Chapter 8

Application of enzymes

1
 The world economy

Contribution to the Global Economy

Bioeconomy
Oil (Modern biotechnology
Bioeconomy economy based)
(Traditional agriculture
based)

1000 2000 3000

Year
2
• Basis for the transition from fossil fuel to bio-based economy?
• Industrial biotechnology (white biotechnology)

3
Industrial (white) biotechnology
• Depend on the use of:
• Microorganisms
• Biocatalysts
• Separately or in combination

• Major application
• Energy
• Chemicals and polymers from biomass

4
 Application of Enzymes
• Bulk
• Large scale use
• Involve use of crude enzymes preparations
• Fine (reagent grade)
• Small quantity needed
• Mostly highly purified enzyme
• Main application areas include
• Research, diagnostics, medical application, etc

5
 Bulk application
• Major areas of application
• Detergent
• Leather tanning
• Starch industry
• Textile industry
• Breweries and alcohol
• Biofuels
• Pulp and paper
• Animal feed industry
• All require large quantities of enzyme

6
Waste degradation
Environment
Detection of toxic
pollutants
Promote cleaner production
(lead to reduction in pollution)

Agriculture Industrial Industry

ENZYMES Leathertanning
Pulp and paper
Agro-processing Starch & fuel
Animal feed additives Chemical &
pharmaceutical
Agro-chemicals
Animal feed from Detergent
agricultural wastes Food & beverages
Textile
Protein hydrolysis
7
Enzyme application in the leather tanning industry

8
 Leather tanning industry
• Enzymes used
• Proteases
• Process
• Soaking
• Dehairing
• Bating
• Waste treatment

9
10
 Table1: Enzymes prepared at AAU and used for bating of sheep skin
Tensile strength & % Average   Tear load Distention and Thickness Porosity & softness
extension tear (N/mm) strength of grain by (mm) (Qualitative
Enzyme load the ball burst test observation)
code (N)
Tensile Elongation Distensio Load at Porosity Softness
strength at breakage n at burst burst (N)
(N/mm2) (%) (mm)

      Commercial enzymes  
 
Enzyme 1 20.0 58.3 21.3   36.9 13.7 333.3 0.59 Excellent Excellent

Enzyme 2 17.4 55.6 21.8   32.5 15.0 329.6 0.67 Excellent Excellent

      New enzyme preparations    

BACC 884 18.1 52.6 30.0   48.0 13.5 330.3 0.62 Excellent Excellent

BACC 41 23.3 62.8 32.7   46.0 14.4 400.6 0.71 Excellent Excellent

BACC 485 26.0 65.8 25.3   42.7 13.7 307.3 0.58 Very good Excellent

11
 Alkaline protease from alkaliphiles
• Several alkaline protease producers
• From soda lakes
(Buffer alone) (Buffer + enzyme)
• Example:
• Vibrio sp R-11 protease
• Interesting application for dehairing

Sheep skin
(Buffer alone) (Buffer + enzyme)

Cow hide
12
 Enzyme production potential benefits
• In enzyme production
• Substrate account for 30 – 40% of the cost
• Here we use hair & mineral salt solution
• Use of the enzyme lead to reduction in pollution
• Na2S
• Lime
• Protein
• Reduction in import
• Na2S
• Other chemicals
13
Other enzymes for detergent application

14
• Cellulase
• Cloth washing
• Lipase
• Assit surfactants
• Amylase
• Dish washing

Amylase from an alkaliphile

15
Detergent

16
 Application of enzymes for detergent
• Enzymes used • Requirement
• Proteases • Stability at high pH
• Lipases • Active at cold temperature
• Cellulases • Active and stable at high
• Source temperature
• Largely bacterial • Other requirement
• Compatibility with chelating
agents
• Oxidation stability

17
 Alkaline proteases from alkaliphiles from Ethiopia

• Two alkaliphilic bacteria isolated from abjata

• Bacillus pseudofirmus
• Nesterenkonia abyssinica
• Produces a calcium independent protease
• Both grow using chicken feather

18
 Applications for detergent and protein hydrolysis

• Detergent industry
• Alkaline proteases
As detergent
• Alkaline amylases addiDve
• Protein hydrolysis
• Microbiological media
• Animal feed
• Food application
• Enzyme production cost
• Considered as key

19
Starch industry

20
The starch industry
• Enzymes
• Amylases (bacterial)
• Pullulanase (deranching enzyme)
• Glucoamylase (fungal)
• Glucose isomerase
• Requirements
• Stability and activity at high temperature
for alpha amylase

