[en] The demand for sustainable and natural alternatives in the flavor and fragrance industries has driven interest toward enzymatic synthetic routes. This study reports the enzymatic production of geranyl benzoate, a monoterpenoid ester with potential bioactivity and commercial applications, catalyzed by immobilized lipases using a solvent-free system. A central composite design was employed to optimize the key process variables: molar ratio, temperature, and enzyme loading using Lipozyme 435 as the biocatalyst. Under optimal conditions (1:7 molar ratio, 80°C, 30% enzyme), a high geraniol conversion (72%) was achieved within 6 hr, as validated by a predictive model. The study also investigated the inhibitory effects of coproduced methanol, demonstrating that open-reactor systems (enabling methanol evaporation) enhanced conversion (>99% in 48 h) compared to closed systems. Additionally, substrate-exposure experiments highlighted methyl benzoate's inhibitory effect on Lipozyme 435, reducing its activity by 54%, whereas Lipura Flex showed greater resilience. Reusability tests indicated a decline in enzyme performance, with Lipozyme 435 retaining 22% activity after five cycles. Cell tests demonstrated no cytotoxicity and proliferative effect at high concentrations. These findings highlight the potential of solvent-free enzymatic processes for sustainable production of geranyl benzoate and provide insights into operational challenges, including methanol management and biocatalyst stability.
Disciplines :
Chemistry
Author, co-author :
Meola, Domenico ; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy
Chaibi, Chaimae ; Université de Liège - ULiège > TERRA Research Centre > Chemistry for Sustainable Food and Environmental Systems (CSFES) ; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy
Aprile, Simona; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy
Cesca, Karina ; Department of Chemical Engineering and Food Engineering, Federal University of Santa Catarina, Florianópolis, Brazil
de Oliveira, Debora ; Department of Chemical Engineering and Food Engineering, Federal University of Santa Catarina, Florianópolis, Brazil
de Paula, Ariela Veloso; Department of Bioprocess Engineering and Biotechnology, São Paulo State University, Araraquara, Brazil
Presini, Francesco ; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy
Zappaterra, Federico ; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy
Giovannini, Pier Paolo ; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy
Lerin, Lindomar Alberto ; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy
Language :
English
Title :
Development of a Sustainable Biocatalytic Process for Geranyl Benzoate Production Using Immobilized Lipase.
C. Ortiz, M. L. Ferreira, O. Barbosa, et al., “Novozym 435: The ‘perfect’ Lipase Immobilized Biocatalyst?,” Catalysis Science & Technology 9 (2019): 2380–2420.
D. Remonatto and L. A. Lerin, “Biocatalysis and Bioactive Molecules: Future and Development,” International Journal of Molecular Sciences 24 (2023): 5571.
L. N. dos Santos, R. F. Perna, A. C. Vieira, A. F. de Almeida, and N. R. Ferreira, “Trends in the Use of Lipases: A Systematic Review and Bibliometric Analysis,” Foods 12 (2023): 3058.
F. Zappaterra, F. Presini, D. Meola, et al., “Green Biocatalysis of Xylitol Monoferulate: Candida antarctica Lipase B-Mediated Synthesis and Characterization of Novel Bifunctional Prodrug,” BioTech 14 (2025): 25.
Y. Liu, X. WeiZhuo, and X. Wei, “A Review on Lipase-Catalyzed Synthesis of Geranyl Esters as Flavor Additives for Food, Pharmaceutical and Cosmetic Applications,” Food Chemistry Advances 1 (2022): 100052.
F. Zappaterra, D. Meola, F. Presini, et al., “Differential Effect of Nine Cinnamic Acid Derivatives on the Biocatalytic Activity of Candida antarctica Lipase Type B,” Current Research in Biotechnology 8 (2024): 100231.
A. A. Abdelaziz, A. M. Abo-Kamar, E. S. Elkotb, and L. A. Al-Madboly, “Microbial Lipases: Advances in Production, Purification, Biochemical Characterization, and Multifaceted Applications in Industry and Medicine,” Microbial Cell Factories 24 (2025): 1–20.
P. K. Robinson, “Enzymes: Principles and Biotechnological Applications,” Essays in biochemistry 59 (2015): 1–41.
P. Chandra, Enespa, R. Singh, and P. K. Arora, “Microbial Lipases and Their Industrial Applications: A Comprehensive Review, ” Microbial Cell Factories 19 (2020): 1–42.
S. Ali, S. A. Khan, M. Hamayun, and I. J. Lee, “The Recent Advances in the Utility of Microbial Lipases: A Review,” Microorganisms 11 (2023): 510.
D. Remonatto, R. H. Miotti, R. Monti, J. C. Bassan, and A. V. de Paula, “Applications of Immobilized Lipases in Enzymatic Reactors: A Review,” Process Biochemistry 114 (2022): 1–20.
