Progress 01/01/14 to 12/31/18
Outputs Target Audience:Like in 2017, this year the results of our research were disseminated through presentations at professional meetings where the target audiences included food scientists, food engineers, electrochemists, and agricultural engineers from academia and industry. Changes/Problems:The main challenge that we faced was that upon depressurization, the enzyme did not retain its increased stability despite the multiple approaches that included hydrophobic modification and chemical crosslinking. However, we discovered that immobilization and high hydrostatic pressure have additive effects on the stability of the enzyme. What opportunities for training and professional development has the project provided?One M.S. student graduated with a M.S. in Food Science this year. Her research focused on the stabilization of glucose oxidase-based biosensors under HHP and by crosslinking with glutarladehyde. A Ph.D. student worked on the fabrication of alcohol oxidase biosensors and the recent B.S. graduate student who worked as technician studying the stability of galactose oxidase started her M.S. program at the PI's laboratory. All students gained research skills, including laboratory skills, data analysis, research report writing. All students prepared and presented at least a poster at professional meetings. How have the results been disseminated to communities of interest?The results of this research were presented at the annual meetings of the Institute of Food Technologists, the American Society of Agricultural and Biological Engineers, and the Electrochemical Society. The PI also presented the resulst of this resesarch at the PI's meeting and 2018 Gordon Research Conference on Nanoscale Science and Engineering for Agriculture and Food Systems. The PI also presented this research at local seminars. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts What was accomplished under these goals?
IMPACT: Through this project we documented the stabilization effect of high hydrostatic pressure on five oxidases that are relevant to the development of enzyme biosensors. The use of chemcial crosslinking of the enzyme under high pressure did not stabilize the enzymes upon depressurization. However, upon enzyme entrapment in a polymer, the application of high hydrostatic pressure enhanced the stability of glucose oxidase more than when the enzyme is just in solution. This means that even though biosensors were not stabilized as initially intended, the use of this approach for enyzmes under high pressure for bioprocessing in foods and agriculture may become ecnomically viable, especially for processes that use expensive enzymes. RELEVANT RESULTS: The temperature-pressure profiles for different oxidases are different. Glucose oxidase immobilized in poly-o-phenylenediamine was 330 times more stable at 180 MPa, 70 deg. Celcius than the enzyme in solution at abmient pressure at the same temperature. Objective 1. Was completed this year including the additional study of study of galactose oxidase, beyond the scope of the proposal. Objective 2. Was completed in 2016 Objective 3. Further studies crosslinking glucose oxidase or alcohol oxidase beforoe or after immobilization in poly-o-phenylenediamine resulted only in modest increase in biosensor stability. However, the stability of biosensors when heated under high pressure was significantly greater than when at atmospheric pressure, which in turn is greater than for the free enzyme. This objective was completed this year for glucose oxidase and alcohol oxidase.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Jackson, J. Reyes-De-Corcuera, J.I. High hydrostatic pressure stabilization of galactose oxidase. In Annual Meeting of the Insititute of Food Technologists.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Olstad, H.E., Reyes-De-Corcuera, J.I. Increased stability of glucose oxidase-based biosensors using high hydrostatic pressure combined with glutaraldehyde cross-linking
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Yang, D., Olstad, H., Reyes-De-Corcuera, J.I. Combined Effects of High Hydrostatic Pressure and Cross-linking with Glutaraldehyde on the Stability of Glucose-Oxidase and Alcohol-Oxidase Based Biosensors. Presented at the Gordon Research Conference on Nanoscale Science and Engineering for Agriculture and Food Systems at Mount Holyoke College in South Hadley, MA, 06/07/18
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Yang, D. Olstad, H.E.,Jenkins, D. Reyes-De-Corcuera, J.I. Stabilization of Glucose Oxidase and Alcohol Oxidase Based Biosensors By Immobilization in Electrochemically Generated Poly-o-Phenylenediamine Under High Hydrostatic Pressure.Presented at the Americas International Meeting on Electrochemistry and Solid State Science, Cancun, Mexico,10/01/18
