USE OF ENZYMES IN THE FOOD INDUSTRY

VII. USE OF ENZYMES IN THE FOOD INDUSTRY

In a historical sense, enzymes were used in food preparation long before their ‘‘discov- ery.’’ Wrapping meat in certain leaves as a tenderizing aid for preparation or cooking serves as one example. Various fermented foods consumed around the world—alcoholic and fermented beverages, cheeses, fermented vegetables, fermented oilseed products, and fermented cereal and grain products—exemplify the use of enzymes in the fermentation or preservation of food. With the advent of modern enzymology, however, isolated and partially purified enzymes have gained increasing use in numerous synthetic, degradative, and analytical reaction.

The successful use of enzymes in any industrial biochemical process is contingent on several factors:

1. The process should be simple and be applicable under conditions that minimize growth of microbial contaminants while reducing viscosity.

2. Enzymes used commercially should be inexpensive, readily available, and ap- proved for use by regulatory agencies.

3. Commercial enzymes must be amenable to use at high substrate concentration and must also be highly active.

4. The interaction of such process parameters as pH, time of residence, tempera- ture, and substrate concentration should be well understood in order to achieve process optimization.

Market statistics on the use of enzymes vary depending on the group that generates the report. In general, 59% of enzymes used are proteases, 28% carbohydrases, and 3% lipases, and all other enzymes combined account for 10% of those used. A majority of the proteases are used in the detergent industry, but rennets account for 10% of all other enzymes used, and all of the rennet usage is attributed to cheese manufacture. Among carbohydrases, amylases, isomerases, and pectinases are important, and these enzymes are used almost exclusively in the food industry. Therefore, one estimate accounts for usage of enzymes as follows: in 1985 the total enzyme market in the United States was $185 million, and the food sector used $55 million worth, while the beverage industry used $53 million worth of enzymes. On the same scale, cleaning products used $25 million worth of enzymes, and all other categories of enzyme use accounted for $49 million (Ci- anci, 1986). The food and beverage sectors used over 58% of the market value of enzymes. Cianci (1986) also reported that the $185 million enzyme sales could be categorized into $88 million for the protease market, while rennets accounted for $35 million and papain for $3 million. The carbohydrase market similarly could be subdivided into glucose iso- merase, $28 million; glucoamylase, $21 million; amylase, $11 million; and pectinases, $4 million. Whereas, only about 43% of the protease market could be attributed to the food industry, over 90% of the carbohydrases are used by the food and beverage industries. The total U.S. enzyme market was estimated to be $260 million in 1990, and of this

Table 1 Number of Enzymes Identified Number of enzymes

Enzyme type

identified

representing a total growth of nearly 28% in the beverage sector and 18% in the food sector over a 5-year period. Table 1 lists the number of enzymes identified (Roberts et al., 1995).

A wide variety of uses of enzymes in the food industry can be cataloged. The follow- ing subsections provide only some representative example. The reader is referred to a series of references to obtain specific details (Wiseman, 1983,1985; Reed, 1975; Birch et al., 1981; Godfrey and Reichelt, 1983; Fogarty, 1983; Schwimmer, 1983).

A. Hydrolysis of Proteins

It is probable that in the future the production of enzymatic hydrolysates from soybean, fish, meat, and microbial protein will become increasingly important. The factors that affect the degradation of proteins by enzymes include enzyme specificity, protein denatur- ation, substrate and enzyme concentration, pH, ionic strength, temperature, and the pres- ence of inhibitors. Native proteins are not generally susceptible to degradation by proteo- lytic enzymes, as their compact conformation makes potentially susceptible bonds inaccessible (Robinson and Jencks, 1965). Denaturation unfolds proteins and makes the peptide bonds accessible to proteolytic cleavage.

Enzymes may be used to hydrolyze proteins in order to improve their functional properties (Whitaker, 1977; Kilara, 1985b) Proteolytic enzymes from Bacillus licheni- formis and B. subtilis have been used to modify soy proteins (Peterson, 1981). Unmodified soy proteins are insoluble at their isoelectric pH of 3–5. Enzymatic hydrolysis improves its usefulness.

Hydrolysis also increases emulsifying and whipping capacities, with the maximum effect being observed at 4 and 5% degree of hydrolysis, respectively. Prior enzyme hydro- lysis can give up to a tenfold increase in the volume of whipping capacities. Proteolytic enzymes can be used to reduce the viscosity of protein solutions and improve handling.

Excessive proteolytic action on certain proteins generates bitter peptides. Proteins with a high proportion of amino acids with hydrophobic side chains has a greater tendency to produce bitter hydrolysates than other proteins (Ney, 1971). It has been shown that bitterness in soy protein increases with the degree of hydrolysis. A balance has to be struck so that high yields of soluble protein are obtained without developing excessive bitterness.

