Proteins and Enzymes

Protein denaturation

Protein denaturation is the loss of the native three-dimensional conformation of a protein without cleavage of its covalent peptide backbone. The primary structure, the sequence of amino acids joined by peptide bonds, stays intact, while the secondary, tertiary and quaternary structures are disrupted. Heat, extremes of pH, organic solvents, detergents, high pressure and mechanical shear can all cause denaturation, because they weaken the non-covalent interactions that hold the folded state together, namely hydrogen bonds, ionic interactions, van der Waals contacts and hydrophobic interactions. Denaturation is therefore not hydrolysis, and it must not be confused with the breaking of peptide bonds.

Denatured chains may aggregate or adopt non-native conformations, so an increase in \alpha-helix or \beta-sheet content can occur, although it is not a universal result. When a globular protein is fully unfolded, the chain approaches a random coil, which is an extended and disordered chain with no defined secondary structure.

Denaturation is not always irreversible. Under mild conditions the native structure can be recovered when the denaturant is removed, as classically shown for ribonuclease, which regains its activity on refolding. Once aggregation, disulfide rearrangement or precipitation occurs, however, the native state is usually not regained, and the process becomes effectively irreversible.

• Denaturation — loss of higher-order structure with the peptide backbone intact. Example: heating egg white denatures its proteins.
• Renaturation — return to the native fold after the denaturing agent is removed, possible only under mild conditions.
• Aggregation — association of unfolded molecules through exposed hydrophobic groups, often following heat denaturation.

In food processing, heat-induced denaturation is the basis of gel formation. In egg white, heating first unfolds the native proteins, which is a reversible step. The unfolded molecules then associate and form a three-dimensional network that traps water and sets as a gel. The denatured intermediate is reached on heating, and the aggregation and gel-network formation steps also proceed on heating. Cooling is not the driving force for these steps, because the gel is a heat-set product.

Enzyme applications in food processing

Food enzymes are chosen for a specific catalytic action on a specific substrate, so each industrial enzyme is linked with one characteristic use. Matching the enzyme with its application is therefore a matter of recognising the substrate it acts upon and the product it forms.

• Chymosin (rennet) — an aspartic protease that clots casein in milk and is the key enzyme in cheese manufacture.
• Thermolysin — a thermostable metalloprotease that catalyses the coupling of N-protected L-aspartic acid with L-phenylalanine methyl ester, the key step in aspartame synthesis.
• \beta-Galactosidase (lactase) — hydrolyses lactose into glucose and galactose, used to produce lactose-reduced milk.
• Lipase — catalyses acylglycerol restructuring (interesterification) to produce structured lipids.
• Pectinase — degrades pectin, removing haze and improving filtration and clarity in fruit juice clarification.
• Papain — a cysteine protease from papaya latex that hydrolyses muscle proteins and is used for meat tenderization.
• \beta-Glucanase — hydrolyses \beta-glucans and is used in brewing to improve filtration and in baking to condition dough.
• \alpha- and \beta-Amylases — hydrolyse starch to fermentable sugars, used in brewing for mashing and in bread making for dough conditioning.

Some enzymes have more than one legitimate use. For example, amylases and \beta-glucanase each appear in both brewing and baking, so an application-matching question may admit more than one correct pairing when each pair is individually valid.

Enzymatic proteolysis in food

Proteolysis is the hydrolysis of peptide bonds by proteases. In food systems, controlled proteolysis gives desirable flavour, texture and functional changes, while uncontrolled proteolysis can cause bitterness and spoilage.

In cheese, the ripening stage depends on the breakdown of casein into peptides and free amino acids, which contribute to flavour and texture. Plasmin, the principal indigenous alkaline protease of milk, enters the curd with the milk and hydrolyses \beta-casein and \alpha_{s}-casein during ageing. Cathepsins from milk and starter bacteria also contribute to proteolysis, but they are not the main enzyme acting on casein in cheese.

Enzymes with different substrates should not be confused with cheese proteases. Myrosinase hydrolyses glucosinolates in Brassica vegetables, and alliinase converts alliin to allicin in garlic. Neither acts on casein, so they have no role in cheese ripening.