Subject Chemistry Paper No and Title Paper 16 Bioorganic

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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

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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

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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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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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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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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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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

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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.

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Documents

Subject Chemistry Paper No and Title Paper 16 Bioorganic