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
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