Exoenzymes

Exoenzymes are enzymes produced within the cell, then released outside of the cell to begin the process of extracellular digestion.  Unless transported into the cell via an energy-using process such as endocytosis, very large macromolecules cannot normally pass through the cell envelope.  Digestive exoenzymes break large molecules into smaller sizes via hydrolysis.  These exoenzymes can be classified on the basis of the kinds of molecules they hydrolyze; carbohydrates, lipids, proteins, or nucleic acids.  Below are examples of tests used in the microbiology laboratory to determine if bacteria produce specific exoenzymes.

Amylase
 

Amylase is an exoenzyme that hydrolyzes starch into mono- and dissacharide subunits.  In this photograph, two microorganisms were streaked on starch agar and allowed to incubate.  After incubation, the plate was flooded with iodine.  Iodine binds to starch but not to its breakdown products, so a clear zone will appear around the streak of organism that produces amylase.
 
 
 

B. subtilis produces the exoenzyme, but S. epidermidis does not.
 
 
 
 
 
 
 
 
 

Lipase
 
 

Lipase is an exoenzyme that hydrolyzes lipids into fatty acids and glycerol.  In this photograph, two organisms were streaked on a plate of spirit blue agar.  This agar contains lipids and spirit blue dye.  If a microorganism produces lipase, the fats around the streak are decomposed, causing a clear zone to appear.  The spirit blue dye also migrates through the plate toward the region lacking complete lipids, resulting in the appearance of a dark blue halo around the organism.
 

S. epidermidis produces lipase.  E. coli does not.
 
 
 
 
 
 
 
 
 

Proteases

Proteases hydrolyze proteins into polypeptides and amino acid subunits.  Below are two examples of digestive proteases.

A.  Caseinase
 

Caseinase is an exoenzyme that hydrolyzes the milk protein casein.  In this photograph, a plate of skim milk agar has been streaked with Bacillus subtilis.  As the casein is hydrolyzed around the streak, a clear halo appears.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

B.  Gelatinase
 
 

Gelatinase is a protease exoenzyme that hydrolyzes gelatin.  These two tubes were produced by stabbing a single microorganism in a nutrient gelatin deep, then incubated at 37o C for 48 hours.

Tube A contains E. coli.  This organism does not produce gelatinase, so the deep remains solid.  Tube B contains Pseudomonas aeruginosa.  This organism produces the exoenzyme, so the deep has liquified.