21
Starch conversion
• Products from starch
• Glucose
• Fructose
• Maltose
• Brewery and alcohol production
• Supplement malt
• Ethiopian breweries import amylase
• Textile industry

22
Breweries and alcohol
• Main applications
• Supplement malt
• To bring about complete hydrolysis
• Usually fungal amylases
• Use adjuncts
• Substitute malt

23
Pulp and paper

24
 Pulp and paper industry
• Biobleacing using xylanases
• Reduce chlorine usage by up to 50%
• Up to 80 -90% reduction in pollution

Chlorinated organic
compounds
White pulp

25
• Enzymes •  Main drive
• Xylanase (biobleaching) •  Environmental concern
• Cellulose (deinking)
•Potentially useful
• Requirement •  Alkaliphiles
• Stability and activity
• at high pH value •  Thermoalkaliphiles

26
 Dairy industry
• Proteases
• Cheese production
• Lactases
• Whey utilization
• Lactose intolerant
• Protein hydrolysis
• Proteases

27
 Textile industry
• Enzymes
• Amylase
• Cellulase
• Laccase
• Peroxidase
• Benefits
• Environmental
• Cost reduction

28
 Raw fabric

α- Amylase De-sizing De-sizing

Neutral
Pectinase Scouring Stone washing cellulase

Bleaching
Bleaching Laccase
Catalase

Dying
Finished blue jeans
Peroxidase (excess
dye removal)
Acid cellulase Finishing

Finished fabric
29
 Animal feed industry
• Xylanases
• Monogastric animals
• Help nutrient absorption
• Phytases
• Increase phosphorus availability
• Environmental application
• Proteases
• Hydrolysis of insoluble proteins
• Amylases

30
Xylanases and phytases for animal feed application
• As supplement for monogastric animals
• Increase feed assimilation efficiency
• Decrease environmental pollution
• Production
• Solid state fermentation
• Whole moldy bran dried
• and used as feed supplement (?) A fungal xylanase

31
 Biofuels
• Starch hydrolysis
• Amylases
• Pullulanases
• Lignocellululose bioconversion
• Cellulases
• Xylanases
• Lignin degrading enzymes
• Enzymatic biodiesl production
• Lipase catalyzed reaction

32
 Fuel application of ethanol
• More attention due to rise in petroleum price
• Important for nonoil producing countries
• Some countries like brazil started decades ago
• Main source of ethanol
• Sugar cane in Brazil
• Corn in the USA

33
Current controversy around biofuel
• Competition with food
• Direct use of grain
• The corn converted to ethanol to fill in a sports car once is enough to feed a man for a
year
• Use of land to be used
• Suggested solution
• Second generation biofuel
• From the fermentation of lignocellulosic

34
Lignocellulose bioconversion
• Substrate abundant
• Agricultural and forestry waste
• Annual plants
• Problem
• Difficulty associated with hydrolysis
• Require expensive pretreatment
• Potential of lignocellulosic fungi

35
The energy situation in Ethiopia and the potential for biofuel

• Import of petroleum a major cost 2.0

16000

Volume of petroleum imported 14000

1.8
Value of petroleum

Petroleum import (million MT)

Value of petroleum in million Birr)

12000
1.6

• Increasing annually with

10000
1.4
8000
1.2

• Population increase 1.0

6000

4000

• Increased economic activity 0.8

2000
0.6
0

7
2

03

8
0/0

1/0

3/0

5/0

6/0

7/0
2/

,/ 05
200

200

200

200

200

200
200

4
200
Fiscal year

36
Share of petroleum import to export earning of the
country
• Petroleum import share on the rise
• Most years above 50%
• Recently >100%
• The country depend on
• Loan
• Foreign aid
• Substitution will have enormous impact
Fiscal Year

37
 Energy sources in Ethiopia
• Main source
• Biomass (95%) 0.70% 4.30%
Electricity

• Implication Petroleum

• Rapid deforestation Biomass

• Environmental degradation
• Expected to rise with population increase
95.00%

38
Share of diesel, benzene, and kerosene

7.20%

10.80%

23.40% Benzene
Diesel
58.60% Kerosene
Others

39
 Liquid biofuels
• First generation biofuels
• Ethanol from grain or sugar cane
• Biodiesel from edible or nonedible vegetable oil
• Competition with food
• Directly
• Indirectly
• Second generation biodiesel
• Ethanol from lignocellulosic
• Biodiesel from algae
• Biocatalysts play a critical role

