# Presentation: 341666

## Nanoscale Biopolymers with Customizable Properties for Heavy Metal Remediation

**U Loi (Ann) Lao, Giri Prabhukumar,** **Jan Kostal, Mark Matsumoto, Ashok Mulchandani, and Wilfred Chen**
**Chemical and Environmental Engineering**
**University of California, Riverside**** **

**Notes:**
I am speaker Ann, from UC Riverside. I am presenting the recent research from our group, Nanoscale Biopolymers with customizable properties for heavy metal remediation

## Heavy Metal Contamination

- 2.4 million tons of metal wastes per year from industrial sources
- 2 million tons per year from agriculture and domestic waste
- Pb2+, Hg2+ and Cd2+ are ranked 2nd, 3rd and 7th, respectively, on the EPA’s priority list

**Notes:**
Heavy metal contamination is a concern for environmental safety. Every year large amounts of metal wastes are produced not only from industrial but also agriculture and domestic sources. Lead, Mercury and cadmium are ranked 2nd, 3rd and 7th respectively on the EPA’s priority list. 
Many 

## Metal Chelating Polymers

- • Requires toxic solvents for synthesis
- • Ultrafiltration is required
- • Membrane clogging

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

## Metal Chelating Biopolymers

- Based on biological building blocks
- Nanoscale biopolymers that are specially pre-programmed within a DNA template
- Economically produced by bacteria
- Environmentally friendly
**Tunable properties based on changes in environmental conditions – pH or temperature**** **

## Elastin Biopolymer

**Structurally similar to the repeating elastometric peptide sequence of the mammalian protein, elastin **
**VPGVG are the most frequently repeating units**
**Undergo a reversible phase transition from water soluble forms into aggregates as the temperature increases**

**Low temperature: ********-turn**

**High temperature: twisted filament of ********-spirals**

## Genetic and Protein Engineering Methodology

**Notes:**
Microbial expressions of artificial genes provides a means of easily producing ELP. amino acid sequences are chosen to create specific folding patterns and desired material properties. The primary amino acid sequence can then be reverse-translated into a corresponding nucleotide sequence. There are 2 methods of obtaining the needed DNA fragments. One possibility is to clone these sequences from an organism that produces the desired structural protein. The second option is to synthesize artificial genes by solid phase techniques. The second method of course allows maximum freedom in designing the target sequence. Because many structural proteins are characterized by repetitive amino acid sequences, it is often possible to multimerize a smaller oligonucleotide sequence to prepare an artificial gene that codes for proteins of high molecular weight.

## Customizable Metal Binding Biopolymers

**Metal Binding Domain**

**Elastin Domain**

## Heavy Metal Removal by Tunable Biopolymer

## Elastin Biopolymers with Metal-Binding Affinity

**  ****A    B   C    D   E**

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

## Phase Transition

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

**Notes:**
Precipitation of Ela38H6 by heating above 30oC (left). The protein resolubilized when placed at a temperature below 26oC (right).

## Cadmium Removal

**50 nmol of biopolymer were incubated with 100 nmol Cd****2+**

## Repeated Cycles of Metal Binding

**Cycle 1     Cycle 2      Cycle 3     Cycle 4**

**5 ********g added each cycle**

## Practical Applications: Soil Flushing/Washing

**Environmental friendly extractant**
**Simple separation and recycling**

**In-situ Soil Flushing**** **

**Ex-situ Soil Washing**

## Soil-Biopolymer Adsorption Characteristics 

**Soil-Biopolymer Sorption Characteristics**
- Freundlich isotherm
- 	Log x/m = Log Kf + 1/n Log Ceq

**Maximum 19% adsorption**
**Biosurfactant – typically in the 70% range**

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

## Biopolymer-Cadmium Extraction Studies 

**Less than 10% adsorption**

## First generation biopolymers  

**Both T****t**** and metal binding capacity can be easily regulated**
**Biopolymers can be recycled**
**Lack specificity and affinity**

**First generation biopolymers  **

## Customizable Metal Binding Biopolymers

**Metal Binding Domain**

**Elastin Domain**

## Bacterial mer Systems

***mer *****genes on *****E. coli***** plasmids or transposons**

**IM**

**OM**

**Mer****T**

**MerP**

**MerA**

**MerA**

**MerB**

**organomercury**

**Notes:**
The regulatory circuit for bacterial mercury detoxifcation

## MerR-Hg complex

**MerR can serve as a specific mercury binding domain**

**  ****C123**
**C79**
** ****C114**

**C114**
**   ****C79**
**C123**

**MerR-Hg complex**

**Notes:**
The high binding affinity of mercury binding by MerR is in accordance with the fact that three different cysteine residues from the two MerR subunits are involved in sequestering mercury, which results in a high-affinity tricoordinate mercury binding site.

## Production and purification of ELP153-MerR

**A, cell free extract**
**B, purified protein (80.1 kDa)**
**C, size marker**

**Production and purification of ELP153-MerR**

**Notes:**
Purification of ELP-MerR.

## Binding of Mercury to ELP153-MerR

**Similar binding from pH 4 to 9**

**Notes:**
Mercury binding stoichiometry of ELP153MR in 50 mM sodium acetate pH 4.0.  One nmol of biopolymers were mixed with various amounts of mercury. Results represented the average of three sets of experiment with error bars showing the standard deviations.

## Selective Binding of Mercury by Ela153-MerR Biopolymer

**Acidic waste water (pH 4)**

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

**Notes:**
Selectivity of the ELP153MR biopolymer. Binding of mercury by ELP153MR in the presence of competing heavy metals. One nmol of biopolymer was mixed with 0.5 nmol of HgCl2 and various amounts of competing heavy metals. 

## Other Metal-binding Domains

**Metallothioneins (MTs)**
- MDRNCSCAACDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA
**Synthetic Phytochelatins (ECs)**
- ECECECECECECECECECECECECECECECECECECECECG

**Higher affinity toward Hg and Cd**

## Cadmium Binding 

## Differential Precipitation and Separation – Strategy

## Customizable Metal Binding Biopolymers

**Metal Binding Domain**

**Elastin Domain**

**Notes:**
In addition to improving the metal-binding affinity and selectivity, we can also control the ELP sequence to fine-tune the transition temperatures for different applications. 

## Differential Precipitation and Separation

- Differential precipitation and recovery by tuning the elastin composition: Lysine as the guest residues

**ELP78**

**ELP-Lys**

**ELP78+ ELP-Lys**

**Notes:**
For example, A regulate ELP biopolymer has a transition temperature around 30oc, while the introduction of a charged lysine residue shifted the transition temperature to around 50 degree Celsius. When these two biopolymers are mixed together, they can undergo distinct phase transition and separate  individually. we can easily take advantage of this idea to achieve differential separation and recovery of two or more heavy metals in a mixture.  

## Differential Precipitation and Separation

**Marker.                        4) 48 ****o****C heat pellet**
**Biopolymer mixture   5) 48 ****o****C supernatant**
**3)  32 ****o****C heat spin pellet **

**Supernatant **
**heat to 48****o****C**

**Mixture**
**Heat to 32****o****C**

**Pellet dissolved**
** ****in cold buffer**

**Centrifuge at 32****o****C**

**Pellet dissolved**
** ****in cold buffer**

**Centrifuge at 48****o****C**

**     ****1       2      3       4       5**

## Conclusions

**Nanoscale ELP biopolymer can be designed with customized properties:**
**Specificity **
**Tunable transition **
**Provide a novel, environmental friendly, and green engineering method for removal of heavy metal from water and soil.**

## Acknowledgement

**Exploratory Research: Nanotechnology**

**R829606**