture microorganisms on an industry scale

What are fermenters/bioreactors?

Sealed, sterile, aseptic units.

Why are they sealed, sterile, aseptic units?

It means that the product is not contaminated and the microorganisms are not in competition with other microorganisms

Limiting factors in growing bacterial colonies:

- Temperature

- Nutrients available

- Oxygen levels

- Change in pH

- Build up of waste

Temperature

Need to maintain the optimum temperature for enzyme controlled reactions such as aerobic respiration

Nutrients Available

Provided through the nutrient medium (usually a liquid broth). The nutrient level will become insufficient to support further growth and reproduction unless more nutrients are added.

Oxygen levels

Needed for aerobic respiration

Change in pH

This will fall as CO₂ is produced.

This impacts enzymes involved in metabolic processes, such as respiration, and therefore a pH buffer is used

Build up of Waste

As bacterial numbers rise, anaerobic respiration may occur, which can lead to the build up of ethanol/lactic acid which can kill the microorganisms

Fermenters

Microorganisms are cultured in fermenters. These cultures can be grown on an industrial scale in a bioreactor.

Aseptic Fermenter Parts:

- Motor

- Paddle

- pH control reservoir

- Gas exhaust outlet

- Filtered air inlet

- Cool water inlet

- Nutrient inlet

Motor

Turns the paddle to mix contents

Paddle

Mixes the contents and creates smaller air bubbles

pH control reservoir

pH buffers are added to prevent the condition from becoming too alkaline, reducing yield

Gas exhaust outlet

Prevents pressure building up

Filtered air inlet

Oxygen for respiraiton

Cool water inlet

Penicillium produces heat during growth, temperature can be monitored and cooling water runs through the water jacket

Nutrient inlet

The medium containing nutrients for the growth of penicillium is added here

Why is asepsis important?

- Avoids any unwanted microbes that would compete for nutrients

- Decrease the yield of product

- Contamination of the product

- Change the conditions in the fermenter

What is batch fermentation?

• Microorganisms starter population is mixed with a specific quantity of nutrient solution, then allowed to grow for a fixed period of time

• The products are then removed and the tank emptied

What is batch fermentation also known as?

A closed culture

When is batch fermentation stopped?

The process is stopped before the death/decline phase and the products harvested (secondary metabolites production higher in stationary phase)

What is continuous fermentation?

Microorganisms are inoculated into a sterile medium, and is added continually to the culture once it has reached its exponential point of growth

What happens to the waste products, desired products, and microorganisms in continuous fermentation?

They are continually removed, keeping the culture volume in the reactor the same

Advantages of Batch Culture

- Easy to set up and maintain

- If contamination occurs, only one batch is lost

- Very useful for process involving the production of secondary metabolite

Disadvantages of Batch Culture

- Growth rate is slower because nutrient level declines with time

- Less efficient, fermenter is not in operation all of the time

Advantages of Continuous Culture

- Growth rate is higher as nutrients are continuously added to the tank

- More efficient, fermenter operates continuously

- Very useful for the processes involving the production of primary metabolites

Disadvantages of Continuous Culture

• Set up is more difficult, maintenance of required growing conditions can be difficult to achieve

• If contamination occurs, huge volumes of products may be lost

Pour plate

Used to identify the number of colony forming units in a solution - may involve a serial dilution

Lawn plate

Use a spreader to spread out the bacterial colonies

These can be used to identify colonies that have been genetically engineered

Streak plate

Used to separate colonies of bacteria

Why do we use aseptic conditions?

To avoid any unwanted microbes that would:

• compete for nutrients

• change the conditions in a fermenter

• decrease the yield of product

• contaminate the batch

Plates should not be incubated at 35 degrees

Could lead to the growth of human pathogens

Flaming the neck of tube

Causes air to expand and push bacteria away, so less likely to settle in the tube.

Also kills bacteria on the neck of the tube

The lid should be held above the dish when adding solutions

Avoids contamination with bacteria from the air

When incubating the plate, they should be kept upside down

Prevents condensation droplets from forming on the lid and dripping onto the agar surface, which can contaminate the culture and interfere with colony growth

What is the problem with the cultured bacteria?

With cultured bacteria, there may be too numerous to count as there are so many colonies, or the colonies overlap, forming a lawn.

What is the solution to this?

Use a serial dilution method to dilute the bacteria in the broth before plating them onto the agar

Apparatus

Sterile agar plates -The agar can be made sterile by boiling

Diluted bacterial broth with a concentration of 1 x 108 CFU mm-3

Pipettes

Spreaders

Bunsen burner

Gloves

Goggles

Incubator

Fridge

Factors Affecting the Growth of Microorganisms Process

1. Spread a sample of the diluted bacterial broth onto the surface of each of the sterile agar plate

2. Tape the lid shut

3. Keep three of the agar plates in the fridge overnight 5°C

4. Keep three of the agar plates in the incubator overnight at 25°C

5. Remove the agar plates the next morning and count the number of colonies, keeping the lid on as you count

6. Calculate the average number of colonies that have formed and compare the results at each temperature

Results

Overnight, the bacterial colonies will grow large enough that they can be easily counted

The bacteria that were cultured at 25°C are expected to have developed at a much faster rate with many more colonies visible

The bacteria cultured at 5°C are expected to have formed fewer colonies which are much smaller in size, or even no colonies at all

What explains the difference in speed bacteria was developed in the different temperatures?

