Nanoscale Biopolymers Nanoscale Biopolymers with Customizable Propewith Customizable Properties for Heavy Metal Rerties for Heavy Metal Re

mediationmediationU Loi (Ann) Lao, Giri Prabhukumar,U Loi (Ann) Lao, Giri Prabhukumar, JaJan Kostal, Mark Matsumoto, Ashok Muln Kostal, Mark Matsumoto, Ashok Mul

chandani, and Wilfred Chenchandani, and Wilfred Chen

Chemical and Environmental EngineeringChemical and Environmental EngineeringUniversity of California, RiversideUniversity of California, Riverside

Heavy Metal Heavy Metal ContaminationContamination

2.4 million tons of metal wastes 2.4 million tons of metal wastes per year from industrial sources per year from industrial sources

2 million tons per year from 2 million tons per year from agriculture and domestic wasteagriculture and domestic waste

PbPb2+2+, Hg, Hg2+2+ and Cd and Cd2+2+ are ranked are ranked 2nd, 3rd and 7th, respectively, 2nd, 3rd and 7th, respectively, on the EPA’s priority list on the EPA’s priority list

Metal Chelating Metal Chelating PolymersPolymers

• Requires toxic solvents for synthesis

• Ultrafiltration is required

• Membrane clogging

Solution: Develop metal-binding materials that can be recovered by environmental stimuli

Metal Chelating Metal Chelating BiopolymersBiopolymers

Based on biological building blocksBased on biological building blocksNanoscale biopolymers that are specially Nanoscale biopolymers that are specially

pre-programmed within a DNA templatepre-programmed within a DNA templateEconomically produced by bacteria Economically produced by bacteria Environmentally friendlyEnvironmentally friendlyTunable properties based on changes Tunable properties based on changes in environmental conditions – pH or in environmental conditions – pH or temperaturetemperature

Elastin BiopolymerElastin Biopolymer Structurally similar to the repeating Structurally similar to the repeating

elastometric peptide sequence of the elastometric peptide sequence of the mammalian protein, elastin mammalian protein, elastin

VPGVG are the most frequently repeating unitsVPGVG are the most frequently repeating units Undergo a reversible phase transition from Undergo a reversible phase transition from

water soluble forms into aggregates as the water soluble forms into aggregates as the temperature increasestemperature increases

Low temperature: -turn

High temperature: twisted filament of -spirals

Genetic and Protein Genetic and Protein Engineering Engineering MethodologyMethodology

Synthetic gene

Recombinantplasmid

Plasmid

Enzyme

ELP biopolymers

Customizable Metal Binding BiCustomizable Metal Binding Biopolymersopolymers

Metal Binding Domain

Fine tune affinity with different binding sequences

Elastin Domain

Fine tune the transition temperature by controlling amino acid sequence and

number of repeating unit (VPGXG)n

Heavy Metal Removal by Heavy Metal Removal by Tunable BiopolymerTunable Biopolymer

+ CdT

Regeneration

soluble protein insoluble protein

Cd2+

Cd2+

Cd2+

Cd2+Cd2+

Cd2+

Elastin Biopolymers with MElastin Biopolymers with Metal-Binding Affinityetal-Binding Affinity

A B C D E

Biopolymer

A. ELP38H6B. ELP58H6C. ELP78H6D. ELP78E. ELP78H12

Kostal et al. Macromolecules, 34, 2257-2261, 2001.

Phase TransitionPhase Transition

Transition temperatures can be fine tuned from 20 to 40oC by controlling the chain length and salt concentration

Low TempHigh Temp

Cadmium RemovalCadmium Removal

Biopolymer

Cd2+ binding ( mol protein : mol Cd2+SD)

Ela38H6 1 : 1.04 0.04

Ela58H6 1 : 0.97 0.01

Ela78H6 1 : 0.95 0.01

Ela78 1 : 0.01 0.00

Ela78H12 1 : 1.50 0.02

SD - standard deviation

50 nmol of biopolymer were incubated with 100 nmol Cd2+

Repeated Cycles of Repeated Cycles of Metal BindingMetal Binding

0.00

1.00

2.00

3.00

4.00

5.00

6.00

S P S P S P S P AS

Ca

dm

ium

(mg

)

Cycle 1 Cycle 2 Cycle 3 Cycle 4

5 5 mmg added each cycleg added each cycle

Practical Applications: Practical Applications: Soil Flushing/WashingSoil Flushing/Washing

Environmental friendly extractantEnvironmental friendly extractant Simple separation and recyclingSimple separation and recycling

In-situ Soil Flushing

Ex-situ Soil Washing

Soil-Biopolymer Soil-Biopolymer Adsorption Adsorption

Characteristics Characteristics Soil-Biopolymer Sorption CharacteristicsSoil-Biopolymer Sorption Characteristics Freundlich isotherm Freundlich isotherm

Log x/m = Log KLog x/m = Log Kff + 1/n Log Ceq + 1/n Log Ceq

-0.1

00.1

0.20.30.40.5

0.60.70.8

-0.2 0 0.2 0.4 0.6 0.8

Log (Ceq)

Lo

g (

x/m

)

Maximum 19% adsorption

Biosurfactant – typically in the 70% range

PrabhukumarPrabhukumar et al. ES&T, 38, 3148-3152, 2004.

