Progress 09/01/07 to 08/31/12
Outputs
OUTPUTS: The objective of this work remains to develop compostable cellulose based nanocomposites with improved performance, specifically weight to strength ratio and wet strength/liquid barrier properties. We have been exploring both proteins and polysaccharide additives to achieve this goal. During this final reporting period, we have developed a new cellulose-dual polysaccharide composition which exhibits exceptional performance. Composite sheets were made by adding a cationic polysaccharide to a cellulose pulp solution and mixing for 2 hours and then adding an anionic polysaccharide and mixing for 2 hours. In the current study the cationic and anionic polymers used were chitosan (CS) and caboxymethyl cellulose (CMC). At first composite sheets were made using CS and CMC solutions at a pH of ~4.5 and ~6.5 which resulted in a final pH of the pulp, CS, and CMC mixture of ~4.5. Composite sheets with 50% CS and 50% CMC (w/w with respect to the dry cellulose weight) showed not only the highest dry and wet mechanical strength but also the highest vapor barrier property. The elastic modulus and stress at break for this particular composition were 4467.7 MPa and 44.4 MPa, respectively, whereas the elastic modulus and stress at break of control sample (pure pulp sheet without any polymer addition) were 2712.7 MPa and 25.78 MPa, respectively. The wet elastic modulus and stress at break of the composite sheet were 39.53 and 2.22 MPa, respectively. The control pure cellulose pulp samples (which included standard copy paper) showed no resistance at all and broke immediately under tensile stress. The specific water vapor transmission rate of the composite sheet was 469.81 g mm/day m2, whereas, for the control pulp sample it was 691.48 g mm/day m2. The impact of pH on the strength of composite sheet was also investigated. Composite sheets with 50% CS and 50% CMC were made at two different pH s where the pH of the pulp, CS and CMC mixture was at 1) pH~ 3 to 3.5, 2) pH~ 5 to 5.5 to see the effect of pH on the wet strength of the composite. When tensile test was done in water medium, composite made at all pHs showed similar strength. However, when creep test was done under 1 MPa stress, using test medium at different pHs such as pH ~3 (acidic), 6.5 (DI Water), and 8.5 (basic), composite sheets made at lower pH (~3 to 3.5) remained intact for 30 mins (longest time tested) without breaking in all the medium. The lowest additive % loading which showed significant improvement over the controls was ~25% CS and ~25% CMC for a total additive loading of 50%. This amount of additive limits the commercialization of this technology due to added cost. To reduce the cost, an alternative implementation of this technology was explored. In this case, cellulose pulp sheets were spray coated with cationic and anionic polysaccharides using a unique process (patent under preparation). Similar or better results have been obtained with as little as ~12% coating (6% on either side) on a ~0.1mm sheet. The current objective is to reduce this loading further. At this loading level, commercialization of the technology may be attractive for many volume disposable applications. PARTICIPANTS: Dr. Jeffrey Catchmark, Dr. Niharika Mishra, Adam Plucinski, Zachory Scorr, and Lee Schlossberg worked on this project. Dr. Catchmark advised Dr. Mishra, a post doctoral scholar, on the development of the composites. Adam Plucinski started as an undergraduate researcher and is now a M.S. student working on the coating formulations described in the report. Zachory Scorr and Lee Schlossberg were undergraduate students working on various aspects of the composite development including characterization of the polysaccharides and solutions used. TARGET AUDIENCES: This project has potentially resulted in the development of a new composite and coating technology useful for making paper based substrates useful for applications where the substrate will be exposed to liquid environments to a temperature as high as 85C and over a pH range of 3-11. Commercialization efforts have focused on food handling and shipping products, although other applications exist. Patent protection on the developed IP is underway. Two patents have been submitted and another is under preparation. Results of research have been shown to visitors and many students. A formal hands-on laboratory experience was developed for a freshman seminar class (BE001S). Results have been presented at several conferences which have resulted in relationships with several companies with the intent of commercialization. In addition, several undergraduate students have been exposed to socially relevant research on compostable composites with the goal of producing ecologically compatible packaging and food handling materials, a key sociotechnological challenge for our world. PROJECT MODIFICATIONS: There has been little modification to the project. This last reporting period a focus was placed on polysaccharides over proteins given the exceptional results observed on dual-polysaccharide formulations. This approach appeared to be the best chance of developing a commercially viable composition to replace plastic, plastic laminate, and styrofoam food handling products such as cups, plates, clam-shell packages, etc. Post-grant efforts will be focused on developing commercially viable formulations and processes with the guidance of corporate partners.
