Microbial Biochemistry Exoenzymes

Microbial Biochemistry I: Exoenzymes

Understanding Biochemistry

• Definition: Biochemistry refers to the chemical reactions occurring within a cell.
  • Example: Glycolysis pathway reactions, where each reaction step is catalyzed by an enzyme.
• Different microorganisms may exhibit distinct biochemical properties, leading to varied test results that help in identification.

Exoenzymes

• Definition: Exoenzymes are enzymes secreted outside the cell to hydrolyze macromolecules into smaller monomers.
• Importance: Large macromolecules (e.g., polysaccharides, lipids, nucleic acids, proteins) cannot pass through cell membranes and must be broken down into monomers for cell uptake.

Macromolecules Utilized by Microbes

• Microbial metabolism can be assessed for:
  • Carbohydrates: Starch
  • Proteins: Casein, Hemoglobin, Gelatin
  • Lipids: Tributyrin
  • Nucleic Acids: DNA

Enzyme Classes

• Proteases: Enzymes acting on proteins
• Lipases: Enzymes acting on lipids
• Nucleases: Enzymes acting on nucleic acids (DNA)

Casein Agar Plate (CAP)

• Characteristics: Casein is a protein found in milk, giving it a white appearance.
• Function: Caseinase (a protease) cleaves casein into smaller fragments, making the medium clear around the colony if the enzyme is produced.
• Differential Test: Clear zone around bacterial growth indicates caseinase production.

Tributyrin Agar Plate (TAP)

• Characteristics: Tributyrin is a type of triglyceride lipid.
• Function: Tributyrinase (lipase) breaks down lipids into fatty acids and glycerol.
• Differential Test: Clearing around growth indicates the enzyme's presence.

Starch Agar Plate (SAP)

• Characteristics: Starch is a large polysaccharide.
• Function: Amylases break starch down into mono- and disaccharides.
• Differential Test: Iodine added to agar turns dark blue in the presence of starch; clearing indicates starch hydrolysis.

DNA Agar Plate (DAP)

• Characteristics: Contains toluidine blue O, showing blue in presence of DNA.
• Function: DNase hydrolyzes DNA into nucleotides, producing a rose/pink color around colonies.
• Differential Test: Blue agar for negative test; rose/pink indicates DNA hydrolysis presence.

Blood Agar Plates (BAP)

• Characteristics: Contains 5% blood to differentiate bacterial hemolytic capabilities.
• Hemolysis Types:
  • Beta Hemolysis: Complete destruction of RBCs; clear zone around colony (e.g., Streptococcus pyogenes).
  • Alpha Hemolysis: Partial RBC lysis; greenish halo (e.g., Streptococcus pneumoniae).
  • Gamma Hemolysis: No lysis; no medium change (e.g., Staphylococcus epidermidis).
• Differential Test: Isolated colonies best for hemolysis determination.

Gelatin Hydrolysis

• Characteristics: Gelatin derived from collagen is used as a semisolid medium.
• Function: Gelatinase (a protease) hydrolyzes gelatin into amino acids, leading to liquefaction.
• Result Identification: Liquid medium at low temperatures indicates positive gelatinase activity.