Food Chemistry

Module 6. Food enzymes

Lesson 15
HYDROLASES, LIPASES AND OTHER IMPORTANT ENZYMES IN FOOD

15.1 Introduction

Enzymes are biological catalysts which are proteinaceous in nature having a specific catalytic site called active centre.

In some cases enzymes contain a nonprotein part called “cofactor”. The protein portion is designated the “apoenzyme”. Without its cofactor it is catalytically inactive. The fully intact enzyme is sometimes referred to as the holoenzyme. The relation expressed in word equation is

Cofactor + apoenzyme ® Holoenzyme (or enzyme)

Cofactor can be a simple divalent metallic ion (e.g. Mg²⁺, Ca²⁺, Zn²⁺, Co²⁺, or Mn²⁺).

Cofactor can be a non protein organic compound. If the cofactor is firmly bound to the apoenzyme it is known as prosthetic group. If the cofactor is loosely bound to the apoenzyme it is known as coenzyme. Cofactors are generally stable to heat, whereas most enzyme proteins lose activity on heating.

Proenzyme or zymogen: Some of enzymes are produced in the inactive form which are called proenzymes or zymogens which can be converted into active form.

Proenzyme – Pepsinogen ; Enzyme - Pepsin

Proenzyme – Trypsinogen ; Enzyme - Trypsin

15.2 Properties of Enzymes

1. Enzyme molecules not only are largely protein in nature but are very often much, much larger than the molecules of the chemical or chemicals whose reactions they catalyze. Since the enzyme molecule is usually much larger than its substrate, it is believed that the latter can occupy a limited area on the enzyme surface. This area to which the substrate becomes bound is known as the active site or active centre of the enzyme and must bear a specific complementary relationship to the structure of the substrate (s) which allows an almost precise fit between the. The active site is made up of a binding site, and a catalytic site. Only a few of the amino acids in the peptide chain take part in the catalytic mechanism, while others, which presumably adjoin or overlap the catalytic site, determining the specificity of the enzyme. As might be expected, the active site usually includes amino acids, such as serine, histidine and cysteine, which have reactive side-chain grouping.
2. Specificity of enzymes for their substrates is one of the most striking proposals of the enzyme molecule. Although this phenomenon is exhibited by inorganic catalysts, the enzymes are far more selective and discriminating in their specificity requirements. Enzyme specificity depends on the particular atomic structure and configuration of both the substrate and the enzyme.
3. The rate of enzyme-catalysed reactions are extraordinarily more rapid than the same or similar reactions subject to nonenzymic catalysis.
4. Enzymes promote reactions under relatively mild temperatures.
5. Enzymes promote reactions at nearly neutral pHs.
6. In contrast to inorganic catalysts enzymes are synthesized under the direction of genes and consequently regulated by factors influencing those genes.
7. One of the distinctive feature observed by enzyme-catalyzed reactions but not usually observed in nonenzymatic reactions is saturation of enzyme with substrate.

15.3 Nomenclature and Classification of Enzymes

Enzymes are classified by the Commission on Enzymes of the International Union of Biochemistry. The basis of classification is the division of enzymes into six major classes and sets of subclasses, according to the type of reaction catalyzed. Each enzyme can be described in three ways –

By a trivial name: usually short and appropriate for everyday use, a

By a systematic name: which identifies the reaction it catalyzes, and

By a number of the Enzyme Commission (EC): which is used where accurate & unambiguous identification of an enzyme is required, as in international research journals, abstracts and indexes.

The six major classes of enzymes are:

1. Oxido-reductases are involved in oxidation-reduction reactions. They oxidize or reduce substrates by transfer of hydrogen or electrons or by oxygen. An example is,catalase (EC 1.11.1.6).

2. Transferases are involved in transfer of functional group. They remove groups from substrates and transfer them to acceptor molecules. An example is, glucokinase (EC 2.7.1.2).

3. Hydrolases are involved in hydrolysis reactions. These enzymes catalyze hydrolysis of ester, thioester, peptide, glycosyl, acid anhydride by the addition of water. For a substrate XY, the reaction can be represented as follows:

XY + HOH ------- HX + YOH

An example is alkaline phosphatase (EC 3.1.3.1).

4. Lyases are enzymes that catalyze the cleavage of C-C, C-O, C-N and other groups by elimination (not by hydrolysis), leaving double bonds, or conversely adding groups to double bonds. An example is fumarate hydratase (EC 4.2.1.2).

