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Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University 2005.12 from Bacillus subtilis FS2
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Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Jan 20, 2016

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Page 1: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Purification of Lipase

NONG Yuan Supervisor: Prof. Jan-Christer Janson

Department of Surface Biotechnology Uppsala Biomedical Center

Uppsala University2005.12

from Bacillus subtilis FS2

Page 2: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Overview

Research Training in B7:3

Department of Surface Biotechnology ;

Period: 1-Nov to 23-Nov;

Mainly work: Purification ; Partially attend: Fermentation.

Page 3: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

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

Page 4: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

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

Page 5: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

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.

Page 6: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

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

Page 7: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

• Desalting figure

Page 8: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

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.

Page 9: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Result

Page 10: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.
Page 11: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Stepwise gradientversusLinear gradient

Stepwise elution would be used afterwards

Page 12: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Results and Discussions

!!!Bottom level shift

Linear elution profiles

Theoretical

VS

Experimental

Page 13: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Results and Discussions

!!! unstable protein binding capacities

Comparison among linear gradient elution profiles

Page 14: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Conclusion

No exactly expected outcome;

Experience accumulation to go further; System control Technology master Project design

Page 15: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

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 .

Page 16: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Acknowledges Special thanks to Prof. Janson and Ms Nguyet

For the happy memory of 2005 Fall

Page 17: Purification of Lipase NONG Yuan Supervisor: Prof. Jan-Christer Janson Department of Surface Biotechnology Uppsala Biomedical Center Uppsala University.

Thank YOU!