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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:
2/1
====
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