5. Isomerases are involved in the catalysis of isomerizations within one molecule. An example is mutase (EC 5.4.2.1).

6. Ligases are involved in the formation of bonds with ATP cleavage. They are involved in the biosynthesis of a compound with the simultaneous hydrolysis of a pyrophosphate bond in ATP.

Alkaline phosphatase

Trivial name : alkaline phosphatase

Systematic name : orthophosphoric monoester phosphohydrolase

Reaction catalysed An orthophosphoric monoestser + H₂O « An alcohol + H₃PO₄

Classification number : EC 3.1.3.1, where EC stands for Enzyme Commission

The first digit (3) for the class name (hydrolases)

The second digit (1) for the subclass (acting on ester bonds)

The third digit (3) for the sub-subclass (phosphoric monoester)

The fourth digit (1) designates alkaline phosphatase

Lipase

Recommended name : lipase

Systematic name : glycerol ester hydrolase

Reaction catalysed A triglyceride + H₂O « A diglyceride + a fatty acid

Classification number : EC 3.1.1.3, where EC stands for Enzyme Commission

The first digit (3) for the class name (hydrolases)

The second digit (1) for the subclass (acting on ester bonds)

The third digit (1) for the sub-subclass (carboxylic ester)

The fourth digit (3) designates lipase

15.4 Hydrolases

Most of the enzymes used in the food industry belong to the class of hydrolase enzymes. Some of them are described below.

15.4.1 Amylases

α-amylase hydrolyzes the α-1,4-bonds of amylose and amylopectin in a random manner, liberating small units with free non-reducing end groups. Low molecular weight dextrins are formed. β-amylase also hydrolyzes the α-1,4-bonds of amylose and amylopectin, removing maltose units from the non-reducing end of starch in an orderly fashion. The α-amylase and β-amylase do not cleave the α-1,6-linkages in amylopectin.

The use of amylases is important in bread making and in the manufacture of corn syrups. In bread making, during fermentation period, α-amylase present in flour catalyzes the dextrinization of the damaged starch granules. These dextrins are further hydrolyzed by β-amylase and converted to maltose, which provides the fermentable sugar for the yeast cells. During baking process, as the oven temperature rises the activity of α-amylase is destroyed. The application of amylases produce a bread with a softer crumb, deeper crust colour, greater volume, and improved grain and texture.

The conversion of starch into sweet syrups e.g. corn syrup is a combination of acid and enzymatic hydrolysis. A fungal amylase preparation consisting of α-, β- and amylo-1,6-glucosidase is used to produce a well flavoured, low viscous syrup consisting of dextrose, maltose, and a small amount of dextrin.

15.4.2 β-D-Fructofuranosidase (Invertase)

This enzyme plays an important role in the confectionary industry. It is involved in hydrolysis of sucrose. The products of hydrolysis, invert sugar consist of equimolar amounts of glucose and fructose and have a much sweeter taste than the original sucrose.

15.4.3 Pectinolytic Enzymes

Pectic enzymes act on pectic substances. They include pectin methylesterase, polygalacturonase, pectate lyases. Pectin methylesterase hydrolyzes the methyl ester bond of pectin to give pectic acid and methanol. Pectic acid flocculates in the presence of Ca2+ ions. Polygalacturonase hydrolyzes the α-1,4-glycosidic bond between the anhydrogalacturonic acid units. Pectinolytic enzymes are used for the clarification of fruit and vegetable juices.

15.4.4 Glucoamylase

Glucoamylase cleaves β-D-glucose units from the non-reducing end of an 1,4-α-D-glucan. The α-1,6-branching bond present in amylo-pectin is cleaved at a rate about 30 times slower than the α-1,4-linkages occurring in straight chains. The enzyme preparation is produced from bacterial and fungal cultures. The removal of transglucosidase enzymes which catalyze, for example, the transfer of glucose to maltose, thus lowering the yield of glucose in the starch saccharification process, is important in the production of glucoamylase. In a purely enzymatic process, the swelling and gelatinization and liquefaction of starch can occur in a single step using heat-stable bacterial α-amylase. The action of amylases yields starch syrup which is a mixture of glucose, maltose and dextrins.

15.4.5 β-D-Galactosidase (Lactase)

β-D-Galactosidase catalyzes the hydrolysis of lactose into glucose and galactose. Enzyme preparations from fungi (Aspergillus niger) or from yeast are used in the dairy industry to hydrolyze lactose. Immobilized enzymes are applied to produce milk suitable for people suffering from lactose intolerance.

