Post on 13-Mar-2022
CHEMISTRY
PAPER 16 Bioorganic and biophysical chemistry
MODULE 29 Enzymes in food
Subject Chemistry
Paper No and Title Paper 16 Bioorganic and biophysical chemistry
Module No and Title 29: Enzyme in food
Module Tag CHE_P16_M29
CHEMISTRY
PAPER 16 Bioorganic and biophysical chemistry
MODULE 29 Enzymes in food
TABLE OF CONTENT
1. Learning outcomes
2. Introduction
2.1 Regulation of Enzyme Reactions
2.2 Nature of Enzymes
3. Food Modification by Enzymes
3.1 Role of Endogenous Enzymes in Food Quality
4. Some Important uses of enzymes in foods
4.1 Production of high-fructose corn syrup and sweeteners
4.2 Specialty products and Ingredients via Enzymology
5. Summary
CHEMISTRY
PAPER 16 Bioorganic and biophysical chemistry
MODULE 29 Enzymes in food
1. Learning outcomes
After studying this module you shall be able to:
Know about regulation of enzyme reactions.
Learn about nature of enzymes.
Understand the food modification by enzymes.
Know the role of endogenous enzymes in food quality.
2. Introduction
Enzymes are proteins with catalytic activity due to their power of specific activation and
conversion of substrates to products:
𝐒𝐮𝐛𝐬𝐭𝐫𝐚𝐭𝐞(𝐬) 𝐄𝐧𝐳𝐲𝐦𝐞𝐬→ 𝐏𝐫𝐨𝐝𝐮𝐜𝐭(𝐬)
Some of the enzymes are composed only of amino acids covalently linked via peptide bonds to
give proteins. Other enzymes contain additional components, such as carbohydrate, phosphate,
and cofactor groups. Enzymes have all the chemical and physical characteristics of other proteins.
Composition-wise, enzymes are not different from all other proteins found in nature and they
comprise a small part of our daily protein intake in our foods. However, unlike other groups of
proteins, they are highly specific catalysts for the thousands of chemical reactions required by
living organisms.
Enzymes are found in all living systems and make life possible, whether the organisms
are adapted to growing near 0°C, at 37°C (humans), or near 100°C (in microorganisms found in
some hot springs). Enzymes accelerate reactions by factors of 103 to 1011 times that of non-
enzyme-catalyzed reaction. In addition, they are highly selective for a limited number of
substrates, since the substrate(s) must bind stereospecifically and correctly into the active site
before any catalysis occurs. Enzymes also control the direction of reactions, leading to
stereospecific product(s) that can be very valuable by-products for foods, nutrition, and health or
the essential compounds of life.
2.1 Regulation of Enzyme Reactions
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PAPER 16 Bioorganic and biophysical chemistry
MODULE 29 Enzymes in food
Enzyme activity can be controlled in a number of ways that are very important to food scientists.
The velocity of an enzyme-catalyzed reaction is usually directly proportional to the active
enzyme concentration, and dependent (in a complex way) on substrate, inhibitor, and cofactor
concentrations, and on temperature and pH. As an example, it is well known that enzyme-
catalyzed reactions occur more slowly when a food is placed in a refrigerator (~ 4°C). But the
reactions do not stop at 4°C (or at 0°C). Most enzyme-catalyzed reactions decrease 1.4-2
times/10°C decrease in temperature. Therefore, at 5°C, the velocities would be 0.5 to 0.25 times
the velocity at 25°C.
Changing the pH of the system by 1 or 2 pH units from the pH-activity optimum can
decrease enzymatic velocity to 0.5 or 0.1, respectively, of that at the pH optimum. Decreasing (by
heating, breeding, or genetic engineering) the concentration of enzyme to 0.1 that normally
present would decrease the enzyme activity to 0.1 that originally present.
2.2 Nature of Enzymes
During the 1870-1890 period, there was a major debate between Pasteur, who believed that
enzymes functioned only when associated with living organisms, and Liebig , who believed that
enzymes continued to function in the absence of cells. This argument was settled in 1897 when
Buchner separated enzymes, including invertase, from yeast cells and showed that they were still
active.
