ELIMINATING UNDESIRED GREENING IN SUNFLOWER SEED DERIVED INGREDIENTS BY HYDROLYSIS OF CHLOROGENIC ACID USING AN ENGINEERED ESTERASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022168
• Grant No.: 2020-67018-31261
• Cumulative Award Amt.: $196,405.00
• Proposal No.: 2019-06807
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2023
• Grant Year: 2020
• Program Code: [A1364]- Novel Foods and Innovative Manufacturing Technologies

Recipient Organization
CHAPMAN UNIVERSITY
ONE UNIVERSITY DRIVE
ORANGE,CA 92866

Performing Department
Food Science & Nutrition

Non Technical Summary
The goal of the proposed research is to develop biotechnology that will enable broader use of sunflower seed-derived ingredients in high pH baking applications. Sunflower seed-derived ingredients represent alternatives to tree nut and legume-based proteins, butters, and spreads, which cause allergic reactions for many people. Where a light color is desired in high pH applications, the use of sunflower seed-derived ingredients (sunflower seed butter, flour, and meal), ishindered by the high concentration of chlorogenic acid in sunflower seeds. Chlorogenic acid reacts with food components such as amino acids at high temperature and alkaline pH, common conditions encountered in baking. This reaction produces an intense green color that can limit the application of sunflower seed-derived ingredients in products with an alkaline pH where a white to beige color is wanted. To date, there is no known method of entirely eliminating this greening without acids or simultaneous removal of beneficial antioxidant compounds that are present in sunflower seed-derived ingredients. To overcome this problem, we propose a new way of removing chlorogenic acid. We will use a chlorogenic acid esterase enzyme to break down chlorogenic acid and thereby eliminate greening. This approach will enable the cleavage of chlorogenic acid without affecting desirable antioxidant compounds.The major innovation of the proposed research is to engineer chlorogenic acid esterase with high activity and stability for use in baking applications. The chlorogenic esterase we will use is from Lactobacillus, a genus of bacteria that is already in use in food manufacturing. We will engineer the esterase based on state-of-the-art computationally-aided protein design strategies for maximal activity and stability. We will then produce the esterase in large quantities in the bacteriumE. coli, lyse the bacterium and purify the enzyme. After that, we will utilize the purified enzyme to break down chlorogenic acid in sunflower meal and flour. This original approach can potentially generate a marketable enzyme for thefood industry. Furthermore, since enzymes are biological molecules, we expect positive consumer perception of the sunflower seed-derived ingredients colored. If successful, work stemming from this proposal will convert sunflower seed ingredients such as flour or the meal, which is currently a low-value byproduct of oilseed-pressing that is mainly used as animal fodder, into a desirable, high-value baking product.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	4010	1000	60%
502	1844	2000	40%

Knowledge Area
501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
4010 - Bacteria; 1844 - Sunflower;

Field Of Science
1000 - Biochemistry and biophysics; 2000 - Chemistry;

Keywords

chlorogenic acid

enzyme engineering

greening

sunflower seed

Goals / Objectives
The overall research goal is to develop an enzyme-based processing strategy utilizing chlorogenic acid esterase (CE) to hydrolyze chlorogenic acid (CGA) and thereby prevent greening in sunflower seed-derived products. The specific objectives are to:(1) Use directed protein evolution strategies to engineer CE from Lactobacillus johnsonii for enhanced activity and stability.(2) Determine the enzymatic activity of engineered CE towards CGA and its ability to degrade CGA in sunflower seed-derived ingredients and prevent greening.(3) Scale-up enzyme production in a fermenter for future industrial application of engineered CE.

