Applied Enzyme Catalysis

Pn Syazni Zainul KamalSchool of Bioprocess Engineering

ERT211 BIOCHEMICAL ENGINEERING

Inside the chapter

1)Survey some of the applications of enzymes ◦ Sources of enzyme◦ Hydrolytic enzymes and applications

2)Examine immobilized enzyme catalyst formulations ◦ which allow sustained, continuous use of the

enzyme.

There are three major sources of enzymea) animalb) plantc) or microbial

Although all living cells produce enzymes, one of the three sources may be favored for a given enzyme or utilization

Sources of enzyme

Some enzymes may be available only from animal sources.

Enzymes obtained from animals may be relatively expensive, e.g., rennin obtain from calf's stomach, ◦ the value depend on demand of lamb or beef, ◦ and their availability.

a) Animal sources

b) Plant sources While some plant enzymes are relatively

easy to obtain e.g., papain from papaya, bromelain from pineapple, actinidin from kiwi fruit◦ their supply is also governed by food demands

c) Microbial enzyme Microbial enzymes are produced by

methods which can be scaled up easily Recombinant DNA technology now provides

the means to produce many different enzymes, including those not normally synthesized by micro organisms or permanent cell lines, in bacteria, yeast and cultured cells.

Due to the rapid doubling time of microbes compared with plants or animals ◦ microbial processes are attuned more easily to

the current market demands for enzymes. On the other hand,

◦ for use in food or drug processes, only those microorganisms certified as safe may be exploited for enzyme production.

Although most of the enzymes used today are derived from living organisms, they are utilized in the absence of life

Example –◦ extracellular enzymes, ◦ secreted by cells in order to degrade polymeric

nutrients into molecules small enough to permeate cell walls.

◦ Grinding, mashing, lysing, or otherwise killing and splitting

◦ intracellular enzymes, ◦ which are normally confined within individual

cells.

The enzyme kinetics study generally carried out with the purest possible enzyme preparations. Such research involves ◦ the fewest possible number of substrates (one if

achievable) ◦ a controlled solution with known levels of

activators (Ca2+, Mg2+,pH etc.), ◦ cofactors, ◦ and inhibitors.

Enzyme Kinetic

Industrial enzymes Many useful industrial enzyme preparations are

not highly purified. They contain a number of enzymes with

different catalytic functions and are not used with either a pure substrate or a completely defined synthetic medium.

Also, the simultaneous use of several different enzymes may be more efficient than sequential catalysis by a separated series of the enzymes.

such enzyme preparations are kinetically more simple than the integrated living organisms from which they are produced

Some industrially important enzymes

Hydrolytic enzyme Enzyme that catalyzes the hydrolysis of a

chemical bond Hydrolytic enzymes are normally associated

with degradative reactions, (break down large molecules into small molecules) e.g., ◦ conversion of starch to sugar, ◦ proteins to polypeptides and amino acids, ◦ and lipids to their constituent glycerols, fatty

acids and phosphate bases

Hydrolytic enzymes 3 major group of hydrolytic enzyme Those involved in the hydrolysis of

◦ Ester (Esterase) – split ester into acid & alcohol◦ Glycosidic (carbohydrase) – act on carbohydrate◦ and various nitrogen bonds – act on proteins and

polypetides Enzymes are named according to the

chemical reactions they catalyze, rather than according to their structure.

Classification of Hydrolytic Enzymes

Since One-enzyme – one-reaction uniqueness does not generally exist, Enzymes from different plant or animal sources which catalyze a given reaction will not always have the same molecular structure or necessarily the same kinetics.

Consequently, ◦ maximum reaction rate, ◦ Michaelis constant, ◦ pH of optimum stability or activity, ◦ and other properties –

depend on the particular enzyme source used.

Application of hydrolytic enzymes In macroscopic degradations such as

◦ food spoilage◦ starch thinning,◦ and waste treatment,

Also in the chemistry of ◦ ripening picked green fruit◦ self-lysis of dead whole cells (autolysis), ◦ desirable aging of meat, ◦ curing cheeses, ◦ preventing beer haze, ◦ texturizing candies, ◦ treating wounds, ◦ and desizing textiles.

In eucaryotes, hydrolases may be stored inside the cell in membrane-enclosed lysosome organelles, reside in the periplasm in microbes like yeast, or be secreted into the environment.

Most hydrolytic enzymes used commercially are extracellular microbial products.

Application of Hydrolytic Enzymes

Hydrolysis of starch Carbohydrase Amylases - extensively applied enzymes

- can hydrolyze the glycosidic bonds in starch and related glucose-containing compounds

(eg. Cellulose). *(glycosidic bond – join carbohydrates to another group) There are three major types of amylases-

◦ α-amylase◦ b-amylase◦ Amyloglucosidase/glucoamylase

Starch contains straight-chain glucose polymers called amylose and a branched component known as amylopectin.