15.4.6 Proteases

The reaction catalyzed by proteases (proteolytic enzymes) is the hydrolysis of peptide bonds of proteins. Most of the proteolytic enzymes used in the food industry endopeptidases. These enzymes are isolated from animal organs, higher plants or microorganisms. They are important in many industrial food processing procedures. Examples of their utilization are as follows. In the dairy industry, in cheese manufacture, the formation of casein curd is achieved with chymosin or rennin. Rennin is present in the fourth stomach of the suckling calf. Rennin can also be produced by genetically engineered microorganism. Proteinases from Mucor miehei, Mucor pusillus and Endothia parasitica are a suitable replacement for rennin. The coagulation of milk by rennin occurs in two stages. In the first, enzymatic stage, the enzyme acts on κ-casein (hydrolysis of peptide bond between Phe₁₀₅-Met₁₀₆) resulting in the formation of insoluble para-κ-casein and a soluble glycomacropeptide. The second stage involves the clotting of the modified casein micelles by calcium ions. Rennin is essentially free of other undesirable proteinases and is, therefore, especially suitable for cheesemaking.

Haze is a result of the combination of polypeptide and tannin molecules in beer giving rise to easily observed particles. Proteolytic enzymes (papain, pepsin, ficin, bromelain and microbial proteases) prevent this type of haze by reducing the polypeptide size. Papain, ficin and bromelain are sulphydryl proteases. These enzymes catalyze the hydrolysis of peptide, ester and amide bonds.

Proteases are added to wheat flour in the production of some bakery products to modify rheological properties of dough and, thus, the firmness of the endproduct. During such dough treatment, the hard wheat gluten is partially hydrolyzed to a soft-type gluten. Proteases are used for tenderization of meat. The enzymes hydrolyze one or more of the muscle tissue components. The enzymes are trypsin, papain, bromelain, ficin, etc.

15.4.7 Lipases

Lipases play a major role in cheese manufacture. Lipases hydrolyze ester linkage in glycerides. Lipase from microbial sources is utilized in cheese ripening for development of aromas. Lipases are responsible for hydrolytic rancidity in dairy products. Staling of bakery products is retarded by lipase, presumably through the release of mono- and diacylglycerols. The defatting of bones, which has to be carried out under mild conditions in the production of gelatin, is facilitated by using lipase-catalyzed hydrolysis.

15.5 Oxidoreductases are involved in oxidation-reduction reactions. They oxidize or reduce substrates by transfer of hydrogen or electrons or by oxygen.

15.5.1 Glucose Oxidase

Glucose oxidase is used to remove traces of glucose and oxygen from food products such as beer, wine, fruit juices, mayonnaise etc. It can be used as an analytical reagent for the specific determination of glucose. Glucose oxidase oxidizes glucose to gluconic acid in presence of oxygen and hydrogen peroxide. Hydrogen peroxide decomposes into water and oxygen in the presence of catalase.The enzyme is produced by fungi such as Aspergillus niger and Penicillium notatum.

15.5.2 Catalase

Catalase catalyzes the decomposition of hydrogen peroxide into water and molecular oxygen. In plants, catalase has the ability to dispose of the excess H₂O₂ produced in oxidative metabolism and to use H₂O₂ in oxidation of phenols, alcohols and other hydrogen donors. Catalase is used in combination with glucose oxidase.

15.5.3 Ascorbic Acid Oxidase

Ascorbic acid oxidase catalyzes the following reaction.

L-Ascorbic acid + ½ O₂ ---------- dehydroascorbic acid + H₂O

The reaction is significant in fruits and vegetables. It is responsible for the initiation of browning reaction, and for the eventual loss of all vitamin C activity.

15.5.4 Lipoxygenase

Lipoxygenase is utilized in the bleaching of flour and the improvement of the rheological properties of dough.

15.5.5 Peroxidase

Peroxidase catalyzes the following reaction

H₂O₂ + AH₂ --------- 2H₂O + A

AH₂ is an oxidizable substrate.

The common plant peroxidases are iron containing peroxidases. Peroxidases of animal tissue and milk (lactoperoxidase) are flavoprotein peroxidases. The peroxidase test is used as indicator of satisfactory blanching of fruits and vegetables.

15.5.6 Phenolases

Phenolases are involved in enzymatic browning. They are also known as polyphenoloxidases or polyphenolases. These enzymes have the ability to oxidize phenolic compounds to o-quinones. High levels of these enzymes are present in potatoes, apples, peaches, bananas, tea leaves, coffee beans etc. The action of phenolases is undesirable when it leads to browning in bruised and broken plant tissue but it is desirable in the processing of tea and coffee.