Impacts
Based on this work 2 additional patent disclosures have been submitted to Penn State during this period. Discussions on development, licensing and commercialization continued with several companies, and several new corporate partners were identified this period. Specifically we are working with PepsiCo, Verso Paper and Ashland Chemicals, and discussions with other companies are underway. We are working with these companies to commercialize new compostable fiber based materials for 2 target markets: food and beverage container packaging (example: packaging for cold beverage cans), and disposable paper cups and plates. If successful, these new paper based materials would replace plastic in these applications, including plastic coated paper products, and provide a compostable alternative. Other applications are also being explored including shipping (paper for cardboard box construction, letter envelopes, etc.), construction materials (paper for barrier applications including backing for insulation, and even new wood particle composites), and specialty paper (archival paper, paper for government and corporate documents, etc.). Other impacts include the introduction of new content into 2 classes: BE 303: Structural Systems in Agriculture (taught by the PI) and BE 001: Freshman Seminar in which the PI presents. In these classes, the issue of sustainability is covered in some detail and advances achieved in the work funded by the USDA is discussed. In-class demonstrations developed and delivered include the polymerization of insoluble fibrous complexes to demonstrate the capabilities of non-covalent electrostatic and ionic interactions. In addition, several undergraduate students were introduced to this topic via undergraduate research projects, where they helped advance the development of compostable composites and participate in the process for solving an important sociotechnological problem. One of these students entered into an M.S. program during this period. Finally, this period, 1 paper was published in Carbohydrate Polymers, 2 papers under final revision for Biomacromolecules and 2 additional papers are under preparation for Carbohydrate Polymers.
Publications
- Guo, J. and J. M. Catchmark. 2012. Surface area and porosity of acid hydrolyzed cellulose nanowhiskers and cellulose produced by Gluconacetobacter xylinus. Carbohydrate Polymers 87(2): 1026.
- Guo, J. and J. M. Catchmark. 2012. Binding Specificity and Thermodynamics of Cellulose-Binding Modules from Trichoderma reesei Cel7A and Cel6A. Biomacromolecules (In Review).
- Guo, J. and J. M. Catchmark. 2012. In Vitro Identification of Peptides Containing Novel Cellulose-Binding Motif. Biomacromolecules (In Review).
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: The objective of this work remains to develop compostable cellulose based nanocomposites with improved performance, specifically weight to strength ratio and wet strength/liquid barrier properties. We have been exploring both proteins and polysaccharide additives to achieve this goal. We have completed the following studies: 1) The expression of CBMCel7A and CBMCel6A protein binders and the study of their binding to cellulose using isothermal titration calorimetry (ITC) and adsorption-isotherm experiments (manuscript under preparation). A key scientific obstacle preventing the quantitative analysis of cellulose binding was resolved relating to the inability to quantify the molarity of different forms of cellulose useful for composite production. To overcome this, we have introduced a new approach based on the available surface area and porosity of the cellulose substrate (manuscript was submitted to Carbohydrate polymers). 2) The available binding surface area and porosity of many forms of cellulose including cellulose nanowhiskers (CNWs) from different origins (plant cotton/bacterium Gluconacetobacter xylinus) and different acid treatments (H2SO4/HCl) were characterized by BET, BJH and HK analysis of N2 adsorption data (manuscript accepted). 3) We have used Phage display to identify the optimal binding peptide limited to 7 amino acids. A conserved polypeptide was clearly identified, but its binding constant was low (~105 M-1). Based on this data we hypothesize that the tertiary structure of CBMs plays a key role in achieving high binding constant and that small peptides my never be able to be engineered to exhibit comparable binding (manuscript under preparation). 4) We have also examined the binding properties of engineered peptides with different number of tyrosines (manuscript under preparation). In addition, two molecular modeling activities were continued. They are 1) Modeling of the binding of new model peptides that are mimicking CBM binding site characteristics, and 2) Modeling of CBM binding to the ends of cellulose chains. In addition, extensive research was conducted on the use of polysaccharides in addition to proteins to create a new cellulose composite with improved wet strength and liquid barrier properties. We have had success with compositions which contain anionic and cationic polysaccharides, where these polysaccharides are added to the cellulose containing solution in specific concentrations. We continue work to further improve the properties by adding particle binders which are coated with anionic proteins or polysaccharides. This will be the focus of the work in the final year of this grant. Specifically, understanding fundamentally the interactions between polysaccharides and proteins, and the application of that understanding to the development of improved cellulose compositions. PARTICIPANTS: Jeffrey M. Catchmark, Associate Professor, PSU; Jing Guo, Ph.D. student (graduated), PSU, Niharika Mishra, post-doctoral scholar, PSU, Drew Wolos, undergraduate student, PSU, Adam Plucinski, undergraduate student, PSU; Rick Lewis, M.S. student, PSU. TARGET AUDIENCES: The research community interested in cellulose-protein interactions and compostable cellulose composites; Companies who produce paper, packaging materials, cellulose composites and wood particle composites; Undergraduate and graduate students learning about cellulose composite properties and polysaccharide-polysaccharide, polysaccharide-protein interactions; Visitors and the general public (including families and their children) interested in advancements in sustainable materials research. PROJECT MODIFICATIONS: There were no major changes to the project. A minor change was additional focus on polysaccharides as additives in the composites in addition to, or, in some cases, in place of, proteins.