3. Food modification by enzymes
Enzymes have a very important impact on the quality of our foods. In fact, without enzymes there
would be no food. But then there would be no need for food, since no organism could live without
enzymes. They are the catalysts that make life possible, as we know it.
For any organism, life begins with enzyme action in the gestation and fertilization processes. The
growth and maturation of our foods depend on enzyme actions. While we have known for some time
that environmental conditions during growing affect the composition including enzymes, of our plant
foods, a recent review has detailed just how much effect moisture deficiency has on the expression of
genes during growth and maturation of plants.
Following maturation, the harvesting, storage, and processing conditions can markedly affect the rate
of food deterioration. Enzymes can also be added to foods during processing change their
characteristics, and some of these changes will be discussed. Microbial enzyme left after destruction
of the microorganisms, continue to affect the quality of processed an reformulated foods. For
example, starch-based sauces can undergo undesirable changes in consistency because of heat-stable
microbial α-amylases that survive a heat treatment sufficient to destroy the microorganisms. Because
of their high specificity, enzymes are also the ideal catalysts for the biosynthesis of highly complex
chemicals.
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PAPER 16 Bioorganic and biophysical chemistry
MODULE 29 Enzymes in food
3.1 Role of Endogenous Enzymes in Food Quality
3.1.1 Color
Color is probably the first attribute the consumer associates with quality and acceptability of foods.
Importance of color in Foods:
A steak must be red, not purple or brown, Redness is due only oxymyoglobin the main
pigment in meat. Deoxymyoglobin is responsible for the purple color of meat. Oxidation of
the Fe(II) present in oxymyoglobin and deoxymyoglobin, to Fe(III) producing metmyoglobin,
is responsible for the brown color of meat. Enzyme-catalyzed reactions in meat can compete
for oxygen, can produce compounds that alter the oxidation—reduction state and water
content, and can thereby influence the color of meat.
The quality of many fresh vegetables and fruits is judged on the basis of their "greenness."
On ripening, the green color of many of our fruits decreases and is replaced with red, orange,
yellow, and black colors. In green beans and English green peas, maturity leads to a decrease
in chlorophyll level. All of these changes are a result of enzyme action. Three key enzymes
responsible for chemical alterations of pigments in fruits and vegetables are lipoxygenase,
chlorophyllase, and polyphenol oxidase.
3.1.1.1 Lipoxygenase
Lipoxygenase (lineoleate:oxygen oxidoreductase; EC 1.13.11.12) has six important effects on
foods, some desirable and others undesirable. The two desirable functions are (a) bleaching of
wheat and soybean flours and (b) formation of disulfide bonds in gluten during dough formation
(eliminates the need to add chemical oxidizers, such as potassium bromate).
The four undesirable actions of lipoxygenase in food are (a) destruction of chlorophyll and
carotenes, (b) development of oxidative off flavors and aromas, often characterized as haylike, (c)
oxidative damage to compounds such as vitamins and proteins, and (d) oxidation of the essential
fatty acids, lineoleic, linolenic, and arachidonic acids.
All six of these reactions result from the direct action of lipoxygenase in oxidation of
polyunsaturated fatty acids (free and lipid-bound) to form free radical intermediate.
Further nonenzymatic reactions lead to formation of aldehydes (including malondialdehyde) and
other components that contribute to off flavors and off aromas. The free radicals and
hydroperoxide are responsible for loss of color (chlorophyll, the orange and red colors of the
carotenoids), disulfide bond formation in gluten of doughs, and damage to vitamins and proteins.
Antioxidants, such as vitamin E, propyl gallate, benzoylated hydroxytoluene, and
nordihydroguaiacetic acid, protect foods from damage from free radicals and hydroperoxides.
3.1.1.2 Chlorophyllase
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PAPER 16 Bioorganic and biophysical chemistry
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Chlorophyllase (chlorophyll chiorophyllido-hydrolase, EC 3.1.1.114 is found in plants and
chlorophyll-containing micro organisms. It hydrolyzes the phytyl group from chlorophyll to give
phytol and chlorophyllide. Although this reaction has been attributed to a loss of green color,
there is no evidence to support this as chlorophyllide is green.
