Molecule of the week-Amylase

Polymers of glucose are important in both plants and animals as energy reserves. In plants these are found as starch whereas glycogen in the liver and muscles of animals is a readily available supply of energy. As there are many glucose units in one starch or glycogen molecule, a series of steps is required for the complete breakdown of a starch molecule. As a result, a number of different enzymes may be required both for the digestion and utilisation of these. The best known of the enzymes is commonly known as α-amylase and this enzyme is important to many different living things, as well as being significant to our health and industrially through its applications in food processing.

Naming of amylases Firstly remember that –ose indicates a carbohydrate whereas –ase is used only for enzymes. As with many areas of science, approaches to naming of enzymes have changed over the years. The terms α- and β-amylase are old names and are trivial in the sense that they do not really tell us what reaction is catalysed by the enzyme. Even though they are non-systematic, these names are accepted, reflecting their long-standing and widespread use. The full systematic name of α-amylase is 4-α-D-glucan glucanohydrolase. Other earlier names are now obsolete include zymase and diastase which were general terms encompassing a number of enzymes involved in starch breakdown. Ptyalin has also been used for the α-amylase in saliva.

Commonly enzymes are referred to by numbers, for example α-amylase has the unique identifier EC 3.2.1.1. These designations are allocated by the Enzyme Commission which also provides a database defining all of the currently recognized enzymes [1].

Pathways of starch breakdown Interestingly there are a number of completely distinct pathways of starch breakdown in living organisms. One of these is phosphorolysis.

[(1→4)-α-D-glucosyl]n + Pi [(1→4)-α-D-glucosyl]n-1 + α-D-glucose 1-phosphate

Where n indicates the number of glucose units in the starch polymer and Pi is a biochemical abbreviation for the inorganic phosphate ion. The reaction is catalysed by phosphorylase (EC 2.4.1.1) and the process is closely linked to activation of the enzyme by ATP (adenosine triphosphate). This breakdown pathway occurs in muscle tissues where rapid mobilisation of glycogen is needed, and in the germination of peas.

The other series of breakdown pathways involve hydrolysis reactions [2, 3]. For complete utilization of granular or molecular starch, a series of distinct enzymes is needed and the various enzymes are relatively specific for substrates of a particular size range. The steps in the hydrolysis of starch are summarised in Figure 1.

Some of the enzymes involved and the steps that these catalyse (indicated by numbers) include α-amylase (EC 3.2.1.1, reactions , and ), β-amylase (EC 3.2.1.2, ), glucoamylase (EC 3.2.1.3, , and ) as well as α-glucosidase (EC 3.2.1.20, and ). In this context, step often occurs during food processing without the involvement of enzymes. If sufficient water is available and the temperature reaches a temperature of around 60C then granules break open in a process referred to as gelatinisation.

Starch granules

Starch molecules

Dextrins

Maltose

Oligosaccharides

Glucose

Figure 1 Pathways of starch hydrolysis

While α and β-amylases both hydrolyse starch chains, their mode of action is quite different. β-amylase removes disaccharide units (maltose) sequentially from one end of the chain. This is referred to as an exo- enzyme (Table 1). In contrast α-amylase randomly breaks bonds between glucose units almost anywhere in the chain, giving dextrins and oligosaccharides and so it is an endo-depolymerase. This explains why researchers have focused on α-amylase: even small amounts change the viscosity, mouth feel and appeal of foods.

Molecular properties of -amylase The enzymes from various sources have been characterised [2] and all are made up of a single polypeptide chain. Typically the molecular weight is approximately 45,000 – 60,000 (often referred as 45-60 kDa, where Daltons are used as a unit of relative molecular mass). As with many proteins, the α-amylase molecules are soluble in aqueous systems and the amino acid chain is folded into a preferred shaped which can be described as globular.

Even though there is some variation between the characteristics of the enzymes from different organisms, there is a similarity in overall size, structure and amino acid sequence. Another common feature is that all of the enzymes have a calcium ion as part of the structure. The ion sits at the active site of the enzyme which is the part of the surface of the globular molecule where the substrate chain binds during the hydrolysis reaction.

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Shape and structure of -amylases The enzymes extracted from some sources have been purified to a high degree and it has been possible to analyse their shape and appearance. Examples are presented in Figure 2 which shows models developed using the technique of X-ray crystallography.

All α-amylases appear to have one or more calcium ions embedded within the protein structure and the X-ray studies show that these are located at the active site. Therefore it is likely that the calcium probably plays a key role in the catalytic process. It has been observed that calcium enhances activity as well as the stability of the enzyme and so, in measuring α-amylase, CaCl2 is usually dissolved in the extracting solution.