The following procedure may be used to produce acceptable soy protein hydroly- sates. An 8% solution of soy protein at pH 8 may be hydrolyzed at 50–55 °C using alkaline Bacillus proteinase (Petersen, 1981). When the degree of hydrolysis is 9–10%, acid is The following procedure may be used to produce acceptable soy protein hydroly- sates. An 8% solution of soy protein at pH 8 may be hydrolyzed at 50–55 °C using alkaline Bacillus proteinase (Petersen, 1981). When the degree of hydrolysis is 9–10%, acid is

1. Milk-Clotting Enzymes Traditionally the enzyme used to clot milk in the formation of cheeses was calf rennet

(EC 3.4.4.3) obtained as a saline extract from the abomasum or fourth stomach of un- weaned calves. The calf enzyme quickly clots milk at pH 6.7 and has been the most intensively studied of the enzymes that coagulate milk protein (Foltmann, 1971). As the slaughter of young animals has decreased, calf rennet has become increasingly difficult to obtain. This has led to the use of rennet substitutes. Some rennet substitutes consist of mixtures of rennet and pepsin. In the last decade milk-clotting enzymes of microbial origin have become important in the manufacture of cheese. The preparations that have become established in cheese manufacture include enzymes from Mucor meihei, M. pusillus, and Endothia parasitica (Sternberg, 1976). Proteases are also produced by A. oryzae and other Aspergillus species. These enzymes are known by Koji and it is widely used in brewing of soy sauce and mainly in the baking industry.

In the enzymatic clotting of milk an especially labile bond between Phe 105 and 106 in k-casein micelles is cleaved to yield two peptides (Dalgleish, 1982). One peptide, comprising the amino acid residues 106–169 and referred to as the glycomacropeptide, is soluble and diffuses away from the micelle. The second peptide comprises the amino acid residues on the micelle. Progressive hydrolysis leads to changes in the micelle struc- ture that eventually lead to aggregation. Both the processes, enzymatic hydrolysis of k- casein and the subsequent aggregation, occur simultaneously. Calcium ions are necessary for the precipitation of para-casein to occur (Sardinas, 1976).

In addition, the hydrolysis of k-casein is dependent on pH ionic strength and temper- ature. Prior heating of milk reduces the susceptibility of k-casein to the action of rennet (Morrissey, 1969; Wilson and Wheelock, 1972). Rennet preparation from Mucor are more affected by variations in calcium ions than calf rennet (Nelson, 1975).

All milk-clotting enzymes are proteolytic enzymes, and their activity is characterized by an increase in soluble nitrogenous compounds in addition to the aggregation of casein micelles to form a gel structure. All are aspartate proteases and depend for their activity on the presence of two reactive aspartate residues at the active sites of the enzymes. En- zymes from different sources appear to share many features of primary structure catalytic activity and three-dimensional structure.

The pH optima for the enzymes are between 1.5 and 5 depending on the enzyme and type of substrate. The acid pH optima arise from the presence of the two aspartate residues that are essential for activity. One of the aspartate residues is protonated, and the second one is ionized. The aspartic acid residues are reactive toward specific reagents. Diazoacetyl norleucine methyl ether and 1,2-epoxy-3-(p-nitro-phenoxy) propane bind to the different aspartate residues and are also inhibited by pepstatin, a peptide from Strepto- myces.

In contrast to microbial enzymes, gastric rennets are secreted as inactive proteins that require activation by removal of a peptide of about 40 amino acid residues from the amino end of the enzyme precursor (Asato and Rand, 1977). Microbial enzymes are pro- duced in active form. Rennets of microbial origin have been found to clot milk in a manner that is generally similar to clotting by calf rennet. However, cheese made with calf rennet is the standard for taste, flavor, consistency, and texture. All commercial preparations of In contrast to microbial enzymes, gastric rennets are secreted as inactive proteins that require activation by removal of a peptide of about 40 amino acid residues from the amino end of the enzyme precursor (Asato and Rand, 1977). Microbial enzymes are pro- duced in active form. Rennets of microbial origin have been found to clot milk in a manner that is generally similar to clotting by calf rennet. However, cheese made with calf rennet is the standard for taste, flavor, consistency, and texture. All commercial preparations of

For milk to be clotted satisfactorily, the clotting enzyme must attack at or close to the Phe 105–Met 106 bond in k-casein. The cleavage destroys the stabilizing properties of k-casein and allows curd to be formed. The capacity for general cleavage must be low in order to avoid nonspecific production of soluble peptides. Excessive proteolytic activity can result in extensive breakdown of milk protein, reduced yield, soft texture, and bitter taste. Compared to calf rennet, microbial enzymes tend to have more unwanted proteolytic activity, which produces more nonprotein nitrogen. It may be necessary to use high-tem- perature scalding to stop enzyme activity.

In some situations the thermal stability of microbial rennets is a disadvantage. Resid- ual enzymes in whey can continue to break down protein and limit the use of whey in foods (Harper and Lee, 1975). At a particular pH, microbial rennets are more resistant to heat treatment in whey than calf rennet (Hyslop et al., 1975). The different proteolytic specificities of microbial rennets may change the maturation characteristics of cheeses to give unfamiliar off-flavours (Cheeseman, 1981). There is a greater degree of variability with batches of microbial rennets due to fermentation and other conditions than with calf rennet.

2. Proteases Used in Meat Tenderizing Proteolytic enzymes from pineapple, fig, and Aspergillus spp. have been used to tenderize

meat (Lawrie, 1974). Papain, isolated from the latex and fruit of Carica papaya, is perhaps the most widely used. Like the other plant proteases, papain is a sulfhydryl protease and will degrade myofibril and connective tissue proteins. Its pH optimum is 5–9. The enzyme is extremely stable even at elevated temperature (Glazer and Smith, 1971).

Papain has little effect at room temperature but will act when the meat is cooked. It shows optimal activity above 50 °C. Undenatured collagen is resistant, but over 50°C collagen fibers are loosened, with maximum solubilization occurring at 60–65 °C. The enzyme is probably not inactivated completely until about 90 °C, but it is usually inacti- vated by oxidizing agents and by exposure to air.