Project Methods
(1) General protein expression and purification methods: A chlorogenic esterase (CE) from L. johnsonii will be cloned into a pET28a vector using PCR and restriction digest methods. CE will be expressed in E. coli, where expression is induced by addition of IPTG. Protein will be purified by nickel affinity chromatography. Protein purity and folding will be assessed by SDS-PAGE and circular dichroism spectroscopy, respectively.(2) Protein engineering and library construction methods: The amino acids that will be targeted for engineering will be chosen based on FoldX predictions and B-factor analysis. We will start with Thr 198 and Asn 237, two residues with low ΔΔGfolding predictions. To engineer CE, mutagenesis will be performed using PCR-based methods, where each target amino acid will be mutated to all other possible amino acids.(3) Screening for activity and stability: We will find the most promisingvariants based on two criteria, CGA degradation and thermal stability. CGA degradation will be measured using a high throughput fluorescence-based activity assay with cell lysate, as CGA and its degradation products have different fluorescence signatures. The most active four variants will then be expressed in 2 L E. coli cultures and purified using methods described in (1). Thermal stability will then be measured using a fluorescence-based thermal unfolding assay and circular dichroism spectroscopy.(4) Enzymatic characterization: Enzymatic characterization will be determined bythe Michaelis-Menten kinetics of the purified CE variants. Assays will be conducted over a range of buffer conditions, and HPLC will be used to quantify CGA and its degradation products. Steps 2-4 will be conducted iteratively to improve CE activity over generations of engineered mutants.(5) De-greening studies in sunflower seed-derived ingredients: Sunflower seed-derived ingredients differing in protein, lipid, and fiber content will be treated with different concentrations of the engineered enzyme at pH 7 to 11 prior to isoelectric precipitation and drying of protein isolates. The proximates and color of the sunflower protein isolates obtained at the various conditions will be measured by the CIE L∗ a∗ b∗ color system, where L∗ denotes lightness (L∗ = 0 denotes black and L∗ = 100 represents white), a∗ is redness (+) or greenness (−), while b∗ denotes yellowness (+) or blueness (−). Response surface methodology will then be used to select the combination of factors that result in the lightest colored protein and determine the impact of enzyme activity on color vs. non-treated controls. Triplicate concentrates and isolates from sunflower seeds will then be tested using the optimal combination of pH, time and enzyme concentration. Analysis of variance and Tukey test will be used to determine the level of statistical significance(6) Scale-up enzyme production for de-greening studies: After a high-performing enzyme is found that is active in small-scale de-greening studies, we will scale-up enzyme production. We will optimize CE expression in E. coli in a 4 L pilot-scale bioreactor and perform mid-scale protein purification and de-greening studies in sunflower ingredients by scaling-up procedures mentioned in (1) and (5).Efforts: Undergraduate and graduate researchers: Carrying out the methods described above will involve training two undergraduate students and a graduate student. Both undergraduate and graduate students will be involved in all aspectsof the project, including hands-on experimental work, experimental design, data analysis, and dissemination of the results.Broader student population: This proposal will benefit not only students working directly on this project but also those enrolled in related courses, including Food Chemistry, which has an annual enrolment of 20-30 students, and Physical Biochemistry Laboratory, which has an annual enrolment of 15-25 students.Students in Physical Biochemistry will carry out laboratory experiments that are based on this research to learn how to study protein stability and enzymatic activity. Using CE mutant protein generated in this project, students will determine protein stability using circular dichroism spectroscopy. Furthermore, students will determine the Michaelis-Menten kinetics of their respective mutants using fluorescence-based analysis of CGA breakdown. These experiments are inquiry-based learning opportunities as the experimental results are unknown to students. Furthermore, the incorporation of these experiments in the biochemistry curriculum represents an opportunity to expose students to biotechnology challenges that are relevant to food scientists.In the Food Chemistry course, findings stemming for this project will be incorporated into the enzymology unit. This project will represent a significant addition to the curriculum by introducing research on the practical use of enzymes in food chemistry. In addition to the Food Chemistry course, the PI teaches Food Ingredients. Students in this course will learn how enzymes affect color and other characteristics in food to facilitate the efficient development of enzymes as processing aids.Evaluation: The ultimate goal of this proposal is to successfully engineer CE to fully prevent greening in baked goods that contain sunflower derived ingredients. Intermediate targets along the way are to:1) Produce pure recombinant protein in high yields (>100 mg/L of culture).2) Demonstrate enzymatic activity against CGA (greater than 95% hydrolysis) in high throughput assays.3) After we demonstrate that CE can effectively break down CGA in vitro, we will apply wild-type and engineered enzymes to food matrices containing CGA. Technical success criteria will be assessed by comparing the degree of de-greening to the processing and ingredient substitutions investigated, to date. Higher de-greening compared to current strategieswill be considered a success. We expect de-greening to be successful if >95% CGA is hydrolyzed. The enzyme will be engineered until this threshold is met.4) Curricular efforts will be evaluated based on departmental student learning goals to measure specific scientific skills, including the ability to design experiments, data analysis, critical thinking and technical communication

