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!