Impacts
Based on this work a U. S. patent has been submitted and 2 additional patent disclosures have been submitted to Penn State. Discussions on development, licensing and commercialization continued with several companies, and a specific corporate partner was identified. A precommercialization grant was awarded to Penn State and this partner by the Pennsylvania Nanomaterials Commercialization Center to optimize a composition and develop a plan for commercialization, if the development activities are successful. This is underway. A specific focus currently is a new paper based packaging material for 2 target markets: food and beverage container packaging (example: packaging for cold beverage cans), and disposable paper cups and plates. If successful, these new paper based materials would replace plastic in these applications, including plastic coated paper products, and provide a compostable alternative. Other applications are also being explored including shipping (paper for cardboard box construction, letter envelopes, etc.) and construction materials (paper for barrier applications including backing for insulation, and even new wood particle composites). Other impacts include the introduction of new content into 2 classes: BE 303: Structural Systems in Agriculture (taught by the PI) and BE 001: Freshman Seminar in which the PI presents. In these classes, the issue of sustainability is covered in some detail and advances achieved in the work funded by the USDA is discussed. In-class demonstrations developed and delivered include the polymerization of insoluble fibrous complexes to demonstrate the capabilities of non-covalent electrostatic and ionic interactions. In addition, several undergraduate students were introduced to this topic via undergraduate research projects, where they helped advance the development of compostable composites and participate in the process for solving an important sociotechnological problem. One of these students is interested in pursuing a Ph.D. in this area. Technically, a highlight of this work includes the development of a new method for quantifying relative binding constants for ligands binding to different forms of cellulose. This new approach uses the molarity of a unit surface area and is adjusted for porosity based on the size of the binding ligand. A manuscript is nearly complete on these results. Significant background technical detail on other outcomes was provided in last year's report. One manuscript was accepted, 2 others are nearly complete and 1 other is in process. This work was also presented at 2 conferences. Finally, Jing Guo, a Ph.D. student funded by this program, was awarded her Ph.D. and is now a Senior Chemist at Dow Wolfe Cellulosics.
Publications
- Guo, J. and J. Catchmark. 2011. Surface area and porosity of acid hydrolyzed cellulose nanowhiskers and cellulose produced by Gluconacetobacter xylius. Carbohydrate Polymers (Accepted).
- Guo, J. and J. Catchmark. 2011. The surface area and porosity of nano-scaled cellulose from acid hydrolysis. ASABE Annual International Meeting paper. Paper number 1111669.