3.1.1.3 Polyphenol Oxidase
Polyphenol oxidase (1 ,2-benzenediol:oxygen oxidoreductase; EC 1.10.3.1) is frequently called
tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, or catecholase, depending on
the substrate used in its assay or found in the greatest concentration in the plant that serves as a
source of the enzyme. Polyphenol oxidase is found in plants, animals and some microorganisms,
especially the fungi. It catalyzes two quite different reactions with a large number of phenols.
Reactions:
The 4-methyl-o-benzoquinone is unstable and undergoes further non-enzyme-catalyzed
oxidation by oxygen, and polymerization, to give melanins. The latter is responsible for
the undesirable brown discoloration of bananas, apples, peaches, potatoes, mushrooms,
shrimp, and humans (freckles), and the desirable brown and black colors of tea, coffee,
raisins, prunes, and human skin pigmentation.
The o-benzoquinone reacts with the E-amino group of lysyl residues of proteins, leading
to loss of nutritional quality and insolubilization of proteins. Changes in texture and taste
also result from the browning reaction.
It is estimated that up to 50% of some tropical fruits are lost due to enzymatic browning. This
reaction is also responsible for deterioration of color in juices and fresh vegetables such as
lettuce, and in taste and nutritional quality. Therefore, much effort has gone into developing
methods for control of polyphenol oxidase activity.
Prevention of Browning: Elimination of 02 and the phenols will prevent browning.
Ascorbic acid, sodium bisulfite, and thiol compounds prevent browning due to reduction of the
initial product, o-benzoquinone, back to the substrate, thereby preventing melanin formation.
When all of the reducing compound is consumed, browning still may occur, since the enzyme
may still be active. Ascorbic acid, sodium/ sulfite, and thiol compounds also have a direct effect
in inactivating polyphenol oxidase due to destruction of the active-site histidines (ascorbic acid)
or in removal (by sodium bisulfite and thiols) of the essential Cu2+ in the active site.
3.1.2 Texture
Texture is a very important quality attribute in foods. In fruits and vegetables, texture is due
primarily to the complex carbohydrates: pectic substances, cellulose, hemicelluloses, starch, and
lignin. There are one or more enzymes that act on each of the complex carbohydrates that are
important in food texture. Proteases are important in the softening of animal tissues and high-
protein plant foods.
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PAPER 16 Bioorganic and biophysical chemistry
MODULE 29 Enzymes in food
3.1.2.1 Pectic Enzymes
Three types of pectic enzymes that act on pectic substances are well described. Two (pectin
methylesterase and polygalacturonase) are found in higher plants and microorganisms and one
type (the Pectate lyases) is found in micro-organisms, especially certain pathogenic micro-
organism that infect plants.
Pectin methylesterase (pectin pectlhydrolase, EC 3.1.1.11) hydrolyzes the methyl ester
bond of pectin to give pectic acid and methanol.
Polygalacturonase (poly-α-1,4-galacturonide glycano-hydrolase, EC 3.2.1.15) hydrolyzes
the α-1, 4-glycosidic bond between the anhydro galacturonic acid units.Both endo- and
exo-polygalacturonases exist; the exo type hydrolyzes bonds at the ends of the polymer
and the endo type acts in the interior. There are differences in opinion as to whether
plants contain both polymethylgalacturonases (act on pectins) and polygalacturonases
(act on pectic acid), since pectin methylesterase in the plant rapidly converts pectin to
pectic acid. Action of polygalacturonase results in hydrolysis of pectic acid, leading to
important decreases in texture of some raw food materials, such as tomatoes.
The pectate lyases (poly 1,4-α-D-galacturonide lyase, EC 4.2.2.2) splits the glycosidic
bonds of both pectin and pectic acid, not with water but by β-elimination they are found
in microorganisms but not in higher plants.
