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

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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

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50

60

VMAX KM

61.7569.88

[S], mM

v,

DS

U/m

l/h

r

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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

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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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