Determination of Lipase Activity

Using Diffraction-Based Sensor

Charisse D. Alcos
 Introduction
LIPASES – enzymes that catalyze the
hydrolysis of triglyceride

triglyceride + H2O Lipase glycerol + 3 fatty acid

 Sources
* Animal (pancreatic, hepatic and gastric)
* Microbial (bacterial, fungal and yeast)
* Plant or seed
 Applications
* Food
* Pharmaceutical
* Fine chemical
* Oil chemical
* Biodiesel
* Industrial detergent industries
 Different Assays
* spectrophotometric
* fluorometric
* calorimetric
* chemiluminescent
* light scattering
* radiometric
* chromatographic assay
* diffraction-based assay
 Diffraction-based sensing

- is a technique for monitoring interactions between

two or more molecules that uses the changes in the
intensity of the diffraction pattern to detect or monitor
specific binding reactions (Goh, 2004)
 Objectives
General: to use diffraction-based sensor to determine
the rate of activity of lipase
from mustard seedling

Specific:
a) to isolate lipase from mustard seedling
b) to demonstrate one application of diffraction sensor
which is the determination of enzyme kinetics
 Objectives
c) to use another method (diffraction
sensing) to determine the rate of activity of
lipase
d) to interpret the results and data given by
the diffraction-based sensor
 Materials and Methods
seedlings will be
soaked in The mixture is After 20 minutes the
homogenizing osterize for one slurry is filtered
buffer (1:4 w/v) minute and later will through single layer
mixture be homogenize of cheesecloth

The fat layer obtained

The filtrate was
This decantate is after centrifugation is
centrifuge for 20
reffered to as the removed using spatula
minutes at 10 000
crude enzyme. and the centrifugate was
rpm at 0°C
decanted

Extraction of Lipase from Mustard Seedling

 Materials and Methods
Put 2-3 drops in
clean and dry stand Wash w/ dH2O
Crude enzyme
polydimethyl silane
(PDMS) stamp

Introduce Attach to glass

substrate (0live Place in diffraction dry
sensor set-up. slide
oil)

Align the Observe and record

Select a diffraction spot instrument the change in the
intensity of light
every 5 or 10 mins.

Diffraction-Based Assay
 Figure 1. Stamping of crude enzyme.*

Figure 2. Attachment to glass slide.*

Note: * Source: GOH, J. B., R. W. LOO and M. C. GOH .2005. Label-free monitoring of multiple
biomolecular binding interactions in real-time with diffraction-based sensing. Sensors and
Actuators B 106 pp.243–248.
 The experimental set-up

Source: LOO, R.W., P. L. TAM, J. B. GOH and M.
C. GOH. 2005. An enzyme-amplified diffraction-
based immunoassay. Analytical Biochemistry
337 338–342.
Source: GOH, J. B., R. W. LOO and M. C. GOH .
2005. Label-free monitoring of multiple
biomolecular binding interactions in real-time
with diffraction-based sensing. Sensors and
Actuators B 106 pp.243–248.
 Analysis of Data
100

60
Intensity
(% increase) 40

0
0 10 20 30

Time (min)
 References
Alonso, F. O. M., Oliveria, E. B. L., Dellamora-Ortiz, G. M. and Pereira-
Meirelles, F. V., Improvement of Lipase Production at Different Stirring
Speeds and Oxygen Levels. Brazilian Journal of Chemical Engineering, 22,
No. 1, 9 (2005).

Castro, H. F., Oliveira, P. C. and Pereira, E. B., Influence of Substrate Partition

Coefficient on the Performance of Lipase Catalyzed Synthesis of Citronellyl Acetate by
Alcoholysis. Brazilian Journal of Chemical Engineering, 17, No. 4- 7, 1 (2000).

Hasan, F., Shah, A. A. and Hameed, A., Industrial Applications of Microbial

Lipases. Enzyme Microbial and Technology, 39, No. 2, 235 (2006).
 References
Goh, Jane Betty, Richard W. Loo, Richard A. McAloney and M. Cynthia Goh.
Diffraction-based assay for detecting multiple analytes. Anal Bioanal
Chem (2002) 374 :54–56
 
Loo, Richard W., Pui L. Tam, Jane Betty Goh, and M. Cynthia Goh. An
enzyme- amplifed diffraction-based immunoassay. Analytical
Biochemistry 337 (2005) 338–342
 
Goh, Jane Betty, Richard W. Loo, Richard and M. Cynthia Goh. Label-free
monitoring of multiple biomolecular binding interactions in real-time with
diffraction- based sensing. Sensors and Actuators B 106 (2005) 243–248