40
 Ethanol from lignocellulose
• Lignocellulose
• Abundant and renewable resource
• Agricultural and forestry residue
• Composition
• Cellulose
• Hemicellulose
• Lignin
• Hydrolysis
• Thermochemical
• Biological

41
 3
Ethanol production (x1000 M ) 100

20
40
60
80

0
200
6/0
7

200
7/0
8

200
8/0
9

200
9/1
0

201
0 /11

201
1/1
2

201
2/1
3
Fiscal year
201
3/1
4

201
4/1
5

201
5/1
6
Projected ethanol production in Ethiopia

42
 What should we do?
• Give serious attention to 140

• Biofuel R&D 120

Population size (million)

• Put appropriate policy 100

80

• Encourage investment 60

• Biotechnologies potential
40

20

• Selection of appropriate strains 0

1960 1980 2000 2020 2040

• Development of new technology Year

• Near to create job

43
Screening for lignin degradation

Manganese peroxidase Laccase

44
Immobilized yeast isolate TAYI 4-2

45
Experimental set up immobilized cell reactor

46
Ethanol production by immobilized yeast
Fermentation conditions Batch Immobilized batch Continuous
(immobilized
yeast)

Fermentation time (h) 48 24 12

Initial sugar concentration(g/l) 110 110 110
Residual sugar (g/l) 2 0.1 2
Final ethanol concentration (g/l) 50.08 51.2 52.14
Efficiency 89 90.07 92
Productivity (g/l/h) 1.04 2.12 14.34
       

47
 Enzymology technology
Application of enzymes II
Application of enzymes
48
Application of enzymes
• As processing aids
• Leather
• Textile, etc
• Pharmaceutical and chemical industries
• Synthesis of optically active isomers
• Reaction under mild reaction condition
• Analytical tools (biosensors)
• Medical, industrial, environmental applications

49
Application of enzymes
• Why use enzymes • Not optimized to work as
• Activity catalysts in industrial reactors
• Selectivity, • Some of their properties are not
well suited for that purpose
• Specificity
• Example:
• Increasingly used in the: • They are water soluble
• Chemical industry: • Unstable physiological conditions
• Food chemistry, • Many inhibited by substrates and
products of the reaction and
• Etc.
• Have rather narrow substrate
specificity

50
 Applications of biosensors
• Medical
• Diagnosis (screening and early detection)
• Useful for enhanced health care delivery to community
• Environmental surveillance
• Monitoring, and
• Regulatory
• Food and public safety
• Monitoring (production or regulatory)
• Diagnosis (mycotoxins, antibiotic residue, hormone, etc
• Biological warfare
• Bioprocess monitoring
• Substrate consumption/ product formation
51
Glucose
• Glucose
• Indicator for diabetes
• Endocrine disorders
• Normal range
• 110 ± 25 mg/dl
• Diabetics of 360 mg/dl
• Diabetics on the rise
• 20 mil in USA
• 200 mil world
• 333 million by 2025
• Need careful control
• Preferably from home
52
Transduction of signal?

Glucose oxidase
Glucose + H2O + O2 gluconic acid + H2O2

53
What to measure
• Oxygen consumed
• Original Clark method
• Enzyme separated by membrane
• As glucose enter the oxygen get depleted

54
Hydrogen peroxide
• Advantage
• Can be oxidized on the surface of the electrode
• Drawback
• Need high potential
• A sorbic acid and other biochemical's also oxidize
• Solution
• Membrane separation

55
Transduction of signal?

Glucose oxidase
Glucose + H2O + O2 gluconic acid + H2O2

H2O2 O 2 + 2H+ + 2e−

56
First generation Glucose Biosensor

57
Limitation
• Not suitable for home use
• Useful only in the lab
• Ideal situation?
• Direct transfer of electrons from enzyme to electrode
• Why not?
• Glucose oxidase a flavoprotein
• Has strongly bound FAD
• Electrons from glucose pass over to the FAD
• Then from FAD to oxygen to form hydrogen peroxide
58
The glucose oxidase reaction
Glucose GOXox
H2O2

Gluconic acid GOXred O2

59
Glucose oxidase homo dimer

60
Glucose oxidase monomer with deeply embedded FAD

61
Limitation of the system
•Barrier for direct electron transfer
• The flavin is deep inside
• No direct contact
• But important to avoid oxygen limitation

62
Second generation glucose sensors
• Use of mediators
• Get oxidized by the enzyme
• Avoid variation due to fluctuation in oxygen tension
• Function of the mediator
• Get oxidized
• Relay electrons to electrode surface

63
The glucose oxidase reaction
Glucose GOXox E
Mediatorred L
E
C
T
R
O
GOXred Mediatorox D
Gluconic acid
E

64
Second generation glucose sensors
• Mediator
• Diffuse to the interior of the enzyme
• Shuttle electrons between the FAD at the center and the surface of the electrode
• No need for hydrogen peroxide formation
• Current produced proportional to glucose conc.

