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

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

Effect Of Non-catalytic Protein Diluent (BSA)

(Kim et al. 2013)

3

Blocking mechanism of BSA motivated this study

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 =

∅

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)

Hypothesis: Linear isotherm (at low concentration)

Equilibrium coefficient KD

Adsorption Isotherm6

Strong adsorption

Weak adsorption

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

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

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

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

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

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)

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

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

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

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)

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

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