Lecture 6-Applied Enzyme

CatalysisDr. A.K.M. Shafiqul Islam

School of Bioprocess Engineering15.01.2010

Survey some of the applications of enzymes ◦ Enzymatic hydrolysis and concept: Starch and

Cellulose Examine immobilized enzyme catalyst

formulations ◦ which allow sustained, continuous use of the

enzyme.

Inside the Chapter

There are three major sources of enzyme• Plant• animal • or microbial

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

Sources Enzymes

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.

Animal Sources

While some plant enzymes are relatively easy to obtain e.g., papain from papaya◦ their supply is also governed by food demands

Plant Sources

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.

Microbial Enzymes

Some Enzymes of Industrial Importance

Some Enzymes of Industrial Importance

Some Enzymes of Industrial Importance

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.

Microbial Enzymes

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.

Microbial Enzymes

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

Many useful industrial enzyme preparations are not highly purified.

They contain a number of enzymes with different catalytic functions and are not used with 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

Industrial enzymes

either a pure substrate

Hydrolytic enzymes are normally associated with degradative reactions, 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

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.

Application of Hydrolytic Enzymes

The three groups of enzymes. Those involved in the hydrolysis of◦ ester, ◦ glycosidic, ◦ and various nitrogen bonds.

Hydrolytic Enzymes

Enzymes are named according to the chemical reactions they catalyze, rather than according to their structure.

Hydrolytic Enzymes

Classification of Hydrolytic Enzymes

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

Many hydrolases are directed to specific compartments separated from the cytoplasm by membranes. This serves the purpose of protecting essential cytoplasmic bipolymers from degradation. Example,

Gram-positive bacteria secrete a variety of hydrolases into their environment. With their double membrane outer envelope, gram-negative bacteria have available the periplasmic space which safely stores a variety of hydrolases.

Application of Hydrolytic Enzymes

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

Amylases are extensively applied enzymes which can hydrolyze the glycosidic bonds in starch and related glucose-containing compounds.

There are two major types of amylases-◦ a-amylase◦ b-amylase

Hydrolysis of Starch and Cellulose

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

Amylopectin Structure

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.

The action of a-amylase reduces the solution viscosity by acting randomly along the glucose chain at a-1,4 glycosidic bonds

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

Hydrolysis of Starch and Cellulose

b-Amylase can attack starch a-1,4 bonds 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 Starch and Cellulose

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.

Hydrolysis of Starch and Cellulose

Common Application of Amylase Preparation

The sources of amylases are very numerous.

Amylases are produced by a number of bacteria and molds – ◦ e. g., amylase produced by Clostridium

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

Hydrolysis of Starch and Cellulose

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.

Hydrolysis of Starch and Cellulose

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.

Hydrolysis of Starch and Cellulose

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 For cellulase

◦ 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

Hydrolysis of Cellulose

Hydrolysis of Cellulose 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

Cellulose Structure Cellulose structure e.g., crystallinity,

specific surface area and degree of polymerization are important

Cellulose structure can be altered by a variety of pretreatments such as ball or compression milling, g-irradiation, pyrolysis, and acidic or caustic chemicals.

Esterase Applications Cleave or synthesize ester bonds to yield an

acid and an alcohol Anaerobic waste digestion Meat processing

Proteolytic Enzymes Attack nitrogen-carrying compounds,

particularly proteins Dry cleaning Detergents Meat processing Cheesemaking