- Guo, J. and J. Catchmark. 2011. The binding affinity and specificity of family 1 Cellulose Binding Modules (CBMs) of Cel7A and Cel6A. IBE Annual International Meeting paper (Abstract). page 7
- Catchmark, J. 2010. Cellulose: natures most widely used nanomaterial. Invited. Texas A&M Inter-disciplinary Distinguished Seminar Series. 74 pages
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: The objective of this work is to develop ecologically compatible cellulose based nanocomposites. We have been exploring both proteins and polysaccharide additives to achieve this goal. Toward this end, we have successfully expressed the cellulose binding modules CBMCel7A and CBMCel6A for use as novel protein binders in cellulose composites. Isothermal Titration Calorimetry (ITC) was performed to obtain a complete thermodynamic profile of the binding between CBMs (as well as other proteins discussed below) and cellulose substrates (manuscript under preparation). While conducting this research, we identified a key scientific obstacle preventing the quantitative analysis of cellulose binding: the inability to quantify the molarity of different forms of cellulose useful for composite production. To overcome this, we have developed a new approach for quantifying binding interactions based on the surface area and porosity of the cellulose substrate (manuscript will be submitted to Biomacromolecules by 11/19/2010). The available binding surface area and porosity of many forms of cellulose including cellulose nanowhiskers (CNWs) were characterized by BET, BJH and KH analysis of N2 sorption data. This technique will be applicable to the analysis of many different binding interactions involving heterogeneous substrates. In addition to CBMs, we have used Phage display to identify the optimal binding peptide limited to 7 amino acids. A conserved polypeptide was clearly identified, but its binding constant was low (~104 M-1). Based on this data we hypothesize that the tertiary structure of CBDs plays a key role in achieving high binding constant and that small peptides my never be able to be engineered to exhibit comparable binding (manuscript under preparation). We have also examined engineered peptides with different number of tyrosines (analysis still underway). Based on this work, we are now designing a novel fusion protein consisting of a CBM and a calcium carbonate binding polypeptide to create a binder for cellulose composites. This engineered molecular linker contains a phosphorylated serine motif believed to bind to calcium carbonate and calcium phosphate (experiments underway). Two Molecular Modeling activities were performed. They are 1. Hydrogen bonding analysis using Quantum Mechanical methods on various cellulose fragments ranging from basic unit cellobiose to two layers of cellulose micro fibrils. 2. Modeling efforts were proposed and carried out to develop new model peptides that are mimicking CBM binding site characteristics. This is because structural and fundamental studies on the structure and functions of representative members of Cellulose Binding Module families reveal that their binding to cellulose fiber can be attributed, at least in part, to their hydrophobic surface, which is composed of several aromatic amino acids. Utilization of these fundamental properties of CBM would be useful in developing a new and efficient model to mimic these properties with better binding. This modeling has initiated the design of engineered cellulose linkers in collaboration with experimental researchers. PARTICIPANTS: Individuals working on project: Penn State Faculty: Jeffrey Catchmark, Nicole Brown, James Kubicki, Ming Tien, Sridhar Komarneni Ph.D. Student: Jing Guo, Niharika Mishra Post-doctoral researcher: Mohamed Naseer Ali Mohamed Undergraduates: Serena Wang Companies contacted to discuss development, licensing and commercialization of patent-pending IP developed under this grant: Appleton Papers, Georgia Pacific, Bayer Material Science, Valspar (renewed contact underway) Economic development organizations assisting in technology transfer: Pennsylvania Nanomaterials Commercialization Center TARGET AUDIENCES: Research results developed through this grant were included in the following activities: Undergraduate education: BE 001 Freshman Seminar; BE 466W Biological Engineering Design High school teacher and student tours: Laboratory demonstration of improved paper composites and discussion on need for sustainable materials in packaging. Alumni and visitor tours. PROJECT MODIFICATIONS: No modifications of project except: 1) we have begun to look at polysaccharide additives also to improve binding properties. 2) We requested an extension to continue the successful work ongoing through this grant.
Impacts
Based on this work a patent has been submitted and discussions on development, licensing and commercialization is underway with several companies including Appleton Papers, Georgia Pacific, Bayer Material Science, and others. In particular, a novel paper composition has been identified through this research which exhibits dramatically improved wet strength and water barrier properties. These are being investigated for applications in food packaging and handling, in addition to applications in shipping, construction and other areas. Technically, our results of cellulose characterization showed that CNWs produced from either H2SO4 or HCl exhibited significantly increased surface area and porosity as compared to the starting material. In particular, both mesopores and micropores were observed in CNWs produced by HCl digestion. It is hypothesized here that some microporosity arises from delamination of the crystalline cellulose sheets at the surface and ends of the CNWs. Our ITC results showed the binding constant (Ka) to insoluble CNWs are ~105 M-1 for CBMCel7A, while ~106 M-1 for CBMCel6A, this result was further confirmed by the adsorption isotherm experiment, which suggests a higher binding affinity of CBM Cel6A to CNWs. By screening Ph.D.-7 phage display library, we have successfully identified one consensus peptide "WHWTYYW" that binds with crystalline CNWs. This peptide appears to bind CNWs in a highly selective manner. In addition, the displayed peptides are enriched in hydrophobic residues, suggesting the major hydrophobic interactions may play a critical role in binding with crystalline cellulose. Affinity constants of isolated heptapeptide against cellulose were determined by ITC and fluorescence titration. Comparing to natural CBMs, which can have Ka as high as 109 M-1, small peptide, like heptapeptide seems to be unable to obtain a high Ka due to the absence of a tertiary structure. Molecular dynamics simulations were conducted for this phage display "WHWTYYW" peptide in two different steps. In the first step, we performed a 100 ns simulation to identify a reasonably folded peptide. In the second step lowest, folded peptide was manually docked onto the cellulose surface to study the interaction mechanism through addition MD simulations. These simulation studies clearly indicate that the complex was stabilized through CH/pi interaction (CH/pi geometry ranging from 2.4 to 2.9 angstroms). Also, from these simulations we found that this peptide exhibits a compact globular shape due to pi- pi stacking between the aromatic groups. All the cellulose binding peptide modeling studies were carried at the molecular mechanics and quantum mechanical level. Among the simulated models, (G)4-Y-Y-(G)4-Y-Y-(G)4, preferentially binds(-159 kJ/mol) with the cellulose surface according to the Amber 94 force field. In addition, from the interaction energy of the model (G)4Y-Y-Y-(G)4, it is noted that the Amber 94 interaction energy is less favorable (-88 kJ/mol) when the gap between the Y residues is zero.