A fourth type of pectin-degrading enzyme, protopectinase, has been reported in a few
microorganisms. Protopectinase hydrolyzes protopectin, producing pectin. However, it is
not clear yet whether the protopectinase activity in plants is due to the combined action of
pectin methylesterase and polygalacturonase or to a true protopectinase.
3.1.2.2 Cellulases
Cellulose is abundant in trees and cotton. Fruits and vegetables contain small amounts of
cellulose, which has a role in the structure of cells. Whether cellulases are important in the
softening of green beans and English green pea pods is still a matter of controversy. Abundant
information is available on the microbial cellulases because of their potential importance in
converting insoluble cellulosic waste to glucose.
3.1.2.3 Pentosanases
Hemicelluloses, which are polymers of xylose (xylans), arabinose (arabans), or xylose and
arabinose (arabinoxylans), with small amounts of other pentoses or hexoses, are found in higher
plants. Pentosanases in microorganism and in some higher plants, hydrolyze the xylans, arabans,
and arabinoxylans to smaller compounds. The microbial pentosanases are better characterized
than those in higher plants.
Several exo- and endo-hydrolyzing pentosanases also exist in wheat at very low
concentrations, but little is known about their properties. It is important that these pentosanases
receive more attention from food scientists.
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3.1.2.4 Amylases
Amylases, the enzymes that hydrolyze starches, are found not only in animals, but also in higher
plants and microorganisms Therefore, it is not surprising that some starch degradation occurs
during maturation, storage, and processing of our foods. Since starch contributes in a major way
to viscosity and texture of foods, its hydrolysis during storage and processing is a matter of
importance. There are three major types of amylases α-amylases, β-amylases, and glucoamylases
They act primarily on both starch and glycogen.
1. The α-amylases, found in all organisms, hydrolyze the interior α-1,4-glucosidic bonds of
starch in both amylose and amylopectin, glycogen, and cyclodextrins with retention of the a-
configuration of the anomeric carbon. Since the enzyme is endo-splitting, its action has a major
effect on the viscosity of starch-based foods, such as puddings, cream sauces, etc. The salivary
and pancreatic a-amylases are very important in digestion of starch in our foods. Some
microorganisms contain high levels of a-amylases. Some of the microbial a-amylases have high
inactivation temperatures, and, if not activated, they can have a drastic undesirable effect on the
stability of starch-based foods.
2. β-Amylases, found in higher plants, hydrolyze the α-1,4-glucosidic bonds of starch at the
nonreducing end to give β-maltose. Since they are exo-splitting enzymes, many bonds must be
hydrolyzed before an appreciable effect on viscosity of starch paste is observed. Amylose can be
hydrolyzed to 100% maltose by β-amylase, while β-amylase cannot continue beyond the first α-1,
6-glycosidic bond encountered in amylopectin. Therefore, amylopectin is hydrolyzed only to a
limited extent by β-amylase alone. "Maltose" syrups, of about DP 10, are very important in the
food industry. β-Amylase, along with α-amylase, is very important in brewing, since the maltose
can be rapidly converted to glucose by yeast maltase. β-Amylase is a sulfhydryl enzyme and can
be inhibited by a number of sulthydryl group reagents, unlike α-amylase and glucoamylase. In
malt, β-amylase is often covalently linked, via disulfide bonds, to other sulfhydryl groups;
therefore, malt should be treated with a sulfhydryl compound, such as cysteine, to increase its
activity in malt.
3.1.2.5 Proteases Texture of food products is changed by hydrolysis of proteins by endogenous and
exogenous proteases.
Gelatin will not gel when raw pineapple is added, because the pineapple contains
bromelain a protease.
Chymosin causes milk to gel, as a result of its hydrolysis of a single peptide bond
between Phe105-Met106 in k-casein. This specific hydrolysis of k-casein
destabilizes the casein micelle, causing it to aggregate to form a curd cottage
cheese). Action of intentionally added microbial proteases during aging of brick
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cheeses assists in development of flavours (flavours in Cheddar cheese vs. Blue
cheese, for example).
Protease activity on the gluten proteins of wheat bread dough during rising is
important not only in the mixing characteristics and energy requirements but also
in the quality of the baked breads.
