Various industrial applictaions of Enzymes

1. Applications of Enzymes
Dr. Kalavati Prajapati

2. Enzymes
 Breakdown substances
 Protein catalyst
 Specific to substrate
Pharmaceutical Industry
 Develops, produces, and
markets medical drugs
Basics

3. Industrial Uses
 Enzymes in the Production of Functional Oligosaccharides and
Other Neutraceuticals
 Enzymes in the Modification of Fats and Oils
 Enzymes in the Animal Feed Industry
 Enzymes in the Pulp and Paper Industry
 Enzymes in the Fruit Juice Processing Industry
 Enzymes in the Meat and Fish Processing Industry
 Enzymes in the Dairy Industry
 Enzymes in Detergents
 Enzymes in the Leather Industry
 Enzymes in the Production of Bulk and Fine Chemicals
 Analytical Applications of Enzymes
 Enzyme-Replacement Therapy

4.  Doctors measure the amount and type of an enzyme to
determine any diseases
Ex. Damaged liver
1. In Diagnosing

5. Enzyme Capsules
 2 practises (Enzyme Therapy and Enzyme
Replacement Therapy)
 Suggested for people over 35 years of age
 Plant-based enzymes are preferred over animal
derived enzymes
 Many different kinds of capsules
 Take once with every meal

6. Protease Capsules
 Provides therapeutic benefits
 Enhance circulatory and immune
systems
 Quicker healing
 Better stamina
 Consume between meals
 The practise is increasing in
popularity

7.  There are supplements for this disorder
 Lactase is the enzyme
 Acidic environment
 Better alternative for some people
Lactose Intolerant

8.  There are enzymes in skin care products
 Rosecea
 Face Wash
Topical Applications

9. Enzymes as diagnostic tools.
Enzyme Disease diagnosed
Choline esterase Paralysis
Alkaline phosphatase Prostrate carcinome
Pancreatic amylase Pancreatitis
Aspartate transaminase Acute hepatitis
Alkaline phosphatase
Gamma glutamyl transferase
Kidney damage
Creatine kinase Skeletal muscle damage
Hydroxy butyrate dehydrogenase
Lactate dehydrogenase
Heart disease
Leucine aminopeptidase Hepatobiliary disease
Transaminase Chronic hepatitis
Pepsin Gastric mucosal damage
Creatine phosphokinase
Aspartate transaminase
Lactate dehydrogenase
Myocardial infraction
Mitchondrial enzymes Tissue necrosis

10. Enzymes used to diagnose inheritable genetic disorder
Enzyme Inherited disease diagnosed
HGPRT (Hypoxanthine guanine
phosphoribosyl transferase)
Lasch Nayan Syndrome
Purine nucleoside
phosphorylase
Adenosine deaminase
Immunodeficiency disease
Glucocerebrosidase Gaucher’s disease
Phosphoribosyl pyrophospate
synthetase
Gout
25-OH-cholecalciferol-1-
hydroxylase
Rickets
Hexosaminidase Tay Sach’s disease

11. The sensitivity and specificity of enzymes makes them useful
tools in medicine for the detection and measurement of chemicals
in fluids such as blood and urine
Because of their sensitivity, enzymes are able to detect the
presence of specific molecules even when they are
present at very low concentrations
The enzyme glucose oxidase is used in an immobilised form
for the detection of glucose in biological fluids
2. Enzymes as Analytical Agents

12. The colour of the pad on the clinistix is compared with
a colour chart to determine the amount of glucose
present in the sample
Increasing amounts of glucose
No
glucose
Glucose Measurement using 'Clinistix'

13. Biosensors
Biosensors are electronic monitoring
devices that make
use of an enzyme’s
specificity and the technique of
enzyme immobilisation

14. A biosensor has been developed for
detecting
glucose in the blood of diabetics
Glucose oxidase
oxidises any glucose
present in the blood to
release electrons – these
are detected by the
transducer and converted
into an electrical current
Transducer
Amplifier
The current generated is
proportional to the amount
of glucose present in the
sample and this is displayed
as a digital read-out
Glucose
molecules
in the blood
Gluco
se
oxidas
e
Biosensors

15. Enzymes used as analytical agent
Enzyme(s) in coupled assay Analyte quantified
Hexokinase
Glucose-6-P dehydrogenase
Glucose
Urease
Glutamate dehydroganase
Urea
Uricase
Catalase
Aldehyde dehydrogenase
Uric acid
Esterase
Cholesterol Oxidase
Peroxidase
Cholesterol

