Progress 09/01/14 to 08/31/17
Outputs
Target Audience:- Graduate Student performing the project activities: enzyme expression and purification; - Undergraduate students performing capstone project (mandatory in the Department of Chemistry) non-funded (credit-based) on activities related to synthesis and analysis of the materials; - 252th American Chemical Society NationalMeeting participants attending the poster session; - Students enrolled in thenew course "Nanotechnology in Food Science and Food Analysis" offered in the Fall 2016 and Fall 2017. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?- Undergraduate students associated with the project have aquiredmaterials synthesis and characterization skills. - Graduate student has been trained on enzyme purification toward enzyme immobilization. Student can express and purify the genetically engineered enzyme and characterize it by molecular biology techniques. -Graduate student is able to prepare enzyme encapsulatedmaterials and test the cascade enzymatic reaction. How have the results been disseminated to communities of interest?- The results obtained in the course of this projecthave been presented at a prominent national conference (American Chemical Sociey Nationa meeting) - project outcomes have been published in a journal with high impact in both materials and biotechnology. - a new course "Nanotechnology in Food Science and Food Analysis" has been developed and introduced in the Fall 2016 and Fall 2017. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Demonstrate porous silica nanospheres (PSN) with controlled-size porosity and rationally designed pore surfaces for dual-enzyme immobilization. Objective Accomplished - A novel material, with larger porosity, Stellate mesoporous silica, offeringa hierarchical pore architecture that enables facile enzyme loading was developed as the dual enzyme host for the project; - The material was successfully synthesized at DSU; - Full characterization of the material was accomplished, encompassing the porosity evaluation (~500 m2/g), transmission electron microscopy (TEM) and scanning electron microscopy); Objective 2. Demonstrate dual-enzyme immobilization in porous silica nanospheres.Objective Accomplished. - The model system we chose is the conversion of cellobiose-to-fructose by a cascade reaction using Stellate-MSN immobilized β-glucosidases and glucose isomerase. - When chosing the enzymes to be loaded in the Stellate MSN, the project team met a feasbility barrier:commercially available enzymes are partially purified and pre-bound to solid supports and cannot be utilized in the project. The solution deployed by the team was genetic engineering,cloning the DNA encoding for the Glucose/Xylose Isomerase, (called r-GI) in bacteria BL21(DE3) , followed by expression of r-GI recombinant proteins in the BL21 CodonPlus (Stratagene) strain of E. coli; - Upon purification, the r-GI enzyme along with β-glucosidase (commercially available) have been succesfully co-loaded in the Stellate-MSN; - The material was characterized by FT-IR and porosity evaluationto demonstrate both presence of enzyme and also the pore residual accessibility toward enabling substrate molecules to reach the enzymes location; Objective 3. Demonstrate proof-of-concept for using the dual-enzyme porous silica nanospheres platform in the conversion of cellulose directly to fructose by a cascade reaction using cellulase and glucose isomerase as bio-nano-catalysts. Objective Accomplished. - The proof-of-concept for the conversion of cellobiose-to-fructose by a cascade reaction using Stellate-MSN-immobilized β-glucosidases and glucose isomerase was demonstrated by converting cellobiose to fructose in a one-step reaction mediated by the Stellate-MSN immobilized dual-enzyme system; Objective 4. Provide education to graduate and undergraduate students in the project's interdisciplinary research. Objective Accomplished - Undergraduate students associated with the project have been trained in developing materials synthesis and characterization skills. -Graduate student has been trained on enzyme purification toward enzyme immobilization. Student can express and purify the enzyme and characterize by molecular biology techniques. • Graduate student is able to load materials and test the cascade enzymatic reaction. - The graduate student associated with the project has contributed to the published journal article as first author. Student also completed his Master's of Science Degree in Applied Chemistry at DSU. Objective 5. Engage in outreach activities to disseminate the project's results to Delaware consumers toward increasing awareness of nanotechnology's impact on food products. The project will provide the opportunity of integrating current food science capabilities at DSU with the novel direction of nanotechnology-enabled enzyme-assisted bioprocessing. Objective Accomplished. - The results obtained in the course of this projecthave been presented at a prominent national conference (American Chemical Sociey Nationa meeting) and have been published in a journal with high impact in both materials and biotechnology. - A new course "Nanotechnology in Food Science and Food Analysis" has been developed and introduced in the Fall 2016 and Fall 2017.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Hao-Hsin Chi, Daniela R Radu, Geraldine Dezayas, Miranda Penney and Cheng-Yu Lai
"Stellate MSN-based Dual-enzyme Nano-Biocatalyst for the Cascade Conversion of Non-Food Feedstocks to Food Products"
Journal of Thermodynamics and Catalysis 2017, 8:185
