Applications of enzymes

Various industrial applictaions of Enzymes

1. Applications of Enzymes
Dr. KalavatiPrajapati

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

3. IndustrialUses
EnzymesintheProductionofFunctionalOligosaccharidesand
OtherNeutraceuticals
EnzymesintheModificationofFatsandOils
EnzymesintheAnimalFeedIndustry
EnzymesinthePulpandPaperIndustry
EnzymesintheFruitJuiceProcessingIndustry
EnzymesintheMeatandFishProcessingIndustry
EnzymesintheDairyIndustry
EnzymesinDetergents
EnzymesintheLeatherIndustry
EnzymesintheProductionofBulkandFineChemicals
AnalyticalApplicationsofEnzymes
Enzyme-ReplacementTherapy

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
Cholineesterase Paralysis
Alkaline phosphatase Prostrate carcinome
Pancreatic amylase Pancreatitis
Aspartate transaminase Acute hepatitis
Alkaline phosphatase
Gamma glutamyltransferase
Kidney damage
Creatine kinase Skeletal muscle damage
Hydroxy butyrate dehydrogenase
Lactate dehydrogenase
Heart disease
Leucine aminopeptidase Hepatobiliarydisease
Transaminase Chronic hepatitis
Pepsin Gastric mucosal damage
Creatine phosphokinase
Aspartate transaminase
Lactate dehydrogenase
Myocardial infraction
Mitchondrialenzymes Tissue necrosis

10. Enzymes used to diagnose inheritable genetic disorder
Enzyme Inherited disease diagnosed
HGPRT (Hypoxanthine guanine
phosphoribosyltransferase)
Lasch Nayan Syndrome
Purinenucleoside
phosphorylase
Adenosine deaminase
Immunodeficiency disease
Glucocerebrosidase Gaucher’sdisease
Phosphoribosyl pyrophospate
synthetase
Gout
25-OH-cholecalciferol-1-
hydroxylase
Rickets
Hexosaminidase TaySach’sdisease

11. The sensitivityand specificityof enzymes makes them useful
tools in medicine for the detectionand measurementof chemicals
in fluidssuch 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 glucosein biological fluids
2. Enzymes as Analytical Agents

12. The colour of the padon the clinistix is compared with
a colour chartto determine theamount 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 biosensorhas been developed for
detecting
glucosein the blood of diabetics
Glucose oxidase
oxidises any glucose
present in the blood to
release electrons –these
are detected by the
transducerand converted
into anelectrical current
Transducer
Amplifier
The currentgenerated is
proportionalto 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 Analytequantified
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. cerevisiaeASP3 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
Fibrinolyticenzymes
Streptokinase, Urokinase, Tissue
plasminogenactivator
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’sdisease
Galactosidase Inherited β-galactosidasedeficiency
Trypsin, Papain, Collagenase Antiinflammatoryagent
Superoxide dismutase Oxygen toxicity (Antioxidant enzyme)
Bromealin (protease) Inflammation & edema
Chymotrypsin Upper respiratory tract disease
Diastase Amyloceousdyspepsia
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 (angiotensinconverting
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 imprvinddough 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 bromatereplacer
Maltase Suitable for rye bread & low sugar doughs
Pentosanase High activity & low activity protease free pentosanaseused in
bread improvers. Eg., endo-xylanasefor bread improvers
Pentosanase, Amylase Increased bake-out volume in bread & rolls
General bread improver suitable for both continental &
chorleywoodprocess
Baking improver specifically formulated for rye bread
Proteinase,
Pentosanase
Metabisulphitereplacer in biscuits & crackers
Complete spectrum
enzyme
For Enlish-style muffins; this product improves the spread of
the dough piece

28. 1.Starch conversions
Starchcontainsabout15–30%amyloseand70–85%amylopectin.
Enzymeshavelargelyreplacedtheuseofstrongacidandhightemperatureto
breakdownstarchymaterials.
Threetypesofenzymesareinvolvedinstarchbioconversion:
1.endo-amylase(a-amylase,EC3.2.1.1),[Bacilluslichiniformis,Bacillus
subtilis,andBacillusamyloliquefaciensandfungisuchasAspergillus
oryzae]
2.exo-amylases
•glucoamylaseorglucan1,4-a-glucosidase,EC3.2.1.3[Endomycopsis,
Aspergillus,Penicillium,Rhizopus,andMucor];
•b-amylase,EC3.2.1.2)[Bacillusmegaterium,Bacilluscereus,Bacillus
polymyxa,Thermoanaerobacterthermosulfurogenes,andPseudomonas
sp.]
3.debranchingenzymes(pullulanase,EC3.2.1.41;isoamylase,EC
3.2.1.68).[Aerobacteraerogenesandisoamylaseisproducedby
Pseudomonasamyloderamosa.]