Progress 06/01/20 to 05/31/23

Outputs
Target Audience: Scientific community. Researchers working on: Related esterases e.g., ferulic acid esterases Protein engineering Upcycling sunflower meal Chlorogenic acid breakdown in foods and biomass Sunflower seed meal processors and sunflower oil producers Producers of alternative flours and sunflower-seed derived protein powders Food processors needing to break down chlorogenic acid such as: Bakers using sunflower seed butter and sunflower seed flour at alkaline conditions. Coffee producers and roasters interested in less bitter coffee. Enzyme manufacturers (makers of enzymes used baking) Changes/Problems:None What opportunities for training and professional development has the project provided?teaching and mentoring of students (10) The grant provided training for eight undergraduate and two M.S. students. Students were involved in experimental design, executing experiments, and data analysis. Students worked independently after being trained in the relevant techniques, and carried out experiments that were used in publications. Eight undergraduate students worked on this project during the semester. One student later continued working on this project as a full-time research technician. Students took part in data dissemination as all eight are coauthors on publications (two accepted, two submitted). Students also disseminated results at conferences. Thus, students received training in science writing and communication. The M.S. level student featured on one paper also received training in science writing, working with enzymes, and data analysis. How have the results been disseminated to communities of interest? Two published papers (Food Research International and LWT) Two papers under review Three presentations at the American Oil Chemist's Society meeting and one at the Society for Industrial Microbiology and Biotechnology and the Protein Society Talks at Carleton College, Chapman University What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our group accomplished the stipulated goals of the grant. 1) Enzyme characterization and CGA breakdown in sunflower meal We cloned several bacterial CGA esterases, determining that L. helveticus (Lh) CGA esterase was most amenable for recombinant expression in E. coli and for easy protein purification. The activity of Lh CGA esterase is very high, having the highest reported kcat of known CGA esterases. Notably, it is much higher than fungal CGA esterases. Lh CGA esterase is thermally stable and can be stored for a long time. Lh CGA esterase is active in a wide range of pH of 6-9. Lh CGA esterase catalyzes complete CGA hydrolysis in sunflower meal. The enzyme is active in both non defatted and defatted meal, meaning that lipids to not compete with CGA for the enzyme's active site. Enzymatic CGA hydrolysis led to the production of non-green alkaline extracted sunflower protein powders. Enzyme activity on the meal was characterized, and was lower than in aqueous solutions, possibly due to the presence of inhibitors in sunflower meal. However, the in-situ enzyme activity was comparable to that of enzymes such as lipases and xylanases that are used in the baking industry. 2) Greening prevention in baked goods and sensory analysis CGA was enzymatically hydrolyzed in sunflower meal and butter. The esterase-treated meal and butter were used to make sunflower butter and sunflower flour cookies. Enzymatically treated cookies were not green, while untreated control cookies were green. Enzymatic greening prevention was compared to chemical de-greening using cysteine and glutathione. These thiols were chosen since they were known to prevent CGA-induced greening in protein powders. Greening prevention was much more effective using CGA-esterase compared to cysteine and glutathione treatment. Proximate analysis determined that enzymatic treatment does not alter composition of sunflower flower cookies other than color, as hypothesized. Enzymatically treated cookies were evaluated using 153 untrained panelists. The taste, texture and flavour between treated and untreated cookies were the same. However, the green color was viewed very negatively by the panelists. The sensory experiment demonstrated that color was an important factor in determining consumer preference as consumers preferred non-green cookies. 3) Structure determination, mutational analysis and protein engineering The structure of Lh CGA esterase was determined, and was found to be similar to related alpha-beta hydrolases, as expected. A lysine residue at the entrance to the active site is important for its function. The lysine residue hydrogen bonds to CGA, giving the enzyme specificity towards CGA vs. other hydroxycinnamic acid derivatives. Mutation of the active site lysine to alanine reduces CGA specificity but increases kcat (enzyme activity). Under conditions where CGA is abundant and the most common hydroxycinnamic acid derivative, such as in sunflower meal, the lysine to alanine mutant is expected to be more active compared to the wild-type enzyme. Iterative saturation mutagenesis (ISM) approaches were used to engineer the enzyme for both higher activity and greater thermal stability. Mutation sites were analyzed using B-factor analysis of the crystal structure. After three rounds of ISM, several mutants with identical activity as wild-type Lh CGA esterase were identified, but none with higher activity. In summary, L. helveticus CGA esterase exhibits the highest CGA hydrolysis activity to date and prevents greening in sunflower seed matrices. We thus present CGA esterase as a "clean label" ingredient that can be used in high pH baking applications.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: 1) Christine Lo Verde, Nana Baah Pepra-Ameyaw, Charlie Drucker, Kate Lyon, Julia Muniz, Tracie Okumara, Chloe Sermet, Lilian Were Senger, Cedric Owens.2022. A Highly Active Esterase from Lactobacillus helveticus Hydrolyzes Chlorogenic Acid in Sunflower Meal to Prevent Chlorogenic Acid Induced Greening in Sunflower Protein Isolates. Food Research International 2022,111996, ISSN 0963-9969, https://doi.org/10.1016/j.foodres.2022.111996. 2) Nana Baah Pepra-Ameyaw, Christine Lo Verde, Charles T. Drucker, Cedric P. Owens, Lilian W. Senger. 2022. Preventing chlorogenic acid quinone-induced greening in sunflower cookies by chlorogenic acid esterase and thiol-based dough conditioners. LWT, 114392, ISSN 0023-6438, https://doi.org/10.1016/j.lwt.2022.114392.
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: 1) Christine Lo Verde; Criselda Pacioles; Natalie Paterson; Jamie Chin; Cedric Owens, Lilian Senger.Hydrolysis of chlorogenic acid in sunflower flour increases consumer acceptability of sunflower flour cookies by improving cookie color. 2)Kellie K. Omori, Tracie L. S. Okumura, Nathaniel B. Carl, Brianna T. Dinn, Destiny Ly, Kylie N. Sacapano, Allie Tajii, Cedric P. Owens. Uncovering structural features that control substrate specificity in a Lactobacillus chlorogenic acid esterase.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: o American Oil Chemists' Society (AOCS).Christine Lo Verde; Criselda Pacioles; Cedric Owens, Lilian Senger. 2023. Valorization of sunflower meal using chlorogenic acid esterase: Antiglycation and Sensory analysis of cookies made with sunflower meal
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 1) K. Omori, B. Dinn, K. Sacapano, D. Ly, C. Monahan, T. Okumura, C. Lo Verde, L. Were, C. Owens. July 9, 2022. Engineering Chlorogenic Acid Esterases for Greater Activity Protein Society Conference. San Francisco, California 2) Christine Lo Verde, Cedric Owens and Lilian Were. 2022. Engineering Lactobacillus helveticus chlorogenic acid esterase for sunflower meal processing. Society for Industrial Microbiology and Biotechnology. San Francisco, California