Papain, in an inactive stabilized form, may be injected into the circulatory systems of animals before slaughter (Dransfield and Etherington, 1981). This ensures an even distribution of enzymes but results in reduced value for the offal, which contains high enzyme levels. Inactivation is effected by oxidizing cysteinyl groups at the active site to form disulfide bridges. The inactivated enzyme remains in this form in the live animal and is excreted if the animal is not slaughtered. In slaughtered animals, continuing glycoly- sis depletes oxygen levels and produces a relatively reducing environment; under these conditions the papain is reactivated.

Conversion is slow in chilled meat but proceeds rapidly on warming. The action of bromelin and ficin is similar to that of papain. The enzymes are inactivated by several reagents such at Hg 2⫹ , parachloromercuribenzoate, iodoacetate, dibromoacetate, and other reagents that react with sulfhydryl groups (Glazer and Smith, 1971).

3. Proteases in Baking and Brewing The major proteins involved in baking processes are glutenins and gliadins. Both the

protein structures are very complex. The major source of enzymes used in the process are fungal proteases. Fungal proteases particularly increase the texture and elasticity of dough.

teins to increase extensibility and workability, of the dough (Samuel, 1972). Proteinases and peptidases improve flavor by splitting the protein chains and forming amino acids res. There is no adverse effect that occurs by increase in concentration of fungal proteases, while there is a structural decomposition due to excess amounts of addition of bacterial or plant proteases. So the excessive proteolytic degradation produces sticky doughs of poor loaf characteristics. The action of fungal proteases on gluten is inhibited by salt. The proteases show little effect on doughs containing 2% salt (Samuel, 1972).

Fermentation and aging follows the filtration of beer. During storage, cloudiness sometimes develops in beer caused by peptides and polyphenolic procyanidins and some metal ions (Hough et al, 1982). In 1910, a cash award was offered by the United States Brewmasters Association to find a solution for this particular problem. Wallerstein in 1911 came up with a solution by using such proteases as bromelin, papain, and pepsin to stabi- lize and chillproof beer. Papain is mainly used to degrade the protein to low molecular weight peptides. The affinity of papain for the soluble protein in beer must be very specific, as there is very little proteolysis that occurs. The process must be carefully controlled so that enough polypeptides of sufficient size remain to entrap carbon dioxide in the formation of a head on the beer.

4. Assay for Proteinases Because their protein substrates are not a homogeneous group and because different pep-

tide bonds are hydrolyzed, the activity of proteolytic enzymes is not easy to measure. Several methods have been used (Ward, 1983). Casein, hemoglobin, or other protein and synthetic peptides or esters can be used as substrates.

In general, the substrate is treated with enzyme for a suitable time. After inactivating the enzyme, the amount of product is measured. This may be done by precipitation of the unhydrolyzed protein with trichloroacetic acid and measuring the absorbance of the supernatant at 280 nm. With milk-clotting enzymes the time required for a given amount of enzyme to start clotting a standard milk sample is a more useful measure of activity than a general measurement of proteolytic activity.

B. Starch-Degrading Enzymes

Starches from various sources contain about 80% amylopectin and 20% amylose. Amylose is a polymer in which glucose units number 300–400 and they are joined through α-1,4 glycosidic bonds. In amylopectin, chains of α-1,4–linked glucose units are joined through β-1,6 glycosidic bonds. Several enzymes can hydrolyze the bonds in starch molecules such as α and β amylases and glucoamylases. Alpha and beta amylases hydrolyze amylose completely to maltose, while glucoamylase hydrolyzes amylose completely to glucose

( Figs. 1 and 2 ). The investigation of amylolytic enzymes is widespread due to applications in industry, such as conversion of starch to different sugars and use as a food sweetners.

1. Alpha Amylase ( α-1,4-D-Glucan Glucanohydrolase) Alpha amylase is obtained from plants, animals, as well as microorganisms. Pancreatic

α-amylases hydrolyze starches of indigested food to oligosaccharides. Bacterial as well as fungal amylases have commercial importance (Fogarty, 1983; Crueger and Crueger, 1977). The amylases produced from bacteria such as Bacillus amyloliquefaciens, B. li- cheniformis , and B. stearothermophilus are endoenzymes that hydrolyze internal α-1,4

Figure 1 Amylose, a straight chain polymer.

maltose. As amylases cannot attack α-1,6 bonds, amylopectin is hydrolyzed to glucose, maltose, and various limit dextrins that consist of four or more glucose units. The nature of the dextrins formed depends upon the amylase used.

These enzymes are stable in a pH range of 5.5–8.0. If the pH is decreased below the optimum range, these enzymes may be inactivated. Above the pH range formation of byproducts increases, which results in a loss of product yield and an increase in refining costs.

