Post on 26-Jun-2015
INDUSTRIAL & ENVIRONMENTAL BIOTECHNOLOGY
Course # KIBGE-707
AFSHEEN AMAN, Ph.D.Assistant Professor
Industrial Biotechnology SectionDr. A.Q. Khan Institute of Biotechnology & Genetic Engineering
(KIBGE)University of Karachi
ENZYME ISOLATION &
ENZYME TECHNOLOGYCourse # 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 ORIGINISOLATION & PURIFICATION
SOURCES OF ENZYMES
PLANT ORIGIN
SeedSeed coatLeafNodulesFruitOther sources
MICROBIAL ORIGIN
BacteriaYeastFungi
ISOLATION/EXTRACTION PLANT SOURCE
Homogenization of plant tissue suspension
- Homogenizer- Waring Blender- Dry Nitrogen Selection of suitable extraction
buffer or water based extract Extraction temperature 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
MICROBIAL SOURCE
Extracellular/Intracellular Centrifugation Homogenization Sonication Osmotic shock Chemicals as lytic agents Bead mill disruption
Continued…..
ASSAY PROCEDURE Selection of suitable assay procedure:
-Spectrophotometeric based (UV/Colorimeteric)-HPLC based -ELISA based
Assay time/Reaction timeSubstrate MaximaSuitable buffer
- pH and ionic strength of bufferTemperature for assay
ENZYME CHARACTERIZATION PARTIAL PURIFICATION USING SPECIFIC
& NON-SPECIFIC PRECIPITATING AGENTSSalt: NH4+> K+> Na+Solvent: Ethanol/Acetone/Methanol/n-Propanol/ i-Propanol/DioxanePolymer: PEGIsoelectric (pI) Precipitation
CONCENTRATING ENZYMEFor concentrating enzymes following techniques will be used:
Ultrafiltration/DiafiltrationDialysisFreeze drying
Continued……
PURIFICATION TO HOMOGENEITYGel Permeation ChromatographySephadex (25-200)Ion Exchange ChromatographyAnionic Matrix: DEAE Sepharose/Q SepharoseCationic Matrix: CM Sepharose/CM Cellulose/
CM Sephadex Affinity ChromatographyHeparin Sepharose 6 Fast Flow/Affi-Gel Protein ARP-HPLCFPLC
Continued….. HOMOGENEITY ANALYSIS
Electrophoresis is used to check and confirm the purity after every step of purification.Native ElectrophoresisSDS PAGEIn-situ Electrophoresis/Zymography
Continued…..
PURE ENZYME KINETICSSubstrate SpecificitySubstrate MaximaTemperature MaximapH MaximaSelection of suitable Buffer
Ionic Strength of bufferThermal StabilityStorage StabilityActivatorsInhibitorsStabilizers
PURE ENZYME STUDIES
Amino Acid AnalysisN-Terminal SequencingC-Terminal Sequencing3-Dimensional Structure StudyImmobilize on various supportsActive Site Studies
0
10
20
30
40
50
60
0 10 20 30 40 50 60 70 80
Enz
yme
Act
ivit
y (D
SU/m
l/hr
)
Time (hr)
Time Course of Glucansucrase Production with and without Calcum Ions in the Culture Medium
With Calcium ions Without Calcium ions
0
10
20
30
40
50
60
0 15 30 45 60 75 90 105 120
Enz
yme
Act
ivit
y (D
SU/m
l/hr)
Time (min)
Effect of Incubation Time on Extracellular Glucansucrase Activity
0
10
20
30
40
50
60
2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5
Enz
yme
Act
ivit
y (D
SU/m
l/hr)
pH
Effect of pH on Extracellular Glucansucrase Activity
0
10
20
30
40
50
60
Citrate Buffer Acetate Buffer Citrate Phosphate Buffer
Succinate Buffer
Enz
yme
Act
ivit
y (D
SU/m
l/hr)
Buffer
Effect of Buffers on Extracellualr Glucansucrase Activity
0
10
20
30
40
50
60
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
En
zym
e A
ctiv
ity
(D
SU
/ml/
hr)
Ionic Strength of Citrate Phosphate Buffer (M)
Effect of Ionic Strength of Buffer on Extracellular Glucansucrase Activity
0
10
20
30
40
50
60
0 10 20 30 40 50
Enz
ym
e A
ctiv
ity
(DSU
/ml/
hr)
Temperature (°C)
Effect of Temperature on Extracellular Glucansucrase Activity
0 100 200 300 400 5000
10
20
30
40
50
60
VMAX KM
61.7569.88
[S], mM
v,
DS
U/m
l/h
r
0
10
20
30
40
50
60
0 20 40 60 80 100
Enz
yme
Act
ivit
y (D
SU/m
l/hr
)
Days
Storage Stability of Extracellular Glucansucrase at Different Temperatures
storage at 4C storage at 30C
storage at -18C
0
20
40
60
80
100
0 10 20 30 40 50 60
% R
ela
tiv
e A
ctiv
ity
Time (min)
Thermal Stability of Extracellular Glucansucrase 30°C
35°C
40°C
45°C
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.
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