Publications
- Mohamed, M. N. A., H. D. Watts, J. Guo, J. M. Catchmark, and J. D. Kubicki. 2010. MP2, density functional theory, and molecular mechanical calculations of C-H/pi and hydrogen bond interactions in a cellulose-binding module-cellulose model system, Carbohyd. Res. 345:1741-1751.
- Guo, J., J. M. Catchmark, M. Tien, and T. H. Kao. 2010. Screening for cellulose nanowhiskers binding peptides by phage display. ASABE Annual International Meeting paper. Paper number 1009575.
- Guo, J. and J. M. Catchmark. 2010. Thermodynamics of Family 1 Cellulose-Binding Modules from T. reesei Cel7A and Cel6A. ASABE Annual International Meeting paper. Paper number 1009577.
- Guo, J., J. M. Catchmark, and M. Tien. 2009. Synthesis and analysis of cellulose binding domains (CBDs). ASABE Annual International Meeting paper. Paper number 096721.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Among the fugal CBMs, the filamentous fungus Trichoderma reesei (also named as Hypocrea jecorina) is one of the most potent cellulase producers, which generally produce 2 different cellobiohydrolases, CBMCel7A and CBMCel6A. We decided to express CBMCel7A and CBMCel6A since they are shorter than the vast majority of other fungal CBMs, they are the best characterized cellulolytic enzyme system today, the crystal structures are well-known, they specifically bind to the non-reducing end and reducing end of cellulose chain, respectively, which makes them good candidates for new cellulose-integrated nanocomposite design and engineering. CBMCel7A and CBMCel6A were heterologously expressed in Escherichia coli, purified under denaturing condition and then refolded by renaturation. Isothermal Titration Calorimetry (ITC) was performed to obtain a complete thermodynamic profile of the binding between CBMs and cellulose substrates. The available binding surface area and porosity of cellulose were characterized by BET and BJH in order to compare binding affinities of CBMs to different cellulose substrates. Phage display is a very powerful selection methodology which has been widely used to identify novel protein or peptide sequences with desired binding traits. Here, small peptides specifically binding to CNWs were screened and identified. A library of approximately 1 billion different polypeptides were constructed and displayed on the surface of bacteriophage, and then been used as sources for selection of variants with desired binding traits to CNWs. The gene encoding the 7 amino acid domain was inserted into a phagemid vector and shown to be functionally displayed on M13 filamentous phage as a protein III fusion protein. The peptides were selected on immobilized cellulose nanowhiskers by bio-panning, by which a pool of randomized peptides are screened based on the binding affinity to the target CNWs. By phage display, we could be able to find new cellulose binders which have both simpler structure and higher binding affinity than natural CBMs to cellulose. Two Molecular Modeling activities were performed during this period: 1. Hydrogen bonding analysis using Quantum Mechanical methods on various cellulose fragments ranging from basic unit cellobiose to two layers of cellulose micro fibrils. 2. Modeling efforts were proposed and carried out to develop new model peptides that are mimicking CBM binding site characteristics. This is because structural and fundamental studies on the structure and functions of representative members of CBM families reveal that their binding to cellulose fiber can be partially attributed, to their hydrophobic surface, which is composed of several aromatic amino acids. Utilization of these fundamental properties of CBM would be useful in developing a new and efficient model to mimic these properties with better binding and inexpensive way. This modeling would initiate the design of engineered cellulose linkers in collaboration with experimental researchers. Patent submitted: Jeffrey Catchmark and Dana Mears. 2009. COMPOSITES CONTAINING POLYPEPTIDES ATTACHED TO POLYSACCHARIDES AND MOLECULES. U.S. Patent Office. PARTICIPANTS: Jeffery Catchmark, Associate Professor, Project Director. Prof. Catchmark directed the biological and materials synthesis aspects of the project and led the design of new cellulose binding polypeptides. Ming Tien, Professor, Co-investigator. Prof. Tien provided input on molecular biology techniques for preparing proteins. Prof. James Kubicki, Associate Professor, Co-investigator . Prof. Kubicki led the quantum mechanical modeling of cellulose and phenol binding simulations. Prof. Nicole Brown, Assistant Professor, Co-investigator. Prof. Brown assisted in the mechanical analysis of nanocomposites. Dr. Douglas Archibald, Research Associate. Dr. Archibald performed IR spectroscopy of cellulose. Prof. Sridhar Komarneni, Professor, Co-investigator. Prof. Sridhar prepared