M. D. Murcia, M. Gómez, E. Gómez, et al., “Kinetic Modelling and Kinetic Parameters Calculation in the Lipase-Catalysed Synthesis of Geranyl Acetate,” Chemical Engineering Research and Design 138 (2018): 135–143.
P. K. Sharma, N. S. Sangwan, S. K. Bose, and R. S. Sangwan, “Biochemical Characteristics of a Novel Vegetative Tissue Geraniol Acetyltransferase from a Monoterpene Oil Grass (Palmarosa, Cymbopogon Martinii Var. Motia) Leaf,” Plant Science 203-204 (2013): 63–73.
V. Venturi, F. Presini, C. Trapella, O. Bortolini, P. P. Giovannini, and L. A. Lerin, “Microwave-Assisted Enzymatic Synthesis of Geraniol Esters in Solvent-Free Systems: Optimization of the Reaction Parameters, Purification and Characterization of the Products, and Biocatalyst Reuse,” Molecular Diversity 28 (2024): 1665–1679.
L. da Silva Corrêa, R. O. Henriques, J. V. Rios, L. A. Lerin, D. de Oliveira, and A. Furigo, “Lipase-Catalyzed Esterification of Geraniol and Citronellol for the Synthesis of Terpenic Esters,” Applied Biochemistry and Biotechnology 190 (2020): 574–583.
D. Remonatto, R. R. Fantatto, R. C. L. R. Pietro, et al., “Enzymatic Synthesis of Geranyl Acetate in Batch and Fed-Batch Reactors and Evaluation of Its Larvicidal Activity against Rhipicephalus (Boophilus) Microplus,” Process Biochemistry 120 (2022): 287–300.
P. D. Tomke and V. K. Rathod, “Ultrasound Assisted Lipase Catalyzed Synthesis of Cinnamyl Acetate via Transesterification Reaction in a Solvent Free Medium,” Ultrasonics Sonochemistry 27 (2015): 241–246.
FAO - Food and Agriculture Organization of the United Nations, Council Directive 88/388/EEC of 22 June 1988 on the Approximation of the Laws of the Member States Relating to Flavourings for use in Foodstuffs and to Source Materials for Their Production (FAO, 1988).
R. A. El-Shiekh, D. E. Ali, A. A. Mandour, and M. R. Meselhy, “Essential Oils of Plumeria Alba L. and Plumeria Rubra L. Growing in Egypt: GC/MS Analysis, Molecular Dynamics and in-Vitro Anti-Cholinesterase Activity,” Industrial Crops and Products 221 (2024): 119316.
A. S. Kamariah, L. B. L. Lim, K. H. C. Baser, T. Ozek, and B. Demirci, “Composition of the Essential Oil of Plumeria Obtusa L,” Flavour and Fragrance Journal 14 (1999): 237–240.
M. Kristiawan, V. Sobolik, M. Al-Haddad, and K. Allaf, “Effect of Pressure-Drop Rate on the Isolation of Cananga Oil Using Instantaneous Controlled Pressure-Drop Process,”Chemical Engineering and Processing: Process Intensification 47 (2008): 66–75.
R. L. Hall and B. L. Oser, Recent Progress in the Consideration of Flavoring Ingredients Under the Food Additives Amendment: III GRAS Substances (The Garrard Press, USA, 1965).
FAO/WHO Expert Committee on Food Additives, WHO Technical Report Series: Evaluation of Certain Food Additives and Contaminants (Genevra, 2002).
M. I. Rodrigues and A. F. Iemma, Experimental Design and Process Optimization (CRC Press, 2014).
R. D. Santos, E. A. Rebelatto, C. Guindani, et al., “Lipase-Catalyzed Synthesis of Poly(ω-Pentadecalactone-Co-Globalide) in Supercritical Carbon Dioxide,” The Journal of Supercritical Fluids 215 (2025): 106409.
R. R. Sousa, A. S. A. Silva, R. Fernandez-Lafuente, and V. S. Ferreira-Leitão, “Solvent-Free Esterifications Mediated by Immobilized Lipases: A Review from Thermodynamic and Kinetic Perspectives,” Catalysis Science & Technology 11 (2021): 5696–5711.
S. D. Shinde and G. D. Yadav, “Insight into Microwave-Assisted Lipase Catalyzed Synthesis of Geranyl Cinnamate: Optimization and Kinetic Modeling,” Applied Biochemistry and Biotechnology 175 (2015): 2035–2049.
R. R. de Sousa, M. M. dos Santos, M. W. R. Medeiros, et al., “Immobilized Lipases in the Synthesis of Short-Chain Esters: An Overview of Constraints and Perspectives,” Catalysts 15 (2025): 375.
