thermos epa rating atps for cutinase extraction

8/7/2019 Thermos Epa Rating ATPS for Cutinase Extraction

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Thermoseparatingaqueous two phasesystem for extraction ofcutinase


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Outline

Introduction

Objectives

Methodology

Results & discussions

Conclusions

Future works


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Introduction

ATPS liquid liquid extraction system.

form when t wo imm iscible polymer solutions @ apolym er solution and a high concentrat ion salt

solution are m ixed together.

Advantages:1. Mild environment

2. Biocompatible

3. Easy to scale up

4. Can be operated at high capacity

5. Capable to integrate clarification, concentration & intermediate purification steps


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continued

key principle solubility of solutes differs significantlybetween the liquids, providing a thermodynamic drivingforce for transfer from one phase to another phase.

Distribution of solute between the two phases describes bypartition coefficient, K

phasebottominionconcentrat

phasetopinionconcentratK=


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Protein partitioning inATPS

Part itioning depends mainly on physicochem icalpropert ies of solute (e.g. size, charge,hydrophobicity).

AND its also affected by polymer type, polymerconcentration, polymer MW, pH & additional salt.

The contribution of these factors to the observed partition coefficient, K can be

expressed in logarithmic term as follows;Ln K = ln K + ln Kelec + ln Khydrophb + ln

Kbiosp +

ln Ksize + ln Kconf +


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Prediction of proteinpartitioning behaviour in

ATPS Theoretically, the protein partition coefficient in ATPS

can be expressed by utilizing thermodynamicmodel.

Thermodynamics model applied for understandingthe general dependence of phase behaviourandprotein partitioning on system variables underdirect experimental control (Habeychaet al.

2009, Tubio et al. 2009, Liaet al. 2010,Filho et al. 2004)


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Prediction of proteinpartitioning using Flory

Huggins model According to Johansson et al. (1993), relative

magnitudes of enthalpic & entropic contributions toprotein partitioning is determined according to Eqsbelow:

( )[ ]

+=

b

b

t

tsbbstts

ss

Vn

Vn

pM)E(w)Ew

RTMlnK

Ks = partit ion coefficient of soluteMs = degree of polymerization of soluteR =T = experiment temperaturent/pVt = no of molecules/ volume unitwts = average interaction between the solute and top phaseEt = top phase self energy


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Effect of additional salt topartitioning behaviour

As simplify by Johansson et al. (1993), for a two phasesystem containing a single electrolyte (salt), thepartitioning can be predicted according to belowequation :

( )[ ]bCtCbAAtionsss w(w)wwRTM)zz z(lnKlnK

=

+

Ln Ks = part i t ion coeff icient by enthalpy and entropy

contribution only

Zs = net charge of soluteZ+ = no of charge for cationZ- = no of charge for cationw tA = direct interaction energy of anion for the top phasesubscript C and b is refer to cation and bottom phase


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Objectives

1. To develop thermoseparating ATPS by usingsuitable recycled copolymer & starch derivative.

2.

3. To optimize the recovery of cut_WP4 by

manipulating parameters of the developedsystem.

4.

5. To study the main driving force for proteinpartitioning and the correlation between

system variables and protein properties.


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Methodology: Construction

of phase diagram

Separate top & bottom phase for analysis6


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Methodology: ATPS

Preparation

Separate top & bottom phase for analysis7


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Results & discussions


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Partitioning of cut_WP4 at

different type of systems


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Estimation of Flory binaryparameters

The estimation of binary parameters was obtainedby correlating the experimental equilibrium data(Tada et al. 2004).

The optimal value of the binary parameters wereobtained through minimizing the followingobjective function:

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====

4r

)(

O.F.

2

1i

2k,expp,

i

k,calcp,

i

2

1p

r

1kww


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Partitioning of cut_WP4 at

different phase compositions


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Influence of additional salt to

cut_WP4 partitioning


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Conclusions

De/dx is suitable for cutinase recovery.

F-H model can be used to estimate the driving forces for phaseseparation and protein partitioning behavior by calculating themagnitude of enthalpy and entropy of the system.

The more negative the direct interaction term, the greater the

tendency for protein to partition to that phase.


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Thank you

thermos epa rating atps for cutinase extraction

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