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Buchholz, M., Halalipour, A., Yang, D., Reyes-De-Corcuera, J.I.* (2019) Increased Stability of Alcohol Oxidase under High Hydrostatic Pressure. J. Food Eng. 249, 95-101
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Reyes-De-Corcuera J.I.,* Olstad, H.E., García-Torres, R. (2018) Stability and stabilization of enzyme biosensors: The key to successful application and commercialization. Ann. Rev. Food Sci. Technolo. 9: 293-322.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2018
Citation:
García-Torres, R., Reyes-De-Corcuera, J.I., Yang, D. (2018) Protection of Enzymes Against Thermal Degradation. In Encyclopedia of Food Chemistry, Varelis, P., Melton, L., Shahidi, F. Elsevier. In Press.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Olstad, H.E. Increased stability of glucose oxidase biosensors using high hydrostatic pressure and cross-linking with glutaraldehyde. University of Georgia, 2018
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Progress 01/01/17 to 12/31/17
Outputs Target Audience:Like in the previous year, in 2017 this research was disseminated through presentations at the annual PI meeting as well as at national and international professional meetings where the target audicences included food scientists, food engineers and agricultural engineers from academia and industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One student graduated with his M.S. degree in Food Science. His research focused on deteremining the effect of HHP on the stability of pyruvate oxidase. Another M.S. student and a Ph.D. student started fabricating glucose and alcohol biosensors based on glucose oxidase and alcohol oxidase. Finally, a recent B.S. graduate started determining the effect of HHP on galactose oxidase. All these students have gained a number of laboratory skills and knowledge about enzymes, electrochemical biosensors, and biocatalysis. One of them presented his research at a professional meeting in the summer 2017. The other two are expected to present their research at professional meetings in 2018. How have the results been disseminated to communities of interest?The results of this projects have been disseminated at professional meetings to the food science (IFT) and the food and agricultural engineering (ASABE) communities. Research findings have also been disseminated by the PI at seminars and other professional meetings. What do you plan to do during the next reporting period to accomplish the goals?We requested at one-year no-cost extension to complete objective three, that is the immobilization, chemical modification, and/or cross-linking of glucose oxidase, alcohol oxidase, xanthine oxidase and galactose oxidase under high hydrostatic pressure on platinized platinum electrodes will be carried out. Biosensor stability will be determined in terms of the rate of loss of sensitivity and operational life at room temperature.
Impacts What was accomplished under these goals?
IMPACT. The main challenge associated with enzyme biosensor development for food and agriculture applications is that enzymes have poor stability and degrade over time, which results in rapid loss of sensitivity and need for frequent calibration and sensor replacement within a few days. The issue of enzyme stability is true for some industrial applications of enzymes as well. Our research uses and combines three strategies to stabilize enzymes: chemical modification, crosslinking, and the application of high hydrostatic pressure and the study of the combined effects on four enzymes: glucose oxidase, alcohol oxidase, xanthine oxidase, and pyruvate oxidase. A fifth enzyme galactose oxidase was added because pyruvate oxidase was very sensitive to pressure and chemical modification. Therefore biosensor fabrication will not be attempted with pyruvate oxidase. RELEVANT RESULTS: While chemical modification increased the melting temperature of glucose oxidase, the rate of inactivation of the enzyme was not different than the native, unmodified enzyme. Pressures around 50 MPa slightly stabilized pyruvate oxidase. Galactose oxidase was stabilized by high hydrostatic pressure up to 300 MPa by up to 5 fold relative to atmospheric pressure. Objective 1. We completed objective 1 for pyruvate oxidase. We added the study of galactose oxidase to this objective and completed about 85% of the study for that enzyme. We completed 95% of this objective overall. Objective 2. That objective was completed in the previous year. Objective 3. Twenty-four sets of platinum electrodes were fabricated. The electrochemical cell for high pressure was validated and used successfully to fabricate alcohol oxidase and glucose oxidase-based biosensors. The effect of high pressure on platinization and electropolymerization was assessed. Preliminary determinations of sensitivity and stability of the resulting biosensors were carried out. Glucose electrodes fabricated under high pressure were more stable that those fabricated at atmospheric pressure. Crosslinking with glutaraldehyde, further stabilized the biosensors. However, chemical modification with phenyl groups does not appear to have impacted the stability of the resulting biosensors.Progress in this objective is approximately 10%.