16. Therapeutic applications of Enzymes
 Therapeutic enzymes have a broad variety of
specific uses
 Oncolytics
 Anticoagulants
 Thrombolytics
 Replacements for metabolic deficiencies
 Digestive aids
 Metabolic storage disorders, etc
 Miscellaneous enzymes of diverse function

17. Oncolytic enzymes
Asparaginase
 A tetrameric enzyme that catalyses the hydrolysis of the
amino acid asparagine

18. Asparaginase
 It may be purified from a wide variety of
microorganisms (yeast, fungi, bacteria such as E.
coli)
 Asn is required for normal metabolic activity
 Most human cells are capable of synthesizing Asn
but certain malignant cells are not
This can be used in the destruction of malignant
cells....

19. Asparaginase
 Source of clinically used asparaginase:
 E. coli: two isozymes of which only one is effective
 Erwinia chrysanthemi
 Treatment of childhood leukaemia
 Side effects: severe allergic reaction, nausea, vomiting, fever,
compromised kidney and liver function
 Allergic reaction is greatly reduced by coupling the asparaginase
with PEG
 Asparaginase production by a recombinant Pichia pastoris
strain harbouring S. cerevisiae ASP3 gene (Ferrera et al,
Enzyme and Microbial Technology 39(7) 2006)

20. Therapeutic enzymes
Enzyme Therapeutic use/ disease cured
Digestive enzymes
Papain, lactase, Pancrealipase, Pepsin
Digestive disorders
Fibrinolytic enzymes
Streptokinase, Urokinase, Tissue
plasminogen activator
Coronary thrombosis
Pulmonary embolism
L-asparaginase Cancer chemotherapy
Actiase, Retavase, Rapilysin Acute myocardial infraction
α1-antitrypsin Neutrophil-induced lung damage Emphysema
DNAse Cystic fibrosis
Dextrase Control of dental plaque
Glucocerebrosidase Gaucher’s disease
Galactosidase Inherited β-galactosidase deficiency
Trypsin, Papain, Collagenase Antiinflammatory agent
Superoxide dismutase Oxygen toxicity (Antioxidant enzyme)
Bromealin (protease) Inflammation & edema
Chymotrypsin Upper respiratory tract disease
Diastase Amyloceous dyspepsia
Hyaluronidase To increase the effect of local anesthesia
Penicillinase Penicillin allergy
Papain Dyspepsia with flatulence
β-galactosidase Lactose intolerance

21. Biologically important enzymes & their roles
Enzyme Biological role
ACE (angiotensin converting
enzymes)
To maintain & regulate blood
pressure
Nucleotidase To catalyze the digestion of
nucleic acids in food.
Cholinesterase To regulate muscle function
G6PD To form NADH in HMP shunt
To maintain GSH cycle
Amylase To catalyze the hydrolysis &
digestion of dietary starch
forming dextrin & maltose

22. According to the intended use food
enzymes are categorized either as:
 Food additives having a technological
function
 Processing aids present only in residual
amounts in food and not having a function
anymore there

23. Historically Speaking…..
 Enzymes (biotech) have been impacting our food
supply for 1000’s of years!!!
 Use of enzymes became an offshoot of a biological
or microbiological discovery (brewing and
alcohol production, vinegar, baking) – involved
fermented foods
 Modern enzyme applications included rennin for
cheese, glucose oxidase for desugaring eggs and
alpha amylase for starch processing

25.  But, typically those products were developed first as
result of a biological processes.
 Foods, as the consumer recognizes them, made directly
by addition of enzymes are limited……

26. Foods Made with Enzymes
Extending shelf life with enzymes

27. Baking enzymes & their applications
Enzyme Application
Alpha amylase Specialtyamylase for French type bread
Bacterial proteinase For processing biscuits, crackers,& wafer biscuits
Fungal proteinase For imprvind dough handling & bread texture
To improve loaf volume, crust color, flavour & crumb softness
Baking lipase To improve dough strength, volume, & crumb whiteness.
Glucose oxidase General baking improver
New generation bromate replacer
Maltase Suitable for rye bread & low sugar doughs
Pentosanase High activity & low activity protease free pentosanase used in
bread improvers. Eg., endo-xylanase for bread improvers
Pentosanase, Amylase Increased bake-out volume in bread & rolls
General bread improver suitable for both continental &
chorleywood process
Baking improver specifically formulated for rye bread
Proteinase,
Pentosanase
Metabisulphite replacer in biscuits & crackers
Complete spectrum
enzyme
For Enlish-style muffins; this product improves the spread of
the dough piece