• Common diffusional mediators

• Ferrocene derivatives
• Ferricyanide,
• Conducting organic salts (eg. tetrathiafulvalene tetracyanoquinodimethane, TTF-TCNQ),
• Phenothiazine
• Phenoxazine compounds, or
• Quinone compounds
65
Second generation glucose sensors
• Use of mediators
• Oxygen partial pressure has no effect on measurement
• Oxidation at lower potential – no interference
• Commercial blood glucose self-testing sensors
• Ferricyanide or
• Ferrocene mediators

66
Required properties for a mediator
• React rapidly with the reduced enzyme
• Minimize competition from oxygen
• Possess good electrochemical properties
• Low redox potential)
• Low solubility in aqueous medium
• Restrict lose by diffusion
• Must be chemically non-toxic and
• No inhibition
• Must be stable
• both reduced and oxidized forms
67
Commercial Glucose Sensor

68
Lactate
• Indicator of
• Respiratory insufficiency
• Shock
• Heart failure, and
• Metabolic disorders

• Lactic acidosis accompanied

• Decreased tissue oxygenation
• Left ventricular failure, and
• Drug toxicity
69
 L-Lactate + O2 Lactate oxidase
Pyruvate + H2O2

Lactate dehydrogenase Pyruvate + NADH + H+

L-Lactate + NAD+

70
Lactate
• Most amperometric biosensors of lactate
• Immobilized lactate dehydrogenase (LDH) or
• Lactate oxidase
• Procedure
• Same general principle for glucose

71
Cholesterol Biosensor
• High levels of cholesterol is associated with:
• Heart disease
• Hypertension
• Coronary artery disease
• Arteriosclerosis cerebral thrombosis
• A major public concern
• Simple and reliable measurement required

72
• Cholesterol oxidase most commonly used

Cholesterol oxidase
• Cholesterol + O2 Cholest -4 -en -3 -one + H2O2

73
Other measurements
• Urea
• Indicator of kidney diseases and disorders
• Creatine and creatinine
• Cancer markers
• Immunosensors
• Nucleic acids

74
Applications of biosensors
• Medical
• Diagnosis (screening and early detection)
• Useful for enhanced health care delivery to community
• Environmental surveillance
• Monitoring, and
• Regulatory
• Food and public safety
• Monitoring (production or regulatory)
• Diagnosis (mycotoxins, antibiotic residue, hormone, etc
• Biological warfare
• Bioprocess monitoring
• Substrate consumption/ product formation
75
Environmental application
• The last few decades
• Thousands of chemicals synthesized
• Different classes of compounds
• Synthetic organic compounds
• Natural compounds (eg. hormones), drugs, etc)
• Heavy metals (Hg, Cr, etc)
• Serious environmental impact
• Human health
• Environmental deterioration
• Legislation put in place
• Monitoring is a challenge
• Fat and cheap analytical required
76
Biosensors for pesticides
• In agriculture chemicals (pesticides) extensively used to control
• Insects, Fungi, Bacteria
• Weeds, Nematodes, Rodents, etc
• Large quantity of residue accumulate in the environment
• Enter the food chain
• Cause serious damage
• Some are carcinogenic or citogenic
• A need to control and also to clean up

77
• In the USA & Europe legislation put in place
• Enforcement require reliable monitoring of the environment for these compounds
• Maximum allowable limit is set
• Recent trend
• Organochlorine insecticides (e.g. DDT, aldrin and lindane) phased out
• Replaced by organophosphorus (e.g. parathion and malathion) and derivatives of
carbamic acid (e.g. carbaryl and aldicarb)
• Low persistence in the environment
• but pose serious risk due to their high acute toxicity

78
• Commonly used analytical methods
• HPLC
• GC
• GC-MS
• ELISA
• Advantage
• Sensitive
• Reliable
• Precise
•

79
Disadvantage
• Expensive instrumentation
• Require skilled technicians
• Time consuming
• Laborious and
• Not easily adoptable for fi eld analysis
• Alternative
• Use biosensors

• How?