This difference is due to the fact that 25°C provides a temperature close to the optimum for enzyme activity in the bacteria. As a result, the rate of growth is much faster than those cultured at 5°C as enzymes' cellular reactions will be very slow

Variations on this investigation

- We can investigate the effect of pH and nutrient availability using a very similar method to the one detailed above

- pH can be altered using different buffer solutions with different pH levels to the broth

- Nutrient availability can be altered by using different agar plates with different nutrient contents

How else can you measure growth instead of counting the colonies?

Use turbidity as a measure

Standard Population Growth Curve

Lag phase

The population size increases slowly as the microorganism population adjusts to its new environment and gradually starts to reproduce

Log phase

With high availability of nutrients and plenty of space, the population moves into exponential growth (the population doubles with each division)

Stationary phase

Occurs when the population reaches its maximum as it is limited by resources e.g. nutrients, toxic substances.

The number of microorganisms dying = the number being reproduced by binary fission

Decline phase

Occurs due to lack of nutrients and death due to toxic substance build up. Death rate exceeds reproduction rate

What is the binary fission process?

1) The single circular DNA molecule undergoes DNA replication. Any plasmids present also undergo DNA replication

2) The parent cell divides into 2 genetically identical daughter cells

3) The 2 daughter cells each contain a single copy of the circular DNA molecule and a variable number of plasmids

Formula to calculate the rate of cell division by binary fission:

N = N₀ x 2ⁿ

N = the final number of bacteria

N₀ = the initial number of bacteria

ⁿ = the number of divisions

Why are logarithmic scales useful when investigating microorganisms?

Allows for a wide range of values to be displayed on a single graph

Log Scale Pros

• The log scale is easily identifiable as there are not equal intervals between the numbers on the y-axis

• The wide range of cell numbers fit easily onto the same scale

What are primary metabolites?

Primary metabolites are molecules needed for the cells normal function, like amino acids, proteins, enzymes

What are secondary metabolites?

Products produced by the microorganisms that are not part of its normal growth (like antibiotics)

Do all microorganisms produce secondary metabolites?

Nah

Primary & Secondary metabolite graph

Advantages

• Animals are less efficient than microorganisms at converting energy into biomass

• Microorganisms reproduce more quickly than plants and animals, so selective breeding is quicker

• Microorganisms can be grown on substrates that are waste products of other industries, such as whey

Advantages of Microorganisms making Food

• Animals are less efficient than microorganisms at converting energy into biomass

• Microorganisms reproduce more quickly than plants and animals, so selective breeding is quicker

• Microorganisms can be grown on substrates that are waste products of other industries, such as whey

• Quick and easy growth of large numbers

• Microorganisms can be grown anywhere at any time, and can be grown on waste products

• Single cell protein is healthier as it has lower fat content

  They can be made to taste like anything

• Microorganisms can be genetically modified and there are no welfare issues to consider when growing them

Disadvantages of Microorganisms making Food

• Growth conditions also ideal for contaminating microorganisms, so aseptic conditions are needed

• If conditions aren't optimal, then microorganisms may produce toxins

• Separation of microorganisms/proteins from nutrient medium and possible contaminants is necessary

• Single-cell protein does not have the same flavour and texture as real meat

• Some people don't like the idea of eating microorganisms grown on waste

• Some people have concerns about eating genetically modified food

• If single-cell protein is consumed in large quantities, then health problems caused by high uric acid may occur

What is biotechnology?

The large-scale commercial use of living organisms for human consumption and produces food, drugs, and other products

Name the Major Applications of Biotechnology

- Healthcare & medical processes

- Agriculture

- Industry

- Food science

Why are microorganisms used to carry out biotechnological processes?

- Wide range of waste materials can be used as cheap growth mediums

- Can be genetically engineered

- Can use low temperature & pressures (cheaper)

- Products can be separated easily from the microorganisms

What are some more reasons why microorganisms are used to carry out biotechnological processes?

- Rapid life cycles

- Fast reproduction

- Growth conditions that are easily created all year round

- Nutrient requirements are cheap

- They grow and synthesise proteins rapidly

Suggest the advantage to an organism of naturally producing an antibiotic as a by-product of its metabolism

Resistance / can grow on unwanted nutrients

Explain why microorganisms are particularly useful in treating water

They break down the organic waste into less harmful substances

What is vegetative propagation?

Natural cloning/asexual reproduction in plants

What are the vegetative organs of plants?

- Root and shoot tips

- Axillary buds

- Vascular cambium

Where are axillary buds?

Where leaves and the stem meet

Where is vascular cambium?

Between xylem and phloem

Vegetative Reproduction Process

1. Over time, a plantlet forms at the vegetative organs and remains attached to its parent plant

2. These plantlets are clones of their parents as no other DNA has been introduced

3. At maturity, the plantlet becomes detached from its parent and can live independently, when it is capable of photosynthesising by itself

4. The new plants all have the same phenotype, so are uniform

What are runners/stolons?

Modified stems that grow horizontally above ground. The buds on these stems produce roots and shoots

What are roots that form under the nodes of runner called?

Adventitious Roots

Will the plantlet be okay when the runner dies?

The plantlet is self sustaining so yh

What is propagation?

The reproduction of organisms (like plants) through methods like cuttings

Why don't methods of propagation require seeds?

It is asexual reproduction

A well as runners/stolons, how else can plants can propagate asexually?

Using tubers, bulbs, suckers