DOI: 10.4172/2157-7544.1000185
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
"Dual-enzyme nano-biocatalyst for the cascade conversion of cellulose-derived oligomers to fructose via a glucose pathway"
Hao-Hsin Chi, Daniela Radu, Gulnihal Ozbay, Cheng-Yu Lai, 252nd American Society Meeting, AGFD: Division of
Agricultural and Food Chemistry, Philadelphia, PA, August 20-25, 2016
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Progress 09/01/15 to 08/31/16
Outputs
Target Audience:- Graduate Student performing the project activities: enzyme expression and purification; - Undergraduate student performing capstone project (mandatory in the Department of Chemistry) non-funded (credit-based) on activities related to synthesis and analysis of the materials; - American Chemical Society meeting 252th participants attending the poster session; Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduate students associated with the project have been trained in developing materials synthesis and characterization skills. Graduate student has been trained on enzyme purification toward enzyme immobilization. Student can express and purify the enzyme and characterize by molecular biology techniques. Graduate student is able to load materials and test the cascade enzymatic reaction. How have the results been disseminated to communities of interest?Results obtained to date have been presented at a prominent national conference and are currently prepared to be submitted in a journal with high impact in both materials and biotechnology. What do you plan to do during the next reporting period to accomplish the goals?Objective 3: Complete conversion of cellobiose to frunctose is sought for the next project period. Objective 4: Provide education to several more undergraduate students in the project's interdisciplinary research Complete training of graduate student and complete M.S. thesis based on the work on this project. Objective 5: Submit at least one journal article in a journal with high impact in both materials and biotechnology; Present further findings at the Materials Research Society meeting Phoenix, AZ, Spring 2017.
Impacts
What was accomplished under these goals?
Objective 1. Demonstrate porous silica nanospheres (PSN) with controlled-size porosity and rationally designed pore surfaces for dual-enzyme immobilization. Accomplished past reporting period. Additional work reported. A novel material, with larger porosity was adopted during this project year. The material, called Stellate mesoporous silica offers a hierarchical pore architecture that enables facile enzyme loading. The successful synthesis was demonstrated by changing Surfactant template changes and Directing agents (Stellate-MSN) Full characterization of material was accomplished. Objective 2. Demonstrate dual-enzyme immobilization in porous silica nanospheres. Accomplished. The model system we chose is the conversion of cellobiose-to-fructose by a cascade reaction using Stellate-MSN-immobilized β-glucosidases and glucose isomerase. Unfortunately, many commercially available enzymes are partially purified and pre-bound to solid supports and cannot be utilized in the project. Therefore, for glucose isomerase we have cloned the DNA encoding for the Glucose/Xylose Isomerase, called r-GI in BL21(DE3) competent cells, followed by expression of r-GI recombinant proteins in the BL21 CodonPlus (Stratagene) strain of E. coli. Upon purification, the enzyme along with β-glucosidase (commercially available) have been loaded in the Stellate-MSN. The material was characterized by FT-IR and nitrogen porosity to demonstrate both presence of enzyme and also the pore loading. Objective 3. Demonstrate proof-of-concept for using the dual-enzyme porous silica nanospheres platform in the conversion of cellulose directly to fructose by a cascade reaction using cellulase and glucose isomerase as bio-nano-catalysts. Ongoing; Partially Accomplished. 85% Completion The proof-of-concept for the conversion of cellobiose-to-fructose by a cascade reaction using Stellate-MSN-immobilized β-glucosidases and glucose isomerase was demonstrated by converting cellobiose to fructose in a one-step reaction mediated by the Stellate-MSN immobilized dual-enzyme system; the challenges to overcome were to fine tune the buffer system to accommodate both reactions. Although the proof-of-concept was already demonstrated complete conversion is sought for the next project period; therefore, we reported the objective as partially accomplished. Objective 4. Provide education to graduate and undergraduate students in the project's interdisciplinary research. Ongoing; partially accomplished Undergraduate students associated with the project have been trained in developing materials synthesis and characterization skills. Graduate student has been trained on enzyme purification toward enzyme immobilization. Student can express and purify the enzyme and characterize by molecular biology techniques. Graduate student is able to load materials and test the cascade enzymatic reaction. Objective 5. Engage in outreach activities to disseminate the project's results to Delaware consumers toward increasing awareness of nanotechnology's impact on food products. The project will provide the opportunity of integrating current food science capabilities at DSU with the novel direction of nanotechnology-enabled enzyme-assisted bioprocessing. Ongoing; Partially Accomplished. Results obtained to date have been presented at a prominent national conference and are currently prepared to be submitted in a journal with high impact in both materials and biotechnology.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