80
• Most based on inhibition of enzymes like;
• Acetylcholinesterase and butyrylcholinesterase
• Method is sensitive
• Main drawback
• Oxidation of the enzymatic product, thiocholine, need a high potential
• Reduce stability of the biosensor
• Other enzymes occasionally used
• Acetolactate synthase
• Acid phosphatase
• Alkaline phosphatase
• Tyrosinase
• Ascorbate oxidase
• Luciferase
• Alternative enzyme
• Organophosphorus hydrolase (OPH)
81
Organophosphorus hydrolase
• Reaction
• Followed spectrophotometrically or
• electrochemically

82
Organophosphorus hydrolase
• Reaction
• Spectrophotometrically or
• Electrochemically
• Direct determination

83
Inhibition-based biosensors
• Organophosphate and carbamate pesticides
• Potent inhibitors of cholinesterase
• Acetylate serine at the active centre
• Commonly used substrate for biosensors
• Acetylthiocholine
• Monitoring of thiocholine produced
• Spectrometric
• Amperometric
• Potentiometric

84
85
Acetycholine + H2O AChE
Choline + acetic acid

Choline + O2 + H2O Choline oxidase

Betaine + H2O2

86
• Advantages
• Sensitive
• Disadvantages
• Poor selectivity
• Slow and tedious
• Analysis involve multiple steps of reaction
• Measuring initial enzyme activity
• Incubation with inhibitor
• Measurement of residual activity
• Regeneration and washing

87
Catalysis-based biosensors
• Organophosphorus hydrolase hydrolyze
• Organophosphorus pesticides
• Insecticides
• Chemical warfare agents (e.g. sarin)

• End product analyzed electrochemically

88
Other methods
• Immunosensors
• Antibodies used
• Detection by labling
• Whole cell biosensor

89
Immunosensors
• Basis
• Antibodies used
• Detection eg. similar to ELISA
• Antibodies have high
• Selectivity
• Analytical sensitivity that
surpass enzymes
• Used for array biosensors

90
Whole cell biosensor
• Hexavalent chromium (Gurung et al 2012)
• Based on sulfur-oxidizing bacteria
• oxidize S0 to H2SO4
• Increased electrical conductivity & low pH
• After 5 h feeding semi continuous 1 min rapid feeding and 29 min batch reaction
• Cr6+ toxicity lead decrease in conductivity and increase in pH (from 2-3 to 6)
• Detection with in minutes to hours

91
Whole cell biosensor

Gurung et al (2012)

92
Application of Biosensors in food
• Composition • Toxins
• Glucose • Mycotoxins
• Lactose • Maltreatment and deterioration
• Sucrose
• Laculose
• Pathogenic microorganisms
• E.coli
• Listeria monocytogenes
• Salmonella
• S. aureus

93
 fructose-­‐5-­‐dehydrogenase
D-Fructose + mediator(ox) 5-keto—fructose + mediator(red)

Lactulose + H2O -galactosidase Β-­‐galactosidase

galactose + fructose
 Formed in alkaline lactose Solutions  
 By heating of milk  
 Used as an indicator  
 Severity of heat treatment of milk  
 Distinguish between pasteurized, ultra-heat treated and
 sterilized milk

94
Multiple-sensing: array biosensors
• Existing biosensors detect
• Single or few analytes
• The trend
• Multiple-sensing
• Example: antibody arrays

95
Application of enzymes in the chemical industry

96
The role of enzymes in the chemical industry
• Generation of fermentable sugars
• Hydrolysis of starch, cellulose, hemicellulose, etc
• Starting materials for fermentation

• Generation of platform chemicals

• Ethanol
• Succinate
• Acetic acid
• Propionates, etc
97
Chemicals and polymers from fermentation products
• Organic chemicals and polymers
• Derived from fossil based feed stocks
• Account for around 9% of all fossil fuel (15% if only oil)
• But nearly 50% of the profit
• Global petroleum based chemicals and polymers
• ~330 million tons
• Demand expected to rise
• Challenges
• Fluctuation in oil price
• Unsustainability of fossil fuel
• Environmental consideration
• For some countries it is a security issue
• Example: acetone during WWI

98
Chemicals and polymers
The chemical industry based • Ethanol ethylene
on few building blocks
• Methanol
• Ethylene Well established technology
• Propylene
• Butadiene
• Benzene
• Toluene and
• Xylene