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Halalipour, A., Duff, M.R., Howell, E.E., Reyes-De-Corcuera, J.I.* (2017). Effects of high hydrostatic pressure or hydrophobic modification on thermal stability of xanthine oxidase. Enzyme Microb. Technol. 103, 18-24
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Reyes De Corcuera, J.I., Olstad, H.E., Garc�a-Torres, R. Stability and stabilization of enzyme biosensors: the key to sucessful application and commercialization. Annu. Rev. Food Sci. Technol.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Buchholz M., Halalipour, A., Reyes-De-Corcuera J.I. Increased Stability and Activity of Alcohol Oxidase under High Hydrostatic Pressure. Innov. Food Sci. Emer. Technol.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Reyes-De-Corcuera, J.I. High Pressure Stabilization of Enzymes Research Seminar presented at the Centro de Investigaciones Biotecnol�gicas del Ecuador, Escuela Superior Polit�cnica del Litoral. Guayaquil, Ecuador August 17, 2017
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Reyes-De-Corcuera, J.I Enzyme Stabilization at High Hydrostatic Pressure for Food Processing and Other Applications. Invited webinar given as a part of a series hosted by the International Society of Food Engineering. August 14, 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Reyes-De-Corcuera, J.I. Enzyme Stabilization at High Hydrostatic Pressure for Food Processing. Presented at the 12th Latin American Symposium of Food Science in Campinas, Brazil, November 5, 2017
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Garcia-Torres, R., Howell, E.E. Reyes-De-Corcuera J.I. Fabrication of stabilized glucose oxidase biosensors under high hydrostatic pressure. Presented at the 2017 Annual Meeting of the Institute of Food Technologists, Las Vegas, NV, 07/26/17
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Wallace, L. The effect of high hydrostatic pressure on stability of pyruvate oxidase from aeroccocus species. M.S. Thesis, University of Georgia, Department of Food Science and Technology.
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Progress 01/01/16 to 12/31/16
Outputs Target Audience:Our research, like last year, was disseminated through presentations at the annual PI meeting as well as at professional meetings where the target audiences include food scientists, food engineers, and agricultural engineers Changes/Problems:Pyruvate oxidase was the most thermally unstable enzyme. We were unable to chemically modify it without causing precipitation and the enzyme inactivated with pressure. Therefore, no further studies will be done on this enzyme. However, the data collected for this enzyme along with the data for the other enzymes that resulted in different levels of pressure induced stabilization will help us better understand the mechanisms by which enzymes are stabilized or inactivated by pressure. What opportunities for training and professional development has the project provided?Two food science graduate students, one Ph.D. and one M.S. graduated in 2016 doing their research in this project, while two additional M.S. students started working on this project in 2016. These students have gained a number of laboratory skills and knowledge about enzymes, biosensors, and biocatalysis. Students have presented their research at professional meetings where they have improved their communication skills. How have the results been disseminated to communities of interest?The results of this projects have been disseminated at professional meetings to the food science and engineering communities. The two students who graudated this year won first and second place at the graduate student competition of the biotechnology division of the Institute of Food Technologists Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?We will work on objective 3. We will immobilized the glucose oxidase, xanthine oxidase and alcohol oxidase at the optimal pressure, using chemically modified or native enzymes. In addition the enzymes will be chemically modified under high pressure to elucidate whether more effective modification can be done at high pressure. We will also use covalent immobilization techniques to further increase the stability of the biosensors. Biosensor stability will be documented.
Impacts What was accomplished under these goals?
