Enzymes for Enzymolysis in Food Applications

Listed below are the food grade enzymes commonly used for enzymolysis (enzymatic hydrolysis) in food applications, with their characteristics. This list is not exhaustive but covers the principal enzymes.

1. Proteases (Hydrolyze Proteins)

• Functionality: Catalyze the hydrolysis of peptide bonds. Used for meat tenderization, cheese making, production of protein hydrolysates (e.g., soy, whey), flavor enhancement, and baking.
• Activities & Specificities:
  • Serine Proteases (e.g., Trypsin, Chymotrypsin): Cleave at specific amino acid residues (Lys/Arg for trypsin; Phe/Trp/Tyr for chymotrypsin).
  • Cysteine Proteases (e.g., Papain, Bromelain, Ficin): Broad specificity; often used for meat tenderization and brewing.
  • Aspartic Proteases (e.g., Pepsin, Chymosin): Pepsin has broad specificity; chymosin (rennet) specifically cleaves κ-casein at Phe105-Met106 to coagulate milk for cheese.
  • Metalloproteases (e.g., Bacillus-derived neutral proteases): Often have endo- and exo-peptidase activity, used in brewing and flavor production.
• Kinetics: Generally follow Michaelis-Menten kinetics. Reaction rates depend on enzyme type, substrate concentration, and degree of hydrolysis (DH). Specific activity is often measured in units like Anson Units (AU) or Tyrosine Units.
• Optimal Conditions:
  • Papain: pH 5.0-7.0, Temp 50-70°C. Cofactor: None required, but reducing agents activate.
  • Bromelain: pH 4.0-6.5, Temp 50-60°C.
  • Ficin: pH 5.0-8.0, Temp 45-65°C.
  • Pepsin: pH 2.0-4.0, Temp 37-40°C.
  • Trypsin: pH 7.0-9.0, Temp 37°C.
  • Microbial Neutral Protease (e.g., from Bacillus subtilis): pH 6.0-8.0, Temp 50-60°C. Cofactor: Often Zn²⁺ or Ca²⁺.
  • Chymosin (Rennet): pH 5.3-6.3 (for milk), Temp 30-40°C. Ca²⁺ is required for milk coagulation.

2. Carbohydrases
a) Amylases (Hydrolyze Starch)

• Functionality: Hydrolyze α-1,4 and/or α-1,6 glycosidic bonds in starch. Used in baking, brewing, starch liquefaction/saccharification, and syrup production.
• Activities:
  • α-Amylase (endo-acting, EC 3.2.1.1): Randomly cleaves internal α-1,4 bonds, producing dextrins and oligosaccharides.
  • β-Amylase (exo-acting, EC 3.2.1.2): Cleaves maltose units from non-reducing ends of starch.
  • Glucoamylase (exo-acting, EC 3.2.1.3): Cleaves glucose units from non-reducing ends of starch and dextrins.
  • Pullulanase (debranching enzyme, EC 3.2.1.41): Hydrolyzes α-1,6 linkages in pullulan and starch.
• Kinetics: Follow Michaelis-Menten kinetics. Activity measured in Dextrinizing Units (DU) for α-amylase or Solubilizing Units (SU).
• Optimal Conditions:
  • Bacterial α-Amylase (e.g., from Bacillus licheniformis): pH 5.5-7.0, Temp 90-110°C, Ca²⁺ stabilizes.
  • Fungal α-Amylase (e.g., from Aspergillus oryzae): pH 4.5-5.5, Temp 50-60°C.
  • Glucoamylase (from Aspergillus niger): pH 4.0-4.5, Temp 55-60°C.
  • Cereal β-Amylase (e.g., from barley): pH 5.0-5.5, Temp 50-60°C.

b) Pectinases

• Functionality: Degrade pectin. Used for fruit juice clarification, extraction, and viscosity reduction.
• Activities:
  • Polygalacturonase (PG, endo- and exo-): Hydrolyzes α-1,4-glycosidic bonds in polygalacturonic acid.
  • Pectin Lyase (PL): Cleaves via β-elimination, producing unsaturated oligogalacturonides.
  • Pectin Esterase (PE): Removes methoxyl groups from pectin.
• Kinetics: Michaelis-Menten. Activity often measured in Polygalacturonase Units (PGU) or Pectin Lyase Units.
• Optimal Conditions:
  • Commercial Pectinase Mixes (from Aspergillus spp.): pH 3.5-4.5, Temp 40-50°C.

