Source: UNIVERSITY OF GEORGIA submitted to
NANOSCALE SCAFFOLDING OF ELECTROCHEMICALLY GENERATED POLYMERS FOR IMMOBILIZATION AND STABILIZATION OF ENZYME BIOSENSORS UNDER HIGH PRESSURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1001899
Grant No.
2014-67021-21604
Project No.
GEO2013-01776
Proposal No.
2013-01776
Multistate No.
(N/A)
Program Code
A1511
Project Start Date
Jan 1, 2014
Project End Date
Dec 31, 2018
Grant Year
2014
Project Director
Reyes De Corcuera, J. I.
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Department of Food Science and Technology, CAES
Non Technical Summary
Rapid detection of pathogen biomarkers, pesticides, fertilizer residues, sugars, alcohol and other compounds is very important to the food and agricultural industries to ensure food safety and quality, as well as to diagnose plant diseases. Because food and agricultural samples are complex mixtures of hundreds of compounds, analyses typically require tedious sample treatment, compound separation, and identification. Enzymes that specifically interact with compounds of interest can be coupled to electrodes to make biosensors that directly and rapidly measure such compounds in complex mixtures. Enzymes are very large proteins consisting of series of amino acids and are folded in a complex manner. Arguably the most difficult challenge that prevents the development of practical, reliable enzyme biosensors is that, over time and under the effect of temperature, enzymes unfold and lose their activity. The stability of enzymes depends in part on the balance between hydrophobic and hydrophilic groups in the protein. In addition, application of high hydrostatic pressure often stabilizes enzymes. This research focuses on the stabilization of enzyme biosensors relevant to food and agriculture using a combination of nanoscale and chemical manipulations of enzymes at high hydrostatic pressure (HHP). Chemical attachment of hydrophobic groups to hydrophilic residues on the enzyme, followed by attachment or entrapment in nanofilms as they grow under of HHP is expected to maximize the stability of the selected enzyme biosensors. The enzymes selected for this research are xanthine oxidase, pyruvate oxidase, alcohol oxidase, and glucose oxidase. These enzymes can be used, for example to measure xanthine in fish to determine freshness, phosphates in water to determine the presence of fertilizer residues, alcohol and glucose in beverages. The specific objectives of this research are: 1) to determine the optimal pressure for maximal stability and activity for each of the selected enzymes; 2) to characterize the effect of attaching hydrophobic groups to the enzymes at atmospheric pressure or at HHP on the stability of each enzyme; and 3) to fabricate stable enzyme biosensors by entrapping modified enzymes and attaching them to nanofilms grown on the surface of platinum electrodes.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7115010100010%
4047299202080%
5017410202010%
Goals / Objectives
The overall objective of this applicationis to stabilize oxidase-based biosensors. The specific objectives of this proejct are: Objective 1: Determine the optimal pressure for maximal stability and activity of xanthine oxidase, pyruvate oxidase, alcohol oxidase, and glucose oxidase. The working hypothesis, strongly supported by our preliminary data on glucose oxidase and other enzymes is that HHP below 400 MPa stabilizes oxidases in buffered solutions. Free oxidase activity will be monitored with spectrophotometric assays. Enzymes, in buffered solutions will be treated at selected HHP. Objective 2: To identify the effect of derivatizing hydrophobic compounds at Patm or HHP. The working hypothesis is that covalently attaching hydrophobic molecules of benzene carboxylic acid or aniline to amino or carboxylate side groups respectively will stabilize the enzymes. Stability and structural changes will be determined as described in Objective 1. Objective 3: To fabricate highly stable amperometric enzyme biosensors using nano-scaffolds of enzymes entrapped and crosslinked in nanofilms electrochemically generated at HHP. The working hypothesis is that under HHP, the enzyme is at its most stable conformation and polymerization under these conditions results in stability retention upon depressurization. Stability of resulting biosensors will be characterized in terms of the amperometric response under continuous operation.
Project Methods
The main milestones of this project are the accomplishement of each objective. In Objective 1 of this project, the selected enzymes will beexposed to selectedhigh pressuresand temperatures combinations. The residual activity aftertreatments for selected amounts of timewill be determined to determine the optimal presure for each enzyme.The kinetics of enzyme inactivation will be characterized. Protein unfolding willbe monitoredby in-situfluorescence determinations in ahigh pressure optical cell. Enzyme conformation after selected treaments willbe studiedbycircular dichroism and differential scanning calorimetry.Although the stability of several enzymes under high hydrostatic pressure has been studied, to the best of our knowledge there are no studies for alcohol oxidase, xanthine oxidase, pyruvate oxidase or glucoseoxidase.Objective 1 will be completed at the end of the first year of this project. Success consists in the determination of the optimal pressure for each enzyme and the relative increase in stability with respect to treatments at atmospheric pressures. Accomplishement of Objective 1 will be documented as publications in refereed journals and in the annual report of this project. In Objective 2, the enzymes will be chemically modified at the optimal presure from objective1 or atmospheric pressure,tochange their stability.Stabilization upon chemical modification will bedeterminedby treating the enzymes atmoderate temperaturesand determining the residual activity at atmospheric pressure.Changes in enzyme structure will be characterzed by circular dichroism. Protein unfolding will be monitored by fluorescence at ambient pressure.To the best of our knowledge, enzymes have not been chemically modified under pressure. Objective2 will be completed at the end of thesecond year of this project. Success consists in enzymes with increased stability as a result of chemica modificationfor each enzymeThe relative increase in stability will be determinedwith respect to native enzymes. Accomplishement of Objective2 will be documented as publications in refereed journals and in the annual report of this project In Objective 3, enzyme biosensors will be fabricated under high hydrostatic pressure by immobilizing the enzymes (native and chemically modified) in electrochemically generated polymers. The stability of the resulting biosensors will be determined by monitoring the sensor amperometric response at constant substrate concentration. Biosensors fabricated using native enzymes will be used as controls.Biosensors that display the greatest stability will be further caracterized in terms of linearity, limit of detection, sensitivity, sensor-to-sensor reproductiblity and drift. To the best of our knowledge, there has been no research on immobilizing enzymes at hight pressure in electrochemically generated polymers. Objective3 will be completed at themiddleof the third year of this project. Success consistsin the development of highly stable enzyme biosensors.Two to ten fold more stable biosensorsare expected. Accomplishement of Objective3 will be documented as publications in refereed journals and in the final report of this project.

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


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.


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


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


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