Enzyme specificity Enzymes are generally highly specific and the amylases have been thoroughly studied. α- and β-Amylases both hydrolyse α(14) linkages but are unable to act upon α(16) linkages in branched starch molecules [2, 3]. In order to hydrolyse the latter links which occur at the branch points in amylopectin, the enzyme glucoamylase (EC 3.2.1.3) is required. Note that further confusion can occur because this enzyme is also commonly referred to as amyloglucosidase.

Among the various enzymes which hydrolyse starch, only α-amylase appears to be able to penetrate the outer regions of intact starch granules. In relation to the breakdown of molecules, α- and β-amylases have very different modes of action and the products formed differ between these amylases. Some of the properties of the two are tabulated and contrasted in Table 1. The hydrolysis products of the two enzymes are listed and interestingly neither can actually produce free glucose as a product. α-Amylases cannot break the last linkage at either end of a chain of glucose units in starch.

Sources and properties of -amylases There has been a considerable amount of research comparing α-amylases and it has been shown that the enzymes extracted from different organisms have significant and useful variations in properties. These include the response to conditions of pH and quite varied optima have been reported. It is well documented that the thermal stability is low for fungal compared to plant α-amylases and those from some bacteria are exceptionally stable to heat. This has particular relevance for a processor selecting the source of an enzyme for incorporation in a food formulation. The bacterial enzyme is permitted in bakery formulations, but this is not fully inactivated during baking in the oven, particularly at the centre of the loaf which may become sticky.

There is also evidence that the typical size of hydrolysis products differs between the α-amylases from different sources. One of the really practical implications of these observations is that there are varying effects of the enzymes incorporated into bread formulations on the rate of staling of loaves after baking.

As with many areas of science, when researchers have looked very closely, the story of α-amylase has been more complicated than we might have ever imagined. So in many organisms a number of quite distinct proteins have been discovered, each having the function of an α-amylase. In the literature the multiple forms of an enzyme from one organism and having the same role are often referred to as iso-enzymes (or isozymes). It is often not obvious why these are produced.

Figure 2 The structure of selected α-amylase moleculesThe enzyme from mammalian pancreas (left and centre) shows the chain of amino acids (left) and the secondary features (centre; helix, β-pleated sheet and random coil) [4]. The calcium ion (arrowed) is located within the active site and the asterisk represents a chloride ion. Barley α-amylase is shown on the right and the three calcium ions are indicated by asterisks [5].

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Where α-amylase is found The enzyme is produced by many animals species for the digestion of food. Bacteria, fungi and plants are also well documented sources of α-amylase. Following extraction, a number of these are used industrially with the best known examples of applications being laundry detergents, brewing as well as in baking as a component of bread improvers.

α-Amylase and digestion of food Humans produce two types of α-amylase, one of which is secreted by the salivary glands and the other by the pancreas. The enzyme molecules have the same pH optima and modes of action. Although similar in many ways, these are coded by distinct genes. Even though these enzymes have been thoroughly characterised, it has only been recognised quite recently that they are unable to break down all of the starch in some foods. This has led to the identification of “resistant” starch (RS) as a significant component in some (but not all) foods. RS is now regarded as a type of dietary fibre and there are well established benefits to health and wellbeing if our diet includes this component. The primary reason that eating baked beans results in gas production is that they are a very good source of RS.

The germination of cereal grains Once a mature grain takes up sufficient water, a complex chain of events is rapidly initiated. The primary purpose is to mobilise the food reserves for the young plant and its growth and development. The pattern during germination is presented in Figure 3. Whereas the enzyme β-amylase is already present in the mature grain, α-amylase is biosynthesized and the activity of this enzyme increases rapidly during germination [6]. These changes can occur as pre-harvest sprouting and may have serious adverse effects on the quality of foods.

One of the most widely used routine measurements of wheat grain quality involves an analysis of α-amylase activity. This is used as an indicator of whether sprouting has been initiated and is more reliable than visual inspection which often does not show that there is a potential problem. The test is important for farmers because sprouting can reduce the financial value of their wheat. For the food processor, the elevated enzyme activity of sprouted wheat can adversely influence product quality, although this varies for different food products.

Figure 3 The activities of α- and β-amylases during

the germination of the wheat grain [6]

In contrast, in commercial malting operations the production of high levels of α-amylase is the purpose of germinating barley or wheat. Malted grains are used widely in baking and brewing as well as other food processing operations.