The effect of proteases in the tenderization of meat is perhaps best known and is
economically most important. After death, muscle becomes rigid due to rigor
mortis (caused by extensive interaction of myosin and actin). Through action of
endogenous proteases (Ca2+ -activated proteases, and perhaps cathepsins) on the
myosin - actin complex during storage (7-21 days) the muscle becomes more
tender and juicy. Exogenous enzymes, such as papain and ficin, are added to
some less choice meats to tenderize them, primarily due to partial hydrolysis of
elastin and collagen.
3.1.3 FIavor and Aroma Changes in Foods
Chemical compounds contributing to the flavor and aroma of foods are numerous, and the critical
combinations of compounds are not easy to determine. It is equally difficult to identify the
enzymes instrumental in the biosynthesis of flavors typical of food flavors and in the
development of undesirable flavors.
Enzymes cause off flavors and off aromas in foods, particularly during storage. Improperly
blanched foods, such as green beans, English green peas, corn, broccoli, and cauliflower, develop
very noticeable off flavors and off aromas during frozen storage. Peroxidase, a relatively heat-
resistant enzyme not usually associated with development of defects in food, is generally used as
the indicator for adequate heat treatment of these foods. It is clear now that a higher quality
product can be produced by using the primary enzyme involved in off flavor and off aroma
development as the indicator enzyme.
It has been determined that lipoxygenase is responsible for off flavor and off aroma development
in English green peas, green beans, and corn, and that cystine lyase is the primary enzyme
responsible for off flavor and off aroma development in broccoli and cauliflower. Evidence to
support the important role of lipoxygenase as a catalyst of off-flavor development in green bean
flavor stability of frozen food (of types mentioned above), blanched to the endpoint of the
responsible enzyme, is better than that of comparable samples blanched to the peroxidase
endpoint.
Naringin is responsible for the bitter taste of grapefruit and grapefruit juice. Naringin can be
destroyed by treating the juice with naraginase. Some research is underway to eliminate naringin
biosynthesis by recombinant DNA techniques.
3.1.4 Nutritional Quality
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There is relatively little data available with respect to the effects of enzymes on nutritional quality
of foods.
Lipoxygenase oxidation of linoleic, linolenic, and arachidonic acids certainly decreases
the amounts of these essential fatty acids in foods.
The free radicals produced by lipoxygenase-catalyzed oxidation of polyunsaturated fatty
acids decrease the carotenoid (vitamin A precursors), tocopherols (vitamin E), vitamin C,
and folate content of foods. The free radicals also are damaging to cysteine, tyrosine,
tryptophan, and histidine residues of proteins.
Ascorbic acid is destroyed by ascorbic acid oxidase found in some vegetables such as
squash.
Thiaminase destroys thiamine, an essential cofactor involved in amino acid metabolism.
Riboflavin hydrolase, found in some microorganisms, can degrade riboflavin.
Polyphenol oxidase caused browning decreases the available lysine content of proteins.
3.1.5 Enzymes Used as Processing Aids and ingredients
Enzymes are ideal for producing key changes in the functional properties of food, for removal of
toxic constituents, and for producing new ingredients. This is because they are highly specific, act
at low temperatures (25-450C), and do not produce side reaction.
3.1.5.2 Immobilized Enzymes
Enzymes in solution are usually used only once. The repeated use of enzymes fixed to a carrier is
more economical. The use of enzymes in a continuous process, for example, immobilized
enzymes used in the form of a stationary phase which fills a reaction column where the reaction
can be controlled simply by adjustment of the flow rate, is the most advanced technique
3.1.5.2 Bound Enzymes
An enzyme can be bound to a carrier by covalent chemical linkages, or in many cases, by
physical forces such as adsorption, by charge attraction, H-bond formation and/or hydro-phobic
interactions. The covalent attachment to a carrier, in this case an activated matrix, is usually
achieved by methods employed in peptide and protein chemistry. First, the matrix is activated. In
the next step, the enzyme is coupled under mild conditions to the reactive site on the matrix,
usually by reaction with a free amino group. This is illustrated by using cellulose as a matrix.