Calcium has a profound effect on enhancing the thermostability of α-amylases. In the presence of calcium ions the enzymes are resistant to inactivation at elevated tempera- Calcium has a profound effect on enhancing the thermostability of α-amylases. In the presence of calcium ions the enzymes are resistant to inactivation at elevated tempera-

Alpha amylases are used for the production of maltose syrups. The enzymatic pro- cess is preferable to acid hydrolysis, because the latter may produce undesirable byprod- ucts such as 5-hydroxymethyl 1-2-furfuraldehyde and anhydroglucose compounds. But some pretreatment methods are required before the enzymatic hydrolysis of starch. The starch is exposed to temperatures above 60 °C with subsequent addition of α-amylase from

B. subtilis , which results in the swelling and disruption of particles due to high temperature and an enzymatic hydrolysis of glycosidic bonds. This process thins the starch slurry. The thinned starch slurry contains 30–40 % solids. The reaction further proceeds in at 103– 107 °C at pH 6.5. Temperature is lowered to 95°C, and the reaction milleu is held for 1–

2 h. This liquefaction process results in a product with a dextrose equivalent of 10–20 and provides a partially hydrolyzed mixture of reduced viscosity (Norman, 1981). These derivatives have commercial value in the food industries.

The pH of the liquefied starch is adjusted to 5, and the temperature is lowered to

50 °C. Fungal β-amylase is added to the mixture, and the reaction is allowed to proceed for 24–48 h. When the desired extent of hydrolysis has occurred, the crude syrup is filtered and decolorized with activated carbon. Ion exchange is then used to remove inorganic ash. The overall process is summarized in Fig. 3. The high maltose syrups produced by this process show a reduced tendency to crystallize and are relatively nonhygroscopic. They find use in the manufacture of candy and frozen desserts and in the baking industry.

It has been observed that the most effective amylases are normally thermostable. Bacteria such as Bacillus species, actinomycetes like micromonospora, thermomonospora, and fungi such as Humicola, Mucor, etc., are excellent test organisms for the study of amylase ( Table 2 ). Heat-labile enzymes, such as the amylase from A. oryzae, cannot be

Table 2 Properties of α-Amylase from Three Microbial Sources

Enzyme source

Property

A. oryzae Optimum pH

B. amyloliquefaciens

B. licheniformis

4–7 Optimum temperature

55 °C End products

70 °C

90 °C

Maltose, maltotriose Maltose, maltotriose Isoelectric pH

Maltotriose

used in the liquefaction process because of the elevated temperatures that are required to bring about starch gelatinization. The A. oryzae enzyme can be used to hydrolyze liquefied starch to maltose and maltotriose (Barfoed, 1976). Generally the liquefaction of starch is carried out in a batch process using free enzyme, but continuous processes with immobi- lized α-amylase to reduce the processing costs have also been attempted (Yi-Hsu-J. et al., 1995).

2. Beta-Amylase (EC 3.21.2.2; β-1,4-D-Glucan Maltohydrolase) Beta amylase is found primarily in microorganisms and such higher plants as barley,

wheat, rye, oats, sorghum, soybeans, and sweet potatoes. Microbial β-amylases have been obtained from B. megaterium (Higashihara and Okada, 1974) and B. cereus (Takasaki, 1976). Plant β-amylases are sulfhydryl enzymes and are sensitive to heavy metal ions and oxidizing agents (Rowe and Weitl, 1962). The bacterial enzymes have higher heat resis- tance and higher pH optima than their plant counterparts. Beta amylases have higher mo- lecular weight than α-amylases.

In the reaction mechanism of action of β-amylase, maltose splits off from amylose. Enzymes have three specific groups on their active sites (X, A, B) which help in binding and transforming the substrate. The X group recognizes and interacts with the hydroxyl group of the fourth carbon atom of the nonreducing end of the polysaccharide chain. Then the other glucosidic linkages of substrate positioned at A and B groups form an enzyme– substrate complex. In the enzyme–substrate complex, the imidazole group donates a hy- drogen to the glycosidic oxygen and forms an intermediate, maltosylenzyme, while the carboxyl group helps in regenerating the enzyme.

3. Glucoamylase (EC 3.2.1.3; α-1,4-D-Glucan Glucohydrolase) The complete hydrolysis of starch to glucose is generally accomplished by fungal gluco-

amylase. A. niger, A. awamori, A. oryzae, and Rhizopus oryzae are good sources of gluco- amylase. The most important commercial application of glucoamylase is the formation of syrups of up to 96% glucose. Such syrups may be used for making crystalline glucose (Kingman, 1969). Glucoamylase hydrolyzes the α-1,4 linkage from the reducing end of amylose, amylopectin, and glycogen to yield glucose residues. The total conversion of starch to glucose can be achieved at 60 °C and pH 4.0 and the reaction time is 3–4 days. After an incubation the crude syrup can be vaccum filtered and purified with activated carbon and ion exchange resins (Norman, 1981).

The low pH reduces unwanted isomerization reactions to fructose and other sugars, and also restricts the contamination of microorganisms during the process. The rate of The low pH reduces unwanted isomerization reactions to fructose and other sugars, and also restricts the contamination of microorganisms during the process. The rate of

D-Glucose ⫹ β-D-Glucose → Disaccharide ⫹ H 2 O

The high dextrose equivalent syrups produced by glucoamylase are used in the brewing, baking, soft drink, canning, and confectionery industries. Glucoamylase is important in light beer production. Under optimum conditions glucoamylase is capable of transforming at least 95% of dextrins to glucose, which in turn can be easily fermented by yeasts during beer production. To measure glucoamylase activity in commercial preparations, different units are used by different manufacturers, but the most common units are comparable in magnitude. A unit may be grams of glucose produced from soluble starch per hour at pH

4.2 and 60 °C.

