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Adsorption Characteristics Of ProteinsOn Lignocellulosic Material By Liquid Chromatography
Leyu Zhang, Eduardo Ximenes, Xingya Liu, Paul Thieme, and Michael Ladisch
Laboratory of Renewable Resources Engineering (LORRE)Department of Agricultural and Biological Engineering
Purdue University38th SBFC, Baltimore, April 25th , 2016
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Acknowledgement-Adviser:
Dr. Michael Ladisch-Committee members:
Dr. Eduardo XimenesDr. Nathan MosierDr. John Morgan
-All LORRE people
-The material in this work was supported by:
Hatch Act Agreements 10677, 10646Department of Agricultural and Biological Engineering
2
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Effect Of Non-catalytic Protein Diluent (BSA)
(Kim et al. 2013)
3
Blocking mechanism of BSA motivated this study
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Inverse Chromatography• Probe characteristics of stationary phase • Relate retention behavior to adsorption
4
to
Det
ecto
r Res
pons
e
twTime
tr
NonretainedSolute
Solute
In this work: Stationary phase = Sugarcane bagasseMobile phase = Citrate bufferProbes = NaCl, BSA, Blue Dextran, Dextran
(*Adapted from http://chemwiki.ucdavis.edu/)
*where ∅ = phase ratio =
∅
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Hypothesis: Linear isotherm (at low concentration)
• Capacity factor k
• Equilibrium coefficient KD
Adsorption Isotherm5
Vm= volume of buffer (mobile phase)Vs = volume of lignocellulosic material (stationary phase)
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Hypothesis: Linear isotherm (at low concentration)
Equilibrium coefficient KD
Adsorption Isotherm6
Strong adsorption
Weak adsorption
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Stationary phase: Sugarcane bagasse (120 mesh)
Mobile phase: Citrate buffer (0.05 M, pH = 4.8)
Materials And Methods7
Temperature: 20 °C or 50 °CFlow rate: 1.0 mL/minColumn volume: 20 mLVoid volume (estimated) : 8 to 10 mL
Sampletested
Concentration(mg/mL)
BSA 20
NaCl 15
Blue dextran 10
Dextran 5
Vanillin 5
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Inverse Chromatography Apparatus8
UV Detector
PC Data Station
Sample
BufferSample Injector
Pump 1
Pump 2
DAQ Board
Column Packed with Sugarcane Bagasse
Waste
RI Detector
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Packing ProcedureMaterial preparation• Suspend 10 g sugarcane bagasse in 1 L buffer• Settle down overnight• Remove fine particles and buffer• Repeat 3 times• Remove buffer to get a total slurry volume of 300
ml
Column Packing• Fill column with buffer • Connect the bulb and fill it with slurry• Turn on pump to pack the column under constant
pressure (~160 psi)• Repeat till column is filled with stationary phase• Settle down for 48 hrs
9
Column
125 mL
PP
Buffer
Pump
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Column Orientation And Equilibrium10
NaCl (14.6 mg/mL)
0.0
0.5
1.0
1.5
0 20 40 60
10 -4
RIU
-FS
Time (min)
Horizontal: tr= 20.7 ± 0.6 min
Vertical: tr= 21.7 ± 1.7 min
Temperature 20 °CFlow Rate 1 mL/minColumn Volume 20 mL
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Column Equilibration 11
NaCl (14.6 mg/mL)
Temperature 20 °C
Flow Rate 1 mL/minColumn Volume 20 mL
700 70 0 70 0 70
Day 1 Day 2 Day 3 & 4tr= 20.7 ± 0.6 min
13 2
123
tr 22.9 29.3 25.9 tr 21.9 24.8 24.9
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BSA Elution Profile12BSA (20 mg/mL)
Temperature 20 °CFlow Rate 1 mL/minColumn Volume 20 mL
tr = 17.4 ± 0.7 min
Abso
rban
ceat
280
nm
Time (min)
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BSA Elution Profile13
0
0.1
0.2
0 20 40 60
Abso
rban
ceat
280
nm
Time (min)
6th 7th
8th 9th
tr = 17.8 ± 1.2 min
BSA (20 mg/mL)Temperature 50 °CFlow Rate 1 mL/minColumn Volume 20 mL
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BSA: 20 °C vs. 50 °C 14BSA (20 mg/mL)
Flow Rate 1 mL/minColumn Volume 20 mL
0
0.1
0.2
0 20 40 60
Abso
rban
ceat
280
nm
Time (min)
50°C
20°C
Sharper peak at 50 °C
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Dextran Elution Profile15
0.0
0.2
0.4
0.6
0 20 40 60
10 -4
RIU
-FS
Time (min)
123
tr= 19.7± 0.5 min
Dextran (5 mg/mL)Temperature 20 °CFlow Rate 1 mL/minColumn Volume 20 mL
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Blue Dextran Elution Profile 16
tr = 16.7 ± 1.9 min
Elution reaches maximumat 4th injection
Blue Dextran (10 mg/mL)Temperature 20 °CFlow Rate 1 mL/minColumn Volume 20 mL
Abso
rban
ceat
280
nm
Time (min)
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Protein Hydrophobicity Accounts For Adsorption Linear correlation exists between the hydrophobic patch score for eachprotein and the total change in adsorbed mass on the lignin surfaces.
(Sammond et al. 2014)
17
β-glucosidase
BSA
Cellulases andhemicellulases
Expect BSA to reduce binding of both cellulaseand β-glucosidase to lignin
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Summary18
• Proof of concept demonstrated
• In terms of tr, difference observed at two temperatures were not obvious.
• Sharper peak (higher number of plates) was obtained at 50°C
• Adsorption of destran, BSA, and blue dextran follows the sequencedextran (20 min) > BSA (18 min) > blue dextran (17 min).
Explanation needed
• Packing procedures need to be improved