and supported analysis of functionalized clay nanoparticles. Jing Guo, Ph.D. Student, created and expressed cellulose binding domains using molecular biology techniques. Dr. Mohamed Naseer Ali Mohamed, Post Doctoral Student, performed quantum mechanical calculations of cellulose crystallization and the binding to phenol end group Heath Watts, Ph.D. Student, Performed ITC experiments and molecular modeling. Dana Mears, Ph.D. Student, Performed ITC and binding assays on cellulose, proteins and other targets. TARGET AUDIENCES: Academics and industry researchers engaged in the development of agriculturally derived sustainable composite materials; Industry representatives interested in collaborative research or licensing of developed technologies; Pre-college, undergraduate and graduate students, as well as visitors to PSU interested in learning about sustainable materials development in the ABE department. PROJECT MODIFICATIONS: The only project modification has been in the expansion of the class of proteins considered for the creation of new cellulose nanocomposites. Cellulose binding domains (CBH1 and CBH2) were initially proposed as the focus of the work. We now are designing proteins and exploring other proteins which may be superior to these CBDs in terms of binding affinity but not in their ability to bind to the reducing and non-reducing ends of cellulose, the key capability of the CBH1 and CBH2 proteins. This was done to both create improved cellulose composite materials and to identify proteins which could be introduced into a volume manufacturing environment more quickly than the CBH1 and CBH2 proteins which would need to be manufactured via fermentation type processes.
Impacts
The binding constant (Ka) for adsorption to insoluble CNWs was ~ 105 M-1 for CBMCel7A, while ~ 106 M-1 for CBMCel6A, this result was further confirmed by the adsorption isotherm experiment. Although small difference of the binding affinity of CBMCel7A and CBMCel6A for CNWs was observed, thermodynamic data showed that the binding mechanisms of CBMCel7A and CBMCel6A were similar. The binding was dominated by a favorable change in entropy, which was partly compensated by unfavorable change in enthalpy change. The entropic driving force was interpreted to arise from ordered water molecules returning to the bulk solution by the dehydration of the polypeptide and the cellulose surface. The hydrophobic nature of the CBMCel7A and CBMCel6A binding site support this, indicating that thermodynamic analysis of CBM-ligand interactions thus provides some understanding of the mechanisms as well as the energetics of binding. The binding constants of these two CBMs were higher for binding to CNWs than Avicel PH101, demonstrating that these CBMs had preference to bind to highly crystallized cellulose. We have screened small 7-mer peptides that have the ability to bind to CNWs. After three round of bio-panning of phage display, a consensus peptide sequence of "WHWTYYW" was found to efficiently bind to CNWs, which is in well agreement with previously proposed model that aromatic amino acids play critical roles in the binding to cellulose. To better understand the nature of the peptide-CNWs binding demonstrated by this experiment, this peptide "WHWTYYW" was synthesized and ITC experiment will be performed to measure the binding affinity to cellulose. Binding of CBMs in cellulases is not only to anchor the catalytic domain but also potentially to aid in the defibrillation process by destabilizing the interchain hydrogen bonds. The result obtained from the QM studies we infer that the four-ring layer H-bond pattern between O3---O5 and O2---O6 and a lateral O6---O3 inter-molecular hydrogen bond pattern were found to conserved in most part of the matrix. Additional H-bonds between cellulose polymers within a given layer were found in these quantum mechanical calculations that were not been observed in previous MD simulations. However, inter-layer H-bonding was not significant suggesting that van der Waal's forces dominate interlayer energetics. To simplify the CBM modeling efforts we started to construct the model peptides by combining a hydrophobic residue viz Tyr and a simple amino acid Gly. This motif is a key component for the hydrophobic nature of the Glycine rich proteins (GRPs). Eight models were generated with the G-Y-X (here X may be G or Y). Here G stands for glycine, Y for Tyrosine and X may be tyrosine or glycine. All the cellulose binding peptide modeling studies were carried at the Molecular Mechanics level. The peptides with the highest interaction energies were G-G-G-Y-Y-G-G-G-G-G-G(-34) and G-G-G-G-Y-Y-G-G-G-G-Y-Y-G-G-G-G(-38). The interaction energies are in kJ/mol. Results indicate that hydrogen bonds are a predominant interacting force with the cellulose surface along with weak hydrophobic interaction.