4. Pullulanases Prolonged action of α-amylases on starch produces limit dextrins that cannot be hy-

drolyzed further. Hydrolysis with β-amylases also stops at α-1,6 branch points and pro- duces limit dextrins (Robyt and Whelan, 1976). Pullulanases produced by Aerobasidium pullulans specifically hydrolyze the α-1,6 glycosidic bonds found in the fungal carbohy- drate pullulan. Pullulanase will also cleave the α-1,6 links found in amylopectin and limit dextrins provided that there are at least two glucose residues on either side of the α-1,6 bond.

Pullulanases are produced by Bacillus cereus, Klebsiella spp, Streptococcus mites, and Escherichia intermedia, but the enzyme from Klebsiella has been the most investigated (Norman, 1981). The pullulanases of industrial importance are produced by K. pneumoniae and B. cereus. The enzymes attack α-1,6 linkages randomly to yield maltotriose and branched maltotriose oligosaccharide. The oligosaccharide may eventually be broken down to maltotriose (Fig. 4). It appears that the primary use of pullulanases in the food industry will be the production of maltose and maltose syrups. The use of these enzymes in combinations yields products in excess of 80% maltose.

5. Glucose Isomerase (EC 5.3.1.5) It is generally accepted that the glucose isomerase used commercially is actually D-xylose

isomerase (Antrim et al., 1979; Bucke, 1983, Hemmingsen, 1979). These enzymes nor- mally catalyze the conversion of D-xylose to xylulose. They also act on glucose and other substrates but at much lower rates.

The conversion of glucose to fructose can be effected chemically at high tempera- tures and in alkaline conditions. In dilute alkaline solutions, reducing sugars are trans- formed into mixtures of aldoses and ketoses. This is an example of a class of reactions known as the Lobry de Bruyn–Alberda van Eckenstein transformation (Fig. 5). In such procedures, however, it is difficult to obtain 40% fructose without the formation of unme- tabolizable products such as psicose and other objectional products. These unwanted mate- rials result in reduced sweetness and may contribute to poor color and off-flavor (Antrim et al., 1979).

Glucose isomerases have been prepared from Lactobacillus (Yamanaka, 1968), Streptomyces, and Bacillus coagulans (Yashimura, 1966). The enzymes are heat stable and operate in the range of 45–65 °C. The optimal temperatures for activity range from

45 °C for the enzymes from L. brevis (Yamanaka, 1968) to 90°C for the Actiniplanes missouriensis enzyme (Scallet et al., 1974), with most having optima around 65 °C. Stabil- ity at elevated temperatures is desirable, not only because the reaction rate is increased, but also because microbial contamination is reduced.

Glucose isomerase requires such metal ions such as magnesium, cobalt, manganese, or chromium for activity (Antrim et al., 1979). The enzymes are inhibited by copper, zinc, nickel (Scallet et al., 1974), calcium (Aschengreen, 1975), silver, and mercury ions (Takasoki et al., 1969). Inactivation by calcium ions may be due to competition with the required magnesium ions for the enzyme’s active site, and inactivation by mercury sug- gests the presence of important thiol groups. These enzymes are also inhibited by sugar alcohols, especially xylitol (Scallet et al., 1974; Young et al., 1975; Yamanaka and Taka- hara, 1977). As trishydroxymethylaminomethane is an inhibitor, it should not be used as

a buffer when glucose isomerase are being studied (Danno, 1970). Glucose isomerases catalyze the production of an equilibrium mixture containing 55–60% fructose; the actual proportions vary slightly with temperature. As it would take

Figure 5 Lobry de Bruyn–Alberda van Eckenstein transformation that converts glucose to fruc- Figure 5 Lobry de Bruyn–Alberda van Eckenstein transformation that converts glucose to fruc-

Glucose isomerase

Because calcium ions are inhibitory to glucose isomerase, it is necessary to deionize syrups after saccharification to remove the calcium ions that are required for α-amylase activity and are always present in starches. Appropriate amounts of magnesium ions are then added for the isomerization step. Alternatively an α-amylase such as that from Bacil- lus licheniformis , which is not dependent on calcium for activity, could be used in the saccharification process (Aschengreen, 1975; Hollo et al., 1975).

Glucose isomerase is an expensive enzyme. It would not have been commercially practicable to use this enzyme were it not for the development of solid supports. The activity of glucose isomerases is measured in terms of the amount of enzyme required to convert 1 µmol or 1 g of glucose to fructose per given time (Antrim et al., 1979). The product fructose is determined spectrophotometrically using cystein carbazole or other ketone-condensing reagents ( Fig. 6 ) (Takasaki, 1966; Lloyd et al., 1972).

C. Pectin-Degrading Enzymes

Along with celluloses and hemicelluloses, pectic substances are the major component of cell wall in higher plants. These plant polysaccharides play an important role in main- taining cell wall structure. The pectic enzymes generally termed as pectinases act on pectic substances. The enzyme sources are commonly derived from Aspergillus, Rhizopus, and some species of Penicillium. The pectin obtained from plant juices, saps, apple pomace, tomatoes, citrus, and beet pulp acts as an inducer during the production of pectic enzymes by submerged fermentation. Their use in the food industry is to improve the filtration rate, increase extraction of fruit pulp, reduce turbidity, and provide clarification. Pectic enzymes reduce haze of grape juice during the wine-making process. Use of pectic enzymes also promotes faster aging of wine. Pectinases also play an important role in coffee and tea fermentation (Jones and Jones, 1984). There are three different types of pectic enzymes; pectinesterase, lyase, and polygalacturonase.