The branched structure is relative more soluble than the linear amylose and is also effective in rapidly raising the viscosity of starch solution.

Glucose Structure

The b(1-4) glycosidic linkage is represented as a "zig-zag" line, but one glucose residue is actually flipped over relative to the other

a(1-4) glycosidic linkage between the C1 hydroxyl of one glucose and the C4 hydroxyl of a second glucose

Starch - polysaccharide of plant - 2 polysaccharide occur together in starch *amylose - α(1-4) glycosidic bond *amylopectin – β(1-6) glycosidic bond

Amylopectin Structure

Hydrolysis of starch

α-amylase The action of α-amylase reduces the

solution viscosity by acting randomly along the glucose chain at α-1,4 glycosidic bonds

α-amylase is often called the starch-liquefying enzyme for this reason.

Hydrolysis of starchβ-amylase b-Amylase can attack starch a-1,4 glycosidic

bond only on the nonreducing ends of the polymer and always produces maltose when a linear chain is hydrolyzed.

Because of the characteristic production of the sugar maltose, b-amylase is also called a saccharifying enzyme.

soluble mixture of starch and b-amylase yields maltose and a remainder of dextrins (starch remnants with 1,6- linkage on the end)

Hydrolysis of starchamyloglucosidase Another saccharifying enzyme,

amyloglucosidase (also called glucoamylase) attacks primarily the nonreducing a-1,4 linkages at the ends of starch, glycogen, dextrins, and maltose. (a-1,6 linkages are cleaved by amyloglucosidase at much lower rates)

Sequential treatment with a-amylase and glucoamylase or enzyme mixtures are utilized where pure glucose rather than maltose is desired, e.g., in distilleries and in the manufacture of glucose syrups (corn syrup) and crystalline glucose.

Sources of amylase The sources of amylases are very numerous Amylases are produced by – microb, plant e. g., 1) amylase produced by Clostridium

acetobutylicum which is clearly involved in the microbial conversion of polysaccharides to butanol and acetone.

2) amylase produced by Aspergillus niger, Penicillium sp.

3) amylase from Bacillus used in clothing and dishwasher detergent

(amylase from microb, not suitable to be used in food industry)

Common Application of Amylase Preparation

Commercial amylase preparations used in human foods are normally obtained from grains, e.g., barley, wheat, rye, oats, maize, sorghum, and rice.

The ratio of saccharifying to liquefying enzyme activity depends ◦ on the particular grain ◦ and upon whether the grain is germinated.

Application of Amylase

In the production of malt for brewing, the ungerminated seeds are exposed to a favorable temperature and humidity so that rapid germination occurs, with resulting large increase in a-amylase.

The germinated barley is then kiln-dried slowly; ◦ this halts all enzyme activity without irreversible

inactivation. The dried malt preparation is then ground, and

its enormous liquefying and saccharifying power is utilized in the subsequent yeast fermentation.◦ to convert starches to fermentable sugars.

Application of Amylase

Invertase hydrolyzes sucrose and poly saccharides containing a b-D-fructofuranosyl linkage.

The hydrolyzed sucrose solution containing fructose and glucose rotates a polarized light beam in the direction opposite that of the original solution.

The partially or completely hydrolyzed solution allows two properties desirable in syrup and candy manufacturing: ◦ a slightly sweeter taste than sucrose ◦ and a much higher sugar concentration before hardening.

Hydrolysis of Disaccharides Maltose

1. Maltose + H2O -*--> glucose + glucose * = enzyme; in this case maltase Enzymes end in -ase

Sucrose Sucrose + H2O -*-> glucose + fructose * = sucrase

Hydrolysis of Lactose Lactose + H2O -*-> galactose + glucose * = lactase

Hydrolysis of Cellulose Cellulose – polysaccharide consisting of a

linear chain of several hundred to over ten thousand β-1,4 linked D-glucose units

Structural component of all plant cells from algae to tree

Hydrolysis of cellulose cellulase – enzyme that hydrolyze cellulose

◦ Trichoderma fungi are commonly used at the present time.

◦ They are thoroughly developed and characterized at present.

◦ There are three major classes of enzymes for different substrates and products1.Exo-b-1,4-cellobiohydrolase (CBH)2.Endo-b-1,4-glucanase 3.b-glucosidase

Hydrolysis of cellulose 3 steps of reaction catalyze by cellulase :1)Breakage of non-covalent interactions

present in the crystalline structure of cellulose by endo-β-1,4-glucanase

2)Hydrolysis of individual cellulose fibers to break it into smaller sugars (cellobiose) by exo-β-1,4-cellobiohydrolase (CBH)

3)Hydrolysis of dissacharides or tetrasaccharides into glucose by β-glucosidase

Hydrolysis of Cellulose

Cellulase sources Many other microorganisms including the

molds bacteria produce cellulases with distinctive activities and properties. e.g.-• Fusarium solani, • Aspergillus niger, • Penicillium funicolsum, • Sporotrichum pulverulentum, • Cellulomonas species, • Clostridium thermocellum, • and Clostridium thermosaccharolyticum