Publications
- Guo, J., J. M. Catchmark, and M. Tien. 2009. Biosynthesis of cellulose binding domains (CBDs). ASABE Annual International Meeting. Reno Nevada. June 21-24. Paper #096721.
- Ali, M. N., L. Zhong, J. D. Kubicki, J. M. Catchmark, N. Brown, M. Tien, D. Archibald, and H. D. Watts. 2009. Cellulosic Ethanol - molecular modeling with multidisciplinary approach. Energy Bridge Inaugural Meeting. July 15-16. Poster #18.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: This program focuses on the development of nanoengineered cellulose materials comprised of proteins which chemically bond to and spatially organize nanodimensional cellulose resulting in a material which consumes less fiber and exhibits superior mechanical properties. Two coupled activities have been proposed and pursued during the reporting period to achieve this: 1) the engineering and synthesis of cellulose binding domain (CBD) proteins and particles which have been functionalized with these proteins; and 2) the quantum and molecular modeling of CBDs with cellulose to understand fundamentally how these proteins bind, and to develop engineering principles which will enable improved proteins to be developed. During the reporting period, plasmid DNA needed for producing both CBH1 and CBH2 CBDs, which bind to reducing and non-reducing ends of a cellulose chain, respectively, were constructed. Specifically, the region of the CBD gene from T. reesei, designed with two restriction digestion sites Nde I, Xho I, was synthesized in the pZERO2 vector; the CBD DNA fragment was then digested by enzymes Nde I and Xho I. Escherichia coli BL21 (DE3) PlysE, was used as the host for expression CBD. The CBD gene from was then cloned into an expression vector Nde I/Xho I-restricted pET21a (with a HIS tag which helps the detection and purification of CBD) and the recombinant plasmid generated was transformed into BL21 (DE3) PlysE to express the CBD. To express the CBD, the bacteria BL21 (DE3) PlysE harboring recombinant plasmid CBD-pET21a plasmids were grown in LB medium to 0.6-0.7 OD600nm at 37C and then isopropyl-1-thio-Beta-D-galactopyranoside was added to a final concentration of 1 mM to induce the CBD expression. Expression was checked by SDS-PAGE and Western blot. Purification of CBDs was also accomplished by NTA-Ni2+ column purification. Extensive quantum mechanical analysis has been performed on the amino acid residue tyrosine which is primarily responsible for the CBD binding to cellulose. The binding topography is characterized by the aromatic side chains of tyrosine (and tryptophan and less commonly phenylalanine). Replacement of these amino acids at the binding site of a CBD has been shown to reduce or completely destroy the affinity for the cellulose target, suggesting that the aromatic side chains play in an important role in CBM-carbohydrate binding through "stacking interaction". To study this phenomenon, modeling has been performed to understand the molecular-level interactions that cause sugar interaction through these residues. The preliminary modeling study includes quantum mechanical (QM) analysis of sugar-tyrosine interactions to explain the role of aromatic residues. Using the QM intermolecular geometries, modeling studies were conducted to study the role of tyrosine residues in fusion model peptides enriched with glycine. Nanocomposite materials were also created using clay nanoparticles which were bonded to cellulose nanowhiskers via CBDs derived from endoglucanases. These studies are in process. This work was presented in several meetings, workshops and educational activities including undergraduate and graduate student seminars. PARTICIPANTS: Jeffery Catchmark, Associate Professor, Project Director. Prof. Catchmark directed the biological and materials synthesis aspects of the project and led the design of new cellulose binding polypeptides. Ming Tien, Professor, Co-investigator. Prof. Tien provided input on molecular biology techniques for preparing proteins. Prof. James Kubicki, Associate Professor, Co-investigator . Prof. Kubicki led the quantum mechanical modeling of cellulose and phenol binding simulations. Prof. Nicole Brown, Assistant Professor, Co-investigator. Prof. Brown assisted in the mechanical analysis of nanocomposites. Dr. Douglas Archibald, Research Associate. Dr. Archibald performed IR spectroscopy of functionalized clay nanoparticles. Prof. Sridhar Komarneni, Professor, Co-investigator. Prof. Sridhar prepared and performed analysis of functionalized clay nanoparticles. Jing Guo, Ph.D. Student, created and expressed cellulose binding domains using molecular biology techniques. Dr. Mohamed Naseer Ali Mohamed, Post Doctoral Student, performed quantum mechanical calculations of cellulose crystallization and the binding to phenol end groups of tyrosine. Dr. Vivek Verma, recent Ph.D. graduate (from Catchmark group) made and tested cellulose nanocomposites containing cellulose binding proteins. TARGET AUDIENCES: The target audiences are the