1. Pectinesterase (EC 3.1.1.1.1) Pectin esterases de-esterify pectin to produce pectic acid and methanol. The enzymes

are specific for the methyl ester of pectic acid and will not attack the methyl ester of

Figure 6 Degradation of starch.

hydrolyzed at reduced rates (Macdonnell et al., 1950). The action of pectinesterase does not proceed to completion but stops at a degree of esterification that has been found to vary between 0.4 and 11%. For substrates containing less than 10 galacturonic acid units, the reaction rate of pectinesterase from orange decreases with substrate chain length until no activity is observed with the triethyl ester of the trimer (McCready and Seegmiller, 1954). There is some support for the hypothesis that pectinesterases act on methyl ester groups that are adjacent to free carboxyl groups (Solms and Denel, 1975). Some microbial enzymes have alkaline pH optima.

The presence of univalent and divalent metal ions enhances the activity of pectin- esterases severalfold. The effect is especially pronounced with enzymes of plant origin. Although the enzymes have been reported to have an unusual resistance to chemicals, inhibition by anionic detergents (McColloch and Kertesz, 1947), sucrose, glycerol, and D-glucose (Change et al., 1965) has been reported. They are also inhibited by polygalactur- onates with a degree of polymerization greater than 8 (Thermote et al., 1977).

Pectinesterase activity can be measured by determining the carboxyl groups that are formed in pectin by the enzyme (Colle and Wood, 1961). The amount of methanol released can also be measured (Holden, 1945). Methanol may be converted to methyl nitrite, which can be analyzed by gas-liquid chromatography (GLC) (Bartolome and Hoff, 1972). Activ-

ity can also be followed by monitoring pH changes in the pH range 7.5–7. [ 14 C]-Methyl pectin has been used as a substrate in pectinesterase assays (Kauss et al., 1969). After enzyme deeesterification, the pectin is precipitated with methanol. The [ 14 C] methol in the supernatant can then be determined in a scintillation counter. One unit of pectinesterase is defined as the amount of enzyme that liberates 1 µmol free carboxyl group (or methanol)

2. Pectin- and Pectate-Depolymerizing Enzymes Depolymerizing enzymes cleave the α-1,4 glycosidic bonds in pectin and pectin acid.

There are two main classes of pectin/pectic acid–depolymerizing enzymes; polygalacturo- nases and lyases. Polygalacturonases are sometimes referred to as pectin or pectic acid hydrolases.

a. Polygalacturonases (EC 3.2.1.15). Polygalacturonases hydrolyze internal bonds in pectic acid, resulting in rapid reduction in viscosity (Kelly and Fogarty, 1978; Rombouts and Pilnik, 1980). The enzymes are specific for high molecular weight pectic acid and show reduced activity with increased esterification. Activity with oligogalacturonates as substrate decreases with decreasing chain length, and little action is observed with the trimer and dimer (Rexova-Baenkova, 1973). Thus when pectic acid is hydrolyzed by endo- polygalacturonases, monomers, dimers, and trimers of galacturonic acid accumulate as end products (Mount et al., 1970). Polygalacturonases are again divided based on the nature of substrate and the activity of enzyme. Enzymes of plant, fungal, and bacterial origin have been described (Rombouts and Pilnik, 1980).

The pH optima of the enzymes are generally in the region 4–5.5 However, the enzyme from Corticium rolfsii has an optimum pH of 2.5 (Kaji and Okada, 1969). The pH optima appear to depend on the degree of polymerization of the substrate (Barash and Eyal, 1970).

Exopolygalacturonases (EC 3.2.1.67) hydrolyze the terminal α-1,4 bonds in pectates at the nonreducing end to release galacturonic acid monomers. Several enzymes from plants have been described (Riov, 1975; Pressey and Avants, 1976; Bartley, 1978). Al- though exopolygalacturonases prefer high molecular weight pectates, they will, unlike the endoenzyme, accept digalacturonic acid as a substrate. The activity of exoenzymes de- pends on the degree of substrate polymerization (Pressey and Avants, 1973; Mills, 1966). The activity of exopolygalacturonases results in only a gradual reduction in viscosity.

b. Lyases. Highly esterified polymethylgalacturonic acid is directly attacked by pectin lyases (EC 4.2.2.10). Most pectin lyases are of fungal origin. All are endoenzymes and cleave highly esterified pectin at random with a rapid decrease in viscosity. Only glyco- sidic bonds adjacent to methyl ester groups are split in an eliminative mechanism. This results in the formation of products with a double bond between CE4 and CE5; conjuga- tion of this double bond of the carboxyl group on CE5 leads to light absorption with a maximum at 235 nm. Because of their preference for highly esterified pectin, pectin lyases can be used in the processing of fruit juices that contain highly esterified pectin (Ishii and Yokotsuka, 1975). The pH optima for the enzyme vary from 5.2 to 8.7 (Fogarty and Kelly, 1983). The activity of most pectin lyases is enhanced in the presence of calcium ions. The extent of stimulation depends on the pH and degree of esterification of the substrate (Edstrom and Phaff, 1964).