Biopolymer-Cadmium Biopolymer-Cadmium Extraction StudiesExtraction Studies

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8

% C

ad

miu

m r

em

ov

ed

1. Single Batch washing with distilled water 2. Two sequential batch washings with distilled water3. Single batch washing with 1.25 mg/ml (0.036 mM) ELP without the histidine tag

4. Single batch washing with 0.036 mM EDTA 5. Single batch washing with 1.25 mg/ml ELPH12 6. Single batch washing with 2.5 mg/ml ELPH12 7. Single batch washing with 5 mg/ml ELPH128. Two sequential batch washings with 1.25 mg/ml ELPH12

Less than 10% adsorption

•His6 or His12 tag can serve as a simple metal binding domain

•Both Tt and metal binding capacity can be easily regulated

•Biopolymers can be recycled

•Lack specificity and affinity

First generation biopolymers

Customizable Metal Binding BiCustomizable Metal Binding Biopolymersopolymers

Fine tune affinity with different binding sequences

Metal Binding Domain

Elastin Domain

Fine tune T by controlling amino acid sequence and number of repeating unit (VPGXG)n

Bacterial Bacterial mermer Systems Systemsm

er g

enes

on

E. c

oli

pla

smid

s or

tra

nsp

oson

s

MerRMerR

Mer

D

IM OM

MerT

Mer

P

MerA MerA

MerB

Hg2+

organomercury

Hg0

Hg2+

Hg2+

MerR can serve as a specific mercury binding domain

C123

C79

C114

C114

C79

C123

MerR-Hg complex

A B C kDaA, cell free extract

B, purified protein (80.1 kDa)

C, size marker

Production and purification of ELP153-

MerR

Binding of Mercury to ELP153-MerR

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0 0.5 1.0 1.5 2.0 2.5 3.0

nmol Hg added

nmol

Hg

boun

d

Ela153-MerR

Added Mercury

Similar binding from pH 4 to 9

Selective Binding of Selective Binding of Mercury by Mercury by

Ela153-MerR BiopolymerEla153-MerR Biopolymer

0.00

0.10

0.20

0.30

0.40

0.50

0 25 50 75 100molar excess of Zn

met

al b

ound

(nm

ol)

Hg

Zn

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0 25 50 75 100

molar excess of Cd

met

al b

ound

(nm

ol)

Hg

Cd

Acidic waste water (pH 4)

Kostal et al. ES&T, 37, 4457-4462, 2003.

Other Metal-binding Other Metal-binding DomainsDomains

Metallothioneins (MTs)

MDRNCSCAACDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA

Synthetic Phytochelatins (ECs)

ECECECECECECECECECECECECECECECECECECECECG

Higher affinity toward Hg and Cd

Cadmium Binding Cadmium Binding

0

2

4

6

8

10

0 5 10 15 20

Cadmium Added (mol/mol ELP)

Cad

miu

m B

ou

nd

(m

ol/

mo

l E

LP

)

ELPH12

ELPEC20

Prefer Hg over Cd

Differential Precipitation Differential Precipitation and Separation – Strategyand Separation – Strategy

Histag

ELP78

Cd2+

+ΔT1

+ΔT2 or NaCl

ELP105K21

Hg2+

EC20

Cd2+

Hg2+

ELP105K21

Hg2+

EC20

Customizable Metal Binding BiCustomizable Metal Binding Biopolymersopolymers

Fine tune affinity with different binding sequences

Metal Binding Domain

Elastin Domain

Fine tune T by controlling amino acid sequence and number of repeating unit (VPGXG)n

Differential Differential Precipitation and Precipitation and

SeparationSeparation Differential precipitation and recovery Differential precipitation and recovery

by tuning the elastin composition: by tuning the elastin composition: Lysine as the guest residuesLysine as the guest residues

0

0.2

0.4

0.6

0.8

1

20 30 40 50 60

Temperature

OD 6

55

ELP78

ELP-Lys

ELP78+ ELP-Lys

Differential Precipitation Differential Precipitation and Separationand Separation

1) Marker. 4) 48 oC heat pellet

2) Biopolymer mixture 5) 48 oC supernatant

3) 32 oC heat spin pellet

Supernatant heat to 48oC

MixtureHeat to 32oC

Pellet dissolved in cold buffer

Centrifuge at 32oC

Pellet dissolved in cold buffer

Centrifuge at 48oC

1 2 3 4 5

ELPH12

ELPKEC20

ConclusionsConclusions

Nanoscale ELP biopolymer can be deNanoscale ELP biopolymer can be designed with customized properties:signed with customized properties:

Specificity Specificity Tunable transition Tunable transition

Provide a novel, environmental frienProvide a novel, environmental friendly, and green engineering method fdly, and green engineering method for removal of heavy metal from wateor removal of heavy metal from water and soil.r and soil.

AcknowledgementAcknowledgement

Exploratory Research: Nanotechnology

R829606