Another possibility is a process of copolymerization with suitable monomers. Generally, covalent
attachment of the enzyme prevents leaching or bleeding.
3.1.5.3 Enzyme Entrapment An enzyme can be entrapped or enclosed in the cavities of a polymer network by polymerization
of a monomer such as acrylamide or N, N'-methylene-bis-acrylamide in the presence of enzyme,
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and still remain accessible to substrate through the network of pores. Furthermore, suitable
processes can bring about enzyme encapsulation in a semipermeable membrane
(microencapsulation) or confinement in hollow fibre bundles.
3.1.5.4 Cross-linked Enzymes
Derivatization of enzymes using a bifunctional reagent. e.g. glutaraldehyde, can result on cross-
linking of the enzyme and, thus, formation of large, still catalytically active insoluble complexes.
Such enzymes preparations are relatively unstable for handling and, therefore, are used mostly for
analytical work.
4. Some important uses of enzymes in foods
There are some major successes in the use of food-related enzymes.
4.1 Production of high-fructose corn syrup and sweeteners
This involves a relatively heat-stable α-amylase, glucoamylase, and glucose isomerase:
𝑆𝑡𝑎𝑟𝑐ℎ α−amylase→ 𝑑𝑒𝑥𝑡𝑟𝑖𝑛𝑠
𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑖𝑠𝑜𝑚𝑒𝑟𝑎𝑠𝑒 → 𝑓𝑟𝑢𝑐𝑡𝑜𝑠𝑒
Starch is heated to 105°C, Bacillus licheniformis a-amylase is added, and dextrins of DP 10-12
are produced by the endo-splitting enzyme. The soluble digest is passed through giant columns
(6-10 ft in diameter and 20 ft high) of immobilized glucoamylase where glucose is produced. The
glucose-containing stream is then run through giant columns of immobilized glucose isomerase
where approximately equimolar concentrations of glucose and fructose are produced. The
fructose is separated from glucose by differential crystallization and is used as a major sweetener
in the food industry (~100 billion tons/year). The glucose or a mixture of glucose and fructose is
used as sweetener, or the glucose is recycled to produce more fructose. Other sweeteners can also
be produced enzymatically. A second example is the use of aminoacylases to separate racemic
mixtures of DL-amino acids, in multi-ton lots. A third example is the use of specific lipases to
tailor-make lipids with respect to melting point, unsaturation, or specific location of a fatty acid in
a triacyiglycerol. This requires a beginning lipid, the required concentrations of free fatty acids,
appropriate enzymes, and appropriate conditions.
4.2 Specialty products and Ingredients via Enzymology
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The following text and tables illustrate some of the current or potential uses of enzymes on an
industrial scale.
4.2.1 Enzymatic Removal of Undesirable Compounds
Raw food materials often contain toxic or anti-nutrient compounds that are sometimes removed
by proper heat treatment, extraction or by enzymatic reactions. There are more than 12,000 plants
in the world that may have potential as food sources. Many are not used because of undesirable
properties, some of which could be overcome by the proper use of enzymes.
4.2.2 Enzymes in Milk and Dairy Products
Bovine milk contains many enzymes, and other enzymes are added during processing. Of most
importance economically is the use of chymosin (rennet) in production of several kinds of cheese.
β-Galactosidase is potentially of great importance in the commercial hydrolysis of lactose in milk
and dairy products, so that these products can be consumed by individuals who are deficient in. β-
galactosidase. β-Galactosidase is also used to convert whey lactose to glucose and galactose,
sweeteners with greater commercial demand.
4.2.3 Enzymes in Baking
Several enzymes are used in breadmaking. Additions of amylases and proteases has been
common for years. Several enzyme preparations are available for the stated purpose of reducing
the rate of staling in bread.