Endopectate lyases (EC 4.2.2.2) randomly split partially or completely de-esterified pectin chains. They are produced by fungi and bacteria, and their pH optima is in the range 8–10. The pH optimum varies with the chain length of the substrate (Ward and Fogarty, 1972). These enzymes require calcium for activity. Although pectates are gener- ally good substrates, some enzymes prefer substrates with a degree of esterification of 21–44%. The activity of endopectate lyases decreases with decrease in the chain length of the substrate (Atallah and Nagel, 1977).

Exopectate lyases (EC 4.2.2.9) liberate unsaturated dimers from the reducing end of pectic acid. The pH optima for the enzymes are in the range 8–9.5. The enzymes prefer pectate over pectins, and completely esterified pectins are not accepted as substrates. The smallest substrate that can be degraded is the trimer.

Enzymatic treatment of soft fruit pulp facilitates pressing and improves juice and anthocyanin pigment yields (Neubeck, 1975). Pectin-degrading enzymes have been used to degrade highly esterified apple pectin and increase juice yields (DeVos and Pilnik, 1973). The enzyme-treated juices have a slightly higher methanol content than juices ob- tained by pressing. The health hazard of such low methanol levels has not been assessed but may be of no consequence.

When pectinases are added to viscous and turbid freshly pressed fruit juices, the cloudy material agglomerates and forms flocs. It has been suggested that the enzymes act by dissolving away the negatively charged pectin coating of cloud particles. Electrostatic interaction of the destabilized cloud material would then result in coagulation (Endo, 1965; Yamasaki et al., 1967). Such clarification and removal of pectin is essential for juices that have to be concentrated. The process is often carried out at elevated temperature to increase the rate of reaction and discourage microbial growth and unwanted fermentation (Grampp, 1977).

Pectinases can be used to recover juices from pulp that is sieved out of freshly pressed juice (Braddock and Kesterton, 1976). Pectic enzymes can also be used in conjuga- tion with mechanical processes to produce macerated suspensions of loose cells from fruit and vegetables. Enzyme treatment can also be used to lower the viscosity of such products. When pectinases are used together with cellulases, effective liquefaction of fruit and vege- table products can be achieved.

The activity of pectin-depolymerizing enzymes may be determined by measuring the rate of increase of reducing groups by titration with hypoiodite. After the oxidation of reducing carbonyl groups with iodine, unreacted iodine is measured by titration with sodium thiosulfate (Phaff, 1960; Mills and Tuttobello, 1961). Reducing groups may also

be determined colorimetrically with 3,5-dinitrosalicylic acid-phenol reagent (Borel et al., 1952) or cuprous reagent (Somogyi, 1952; Spiro, 1966; Milner and Avigad, 1967). With highly esterified substrates, alkaline conditions should be avoided because under these conditions these substrates may be split by β elimination, leading to high values for reduc- ing group content (Albersheim et al., 1960a,b). For esterified substrate the methods of Launer and Tomimatsu (1959) are suitable.

The activity of exoenzymes can be monitored by measuring the amount of D-galac- turonate degraded in the internal positions. The activity of endoenzymes can also be fol- lowed by measuring the decrease in substrate viscosity (Pressey and Avants, 1973; Wim- borne and Richard, 1978; Mills and Tuttobello, 1961). As viscosities of pectic acid materials are influenced by temperature, pH, and ionic strength, these factors must be carefully controlled. Viscosity changes can be used to distinguish between exoenzymes and endoenzymes. A greater proportion of glycosidic bonds need to be cleaved by exoen- zymes to achieve the same reduction in viscosity effected by endoenzymes.

In commercial applications enzymes may be compared by determining the minimal amounts of an enzyme required to perform a given task (Bauman, 1981). The double bond introduced by the action of lyases leads to maximal absorption at 235 nm (Albersheim et al, 1960a,b) or 230 nm (Starr and Moran, 1962). Increase in absorbance at the two wave- lengths can therefore be used to follow lyase activity (Saio and Kaji, 1980; Rombouts et In commercial applications enzymes may be compared by determining the minimal amounts of an enzyme required to perform a given task (Bauman, 1981). The double bond introduced by the action of lyases leads to maximal absorption at 235 nm (Albersheim et al, 1960a,b) or 230 nm (Starr and Moran, 1962). Increase in absorbance at the two wave- lengths can therefore be used to follow lyase activity (Saio and Kaji, 1980; Rombouts et

D. Cellulases

Cellulose is a linear polymer of a simple sugar or saccharide which does not occur in pure form in any natural resource. Most forms of cellulose which are available in nature contain about 10% by weight noncellulosic polysaccahrides, proteins, and mineral ele- ments. Most cellulosic materials are generally found in association with hemicellulose, pectin, and lignin. In cellulose, glucose units are joined by β-1,4 glycosidic bonds. The degree of polymerization varies from 15 to 14,000, but most chains are 3000 glucose units long (Cowling and Brown, 1969). The linear polymers are held together by hydrogen bonding to form crystalline fibrils. Crystalline regions may be interspaced with less- or-dered amorphous regions. Although cellulolytic enzymes from bacteria, fungi, plants, and invertebrates have been described (Whitaker, 1971), only enzymes of microbial origin have industrial potential. Several bacteria—cellulomonas, clostridium, acetobutlylcum cellovibrio, Clostridium thermomonospora—and fungi like Trichoderma reesei, Penicil- lium fusicolsum , and A. niger are good producers of this enzymes.