IMPACT. The main challenge associated with enzyme biosensor development for food and agriculture applications is that enzymes have poor stability, degrade over time, which results in rapid loss of sensitivity and need for frequent calibration and sensor replacement within a few days. The issue of enzyme stability is true for some industrial applications of enzymes. Our research uses and combines two strategies to stabilize enzymes: chemical modification and the application of high hydrostatic pressure and the study of the combined effects on four enzymes: glucose oxidase, alcohol oxidase, xanthine oxidase, and pyruvate oxidase. RELEVANT RESULTS. The stability of glucose oxidase, xanthine oxidase and alcohol oxidase increased by a factor of 50, 9.5, and 3.2 respectively at their optimal hydrostatic pressures of 240, 160, and 160 MPa respectively. Chemical modification of glucose oxidase increased the temperature of denaturation by 8 ºC at atmospheric pressure. Chemical modification also stabilized alcohol oxidase but not xanthine oxidase. Pyruvate oxidase could not be modified chemically without inactivating it. Also, pyruvate oxidase was not stabilized by pressure, on the contrary, pressure inactivated that enzyme. These results provide the pressure and chemical conditions to immobilize these enzymes on electrodes which is the next step for this project. Objective 1. We completed the study of the effect of high pressure on the stability of alcohol oxidase and did about 50% of the study on the effect of high pressure on the stability of pyruvate oxidase. Objective 2. We completed the derivatization of glucose oxidase, xanthine oxidase and alcohol oxidase. We were not successful at derivatizing pyruvate oxidase as the enzyme precipitates under derivatization conditions. We completed the studies on the effects of high hydrostatic pressure on the stability and the activity of chemically modified glucose oxidase, alcohol oxidase, and xanthine oxidase. Objective 3. We further improved the electrode and electrochemical cell prototype to carry enzyme immobilization at high hydrostatic pressure and successfully tested it. Six set of platinum electrodes were fabricated using the improved design.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Halalipour, A., Duff, M. R., Howell, E. E., & Reyes-De-Corcuera, J. I. (2016). Glucose oxidase stabilization against thermal inactivation using high hydrostatic pressure and hydrophobic modification. Biotechnology and Bioengineering. doi:10.1002/bit.26185
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Halalipour, A., Duff, M., Howell, E., & Reyes De Corcuera, J. (2016). Effect of high hydrostatic pressure and hydrophobic modification on thermal stability of xanthine oxidase. Enzyme and Microbial Technology
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Reyes De Corcuera, J. (2016). Enzyme Stability. The Missing Link in Enzyme Biosensor Development. In Conference of Food Engineering
International, Invited
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Reyes De Corcuera, J. (2016). Immobilization and Stabilization of Enzyme Biosensors. In Annual Meeting of the Institute of Food Technologists
International, Invited
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Halalipour, A., Duff, M., Howell, E., & Reyes De Corcuera, J. (2016). High Hydrostatic Pressure: A Novel Method of Glucose Oxidase Enhancement Combined with Hydrophobic Modification. In Annual Meeting of the Institute of Food Technologists
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Buchholz, M., Halalipour, A., Howell, E., & Reyes De Corcuera, J. (2016). Increased Stability and Activity of Alcohol Oxidase at High Hydrostatic Pressure. In Annual Meeting of the Institute of Food Technologists
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Buchholz, M., Halalipour, A., Howell, E., & Reyes De Corcuera, J. (2016). Stabilization of Alcohol Oxidase at High Hydrostatic Pressure. In 7th Annual Southeast Enzyme Conference
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Halalipour, A., Duff, M., Howell, E., & Reyes De Corcuera, J. (2016). Increased Activity of Glucose Oxidase under High Hydrostatic Pressure for Food Processing Applications. In 7th Annual Southeast Enzyme Conference
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2016
Citation:
Halalipour, A. High hydrostatic pressure combined with hydrophobic modification to enhance stability and activity of glucose oxidase and xanthine oxidase, Ph.D. Dissertation, Department of Food Science and Technology University of Georgia, 2016
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2016
Citation:
Buchholz, M.I. Increased stability and activity of alcohol oxidase under high hydrostatic pressure. M.S. Thesis, Department of Food Science and Technology, University of Georgia. 2016
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Progress 01/01/15 to 12/31/15