Table 1: Enzymatic production of Specific Compounds or Creation of Desirable flavours
Enzymes Purpose
Aminoacylases Resolve DL-amino acids
Aspartase Produce aspartate
Proteases Surfactants
Peroxidase Phenol resins
5'-Phosphodiesterases 5'-Nucleotides for flavor enhancers
5'-Adenylic deaminase Produce 5'-inosinic acid for flavour
Lipases (pregastric) Cheese/butter flavors
Proteases Decrease ripening time of cheeses
Tenderization of meat
Lipases/esterases Flavor esters
Proteases, nucleases Meaty flavors from yeast hydrolysis
Fumarase Fumaric acid as acidulant
Cyclomaltodextrin glucanotransferase Cyclodextrins for inclusion complexation
Tannase Antioxidants, such as propylgallate
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α-Galactosidase Modified food gums
Table 2: Enzymatic Removal of Unwanted Constituents
Enzymes Unwanted constituent
α-galactosidase Raffinose
β-galactosidase Lactose
Glucose oxidase Glucose
O2
Phytase Phytic acid
Thioglycosidases Thioglycosides
Oxalate oxidase O2
Alcohol oxidase O2
Oxyrase O2
Catalase H2O2
Sulfhydryl oxidase Oxidized flavours
Urease Carbamates
Cyanidase Cyanide
Pepsin,Chymotrypsin,carboxypeptidease A Bitter peptides
Naringinase Bitter compounds in citrus
Proteases Phenylalanine
α –Amylases Amylase inhibitors
Proteases Protease inhibitors
Table 3: Enzymes in Milk and Dairy Products
Enzymes Function
Chymosin Milk coagulation
For rennet puddings
Chymosin, fungal proteases For cottage cheese
For brick cheeses
Proteases Flavor improvement, decrease
ripening time of cheeses
Lipases
Flavor improvement; decrease
ripening time of cheeses
Sulfhydryl oxidase Remove cooked flavour
β-Galactosidase Lactose removal
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Microbial proteases Soybean milk coagulation
4.2.4 Enzymes in Brewing
Several recent advances have been made with regard to the use of enzymes in brewing. There is
increasing interest in the possible use of blended a-amylase and protease preparations as
replacements for malt in brewing. This is attributable to the expense and limited supplies of malt
and the possibility that better quality control might be achieved. The industry has also used
amyloglucosidases recently to make "light" beer. Amyloglucosidases hydrolyze the α-1,6-
glucosidic bonds of the amylopectin fraction, permitting the complete fermentation of starch. Use
of β-glucanases may solve the high viscosity/slow filtration rate problems caused by mannans
from cell walls. The most exciting advance, however, is the use of acetolactate decarboxylase,
now cloned into brewers yeast, to shorten the fermentation time by avoiding diacetyl formation.
4.2.5 Enzymes for Control of Microorganisms
Enzymes have potential for destroying microorganisms by several means. The means range from
hydrolysis of cell-wall compounds, such as β-glucans, chitin, and peptidoglycans, to production
of H202 and O2, which oxidize the essential - SH group of key sulfhydryl enzymes or
polyunsaturated fatty acids in cell walls. These are interesting possibilities, worthy of being tested
at the commercial level.
Table 4: Enzymes for Control of Microorganisms
Enzymes Function
Oxidases
Removal of 02, NADH or NADPH, produce
H202 and O2, which oxidize -SH groups
and polyunsaturated lipids
Xylitol phosphorylase
Conversion of xylitol to xylitol 5-phosphate,
which kills microorganism
Lipases
Liberation of free fatty acids, which are
toxic to protozoa Giardia lamblia
Lactoperoxidase
Uses O2 & H202, produced by oxidase, to
convert SCN– to SCNO– and -SH groups to -
S-S-, -S-SCN, or -S-OH
Myeloperoxidase
With added H202 and Cl-, produces HOCI
and chioroamines
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Lysozyme
Effective against a number of gram-positive
organisms via hydrolysis of cell-wall
peptidoglycans; hydrolysis of protein-
mannan outer layer of yeast when added
with an endo- β-1, 3-glucanase
Mannanase
Lysis of β-glucans-protein cell wall of yeast
Chitinase
Effective against chitin in cell wall of
several fungi
Antienzymes
Proteases, sulfhydryl oxidase,
dehydrogenases, lipoxygenases
4.2.6 Enzymes in Waste Management
It is estimated that the annual production of major carbohydrate feedstock for use as potential
fuels or for manufacturing chemicals in the United States is about 1160 million tons, versus 50
million tons of organic chemical feedstocks. This number includes 160 million tons of municipal
solid waste, 400 million tons of agricultural residue, 400 million tons of forest residue, and 200
million tons of corn and grains. These residues pose major environmental problems, since a large
amount of the agricultural residues are burned (where permitted), the forest residues result in
major fires each year, and the massive municipal landfills are often sources of groundwater
pollution. Therefore, if some of these materials could be converted to other forms of fuel (ethanol
and methanol) or to fermentation feedstock (glucose) to produce proteins, ethanol, and CO2,
disposal problems would be alleviated and nonrenewable fuel supplies would be conserved. The
U.S. government is spending considerable money on research on how to efficiently tap these
potential sources of fuel and chemicals.