Three major types of enzyme activity are now generally accepted as acting in concert or in sequence to bring about the degradation of cellulose (Enari, 1983). Endoglucanase (EC 3.2.14; 1,4- β-D-glucan-4-glucanohydrolase) hydrolyzes β-1,4 glycosidic bonds in a random fashion. Cellobiose is not a substrate for this enzyme. Otherwise the enzyme does not appear to be highly specific for any substrate.

Cellobiohydrolase (EC 3.2.14.1; 4- β-glucan-4-glucanohydrolase) cleaves off cello- biose units from the nonreducing ends of cellulose polymers. Substituted celluloses and cellobiose are not hydrolyzed. The third enzyme, β-D-glucoside glucohydrolas (EC 3.2.1.21), hydrolyzes cellobiose and oligosaccharides derived from cellulose to yield glu- cose.

The association of cellulose with hemicellulose and lignin in most cellulose sources makes the commercial degradation of cellulose difficult as the native cellulose is highly resistant to hydrolysis. The associated materials have to be removed and the crystalline structure destroyed if the enzymatic hydrolysis is to be improved. Treatment with alkali, acid, or steam and milling treatments may be used to prepare cellulosic materials for enzymatic hydrolysis (Enari, 1983).

Several methods are available to determine cellulose-degrading activity (Goksyr and Eriksen, 1980; Enari, 1983). A variety of substrates including cotton fiber, filter paper, and cellulose derivatives such as carboxymethylcellulose, hydroxyethylcellulose, and Avi- cel have been reported in the literature.

Alpha-glucosidase activity can be measured using cellobiose, saiicin, or p-nitrophe- nyl- α-D-glucoside as substrate (Selby and Maitland, 1967). The reactions may be followed by determining the reducing sugars produced or by measuring changes in viscosity or absorbance of light.

For commercial purposes it is the total solubilizing effect that is of interest. The amount of reducing sugars present is not a reliable measure of enzyme activity. Total solubilizing effect may be conveniently measured using dyed substrates such as Avicel, For commercial purposes it is the total solubilizing effect that is of interest. The amount of reducing sugars present is not a reliable measure of enzyme activity. Total solubilizing effect may be conveniently measured using dyed substrates such as Avicel,

a cotton or Avicel substrate.

E. Lipases

Lipases bring about the hydrolysis of insoluble triacylglycerols to produce glycerol and fatty acids. The hydrolysis of triglycerides is reversible and triglycerides may be formed from free fatty acids and glycerol. Their preference for emulsified substrates distinguishes lipases from esterases, which attack substrates in solution. Solid triglycerides are poor substrates for lipases (Suigara and Isobe, 1975). Simple alkyl esters are hydrolyzed by lipases but at reduced rates. The substrate specificity of various lipases has been reviewed (Shahani, 1975; MacRae, 1983).

A group of enzymes will hydrolyze triglycerides at positions 1 and 3 only to produce 1,2-diglycerides and 2-monoglycerides by migration of the fatty acid moiety. Because of this migration, the complete hydrolysis of some triglycerides can be achieved even though no lipases are known to release fatty acids from the 2 position.

Lipases from Candidum, Candida cylindracae (Candida rugosa), Corynebacterium acnes , Chromobacterium viscosum, Penicillium cyclopium, and Humicola lanuginosa have been reported to be nonspecific, whereas lipases from Aspergillus niger, Mucor ja- vanicus , Rhizopus, and Pseudomonas fragi, and pancreatic lipase, show 1,3 specificity (MacRae, 1983). Most microbial enzymes do not appear to show specificity for fatty acids. However, an enzyme from G. candidum preferentially releases fatty acids with a cis double bond in position 9 (Jensen, 1974). A lipase from P. cyclopium has been shown to hydrolyze the partial glycerides diolein and monolein faster than the triglyceride (Okumura et al., 1976).

Lipases may be used instead of sodium or sodium methoxide to effect interesterifi- cation reactions. Because the lipase reaction is reversible, the triglycerides may be hy- drolyzed and resynthesized. Resynthesis to yield a product with rearranged fatty acids occurs under conditions of reduced water. With nonspecific enzymes the interesterified products are similar to those obtained by chemical means. With 1,3-specific enzymes, interesterification is limited to positions 1 and 3.

Mixtures of free acids and glycerides may be used for interesterification reactions. There is thus the possibility of producing novel triglycerides, some of which may have desirable properties. Using such procedures it is possible to introduce unsaturated fatty acids such as linoleic acid into saturated fats.

The hydrolysis of milk fat can result in rancid flavors in milk, cream, and other dairy products (Arnold et al., 1975). Controlled lipolysis with enzymes specific for short- chain fatty acids makes it possible to develop desirable flavors (Nelson, 1972). The pre- pared fat substrate is combined with the enzyme preparation and the mixture is blended into a stable emulsion. After incubation at a suitable, temperature to achieve the desirable extent of lipolysis, the enzyme can be inactivated by pasteurization. Enzymes specific for short-chain fatty acids have also found application in generating flavors in Italian blue and cheddar cheese (Moskovitz et al., 1977)

The activity of lipases may be determined using emulsified triglycerides. Soluble glycerides are not suitable because they are hydrolyzed slowly by lipases and may be hydrolyzed faster by other esterases. Triolein, the best substrate, is too expensive; it may The activity of lipases may be determined using emulsified triglycerides. Soluble glycerides are not suitable because they are hydrolyzed slowly by lipases and may be hydrolyzed faster by other esterases. Triolein, the best substrate, is too expensive; it may