28. 1. Starch conversions
Starch contains about 15–30% amylose and 70–85% amylopectin.
Enzymes have largely replaced the use of strong acid and high temperature to
break down starchy materials.
Three types of enzymes are involved in starch bioconversion:
1. endo-amylase (a-amylase, EC 3.2.1.1), [Bacillus lichiniformis, Bacillus
subtilis, and Bacillus amyloliquefaciens and fungi such as Aspergillus
oryzae]
2. exo-amylases
• glucoamylase or glucan 1,4-a-glucosidase, EC 3.2.1.3 [Endomycopsis,
Aspergillus, Penicillium, Rhizopus, and Mucor];
• b-amylase, EC 3.2.1.2) [Bacillus megaterium, Bacillus cereus, Bacillus
polymyxa, Thermoanaerobacter thermosulfurogenes, and Pseudomonas
sp.]
3. debranching enzymes (pullulanase, EC 3.2.1.41; isoamylase, EC
3.2.1.68). [Aerobacter aerogenes and isoamylase is produced by
Pseudomonas amyloderamosa.]

29. In amylose these are linked  -(1, 4)-, with the ring oxygen atoms all on
the same side.

30. In amylopectin about one residue in every twenty or so is also linked -
(1,6)- forming branch-points.

31. Enzymatic hydrolysis of amylopectin
cleaves glucose units from the nonreducing
end of starch and it can hydrolyze both -1,4
and -1,6 linkages of starch, slower

32. Dextrins: A group of low-molecular-
weight carbohydrates produced by the
hydrolysis of starch.
Dextrins are mixtures of linear a-(1,4)-
linked D-glucose polymers starting with
an a-(1,6) bond.

33. Amylases break starch into sugars
All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds.
-Amylases
Both the salivary and pancreatic amylases are α-Amylases. They are Ca
metalloenzymes,completely unable to function in the absence of calcium.
They act at random locations along the starch chain hence faster than
b-amylases
Working from the non-reducing end, β-amylase catalyzes the hydrolysis
of the second α-1,4 glycosidic bond, cleaving off two glucose units
(maltose) at a time. During the ripening of fruit, β-amylase breaks
starch into sugar, resulting in the sweet flavor of ripe fruit. Both are
present in seeds; β-amylase is present prior to germination, whereas α-
amylase and proteases appear once germination has begun.

34. a-amylase randomly hydrolyse a-1,4 linkages in both amylose and
amylopectin to yeild mixture of glucose, maltose, maltotriose and series of a-
limit dextrins.
b-amylase sometimes used in place of a-amylase. They hydrolyze alternate a-
1,4 linkages and yield maltose residues and b-limit dextrins
Glucoamylase hydrolyses a-1,3. a-1,4 and a-1,6 linkages but is less efficient
than a-amylase. Major role is to break cross links of amylopectin resulting in
complete breakdown to glucose. Generally used to reduce CHO content of
beers. Industrially obtained from fungus Aspergillus niger.
Glucose isomerase is used for conversion of glucose obtained after processing
to fructose.
Pullulanase (pullulan a-1,6-glucanohydrolase) or isoamy- lase (glycogen a-
1,6-glucanohydrolase) cleaves the a-1,6- linked branch points of starch and
produces linear amylosaccharides of varying lengths.
1. Enzymes for starch conversion

35. 1. Production of glucose syrup
2. Production of high fructose corn syrup
3. Production of high maltose conversion syrups
4. Production of cyclodextrins
1. Production of D-glucose from starch by acid hydrolysis (chemical) produces
undesirable bitter sugar (gentiobiose), and the inevitable formation of salt
(from subsequent neutralization with alkali) and coloring materials.
With the discovery and development of
thermostable a-amylase from Bacillus
licheniformis, an enzymatic process has replaced
the acid hydrolysis process.
liquefaction and saccharification
Typically, glucose syrups (DE 97–98) having 96% glucose contain 2–
3% disaccharides (maltose and isomaltose) and 1–2% higher
saccharides.