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:1) Sunflower seed meal processors 2) Producers of alternative flours and sunflower-seed derived protein powders Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the summers, we have students from the Chapman University Summer Undergraduate Research Fellowship work in the lab. They are taught basic wet lab skills including protein purification, scientific baking with sunflower seed derived ingredients, and HPLC protocols. There fellowship requires them to present a poster at the end of the summer with which we help advise them. During the semesters we have undergraduate students that work in the lab. Most participate in the Chapman University Student Scholar Symposium offered twice a year. During the semester each student has a developed curriculum with goals that are to be met by the end of the semester and work with other students and the research assistant to progress the project. In an outreach effort, we take students from the SURFEE program, which brings students from various community colleges to come collaborate and learn research skills. Absorbance spectrometry was used to quantify Michaelis-Menten parameters of CGA esterase. Cholorogenic/CGA and caffeic acid have different absorbance spectra which allowed us to measure the depletion of CGA, seen by a change in absorbance at 340 nm. Then we utilized that absorbance change and used a derived equation that ultimately measures the rate of the reaction. This allowed us to compare our enzyme's activity with other esterases that have been previously studied. It also allowed us to calculate the amount of enzyme needed to break down CGA in either sunflower meal or sunflower butter. We further tested Michaelis-Menten parameters in sunflower meal. Here we used HPLC which allowed us to separate the CGA from the caffeic acid. Further, this method allowed us to calculate concentrations of both CGA and caffeic acid. For samples treated with enzyme, there was no CGA peak evident on HPLC, and there was a large peak eluting at 5.4 min which was indicative of caffeic acid. To track reaction progression, we stopped the activity of the enzyme by adding HCl at different points in time. To evaluate the efficacy of CGA esterase when making sunflower seed derived cookies, CIE L*a*b* values were taken at regular intervals after baking. Enzyme-treated cookies were compared to control cookies that have not received the enzyme. Control cookies exhibited a green pigment (indicated by a negative a* value) while enzyme treated cookies hold positive a* values. 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?Revise the manuscripts submitted and present work at a conference