V. Venturi, E. Marchesi, D. Perrone, et al., “Enzymatic Synthesis of New Acetoacetate–Ursodeoxycholic Acid Hybrids as Potential Therapeutic Agents and Useful Synthetic Scaffolds as Well,” Molecules 29 (2024): 1305.
M. Cao, F. Yang, Y. Zhang, et al., “Efficient Method of Synthesizing Sn-2 Eicosapentaenoic Acid (EPA) Monoacylglycerols Using Circular Ethanolysis and Glycerolysis,” Food Chemistry 474 (2025): 143047.
D. Remonatto, S. R. Amaral, J. F. C. do Nascimento, et al., “Geraniol Ester Loaded-Nanoemulsions: Exploring Enzymatic Synthesis, Biocompatibility, Antioxidant Potential, and Antimicrobial Activity,” Colloids and Surfaces A: Physicochemical and Engineering Aspects 718 (2025): 136917.
M. Y. Liow, E. S. Chan, W. Z. Ng, and C. P. Song, “Stabilization of Eversa Transform 2.0 Lipase with Sorbitol to Enhance the Efficiency of Ultrasound-Assisted Biodiesel Production,” International Journal of Biological Macromolecules 276 (2024): 133817.
W. Gourich, C. P. Song, and E. S. Chan, “Overcoming the Equilibrium Constraints in the Enzymatic Hydrolysis of Methyl Esters via a Sequencing Batch Process to Improve Conversion into Fatty Acids,” Journal of Molecular Liquids 423 (2025): 127023.
T. Kulschewski, F. Sasso, F. Secundo, M. Lotti, and J. Pleiss, “Molecular Mechanism of Deactivation of C. antarctica Lipase B by Methanol,” Journal of Biotechnology 168 (2013): 462–469.
G. Ceni, P. C. D. Silva, L. Lerin, J. V. Oliveira, G. Toniazzo, H. Treichel, E. G. Oestreicher, D. de Oliveira, “Ultrasound-Assisted Enzymatic Transesterification of Methyl Benzoate and Glycerol to 1-Glyceryl Benzoate in Organic Solvent,” Enzyme and Microbial Technology 48 (2011), 169-174, https://doi.org/10.1016/j.enzmictec.2010.10.004.
S. D. Shinde and G. D. Yadav, “Microwave Irradiated Immobilized Lipase Catalyzed Synthesis of Alkyl Benzoate Esters by Transesterification: Mechanism and Kinetic Modeling,” Industrial & Engineering Chemistry Research 53 (2014): 8706–8713.
L. Lerin, G. Ceni, A. Richetti, et al., “Successive Cycles of Utilization of novozym 435 in Three Different Reaction Systems,” Brazilian Journal of Chemical Engineering 28 (2011): 181–188.
C. Laane, S. Boeren, K. Vos, and C. Veeger, “Rules for Optimization of Biocatalysis in Organic Solvents,” Biotechnology and Bioengineering 30 (1987): 81–87.
R. R. de Sousa, M. C. C. Pinto, E. C. G. Aguieiras, et al., “Comparative Performance and Reusability Studies of Lipases on Syntheses of Octyl Esters with an Economic Approach,” Bioprocess and Biosystems Engineering 45 (2022): 131–145.
D. Kowalczykiewicz, K. Szymańska, D. Gillner, and A. B. Jarzębski, “Rotating Bed Reactor Packed with Heterofunctional Structured Silica-Supported Lipase. Developing an Effective System for the Organic Solvent and Aqueous Phase Reactions,” Microporous and Mesoporous Materials 312 (2021): 110789.
K. T. Desta and A. M. Abd El-Aty, “Millettia Isoflavonoids: A Comprehensive Review of Structural Diversity, Extraction, Isolation, and Pharmacological Properties,” Phytochemistry Reviews 22 (2023): 275–308.
W. Chen and A. M. Viljoen, “Geraniol—A Review of a Commercially Important Fragrance Material,” South African Journal of Botany 76 (2010): 643–651.
L. Benvenutti, B. C. Rovaris, K. Cesca, et al., “A Water-Based Ultrasound-Assisted Extraction System to Obtain Natural Blue Colorant from Genipap (Genipa americana L),” Food Chemistry Advances 4 (2024): 100704.
International Standards Organization (ISO), ISO, 10993-5: 2009 - Biological Evaluation of Medical Devices—Part 5: Tests for In Vitro Cytotoxicity(2009).
M. H. O. de Sousa, J. M. S. Morgan, K. Cesca, A. Flach, and N. F. de Moura, “Cytotoxic Activity of Cunila Angustifolia Essential Oil,” Chemistry & Biodiversity 17 (2020): e1900656.