36. The process of making ethanol from starch involves three
basic steps:
(1) preparation of the glucose feedstock,
(2) fermentation of glucose to ethanol, and
(3) recovery of ethanol.
Production of ethanol
Enzymes have major role in preparation of feedstock:
Corn kernels contain 60–70% starch

37. Milled grain
Gelatinized material
Glucoamylase
Liquefied material
-amylase
cool
Saccharified material
Fermentation
Fructose
Glucose isomerase
Alcohol
Yeast
Bacillus amyloliquefaciens
Industrial production of alcohol and fructose from starch
steam

38. 2. Lignocellulosic Biomass conversions
Various agricultural residues (straws, hulls, stems, cobs, stalks), deciduous
and coniferous woods, municipal solid wastes (paper, cardboard, yard
debris, wood products), waste from the pulp and paper industry, and
energy crops (switchgrass, miscanthus).
These materials are structurally diverse and compositions vary widely
(cellulose, 35–50%;
hemicellulose, 20–35%;
lignin, 10–25%;
proteins, oils, and
ash, 3–15%).
Native lignocellulosic biomass is resistant to enzymatic hydrolysis
hence Pretreatment is required like
steam explosion, dilute acid, concentrated acid, alkali, SO2, alkaline
peroxide, ammonia fiber expansion, and organic solvents.

39. • hemicellulose to simple sugars (xylose, arabinose, and
other sugars) and acids (acetic, glucuronic), which
are water-soluble.
• insoluble residue contains cellulose and lignin.
• The lignin can be extracted with solvents such as
ethanol, butanol, or formic acid.
• Alternatively, enzymatic hydrolysis of cellulose with
lignin present produces glucose, and the residues are
lignin plus any unreacted materials.

40. Lignocellulose: structural support system for all terrestrial plants
Lignocellulosics
Primary
cellulosics
Agricultural
waste
cellulosics
Municipal
Waste
cellulosics
Plant biomass comprises
of Lignin, hemicellulose
and cellulose combined in
different proportions
Plants harvested for
cellulosic content,
structural use or feed
Cotton, timber, hay
Plant material that
remain after
harvesting and
processing Straw,
corn, rice hulls,
sugarcane baggase,
animal manure,
timber residues
Waste paper and
discarded paper
products

41. Lignin
•Complex chemical compound most commonly derived from wood and an integral part
of the cell walls of plants.
•one of the most abundant organic polymers on Earth, superseded only by cellulose,
employing 30% of non-fossil organic carbon and constituting from a quarter to a
third of the dry mass of wood
LIGNIN: 3D, GLOBULAR, IRREGULAR, INSOLUBLE, HIGH MW POLYMER MADE
OF PHENYLPROPANE SUBUNITS
NO CHAINS OF REPEATING UNITS OR BONDS THAT ARE EASILY
HYDROLYSABLE
In plants lignin is bonded to hemicellulose and wraps around fibres composed of
cellulose
Gives rigidity, resistance to mechanical stress and microbial attack
Fungus: Phanerochaete chrysosporium

42. Endo-1,4-b-glucanse: hydrolyzes b-1,4 linkages b/w adj glu mocs
(cellulase, EC 3.2.1.4)
Exo-1,4-b-glucanase: degrades nicked cellulose chains from non reducing
ends and produced glucose, cellobiose (2 glu units) and cellotriose (3 glu
units)
1,4-b-Cellobiohydrolase; type of exoglucanase removes units of 10 or
more glu residues from non reducing ends (found in cellulolytic fungi)
b-glucosidase or cellobiase converts cellobiose and cellotriose to glucose
Enzymes for cellulose hydrolysis
cellobiose
Fungus: Trichoderma reesei
Cellulomonas fimi
Aspergillus

43. Crystalline region Amorphous region
Endoglucanase
cellotriose Exoglucanase
Cellobiohydrolase
cellobiose
Endoglucanase
Enzymatic
biodegradation
of cellulose
Exoglucanase
glu
Removal of oligosacc.
from reducing ends
b-glucosidase

44. Decrease amount of
cellobiose which prevents
end product inhibition of
exo and endo
b-glucosidase
Cloning of gene in host cell
To increase rate and extent of degradation , addition of
Cellulose
Cellobiose
Glucose
Inhibits
Feedback Inhibitor of
cellobiose
Feedback Inhibitor of
cellulose hydrolysis
Inhibits
Fermentation
b-glucosidase enhances enzymatic utilization of cellulose
b-glucosidase not only produces glucose from
cellobiose but also lowers cellobiose inhibition,
allowing the cellulolytic enzymes to function more
efficiently.
However, like b-glucanases, most b-glucosidases
are subject to product (glucose) inhibition.

