thermal unfolding kinetics of b-glucosidase of pichia etchelsii

8/2/2019 Thermal Unfolding Kinetics of B-glucosidase of Pichia Etchelsii

1/23

Thermal unfolding kinetics ofb-glucosidase ofPichia etchelsii

BED 851 (MTP Part I)

Supervisor- Prof. Saroj Mishra

Dhananjay Beri

2006BB50006


2/23

Introduction

Pichia pastoris is an efficient system for

production of heterologous proteins.

Grows on minimal medium to very high cell

densities.

Performs post-translational modifications

Promoter is strong and tightly regulated. Fermentation media and techniques for

manipulation have been optimised.


3/23


4/23

Biotechnology Applications

Ethanol production from cellulose. Flavor enhancement of food products.

Biosynthetic reactions to form a number of

glucosides such as alkyl-glucosides


5/23

Pichia etchelsii

P. etchelsiiis a thermo-tolerant yeast, growsoptimally at 40-45 0C.

It produces high levels of cell wall-bound b-

glucosidases. Two b- glucosidases have been identified from this

yeast namely BGL I and BGL II.

BGL I has been shown to be more resistant to

denaturation and loss of activity (Mishra et al.,

2011)

BGL I has been cloned and expressed in P.pastoris.


6/23

BGL I

BGL I belongs to the GH 3 family ofb-

glucosidases.

This family has a two-domain structure, a

(b/a)8-barrel followed by an a/b sandwich.

The active site of GH3 enzymes is situated

between the (b/a)8 and (a/b)6 sandwich

domains, each of which contributes onecatalytic carboxylate residue.


7/23


8/23


9/23

Objectives

Biophysical characterization of the

recombinant Pichia etchelsii b-glucosidase I

(BGL I)cloned in Pichia pastoris.

Thermal unfolding kinetics of BGL I. To evaluate parameters responsible for the

thermo-tolerance phenotype.


10/23

Steps to be undertaken

1. Production of recombinant P. etchelsii b-glucosidase I cloned in P.pastoris using shake flask cultivation.

2. Purification of the recombinant protein from the harvest.

3. Carrying out Circular-dichroism and fluorescence studies on the

recombinant protein as part of its biophysical characterization.

4. Studying the thermal unfolding kinetics using CD spectroscopy.

5. Evaluation of structural properties responsible for the thermo-tolerant

phenotype.

6. Comparison of biophysical properties of native protein and

recombinant protein to study the effect of post-translational

modifications.


11/23

Literature review


12/23

Post-translational modifications

P.pastoris carries out various types of post-translational modifications (PTM) like highereukaryotes.

PTM pattern is organism specific.

Glycosylation- O and N.

In mammals, O-oligosaccharides arecomposed of NAG, Gal and sialic acid.

In P.pastoris only mannose are present.

Glycosylation pattern will be different.


13/23

The outer chain of the N-oligosaccharides ofsecreted proteins in P.pastoris is mostly

unaltered and consists of Man89GlcNAc2. Glycosylation can play an important role in

thermal stability as there is a higher tendency

for deglycosylated enzymes to aggregate. Properly glycosylated enzymes have shown a

higher optimum temperature for activity(Olsen and Thomsen,1991)

Enzymes can be engineered to prevent the ill-effects of PTM.


14/23

Work done so far

Shake flask cultivation Cultivation ofP.pastoris is usually carried out by

following the fed-batch strategy proposed by

Invitrogen. Three phases are used

Glycerol Batch phase

Transition Phase

Methanol Induction Phase


15/23

The medium employed was BufferedMethanol-complex Medium (BMMY) and

BMGY for the inoculum. 1l flasks used for inoculum, incubated at 280C,

2l flasks used for protein expression,incubated at 250C to minimise proteindegradation.

A small quantity of sample is taken every dayfor further analysis.

Samples analysed for protein content(Bradford assay), pH, cell growth (OD600) andb-glucosidase activity (PNPG assay)


16/23

Results

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 20 40 60 80 100 120 140 160

Activity(IU)

Time (hr)

Activity(IU)


17/23

0

20

40

60

80

100

120

140

160

180

200

0 20 40 60 80 100 120 140 160

ProteinConc(mg/l)

Time (hr)

Protein Conc


18/23

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160

OD600

Time (hr)

Cell Growth


19/23

5.5

5.7

5.9

6.1

6.3

6.5

6.7

6.9

7.1

7.3

7.5

24 48 72 96 120 144

pH

Time (hr)

pH


20/23


21/23

The parameters monitored throughout the

cultivation are consistent with the valuesobserved before in the lab.

Consistently high values of activity now being

achieved.

Assisting in standardization of purification

method.

Maximum activity- 1.52 IU

Maximum Cell OD600- 51

Maximum Extracellular Protein Conc.- 163.2 mg/l


22/23

Work to be done next semester

Purification of the BGL I enzyme to homogeneity from theharvest obtained from the shake flask cultivation using thepurification strategy standardized in the lab.

Carrying out Circular-dichroism (CD) and fluorescencespectroscopy on the recombinant protein as part of itsbiophysical characterization.

Studying the thermal unfolding kinetics of the protein usingCD spectroscopy.

Evaluation of structural properties responsible for the

thermo-tolerant phenotype by comparison of the post-translational modification patterns between the P. etchelsiib-glucosidase and the recombinant b-glucosidase (BGL I)expressed in P.pastoris.


23/23

References

Structural stability and unfolding transition of b-glucosidases:

a comparative investigation on isozymes from a thermo-

tolerant yeast- Mohammad Asif Shah, Saroj Mishra, Tapan

Kumar ChaudhuriEur Biophys J

thermal unfolding kinetics of b-glucosidase of pichia etchelsii

Documents