Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface...
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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
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Overview
Research Training in B7:3
Department of Surface Biotechnology ;
Period: 1-Nov to 23-Nov;
Mainly work: Purification ; Partially attend: Fermentation.
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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
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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
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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.
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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
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• Desalting figure
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What is expected in cation exchange?
Sampleapplicationand wash
ElutionEquilibration Cleaning/Regeneration
-
-- -
++
+ ++
++ - -
++
+ ++
+ +
-+ ++
++
+
-
+ ++
-
++
+ +
+- ++
+ ++ ++
-- --+
+ --
++
+
+
++
-
-
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---
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--- -
-
-
---- -
-
-
-- - -
-
-
-- -
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.
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Result
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Stepwise gradientversusLinear gradient
Stepwise elution would be used afterwards
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Results and Discussions
!!!Bottom level shift
Linear elution profiles
Theoretical
VS
Experimental
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Results and Discussions
!!! unstable protein binding capacities
Comparison among linear gradient elution profiles
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Conclusion
No exactly expected outcome;
Experience accumulation to go further; System control Technology master Project design
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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 .
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Acknowledges Special thanks to Prof. Janson and Ms Nguyet
For the happy memory of 2005 Fall
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Thank YOU!