α-Amylase in baking and brewing The partial breakdown of starches is important in baking and brewing. This is facilitated by the co-operative action of α- and β-amylases, reflecting the very different modes of action of the two enzymes (Table 1). In bakery formulations there is sufficient β-amylase already present in cereal grains, but it is usually necessary to add α-amylase. This can be from germinated grains (malt) or microbial sources and these are either fungal (from Aspergillus sp) or bacterial (Bacillus sp). One of the roles of the enzymes is to provide a supply of low molecular weight sugars to the yeast which do not produce amylases of their own. In brewing it is thought that β-amylase is the most significant [3] and the resultant maltose is utilised by the yeast for growth and production of the ethanol in the beer.

Table 1 A comparison of the properties and characteristics of - and -amylases

Property/characteristic -Amylase -Amylase

Action on intact starch granules relatively slow noneProducts of hydrolysis chains of many sizes and -limit dextrins maltose and a -limit dextrinEffect on starch paste viscosity rapid loss in gel strength virtually no effectPosition of attack in chain almost anywhere sequentially from one end Mode of action endo-depolymerase exo-depolymeraseActivity in sound wheat/flour relatively low but detectable mediumChange during grain germination rapid increase only minor changes

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Use of -amylases in food analysis Over recent decades advances in the purification of enzymes has resulted in increasing application of amylases as reagents in the analysis of foods. One supplier of purified enzymes, and kits using these, is Megazyme International (www.megazyme.com.au). A purified bacterial α-amylase is one of four enzymes used in the analysis of starch in foods by the international standard method. In this analysis the measurement of starch is based upon the complete breakdown of the polymer to give only glucose. For this, the pathway shown in Figure 1 (steps to ) is used where α-amylase is applied in conjunction with a purified glucoamylase.

Amylases are also very useful tools in the disintegration of samples for vitamin analysis. For cereal grain samples, amylase is often used in conjunction with proteolytic enzymes. Examples include the preparation of Asian noodles for measurement of total folates [7], folic acid [8] and most recently vitamin B6 [9].

RMIT research on -amylases Some of our own research has been focussed upon α-amylase and has investigated its use as a processing aid in the manufacture of noodles [10]. These studies showed that various sources of the enzyme have a softening effect on noodle texture. However the results raise serious doubts about the long held view that α-amylase is the primary reason that flour milled from sprouted wheats cannot be used for processing of Asian noodles.

Current studies are focussed upon the activity of the bacterial enzyme under low moisture-low temperature conditions. Other projects are on digestion, the analysis of carbohydrate utilisation and the glycaemic index of foods. As part of the latter project we have looked at the pattern of amylase action on intact starch granules and some of the electron micrographs are shown in Figure 4. Initially the attack appears to take the form of pitting, particularly at the equator of the granules. Further breakdown results in formation of channels leading to the centre of the structure while leaving some of the external “shell” in place.

References and further reading

[1] Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. 2011. Enzyme nomenclature. [Online. Internet.] Available from: http://www.chem.qmul.ac.uk/iubmb/enzyme/.

[2] Wong DWS, Robertson GH. 2002. α–Amylases. In: Whitaker JR, Voragen AGJ, Wong DWS, editors. Handbook of food enzymology. New York: Marcel Dekker. 56 p1-12.

[3] Ziegler P. 1999. Mini review: cereal beta-amylases. J Cereal Sci 29:195-204.

[4] Qian M, Haser R, Payan F. 1993. Structure and molecular model refinement of pig pancreatic α-amylase at 2.1Å resolution. J Mol Biol 231:785-99.

[5] Kadziola A, Abe J, Svensson B, Haser R. 1994. Crystal and molecular structure of barley α-amylase. J Mol Biol 239:104-21.

[6] Corder AM, Henry RJ. 1989. Carbohydrate-degrading enzymes in germinating wheat. Cereal Chem 66(5):435-39.

[7] Bui LTT, Small DM. 2007. Folates in Asian noodles: I. Microbiological analysis and the use of enzyme treatments. J Food Sci 72(5):C276–C82.

[8] Hau Fung Cheung R, Morrison PD, Small DM, Marriott PJ. 2008. Investigation of folic acid stability in fortified instant noodles by use of capillary electrophoresis and RP-HPLC. J Chromatog 1213(1):93–99.

[9] Bui LTT, Small DM. 2012. The stability of pyridoxine hydrochloride used as a fortificant in Asian wheat flour noodles. Food Chem 130(1):841-46.

[10] Cato L, Halmos AL, Small DM. 2006. Impact of α-amylase on quality characteristics of Asian white salted noodles made from Australian white wheat flour. Cereal Chem 83(5):491-97.

Figure 4 Starch granules from wheat. Images show a) intact granules b) their appearance following a short period of exposure to pancreatic α-amylase and c) more extensive hydrolysis by the enzyme. Images were prepared by Mee-Lin Lim Chai Teo using the environmental scanning electron microscope.

Prepared by Assoc Prof Darryl Small – send feedback and questions to <darryl.small@rmit.edu.au>