Microbial Enzymes

1
Microbial Enzymes

• Used in applications from food production to
  molecular biology research
• Microbial enzymes are active in a pH range of
  3.0 - 9.0, whereas animal enzymes require a pH
  over 5.0 to work effectively
• Some of the first commercially available enzymes
  were DNA polymerases and restriction enzymes

• DNA Polymerases were initially isolated primarily
  from Escherichia coli and became available for
  rDNA techniques

2
Taq polymerase

• Thermostable enzyme essential for PCR reactions
• Isolated from hot-spring dwelling species
  Thermus aquaticus
• Species is a thermophile (heat-loving) because
  of its ability to grow thrive under extreme
  heat
• Several companies have permission to inspect
  geysers in Yellowstone

3
Cellulase

• Enzyme produced by E. coli that degrades
  cellulose
• Widely used in the biotech industry, including
• Making animal food more easily digested
• Makes faded jeans by digesting cellulose fibers
  in cotton
• Processing of coffee beans
• Fermentation processes to create biofuels

4
Subtilisin

• Derived from Bacillus subtilis
• Protease (breaks down protein)
• Valuable component of laundry detergents
• Degrades removes protein stains from clothing

5
Amylase

• Found in a variety of organisms, including the
  saliva of humans
• Aspergillus oryzae (fungal) and Bacillus
  subtilis (bacterial)
• Sugar-digesting enzyme
• One of several enzymes used to manufacture food
• Used to create various syrups
• Degrades starch in order to produce corn syrup
  (fungal)

6
Lactase

• Produced by Saccharomyces fragilis
• Enzyme used to degrade lactose into glucose and
  galactose
• Given to lactose intolerant
• Used in the production of ice cream and frozen
  desserts

7
Invertase

• Derived from Saccharomyces cerevisiae
• Also known as sucrase
• Enzyme digests sucrose into glucose and fructose
  
• Used to create candies with a soft center
• Most commonly used to make chocolate covered
  cherries

8
Bacterial Transformation
Transformation is the ability to take DNA from
the environment.

• It is an essential step in the rDNA cloning
  process.
• Recombinant plasmids are introduced into
  bacterial cells through transformation so they
  can replicate plasmids.
• Cells that have been treated for transformation
  (so they are more receptive to take up DNA) are
  called competent cells.

9
Competent Cell Formation

• One technique involves treating cells with
  ice-cold calcium chloride
• Cations disrupt bacterial cell wall membrane
  to create small holes (so DNA can enter)
• Cells are frozen to maintain competent state
• Once prepared, they can be transformed
  relatively easily

10
Competent Cell Formation

• Target DNA inserted into plasmid with one or
  more antibiotic resistance genes
• Mixed with competent cells put on ice
• Cells briefly heated (37-42C) so DNA can enter
  cell during heat shock
• Cells plated on agar with antibiotics (only
  those with plasmid will grow into colonies)

11
Electroporation

• Another technique for transforming cells that is
  sometimes preferred over calcium chloride
• Rapid
• Requires fewer cells
• Used to introduced DNA into many cell types
  (yeast, fungi, plant, animal)
• More efficient (greater of cells get DNA)

12
Why transform?

• One reason for transformation is to replicate
  the recombinant DNA of interest.
• They can be used to mass produce proteins for a
  variety of purposes.
• One way to express and isolate a recombinant
  protein is to make a fusion protein.

13
Fusion proteins
A fusion protein is a hybrid protein consisting
of a protein from a gene of interest connected to
another, well-known protein that serves as a
tag for isolating the recombinant protein.

• Tag protein allows for isolation and
  purification of recombinant protein as a fusion
  protein
• Expression vectors are used to make fusion
  proteins.

14
Expression Vectors

• Expression vectors are plasmid vectors that
  enable bacterial cells to produce or express
  large amounts of protein
• Common expression vectors include those that
  make luciferase, maltose-binding protein (MBP)
  and glutathione S-transferase (GST).
• Luciferase derived from Photinus pyralis
  (firefly), while other 2 are from E. coli.

15
Isolating a fusion protein

• Bacterial cells are lysed homogenized to
  create an extract
• Affinity chromatography used (beads attach
  fusion proteins) then proteases cleave protein to
  release protein of interest

16
Microbial Proteins

• Close to 75 of marine organisms can release
  light through bioluminescence (release of light
  by a living organism)
• Used to attract mates in dark environment
• Bioluminescence in marine organisms is created
  by bacteria (Vibrio fischeri) with bioluminescent
  properties that use marine organism as host

17
lux genes

• Create light through lux genes (encode subunits
  that make luciferase - same enzyme as fireflies)
• Light is made by oxidation of luciferin (pigment)

• Used in fusion proteins as tag
• Can also serve as reporter genes (genes used to
  track or follow expression of other genes)
• These genes now being used to create diagnostic
  tests (tuberculosis)