Impacts
What was accomplished under these goals? Biochemical characterization of CGA esterase indicated a mix of α-helices and β-sheets comprised its secondary structure CGA esterase was active across a wide range of temperature (20°C-50°C) and pH (6.0-9.0) will increase its interest in food industrial applications Activity of CGA esterase had a Km of 0.090 mM and a kcat of 82 s-1 L. helveticus CGA esterase exhibited a 8500-fold higher Vmax compared to fungal esterases Our work presents the sole use of CGA esterase, without need for acid extraction, and ability to work in alkaline conditions First attempt to accurately test Michaelis Menten kinetics in sunflower meal Full hydrolysis of CGA indicates full prevention of greening in sunflower protein isolates First attempt to fully prevent greening in cookies made with sunflower seed ingredients CGA hydrolysis occurs within sunflower cookies made with either sunflower butter or sunflower flour CGA esterase has potential use as an ingredient for baking applications CGA esterase represents a "clean label" approach to prevent CGA-induced greening compared to currently utilized methods for greening prevention that requires harsh chemicals or eliminates beneficial phenolic capacity Provides an alternative use for sunflower meal other than discarding it or using it as a fertilizer

Publications

• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: 1) Lo Verde, C., Pepra-Ameyaw, N., Drucker, Okumara, T; Lyon. K; Muniz, J; Sermet, C C. Were, L, Owens, C.P. (submitted in May). A Highly Active Esterase from Lactobacillus helveticus Breaks Down Chlorogenic Acid in Sunflower Meal and Prevents Chlorogenic Acid Greening in Sunflower Protein Isolates. Food Research International
• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: 2) Pepra-Ameyaw, N., Lo Verde, C., Drucker, C. Owens, C.P. Were, L. (submitted in May). Preventing chlorogenic acid quinone-induced greening in sunflower cookies by chlorogenic acid esterase and thiol-based dough conditioners. Food Research International

Progress 06/01/20 to 05/31/21

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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?While we have completed characterizing their Michaelis-Menten kinetics, we still need to solve the structures ofL. helveticusandL. gasseriesterases and determine the enzymes' thermodynamics. This work will be completed within about two months. Finally, we improved a fluorescence-based assay that will allow us to screen for enzymatic activity more rapidly. Together this work will allow us to start iterative scanning mutagenesis for enzyme engineering. We will begin the first round of iterative scanning mutagenesis in early summer to assess the efficacy of our PCR methods, and to determine the number of reactions required to create the needed number of mutations. We expect to begin the enzyme design portion of the project in July 2021 and complete it by the end of the year. Before the next reporting period, we also intend to work on our second objective toDetermine the enzymatic activity of engineered CE to degrade CGA in sunflower seed-derived ingredients and prevent greening.

Impacts
What was accomplished under these goals? Activities during the campus shutdown (June 1st2020 - Jan. 4 2020) We used this time to plan future work as much as possible, for example ordering plasmids and other reagents. We were also able to train an undergraduate student between October and early December 2020 on general lab techniques, but did not start actively working on the project. Activities since January 2021 (January 4st- Current) Starting in January 2021 it became easier to carry out research on campus and we have made steady progress since then. We focused our work on completing the first two goal of the proposal, to create a mutant library for improving the activity of bacterial chlorogenic acid esterases, and to determine large-scale expression and purification conditions. To do so, we first cloned and purified three wild type esterases fromLactobacillus helveticus, L. gasseriand L. johnsonii. We have tested large scale expression methods with wild-type enzyme, finding thatL. helveticusandL. johnsoniienzymes are expressed in high yield whereasL. gasserichlorogenic acid esterase isn't. Currently, we are determining how to improve theL. gasseriesterase yield. We have also been working on completing the initial characterization of the wild-type enzymes.

Publications