What are the compounds that need to be converted? They are largely starch, cellulose, lignin,
lipids, nucleic acids and proteins. What enzymes are needed? Primarily the amylases, cellulases,
lignin peroxidases, lipases, nucleases, and proteases. These are enzymes we know quite a lot
about. So, what is the problem? The major problems are the insolubility of the potential substrates
just listed, and the poor stability and catalytic inefficiency of the enzymes required. Some
methods exist for increasing the solubility of the substrates and thereby the rate of hydrolysis. But
all methods require considerable energy expenditure that makes the process uneconomical at the
moment, when petroleum and coal are relatively inexpensive.
Do commercially feasible solutions exist for converting biomass to valuable chemicals? Nature
does this efficiently, given the huge 1160 million tons to be converted annually. The natural
conversion of these compounds occurs by action of the usual classes of enzymes, but the process
CHEMISTRY
PAPER 16 Bioorganic and biophysical chemistry
MODULE 29 Enzymes in food
is slow. Improvements in rate are possible through redesign of enzymes with greater efficiency
and synergistic properties.
Rapid, economical biomass conversion is a difficult task so the approach taken must be
multifaceted. More efficient enzymes are needed, and this requires knowledge of why these
enzymes are so inefficient in attacking insoluble substrates. Once this is better understood, better
enzymes can be searched for or developed by recombinant DNA techniques. One needed property
is greater stability, especially at temperatures sufficiently high microbial growth will not be a
problem. The pentose-containing polymers are especially difficult to hydrolyze and even more
difficult to ferment to ethanol and CO2. Lignin severely limits hydrolysis and fermentation of
cellulose, because it is so inert. Enzymes that efficiently attack these substrates are needed.
Solution of the biomass conversion problem is a must. Conversion processes must be
environmentally acceptable, and all components, even minor ones, must be economically
converted to usable or innocuous products to meet waste minimization goals pursued by the U.S.
Environmental Protection Agency.
5. Summary
Enzymes are proteins with catalytic activity due to their power of specific activation and
conversion of substrates to products.
Some of the enzymes are composed only of amino acids covalently linked via peptide
bonds to give proteins.
Enzymes have all the chemical and physical characteristics of other proteins.
Composition-wise, enzymes are not different from all other proteins found in nature and
they comprise a small part of our daily protein intake in our foods.
Several recent advances have been made with regard to the use of enzymes in brewing.
There is increasing interest in the possible use of blended a-amylase and protease
preparations as replacements for malt in brewing. Use of β-glucanases may solve the high viscosity/slow filtration rate problems caused by
mannans from cell walls. The most exciting advance, however, is the use of acetolactate
decarboxylase, now cloned into brewer’s yeast, to shorten the fermentation time by
avoiding diacetyl formation.
Enzymes have potential for destroying microorganisms by several means. The means
range from hydrolysis of cell-wall compounds, such as β-glucans, chitin, and
peptidoglycans, to production of H202 and O2, which oxidize the essential - SH group of
key sulfhydryl enzymes or polyunsaturated fatty acids in cell walls.
It is estimated that the annual production of major carbohydrate feedstock for use as
potential fuels or for manufacturing chemicals in the United States is about 1160 million
tons, versus 50 million tons of organic chemical feedstocks.