Converted to
• Polymers and plastics
• A huge array of of different fine and
specialty chemicals

Can be produced from

biomass
•  Role of fermentation

99
Some bio-based chemicals & counterparts from petroleum

Bio-based chemical Reference petrochemicals

Ethyl lactate Ethyl acetate
Ethylene Ethylene
Acetic acid Acetic acid
n-Butanol n-Butanol
Polyhydroxyalkanoate HDPE
(PHA)
Polylac2c acid( PLA) PET and PS
Succinic acid Maleic anhydride

100
Market value of selected fermentation derived chemicals (2013)

Chemical Value in US$ (2013)

Amino acids 7,821
Enzymes 4,900
Organic Acids (Lactic acid 20%) 4,036
Vitamins and related 2,286
compounds
Antibiotics 2,600
Xanthan 708
Total 22,351
101
Other applications in the chemical industry
• Synthesis in nonconventional media
• Lipases
• Proteases
• Oxidoreductses
• Resolution of optical isomers
• Pharmaceutical intermediates
• Intermediates for the synthesis of agrochemicals

102
Application of enzymes in the food industry
• For processing
• Starch processing
• Production of invert sugar
• To improve quality
• Meat tenderization
• Beer chill proofing
• Analysis
• Biosensors

103
Environmental application
• Reduction of pollution
• Example: Pulp and paper industry
• Assist degradation
• Biological polymers (proteins, lipid, etc)
• Monitoring the level of pollution
• Biosensors

104
Synthetic application (fine chemicals)
• Lipases
• Most popular
• Under organic solvents
• Esterases
• Same as lipase
• Proteases
• Same as lipase
• Aspartame production

105
Synthetic application of enzymes
• Some reactions (eg. steroids transformation)
• Reactants not soluble in water
• Soluble in organic solvents than in water
• Products uunstable in aqueous solvents
• Better
• If the reaction take place in organic solvents
• Advantages of reactions in organic solvents
• Solubility
• Absence of microbial contamination
• Increased product stability
• Keq of a reaction change
• Hydrolytic reactions
• Carry out synthetic reactions
• Water acceptor in hydrolytic reactions
• Always in molar excess and no reversal
• At low water activity other acceptor are used

106
Enzyme stability in organic solvents
• Enzymes
• Some are hydrophilic
• Some are hydrophobic ( those associated with lipids and membranes)
• Hydrophilic enzymes
• Require the presence of a thin layer of water (50 and 500 molecules of water
per enzyme molecule)
• Help to stabilize the conformation
• If they have this water enzymes
• Enzymes operate under anhydrous condition

107
• Effect of pH on enzyme activity
• Enzyme reaction pH dependent
• pH a function of [H+]
• Determine catalytic activity
• How about in organic solvents?
• Enzymes ‘remember’ the last pH

108
• Removal of enzyme-bound water
• Affect stability
• Occur in water-soluble, or miscible, organic solvents
• Lead to enzyme inactivation
• In the presence of the water shell
• Low water activity
• Reduction in thermo-inactivation
• Half life of porcine pancreas lipase at 100°C
• >12 hours in 0.02% water in tributyrin
• 12 minutes at a 0.8% water content
• Instant inactivation in 100% water
109
• Solvent polarity determine stability
• Solvents with low polarity can not disrupt the water shell
• Polarity
• measured by logarithm of the partition coefficient
• (LogP)
• partition coefficient of the solvent between n-octanol and water

110
LogP of different solvents
Butanone 0.3
Ethyl acetate 0.7
Diethyl ether 0.8
Benzene 2.0
Toluene 2.7
Hexane 3.5
Heptane 4.0
Hexadecane 8.7
111
Enzyme stability in organic solvents
• Stability
• Correlated with LogP
• Solvents with logP < 2 denature
enzymes
• Enzymes are stable in solvents
with LogP >

112
Fine application

113
Enzymes in research and diagnostics
• Research and medical/diagnostic
• DNA polymerases
• Restriction enzymes
• Peroxidases
• Diagnosis
• Glucose oxidase
• Others
• Others
• Pectinases for fruit juice clarification
• Xylanases for animal feed (fungal origin)

114
Medical application of enzymes
• Therapeutic
• As supplements
• Pure therapeutic (eg arginase for cancer patients
• Diagnosis
• Monitoring blood glucose level
• ELISA
• Western blotting
• Biosensors

115