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

30. Inamylopectinaboutoneresidueineverytwentyorsoisalsolinked-
(1,6)-formingbranch-points.

31. Enzymatic hydrolysis of amylopectin
cleavesglucoseunitsfromthenonreducing
endofstarchanditcanhydrolyzeboth-1,4
and-1,6linkagesofstarch,slower

32. Dextrins:Agroupoflow-molecular-
weightcarbohydratesproducedbythe
hydrolysisofstarch.
Dextrinsaremixturesoflineara-(1,4)-
linkedD-glucosepolymersstartingwith
ana-(1,6)bond.

33. Amylasesbreak starch into sugars
Allamylasesareglycosidehydrolasesandactonα-1,4-glycosidicbonds.
-Amylases
Boththesalivaryandpancreaticamylasesareα-Amylases.TheyareCa
metalloenzymes,completelyunabletofunctionintheabsenceofcalcium.
Theyactatrandomlocationsalongthestarchchainhencefasterthan
b-amylases
Workingfromthenon-reducingend,β-amylasecatalyzesthehydrolysis
ofthesecondα-1,4glycosidicbond,cleavingofftwoglucoseunits
(maltose)atatime.Duringtheripeningoffruit,β-amylasebreaks
starchintosugar,resultinginthesweetflavorofripefruit.Bothare
presentinseeds;β-amylaseispresentpriortogermination,whereasα-
amylaseandproteasesappearoncegerminationhasbegun.

34. a-amylaserandomlyhydrolysea-1,4linkagesinbothamyloseand
amylopectintoyeildmixtureofglucose,maltose,maltotrioseandseriesofa-
limitdextrins.
b-amylasesometimesusedinplaceofa-amylase.Theyhydrolyzealternatea-
1,4linkagesandyieldmaltoseresiduesandb-limitdextrins
Glucoamylasehydrolysesa-1,3.a-1,4anda-1,6linkagesbutislessefficient
thana-amylase.Majorroleistobreakcrosslinksofamylopectinresultingin
completebreakdowntoglucose.GenerallyusedtoreduceCHOcontentof
beers.IndustriallyobtainedfromfungusAspergillusniger.
Glucoseisomeraseisusedforconversionofglucoseobtainedafterprocessing
tofructose.
Pullulanase(pullulana-1,6-glucanohydrolase)orisoamy-lase(glycogena-
1,6-glucanohydrolase)cleavesthea-1,6-linkedbranchpointsofstarchand
produceslinearamylosaccharidesofvaryinglengths.
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.ProductionofD-glucosefromstarchbyacidhydrolysis(chemical)produces
undesirablebittersugar(gentiobiose),andtheinevitableformationofsalt
(fromsubsequentneutralizationwithalkali)andcoloringmaterials.
Withthediscoveryanddevelopmentof
thermostablea-amylasefrom Bacillus
licheniformis,anenzymaticprocesshasreplaced
theacidhydrolysisprocess.
liquefactionandsaccharification
Typically,glucosesyrups(DE97–98)having96%glucosecontain2–
3%disaccharides(maltoseandisomaltose)and1–2%higher
saccharides.

