INDUSTRIAL & ENVIRONMENTAL

BIOTECHNOLOGY
Course # KIBGE-707

AFSHEEN AMAN, Ph.D.

Assistant Professor
Industrial Biotechnology Section
Dr. A.Q. Khan Institute of Biotechnology & Genetic Engineering
(KIBGE)
University of Karachi
 ENZYME ISOLATION
&
ENZYME TECHNOLOGY
Course # KIBGE-707
 ENZYME TECHNOLOGY
 The use of purified enzymes for generating a useful
product or service constitutes enzyme technology.
HISTORICAL BACKGROUND
• 1833: first observation of enzyme activity in a test tube
was reported by Payen and Persoz
• 1878: The term 'enzyme' was introduced by Kiihne
• 1890: Fisher suggested the 'lock and key' model of
enzyme action
• 1913: mathematical model of enzyme action was
proposed by Michaelis and Menten
• 1926: Sumner crystallized, for the first time, an enzyme
(urease)
 Continued.....

• 1948: The transition state theory of enzyme action was put

forth by Pauling
• 1951: Pauling and Corey discovered the X-helix and sheet
structures of enzymes
• 1953: Sanger determined the amino acid sequence of a protein
(insulin)
• 1986: Cech discovered catalytic RNA, while Lerner and Schutlz
developed catalytic antibodies
ENZYMES OF PLANT ORIGIN
ISOLATION & PURIFICATION
 SOURCES OF ENZYMES
PLANT ORIGIN MICROBIAL ORIGIN

Seed Bacteria
Seed coat Yeast
Leaf Fungi
Nodules
Fruit
Other sources
 ISOLATION/EXTRACTION
PLANT SOURCE MICROBIAL SOURCE
 Homogenization of plant Extracellular/Intracellular
tissue suspension  Centrifugation
- Homogenizer  Homogenization
- Waring Blender  Sonication
- Dry Nitrogen  Osmotic shock
 Selection of suitable extraction  Chemicals as lytic agents
buffer or water based extract
 Extraction temperature  Bead mill disruption
 pH and ionic strength of buffer
 Use/ not use phenolic scavenger
e.g. PVP and serine protease
inhibitor e.g. PMSF
 Addition of protein stabilizers
 Salt stress
- Various concentrations
- Different time intervals
 Continued…..

ASSAY PROCEDURE
 Selection of suitable assay procedure:
-Spectrophotometeric based (UV/Colorimeteric)
-HPLC based
-ELISA based
Assay time/Reaction time
Substrate Maxima
Suitable buffer
- pH and ionic strength of buffer
Temperature for assay
 ENZYME CHARACTERIZATION
 PARTIAL PURIFICATION USING SPECIFIC &
NON-SPECIFIC PRECIPITATING AGENTS
Salt: NH4+> K+> Na+
Solvent: Ethanol/Acetone/Methanol/n-Propanol/
i-Propanol/Dioxane
Polymer: PEG
Isoelectric (pI) Precipitation
 CONCENTRATING ENZYME
For concentrating enzymes following techniques
will be used:
Ultrafiltration/Diafiltration
Dialysis
Freeze drying
 Continued……

 PURIFICATION TO HOMOGENEITY
Gel Permeation Chromatography
Sephadex (25-200)
Ion Exchange Chromatography
Anionic Matrix: DEAE Sepharose/Q Sepharose
Cationic Matrix: CM Sepharose/CM Cellulose/
CM Sephadex
Affinity Chromatography
Heparin Sepharose 6 Fast Flow/Affi-Gel Protein A
RP-HPLC
FPLC
 Continued…..
 HOMOGENEITY ANALYSIS
Electrophoresis is used to check and confirm
the purity after every step of purification.
Native Electrophoresis
SDS PAGE
In-situ Electrophoresis/Zymography
 Continued…..

 PURE ENZYME KINETICS

Substrate Specificity Ionic Strength of buffer
Substrate Maxima Thermal Stability
Temperature Maxima Storage Stability
pH Maxima Activators
Selection of suitable Inhibitors
Buffer Stabilizers
 PURE ENZYME STUDIES

Amino Acid Analysis

N-Terminal Sequencing
C-Terminal Sequencing
3-Dimensional Structure Study
Immobilize on various supports
Active Site Studies
 Time Course of Glucansucrase Production with and without Calcum Ions in the
Culture Medium

60

50
Enzyme Activity (DSU/ml/hr)

With Calcium ions Without Calcium ions

40

30

20

10

0
0 10 20 30 40 50 60 70 80

Time (hr)
 Effect of Incubation Time on Extracellular Glucansucrase Activity
60

50
Enzyme Activity
(DSU/ml/hr)

40

30

20

10

0
0 15 30 45 60 75 90 105 120

Time (min)
 Effect of pH on Extracellular Glucansucrase Activity

60

50
Enzyme Activity (DSU/ml/hr)

40

30

20

10

0
2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5
pH
 Effect of Buffers on Extracellualr Glucansucrase Activity

60

50
Enzyme Activity
(DSU/ml/hr)

40

30

20

10

0
Citrate Buffer Acetate Buffer Citrate Phosphate Succinate Buffer
Buffer
Buffer
 Effect of Ionic Strength of Buffer on Extracellular Glucansucrase
Activity
60
Enzyme Activity (DSU/ml/hr)

50

40

30

20

10

0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Ionic Strength of Citrate Phosphate Buffer (M)

 Effect of Temperature on Extracellular Glucansucrase Activity

60

50
Enzyme Activity (DSU/ml/hr)

40

30

20

10

0
0 10 20 30 40 50

Temperature (°C)
 60

50
v, DSU/ml/hr

40

30
VMAX 61.75
20
KM 69.88
10

0
0 100 200 300 400 500
[S], mM
 Storage Stability of Extracellular Glucansucrase at Different
Temperatures

60

storage at 4C storage at 30C

50
storage at -18C
Enzyme Activity (DSU/ml/hr)

40

30

20

10

0
0 20 40 60 80 100
Days
 Thermal Stability of Extracellular Glucansucrase 30°C

35°C

40°C
100
45°C
% Rela tiv e Activ ity

80

60

40

20

0
0 10 20 30 40 50 60

Time (min)
SDS-PAGE profile of glucansucrase: Lane A, high molecular weight
standards; Lane B, partially purified glucansucrase; Lane C purified
glucansucrase (Coomassie blue staining); Lane D, assay for soluble
glucan-synthesis corresponding to lane C.
THANK YOU