c) Cellulases & Hemicellulases

• Functionality: Hydrolyze cellulose and hemicellulose. Used for fruit/vegetable processing, brewing, and extraction of oils/compounds.
• Activities: Complex enzyme systems (endoglucanases, cellobiohydrolases, β-glucosidases; xylanases, β-xylosidases).
• Optimal Conditions:
  • Commercial Cellulase (from Trichoderma reesei): pH 4.5-5.0, Temp 50°C.
  • Xylanase: pH 4.5-6.0, Temp 50-60°C.

d) β-Glucanase

• Functionality: Hydrolyzes β-glucans in cereals (e.g., barley, oats). Primarily used in brewing and animal feed to reduce viscosity.
• Optimal Conditions:
  • Bacterial β-Glucanase: pH 5.5-7.0, Temp 50-60°C.
  • Fungal β-Glucanase: pH 4.5-5.5, Temp 50-55°C.

3. Lipases (Triacylglycerol Acylhydrolases, EC 3.1.1.3)

• Functionality: Hydrolyze ester bonds in triglycerides, releasing free fatty acids, di-/monoglycerides, and glycerol. Used for cheese flavor development (lipolysis), dairy flavor production, and interesterification of fats/oils.
• Activities: Vary in positional specificity (sn-1,3 specific or non-specific) and fatty acid chain length preference.
• Kinetics: Michaelis-Menten, often influenced by interfacial activation. Activity measured in Lipase Units (LU) or International Units.
• Optimal Conditions:
  • Pancreatic Lipase: pH 7.0-8.5, Temp 37°C.
  • Fungal Lipase (e.g., from Rhizomucor miehei): pH 7.0-9.0, Temp 30-40°C (for cheese).
  • Bacterial Lipase (e.g., from Candida antarctica - used immobilized): pH 7.0-8.0, Temp 60-70°C (for interesterification).

4. Phospholipases (e.g., Phospholipase A1, A2, C)

• Functionality: Hydrolyze phospholipids. Phospholipase A2 (PLA2) is used for dairy cream restructuring and in egg yolk processing to improve emulsification properties (e.g., mayonnaise).
• Optimal Conditions:
  • Commercial PLA2 (from porcine pancreas or microbial): pH 5.0-8.0, Temp 40-55°C. Ca²⁺ is often required as a cofactor.

5. Naringinase (α-L-Rhamnosidase + β-Glucosidase complex)

• Functionality: Hydrolyzes naringin (bitter compound in grapefruit) to non-bitter compounds. Used for citrus juice debittering.
• Optimal Conditions:
  • Fungal Naringinase: pH 3.5-5.0, Temp 40-60°C.

General Kinetic & Reaction Condition Notes:

• Kinetics: Most food enzymolysis reactions are modeled using the Michaelis-Menten equation for initial rates. For extended hydrolysis (e.g., of proteins or polysaccharides), the progression is described by parameters like Degree of Hydrolysis (DH), and kinetics can become more complex due to product inhibition and shifting substrate specificity.
• Optimal Conditions: The stated pH and temperature ranges are general optima for activity. Stability may differ. Industrial processes often balance activity with enzyme stability and process constraints.
• Cofactors: Many metalloenzymes (e.g., neutral proteases, some amylases) require Ca²⁺, Zn²⁺, or Mg²⁺ for structural stability and activity. Chelating agents inhibit them.
• Units: Enzyme activity is quantified in standardized units (U), typically defined as the amount of enzyme that catalyzes the conversion of 1 μmol of substrate per minute under defined conditions (pH, temperature, substrate).

Important Consideration: Commercial enzyme preparations for food use (e.g., from Novozymes, DuPont, Amano, Kerry) are often complex mixtures of multiple enzyme activities (protease, amylase, etc.) tailored for specific substrates (wheat, meat, fruit) and process conditions. The precise specifications are provided by the manufacturer for each product.