36. Theprocessofmakingethanolfromstarchinvolvesthree
basicsteps:
(1)preparationoftheglucosefeedstock,
(2)fermentationofglucosetoethanol,and
(3)recoveryofethanol.
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
Variousagriculturalresidues(straws,hulls,stems,cobs,stalks),deciduous
andconiferouswoods,municipalsolidwastes(paper,cardboard,yard
debris,woodproducts),wastefromthepulpandpaperindustry,and
energycrops(switchgrass,miscanthus).
Thesematerialsarestructurallydiverseandcompositionsvarywidely
(cellulose,35–50%;
hemicellulose,20–35%;
lignin,10–25%;
proteins,oils,and
ash,3–15%).
Nativelignocellulosicbiomassisresistanttoenzymatichydrolysis
hencePretreatmentisrequiredlike
steamexplosion,diluteacid,concentratedacid,alkali,SO2,alkaline
peroxide,ammoniafiberexpansion,andorganicsolvents.

39. •hemicellulosetosimplesugars(xylose,arabinose,and
othersugars)andacids(acetic,glucuronic),which
arewater-soluble.
•insolubleresiduecontainscelluloseandlignin.
•Thelignincanbeextractedwithsolventssuchas
ethanol,butanol,orformicacid.
•Alternatively,enzymatichydrolysisofcellulosewith
ligninpresentproducesglucose,andtheresiduesare
ligninplusanyunreactedmaterials.

40. Lignocellulose: structural support system for all terrestrial plants
Lignocellulosics
Primary
cellulosics
Agricultural
waste
cellulosics
Municipal
Waste
cellulosics
Plantbiomasscomprises
ofLignin,hemicellulose
andcellulosecombinedin
differentproportions
Plantsharvestedfor
cellulosic content,
structuraluseorfeed
Cotton,timber,hay
Plantmaterialthat
remain after
harvesting and
processing Straw,
corn,ricehulls,
sugarcane baggase,
animal manure,
timberresidues
Waste paper and
discarded paper
products

41. Lignin
•Complexchemicalcompoundmostcommonlyderivedfromwoodandanintegralpart
ofthecellwallsofplants.
•oneofthemostabundantorganicpolymersonEarth,supersededonlybycellulose,
employing30%ofnon-fossilorganiccarbonandconstitutingfromaquartertoa
thirdofthedrymassofwood
LIGNIN:3D,GLOBULAR,IRREGULAR,INSOLUBLE,HIGHMWPOLYMERMADE
OFPHENYLPROPANE SUBUNITS
NOCHAINS OFREPEATING UNITSORBONDS THATAREEASILY
HYDROLYSABLE
Inplantsligninisbondedtohemicelluloseandwrapsaroundfibrescomposedof
cellulose
Givesrigidity,resistancetomechanicalstressandmicrobialattack
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 cellobiaseconverts 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
Removalofoligosacc.
fromreducingends
b-glucosidase

44. Decrease amount of
cellobiosewhichprevents
endproductinhibitionof
exoandendo
b-glucosidase
Cloning of gene in host cell
To increase rate and extent of degradation , addition of
Cellulose
Cellobiose
Glucose
Inhibits
FeedbackInhibitorof
cellobiose
FeedbackInhibitorof
cellulosehydrolysis
Inhibits
Fermentation
b-glucosidase enhances enzymatic utilization of cellulose
b-glucosidasenotonlyproducesglucosefrom
cellobiosebutalsolowerscellobioseinhibition,
allowingthecellulolyticenzymestofunctionmore
efficiently.
However,likeb-glucanases,mostb-glucosidases
aresubjecttoproduct(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 H
2O
2into
harmless by-products
Glucose oxidase Prevents browning
Phospholipase(Cakezymes) Improves the emulsification &
gelationproperties 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 Debitteringof 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 perfomancepeptidase 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 anmalfat 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 includigcarrots & mangoes
Alpha-amylase Hydrolysis of starches during fruit processing
Ferulic acid esteraseMacerating enzyme containing ferulicacid esterase for
improved digestion of plant cell walls
Beta-glucanase To improve mash & fermentation performance
Glucose oxidase For the reovalof glucose from soft drinks
Pectinases High active formulation for general depectinisingapplication In
wide range of fruit juice extraction
Improve extraction rates & flavor inhancementof white wines
Cost-efficient peeling of citrus fruits using an automated
process
Unsurpassed release of color from cranberries
Pectinases, CellulasePeeling 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 oryzaeis 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 thermostableenzyme 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)Urokinaseb) Lysozymec) Glucose oxidased) catalase
7.Actiase, Retavasemand Rapilysinused 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