scientific community in the area of cellulose composite materials and cellulose binding proteins, the industrial community which make cellulose based materials including paper and wood chip and fiber composites, and the national and international professional and economic development communities which shape research and development investment into plant/forest based materials. Several comapnies and organizations have been engaged to form collaborative relationships including Rohm and Haas, Specialty Minerals, Valspar, Integran, Appleton Paper and Weyerhaeuser. Other organizations include the Forest Products Laboratories, FPInnovations (Canada), University of Alberta, The Alberta Forestry Research Institute, Alberta Region of Western Economic Diversification Canada, NanoAlberta, KCL (Finland) and the North Carolina State University. Much of this interaction was done through the Penn State Center for NanoCellulosics. PROJECT MODIFICATIONS: The only project modification has been in the expansion of the class of proteins considered for the creation of new cellulose nanocomposites. Cellulose binding domains (CBH1 and CBH2) were initially proposed as the focus of the work. We now are designing proteins and exploring other agricultural proteins which may be superior to these CBDs in terms of binding affinity but not in their ability to bind to the reducing and non-reducing ends of cellulose, the key capability of the CBH1 and CBH2 proteins. This was done to both create improved cellulose composite materials and to identify proteins which could be introduced into a volume manufacturing environment more quickly than the CBH1 and CBH2 proteins which would need to be manufactured via fermentation type processes.
Impacts
The work performed during this reporting period had several outcomes and impacts. Knowledge based outcomes include the development of a new polypeptide architecture potentially allowing proteins to be engineered with specific cellulose binding affinities. In particular low molecular weight proteins containing engineered sequences comprised of flexible hydrophilic linkers coupled to regions of cellulose binding amino acids have been engineered. The His binding tag mentioned above will be used to couple these polypeptides to various commercially available nanoparticles for constructing new cellulose nanocomposites. This work was inspired in part by the QM analysis of the interaction of the binding residue of tyrosine to cellulose. That work has revealed that CH-pi interactions in methyl-Beta-D-Glc-Tyr as sugar-CBM exhibit interaction energies which range from -25 kJ/mol to -38 kJ/mol. The variation of potential energies as a function of interplanar distance is almost flat near the minimum and the shallow potential of these interactions arises due to existence of classical intermolecular H-bond of the phenyl hydroxyl of tyrosine side chain with O4 of glucose. To compute the energy contribution due to this hydroxyl interaction with sugar, dihedral scanning was performed about C epsilon-C zeta-O eta-H of tyrosine in the vicinity of sugar. Computations were also performed to study the pattern of H-bond network with a tetramer bi-layer cellulose fragment. Calculations suggest that the four-ring layer H-bond pattern between O3-O5 and O2-O6 and a lateral O6-O3 inter-molecular H-bond pattern are conserved in the matrix. Inter-layer H-bonding was not significant suggesting that van der Waal's forces dominate interlayer energetics. Understanding of the structure of cellulose provides insight into the evolution of the CBD architecture. This work is beginning to reveal the exact binding dynamics and may lead to new binder chemistries which may not incorporate expressed proteins but just the specific binding chemistries. This work has also been leveraged by other USDA funding. A USDA National Needs Fellowship recipient is working on other agriculturally derived proteins we have discovered which bind to cellulose. One class of protein exhibit binding affinities 100 times greater than that exhibited by most CBDs. This interesting development has also guided the design of the previously mentioned polypeptides, and new engineering principles for the design of cellulose binding biochemistries are emerging. Other outcomes include new industrial interaction and student education. This work has been presented to several companies and has stimulated interactions with several major agriculturally related materials manufacturers and additional collaborative grants have been awarded and are pending. Faculty in this program have also used this work to augment their teaching and both undergraduates and graduate students have been exposed to the key molecular biochemistry which is at the core of how nature forms and degrades cellulose. It is also an example of biologically inspired nanotechnology which is inspirational to young students seeking exciting new careers.
Publications
- No publications reported this period
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