"Dual-enzyme nano-biocatalyst for the cascade conversion of cellulose-derived oligomers to fructose via a glucose pathway"
Hao-Hsin Chi, Daniela Radu, Gulnihal Ozbay, Cheng-Yu Lai, 252nd American Society Meeting, AGFD: Division of Agricultural and Food Chemistry, Philadelphia, PA, August 20-25, 2016
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:- Undergraduate student performing summer research on activities related to synthesis and analysis of the materials. - Undergraduate student performing capstone project (mandatory in the Department of Chemistry) non-funded (credit-based) on activities related to synthesis and analysis of the materials. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training activities : PD Lai has assisted the undergratuate students associated with the project in developing materials synthesis and charactereization skills. Graduate student joined the team in July 2015 and started training on enzyme preparation toward enzyme immobilization. Professional development: PD Lai particpation at the 250th ACS National Meeting & Exposition August 16-20, 2015. Boston, Massachusetts C. Lai, D.R. Radu: Paper CHED 147. "Infusion of sustainable chemistry concepts in the undergraduate; curricula through multiple interventions. " How have the results been disseminated to communities of interest?Presentation: 250th ACS National Meeting & Exposition August 16-20, 2015. Boston, Massachusetts C. Lai, D.R. Radu: Paper CHED 147. "Infusion of sustainable chemistry concepts in the undergraduate; curricula through multiple interventions. " What do you plan to do during the next reporting period to accomplish the goals?Objective 2. Demonstrate dual-enzyme immobilization in porous silica nanospheres. Started; to be continnued on two fronts: enzyme purification enzyme immobilization Materials hosting enzymes will be thoroughly charterized for measuring enzyme loading (enzymatic assay) and material stability. Objective 3. Demonstrate proof-of-concept for using the dual-enzyme porous silica nanospheres platform in the conversion of cellulose directly to fructose by a cascade reaction using cellulase and glucose isomerase as bio-nano-catalysts. Enzymatic conversions will be performed with the newly created host-guest material and the products will be characterized by U-HPLC (Aquired and installed May 2015). Results will be disseminated through journal articles and conference presentation Objective 4. Provide education to graduate and undergraduate students in the project's interdisciplinary research. Continue traiuning of graduate and undergraduate students in research and outreach (the later refered below; Obj. 5) An elective course in bioprocessing with specific emphasis on utilizing nanotechnology in bioprocessing is in preparation and will be offered Spring 2016. The course will be offered as elective to all DSU students and will be recommended to all Food Science students. Objective 5. Engage in outreach activities to disseminate the project's results to Delaware consumers toward increasing awareness of nanotechnology's impact on food products. Workshops open to public will be held at DSU. Advertisement will be done through local media distributors. Dr. Ozbay, who is a specialist in outreach and extension, will conduct workshop organizations and evaluation survey activities. She will be assisted by an undergraduate student, whose training pertains to Objective 4.
Impacts
What was accomplished under these goals?
Objective 1 was accomplished. Demonstration of porous silica nanospheres (PSN) with controlled-size porosity and rationally designed pore surfaces for dual-enzyme immobilization was completed. 1. Preparation of Porous Silica Nanospheres (PSN). In porous silica preparation, reaction conditions govern particle size, microstructure, porosity, morphology etc. A systematic study of two parameters was conducted: reaction time and aqueous/organic phase ratio. 2. Characterization of Porous Silica Nanospheres (PSN). To determine the structure of the obtained materials, a set of analytical tests was performed: the particles resulted from each synthesis are characterized by nitrogen sorption analysis to understand surface area per gram of material and average pore size, Scanning Electron Microscopy (SEM) for understanding microstructure and pore structure on the surface, and particle size; and Transmission Electron Microscopy (TEM) for pore structure and other microstructural features. The study conducted under this objective resulted in a thorough understanding of reaction conditions that control PSN surface area and pore size distribution, thus enabling optimization of current enzymes loading and application of PSN platform to other enzymatic bioprocesses. Two methodologies have been generated for creating materials with large surface area and further functionalization toward enzyme conjugation. One journal article is in preparation for reporting the unique properties of the synthesized materials. Objective 4. Provide education to graduate and undergraduate students in the project's interdisciplinary research. Ongoing; partially accomplished; Undergratuate students associated with the project have been trained in developing materials synthesis and charactereization skills. Graduate student started training on enzyme purification toward enzyme immobilization. Currently student learned to characterize protein purity. (SDS PAGE gel)
Publications
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