Outputs Target Audience:Our research efforts have been disseminated through presentations at professional meetings where the target audiences include the scientific communities of Food Scientists and Agricultural Engineers. Changes/Problems:The two main challenges that we faced this year dealt with the solublity of derivatized pyruvate oxidase and xanthine oxidase. This year we will continue working on these protocols. Relative to alcohol oxidase, studying what appears to be two populations of enzymes with different thermal stability, essentially duplicated the work for that enzyme, which delayed our study with pyruvate oxidase. These two challenges may delay the delivery of final results. We do not belive at this point that such challenges will require an change in scope. An addition to the project was the development of a pH sensor for high pressure. Although out of the scope of the proposal, the development of such sensor will help better understand whether pH shifts contribute to the observed stabilization of the enzyme under high pressure and the extent of that contribution. What opportunities for training and professional development has the project provided?Three graduate students were trained under this project. Students have learned enzyme reaction kinetics, enzyme inactivation kinetics, computer programming with LabVIEW, electrochemistry, and have learned to write scientific reports in the form of posters and oral papers presented at professional meetings as well as drafts of scientific journal papers. Students involved in this project have startes writing their thesis/dissertations. How have the results been disseminated to communities of interest?The results of this research have been disseminated through presentations at three professional meetings/ scientific conferences What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will complete the studies on the stabilization of alcohol oxidase and pyruvae oxidase at high pressure and publish the results for glucose oxidase, xanthine oxidase and alcohol oxidase in refereed journals. Objective 2. We will complete the stabilization of pyruvate oxidase and xanthine oxidase by derivatization wiht phenyl groups and determine the thermal inactivation of all derivatized enzymes at high pressure. We intent to publish the results of the stabilization by derivatization of glucose oxidase and xanthine oxidase in refereed journals. Objectve 3. We will build stabilized biosensors under high pressure. We expect to achieve this objective by the end of 2016 for glucose oxidase and alcohol oxidase.
Impacts What was accomplished under these goals?
Objective 1. The studies on the thermal stability at high pressures of glucose oxidase and xanthine oxidase were completed. A first draft of a researh paper was written. The studies on the thermal stability at high pressures of alcohol oxidase was initiated. Alcohol oxidase displayed an unusual rate of inactivation suggesting two populations with different thermal stability. The difference was exacerbated by pressure. This required doubling the number of experiments. Objective 2. The effects of derivatzing phenyl groups on pyruvate oxidase and xanthine oxidase were studied. Derivatization of these enzymes proved more difficult than the other two becuase of precipitation of the protein upon derivatization. Objective 3. High pressure electrochemical cells were fabricated.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Halalipour, A, Howell, E., Reyes-De-Corcuera, J.I. Glucose Oxidase and Alcohol Oxidase Stabilization under Optimized High Hydrostatic Pressure. Presented at the 2016 Annual International Meeting of the American Society of Agricultural and Biological Engineers. New Orleans, LA, July 28, 2015
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Halalipour, A, Duff, M., Howell, E., Reyes-De-Corcuera, J.I. Optimized High Pressure Stabilization of Glucose Oxidase for Biosensor Construction. Presented at the Nanoscale Science & Engineering for Agriculture & Food Systems Conference, Waltham, MA, June 8, 2015
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Reyes-De-Corcuera, J.I., Halalipour, A. Enzme Stabilization at High Hydrostatic Pressure. Presented at the 10th International Congress of Chemical, Food, and Environmental Engineering, Puebla, Mexico, April 10, 2015
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Progress 01/01/14 to 12/31/14
Outputs Target Audience: We are at the stage of collecting the first sets of data. Changes/Problems: The only major problem we faced this year was the delayed delivery of the high pressure equipment. To compensate for this, we recently pruchased also a high pressure manual pump that will be used to carry out experiments that do not require reproducible, fast pressurization. This will allow us to work on objectives 1 and 3 in parallel. What opportunities for training and professional development has the project provided? Two food science students have been learning about pressure stabilization of enzymes, design of cells for electrochemical experiments and enzyme kinetics How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Objective 1. We will complete the determination of optimal pressures for enzyme stability for all four enzymes Objective 2. We will complete the determination of the effect of phenyl group derivatization for the remaining enzymes (pyruvate oxidase and xanthine oxidase) Objective 3. We will complete the design and testing of a new electrochemical cell prototype and produce stabilized glucose oxidase electrodes
Impacts What was accomplished under these goals?
Objective 1. The pressure for optimal stability of glucose oxidase was determined. We sarted experiments with alcohol oxidase. Objective 2. The effects of derivatization of phenyl groups onto amino or carboxy groups were characterized for glucose oxidase and alcohol oxidase. Objective 3. Nothing to report
Publications
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