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Purification of Lipase
NONG Yuan Supervisor: Prof. Jan-Christer Janson
Department of Surface Biotechnology Uppsala Biomedical Center
Uppsala University2005.12
from Bacillus subtilis FS2
Overview
Research Training in B7:3
Department of Surface Biotechnology ;
Period: 1-Nov to 23-Nov;
Mainly work: Purification ; Partially attend: Fermentation.
Introduction
Why interesting?
Glycerol ester hydrolases, catalyze hydrolysis of triacylglycerols free fatty acids + glycerol ; High biotechnological potential; Widely application in bioindustry.
Purpose of this project
Research on Lipase from Bacillus Subtilis FS2;
First phase: purify lipase!
Purification of Lipase from Bacillus subtilis FS2
B. Subtilis FS2 -- lipase producing bacteria
novel strain, isolated from traditional fish source in Vietnam; 97% homology with that of B. subtilis 168, which has become an attractive enzyme for industry;
Remarkable properties of Lipase
Molecular Weight: 19 kDa;pI = 9.9 High activity under alkaline conditions
Fermentation of Bacillus subtilis FS2 (from Vietnam)
Ammonium Sulphate 60% (w. v) Precipitation
Cation Echange Chromatography
Material and Method
Collection of supernatant contained extra-cellular lipase
Desalting
Purification
ÄKTAdesign, UNICORN.
6
Desalting proteins
2Elution volume (ml)
0 4 6 8 10 12
Desalted samplesalt
Column:
Sample:
Buffer:
Hi_Trap Desalting
B.Subtilis FS2
20mM Sodium phosphatepH7.5
• Desalting figure
8
What is expected in cation exchange?
Sampleapplicationand wash
ElutionEquilibration Cleaning/Regeneration
-
-- -
++
+ ++
++ - -
++
+ ++
+ +
-+ ++
++
+
-
+ ++
-
++
+ +
+- ++
+ ++ ++
-- --+
+ --
++
+
+
++
-
-
-
---
-
-
--- -
-
-
---- -
-
-
-- - -
-
-
-- -
Cation exchanger bead
++
++++
+ +
+++
Column: HiTrap SPFF 1ml, Sample: B. subtilis FS2, ÄKTAdesign, UNICORN. Start buffer: 20mM Sodium Phosphate pH 7.5. Elution buffer: 20mM Sodium Phosphate pH 7.5 with 1M NaCl.
Result
Stepwise gradientversusLinear gradient
Stepwise elution would be used afterwards
Results and Discussions
!!!Bottom level shift
Linear elution profiles
Theoretical
VS
Experimental
Results and Discussions
!!! unstable protein binding capacities
Comparison among linear gradient elution profiles
Conclusion
No exactly expected outcome;
Experience accumulation to go further; System control Technology master Project design
Future work
Enzyme activity assay• Suitable method for lipase activity test;Purification work• Gel Fltration (Homology protein before
application to ion exchanger column);• Hydrophobic Interaction Chromatography
(recommended by previous researcher);Consider back to materials• Optimization of lipase producing----good
sample .
Acknowledges Special thanks to Prof. Janson and Ms Nguyet
For the happy memory of 2005 Fall
Thank YOU!