45. Dietetics enzymes & their applications
Enzyme Application
Amylase Aid for digestion of dietary starch
Proteinase High effective proteinase for acid
environments
Cellulase Aid for digestion of dietary
cellulose
Xylanase Aid for digestion of dietary
hemicellulose
Lactase Aid for digestion of dietary lactose
Lipase, Esterase Aid for digestion of dietary fats &
lipids
Amylase, Lipase, Proteinase General aid for digestion
Amylase, Lipase, Proteinase General aid for digestion
Proteinase Broad spectrum protease for
aiding the digestion of dietary
proteins
Sucrase Digestive aid for breaking down
sucrose

46. Egg-processing enzymes & their applications
Enzyme Application
Catalase Beaks down residual H2O2 into
harmless by-products
Glucose oxidase Prevents browning
Phospholipase (Cakezymes) Improves the emulsification &
gelation properties of yolks
Lipase Breaks down lipid complexes to
ensure egg whites maintain
foaming capacity
Proteinase Improves foaming ability by
modifying the egg white protein

47. 9.Flavoring enzymes & their applications
Enzyme Application
Cellulase, Pectinase,
Beta-Glucosidase
Versatile formulation for extraction from a wide range of plants material
including vanilla
Peptidases Debittering of protein hydrolysates.
Esterase Protease- free lipase for enzyme modified cheese (EMC) production & Cheddar
flavors
Esterase (Proteinase) Enzyme modified cheese (EMC) production & Cheddar flavors
Esterase, Lipase Produces typical Cheedar-type flavor
Esterase Protease- free lipase for enzyme modified cheese (EMC) production
Blue cheese flavors
Endo-Proteinase For use with protease-free lipase in EMC productions
Introduces protein notes
Peptidases High perfomance peptidase used to control the bitterness in EMC cheese
production
Lipase, Esterase Protease- free high activity lipase for hydrolysis of oils,tallow & fats including
butterfat
Meaty flavors from soft anmal fat like chicken
For the production of low level blue flavor notes. Suitable for vegetarian &
kosher markets
Cost-effective mixed enzyme for producing Cheddar-type EMC

48. 10.Fruit & vegetable processing enzymes & their applications
Enzyme Application
Cellulase, Pectinases,
Beta-glucosidases
Versatile formulation for maceration & extraction in a wide
range of fruits & vegetables includig carrots & mangoes
Alpha-amylase Hydrolysis of starches during fruit processing
Ferulic acid esterase Macerating enzyme containing ferulic acid esterase for
improved digestion of plant cell walls
Beta-glucanase To improve mash & fermentation performance
Glucose oxidase For the reoval of glucose from soft drinks
Pectinases High active formulation for general depectinising application In
wide range of fruit juice extraction
Improve extraction rates & flavor inhancement of white wines
Cost-efficient peeling of citrus fruits using an automated
process
Unsurpassed release of color from cranberries
Pectinases, Cellulase Peeling of citrus fruit, especially grapefruit, into individual
segments
Papain For prevention of chill haze in brewing

49. Multiple choice questions
1. Therapeutic enzymes:
(A) Streptokinase (B) Asparaginase
(C) Riboflavinase (D) All of these
2. Which enzyme hydrolyses starch?
(A) Invertase (B) Maltase
(C) Sucrase (D) Diastase
3.Penicillin acylase also called as
(A) Penicillin esterase (B) penicillin amidase
(C) Penicillin kinase (D) penicilinase
4.The enzyme of Aspergillus oryzae is used to resolve D and L Amino
acids from the racemic mixture
(A) Aminoacid Acylase (B) Racemase
(C) Aminoacid isomerase (D) Aminoacid esterase

50. 5.Which thermostable enzyme from Bacillus licheniformis, an enzymatic
process has replaced the acid hydrolysis process.
(A) Sucrase (B) lipase
(C) Amylase (D) none of these
6. Enzyme that removes blood clots in heart disease patients is ______.
a)Urokinase b) Lysozyme c) Glucose oxidase d) catalase
7. Actiase, Retavasem and Rapilysin used in the therapy of
a) Coronary thrombosis b)Pulmonary embolism
c) Cancer chemotherapy d)Acute myocardial infraction
8. enzyme which is used in the treatment of tumors.
(A)Transaminase (B) pepsin
(C) L- Asparginase (D) none of these

51. 9.The enzymes which hydrolyse the neurotransmitter
(A)Alkaline phosphatase (B) choline esterase
(C) L- Asparginase (D) none of these
10. Enzymes used in cake processing
(A)Amylase (B) pepsin
(C) protease (D) phospholipase
11.Enzymes as oxygen scavengers
(A)Catalase (B) SOD
(C) A and B (D)Glucose oxidase
12. Whichenzyme used to maintain the blood pressure
(A)Transaminase (B) Nucleotidase
(C) L- Asparginase (D) ACE