Applications of cellulase Alcohol fermentation from biomass Brewing Waste treatment Cereal processing Pulp and paper industries

Proteolytic enzymes Enzyme that catalyze the splitting of protein

into smaller peptide fractions and amino acids by a process known as proteolysis

Eg. Proteinase + protein polypeptides polypeptidase + protein amino

acids Some can detach the terminal amino acids

from the protein chain i.e Exopeptidase – aminopeptidase,

carboxypeptidase A Others attack internal peptide bonds of a

protein i.e Endopeptidase – trypsin,pepsin,papain

Sources of proteolytic enzyme Animal, plant, microb Trypsin – animal pancrease Papain – papaya Protease – Bacillus sp., Aspergillus sp.

Application of proteolytic enzyme1) Detergents- Enzyme used in laundry aid as early 1913- Protease-contain a mixture of bacterial

neutral-alkaline protease/lipase active at pH6-10 and 30-60°C

- facilitate spot removal, so that it can be wash easily

- Since one enzyme molecule can act on many substrate (i.e., soil) molecules, a small amount of enzyme added to a laundry detergent can provide a big cleaning benefit to the consumer

2) Meat tenderization- eg. Bromelain and papain- breaking the peptide bonds between

amino acids found in complex proteins - Meat is held together by a complex protein

called collagen- Meat is often tenderized before cooking, to

make it less tough and more suitable for consumption

- If meat tenderizers are allowed to act for too long, the meat can become squishy and lose its special texture.

3) Tanning- Making leather process from animal skin- Ground pancreases contain digestive

proteases eg trypsins, lipases- Use for – dehairing animal hides & removal

of noncollagen protein- Environmental friendly rather than using

chemical

4) Dairy industries- Coagulation of milk- Rennin remove glucopeptide from soluble

calcium casein to yield paracaseinate- Paracaseinate precipitate to form curd- Curd further process to make cheese

5) Clinical & medical application- eg. trypsin- Reduce inflammation & swelling (internal

injuries & infection)- Dissolve blood clots & extracellular protein

precipitates

Esterase

Cleave or synthesize ester bonds to yield an acid and an alcohol

R1COOR2 + H2O R1COOH + R2OH

eg. Lipases – hydrolyze fats into glycerol & fatty acids

lipase Most important enzyme in esterase group Hydrolyze triglycerides into diglycerides,

monoglycerides, fatty acids and glycerols Applications in food, detergent,

pharmaceuticals, leather, textile, cosmetic

Applications of lipase

1)Dairy industries- Lipase used for hydrolysis of milk fat- Current applications ; flavour

enhancement of cheese, acceleration of cheese ripening (lipase degrade protein, fat, lactose)

- Addition of lipase to cow’s milk, generate flavour similar to that of ewe’s 0r goat’s milk

2)Detergents- Lipase add to detergents- Remove fat and oils based stains

3) Lipase in oleochemical industry- Before this used organic solvent and

emulsifier in oleochemical industry- Now using immobilized lipase from Candida

cylindracea in production of soap- Resulted in high productivity & continuous

running of the process- Reduce cost for expensive equipment &

thermal energy

4) Meat processing industry- to produce fat free meats- Partial fat hydrolysis of the meat cut using

lipase

5) Pulp & paper industry- Deinking – removal of ink process from

surface of paper- Conventional method – used chemical to

remove ink ; cause water pollution & high cost

- Deinking by enzyme – Lipase used to remove oil based ink

Enzyme mixtures Mixture of enzymes may contain : a) same general type = α- & β- amylase and

amyloglucosidase b) Different type = found in pancreas extract

(trypsin, lipase, amylase) are often used more successfully than

single enzyme preparations eg. Blend of diff. amylase yields large amount of

saccharified starch suitable for yeast fermentation

Other applications of enzyme in solution

Hydrolysis enzymes applications dominate past and present enzyme technology

Other enzyme processes currently serve important function in food, pharmaceutical and biochemical industries

Medical applications of enzymes Recently free or extracellular enzymes were

used in medicine1)Lysozyme (in nasal mucus, saliva, tears) It hydrolyze mucopolysaccharides of

bacterial cell walls. Used as an antibacterial agent, treatment of

ulcer, skin disease

2) Asparaginase Used as anticancer agent Enzyme that catalyze hydrolysis of

asparagine to aspartic acid Some cancer cells require asparagine

(nurient), their growth can be inhibited using asparaginase

Can be given to patient as intramuscular, subcutaneous or intravenous injection (differ from other chemo agent, no tissue irritation)

3)Penicillinase 1st isolated from gram negative bacteria

E.coli human beings do not produce penicillinase Remove allergenic form of penicillin from

allergic individuals Convert the drug into nonallergic form

Thank You