Question

Give at least two possibilities for why enzymes could be partially protected from heat denaturation through high substrate concentrations.

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Answer 1

The two possibilities for why enzymes could be partially protected from heat denaturation through high substrate concentrations.

Hydrolases

The ubiquitous presence of hydrolases in foodstuffs and their propensity to liberate short-chain acids (<C14) which have low odour and aroma thresholds, from the tasteless tryacylglycerol components means that this is an extremely important mechanism of introducing sensory alteration in food. Moreover, the fact that microorganisms also use hydrolases for food digestion means that also these will be actively involved in changing food properties. Changes due to hydrolysis may range from extremely desirable such as the soapy notes imparted upon blue cheeses by hydrolases originating from the mould, to extremely unpleasant high butyric notes in butter and lauric acidity in coconut oil.

In commonly used vegetable oils, the polyunsaturated C18 acids which are much better substrates than the tryacylglycerols from which they originate, will, when liberated by hydrolysis, readily be oxidised into very odoriferous compounds.

In fruits and vegetables and especially when tissues are sliced or homogenised during processing, oxidation and lypolysis will generally be very quick. Free acids are responsible for off flavours and enhanced oxidability. Also, enzymatic hydrolysis of a small amount of the acyl lipids present can not be avoided during disintegration of oil seeds, a characteristic which makes further processing mandatory for separation and/or denaturation of those enzymes.

Lipid hydrolysis is catalysed by some hydrolases, whose action and specificities differ. In particular, lipases will hydrolyse triacylglycerol and related moieties when emulsified and other hydrolases are specific for polar lipids.

Lipases

Lipases are active on a water/lipid interface and in this respect differ from esterase enzymes which cleave only water-soluble esters, such as triacetylglycerol.

Lipase activity is detected in, for example, milk, oilseeds (soybean, peanut), cereals (oats, wheat), in fruits and vegetables and in the diggestive tract of mammals. Many microorganisms release lipase-type enzymes into their culture media, and these may contribute to enhance the deterioration of foods.

As to specifity, fat-splitting enzymes, which preferentially cleave primary HO-group esters are distinguished from those which indiscriminately hydrolyse any ester bonds of acyl glycerols.

The 48 kcal lipase secreted by pig pancreas has probably been the best studied. It cleaves all acyl glycerols but preferred substrates are triacylglycerols, and worst of all monoacylglycerols, and in any case breaks the ester bonds at positions 1 or 3 only.

Oat and Aspergillus flavus lipases present no positional specificity whatsoever, whereas Geotrichum candidum lipase is specific for oleic and linoleic residues in any position, and Mucor miehei and Penicillium roqueforti lipases also show 1,3 specificity.

Acyl migration from position 2 to 1 is thermodinamically favoured and normally precedes enzymatic hydrolysis of that acyl residue; longer hydrolysis times are needed for completeness of this reaction unless an unspecific lipase is used.

At an estimated physiological concentration (14 mM), maltose was able to partially protect SspI function. Not surprisingly, this protection increased with increasing maltose concentration . At 200 and 400 mM, the intensity of the top band was much reduced compared to 0 mM maltose.