Industrial Applications of Enzymes

Chapter 4

Carbohydrates

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Classification of Carbohydrates

Carbohydrates

Monosaccharides

Glucose Fructose Galactose

Oligosaccharides

Sucrose Lactose Maltose

Polysaccharides

Starch Glycogen Cellulose

Monosaccharides• Simple sugars that cannot be hydrolyzed further

Open form to Ring form• Aldehydes and hydroxyls in a sugar

molecule can react in a solution to form a ring.

• H from the OH at fifth carbon joins the aldehyde and the O from the same OH bonds to the first carbon.

Fischer projection and Haworth projection

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Disaccharides

•Contain two molecules of same or different monosaccharide units.•On hydrolysis they give two monosaccharide units.•Monosaccharide units are joined by glycosidic bond.

Disaccharide Monosaccharide 1 Monosaccharide 2 Bond

Sucrose

Lactose D-Glucose

Maltose

Cellobiose

Polysaccharides

•polysaccharides are high molecular mass carbohydrates composed of many monosaccharide units joined by glycosidic bonds.• Polysaccharides serve a number of crucial biological functions in

organisms such as • cell wall support (structural polysaccharides) Ex. Cellulose• food storage (storage polysaccharides) Ex. Starch, Glycogen

Starch

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Digestion of Starch

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Glycogen

• In glycogen (left), branches occur every 8 to 12 residues whereas in amylopectin (right), branches occur every 25 to 30 residues

Cellulose

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Corn kernel

Starch Conversion

•Cleaning• First we clean the shelled corn shipments to ensure that they are free from

dust and foreign bodies.

• Steeping• Once clean, the corn is soaked in water, called steep water, at 50˚C for

between 20 and 30 hours, during which time it doubles in size. • Sulphur dioxide is added to the water to prevent excessive bacterial growth.• As the corn swells and softens, the mildly acidic steep water starts to loosen

the gluten bonds with the corn, and to release the starch. • The corn goes on to be milled.• The steep water is concentrated in an evaporator to capture nutrients, which

are used for animal feed and fermentation.

•Milling and separation• The corn is coarsely milled in the cracking mills to separate the germ from the

rest of the components (including starch, fiber and gluten). Now in a form of slurry, the corn flows to the germ or ‘cyclone’ separators to separate out the corn germ.

• The corn germ, which contains about 85% of the corn’s oil, is removed from the slurry and washed. It is then dried and sold for further processing to recover the oil.

• Fine grinding and screening• The remaining slurry then leaves the separation step for fine grinding. • After the fine grinding, which releases the starch and gluten from the fiber,

the slurry flows over fixed concave screens which catch the fiber but allow the starch and gluten to pass through.

• The starch-gluten suspension is sent to the starch separators.• The collected fiber is dried for use in animal feed.

• Separating the starch and gluten• The starch-gluten suspension passes through a centrifuge where the gluten,

which is less dense than starch, is easily spun out.• The gluten is dried and used in animal feed.• The starch, which still has a small percentage of protein remaining, is washed

to remove the last traces of protein and leave a 99.5% pure starch. • The starch can either be dried and sold as corn starch, or it can be modified to

turn into other products, such as corn sweeteners, corn syrups, dextrose and fructose.

Corn refining process

• Starch slurry from wet milling process is broken down to glucose and isomerized to fructose.

Cellulose

• The cellulose chains are grouped together to form microfibrils, which are bundled together to form cellulose fibers.• The cellulose microfibrils are mostly independent but the ultrastructure of

cellulose is largely due to the presence of covalent bonds, hydrogen bonds and Van der Waals forces.• Hydrogen bonding within a cellulose microfibril determines ‘straightness’

of the chain but inter-chain hydrogen bonds might introduce order (crystalline) or disorder (amorphous) into the structure of the cellulose (Klemm et al., 2005). • In nature, cellulose appears to be associated with other plant compounds

and this association may affect its biodegradation.

Schematic of cellulose microfibrils

Lignocellulosic Materials

• Lignocellulosic materials contain:• Lignin• Cellulose• Hemicellulose

• Cellulose provides strength and flexibility.• Lignin supports and protects the cellulose from biological and

chemical attack.• Hemicellulose bonds lignin to cellulose.

Lignin

• It is present in plant cell walls and confers a rigid, impermeable, resistance to microbial attack and oxidative stress. • Lignin is a complex network formed by polymerization of oxidatively

formed radicals of p-hydroxycinnamyl alcohols.• The complexity of the chemical structure of lignin makes it very difficult to

use except as a fuel.• Because of its relatively high calorific value (12,700 BTU/lb), most of waste

lignin is being used as fuel in the chemical recovery processes of the pulp plants.• Only a small part of lignin is utilized in adhesives, structural polymers,

coating, dispersants, soil conditioner, pesticide carrier e.t.c.

Hemicelluloses• Hemicelluloses are branched, heterogeneous polymers of pentoses (xylose,

arabinose), hexoses (mannose, glucose, galactose) and acetylated sugars.• They have lower molecular weight compared to cellulose and branches

with short lateral chains that are easily hydrolyzed (Saha, 2003; Scheller & Ulvskov, 2010).• Hemicelluloses are bound via hydrogen bonds to the cellulose microfibrils

in the plant cell wall, crosslinking them into a robust network. • Hemicelluloses are also covalently attached to lignin, forming together with

cellulose to form a highly complex structure. • Hemicelluloses in agricultural biomass like straws and grasses are

composed mainly of xylan, while softwood hemicelluloses contain mainly glucomannan.

Cellulose Pretreatment

Cellulases

•Cellulases are responsible for the hydrolysis of cellulose.•Cellulases are composed of a complex mixture of enzymes with

different specificities to hydrolyze the β-1,4-glycosidic linkages. •Cellulases can be divided into three major enzyme activity classes (Goyal

et al., 1991; Rabinovich et al., 2002 ).

• Endoglucanases or endo-1-4-β-glucanase (EC 3.2.1.4)• Exoglucanase or cellobiohydrolase (EC 3.2.1.91)• β-glucosidase (EC 3.2.1.21).

Cellulases

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Kinetics of Enzymatic Hydrolysis of Cellulose

Reaction Mechanism