Ultrasound-assisted butyl acetate synthesis catalyzed by Novozym 435: Enhanced activity and operational stability
Highlights
► Ultrasound energy was used as mixing system in the esterification reaction of flavor esters. ► Synthesis of butyl butyrate catalyzed by Novozym 435 was optimized. ► Under the optimal conditions over 94% of conversion was obtained in 2.5 h. ► Enzyme was reused for 14 reaction cycles, keeping 70% of its original activity. ► Enzyme stability in acetic acid was improved under ultrasound-assisted reaction.
Introduction
Lipases are versatile biocatalysts, mainly used to perform the hydrolysis of oils and fats and, under specific conditions, also capable of catalyzing esterification, transesterification, and interesterification reactions [1]. These enzymes can be easily obtained from microorganisms, and are very active in organic solvents, which is needed to obtain low water activity used in esterification reactions [2]. In particular, the application of these enzymes for the synthesis of short chain esters has attracted the interest of a broad range of industrial fields like foods, pharmaceuticals and cosmetics industries [3].
Lipases are of great interest for the synthesis of natural flavor esters [4]. These reactions occur between short chains alcohols and carboxylic acids. Presently, international legislation have defined that “natural” flavor substances can only be prepared by either physical processes (extraction from natural sources), or by enzymatic or microbial processes. From the consumers’ perception, compounds labeled “natural” are more readily acceptable than products labeled “nature-identical” because these are associated (and produced by) to chemical methods [5]. Thus, lipase-catalyzed esterification is the target of many current researches aiming at finding a suitable biocatalyst to make this process viable in large scales [3], [6], [7], [8], [9], [10].
Ultrasound is a sound of a frequency higher than that the human ear can perceive which is being used in several technical fields. It is considered a “green” technology because of its high efficiency, its economic performance and low instrumental requirements. It significantly reduces process time compared with other conventional mixing techniques [11]. Ultrasound technology has been used due to its capacity of increasing the interaction between phases by cavitation caused by the collapse of bubbles, and the ultrasonic jet, that disrupts the boundary phase and causes emulsification [11], [12]. Furthermore, when ultrasound is applied to an aqueous solution or suspension, increases in mixing, shearing, and mass transfer are observed [13], [14]. Until recently, it was unusual the use of ultrasound in biological reactions, but some studies pointed that this technology could be used for biotechnological applications [13], [15], [16].
According to Kwiatkowska et al., [13] the influence of the low frequency ultrasound is not able to inactivate enzymes, but can affect these sensitive catalysts in some processes. However, it must be remarked that the influence of sonic radiation on the activity and stability of enzymes depends on the sonication parameters and the specific enzyme preparation. In this sense, some researchers reported the influence of ultrasound in the kinetic parameters and reduction of esterification reaction time [17], [18]. Nevertheless, its application to enzymatic-catalyzed reactions is still scarcely explored, and it was not found in the literature any research on the application of ultrasound-assisted technology for lipase-catalyzed flavor esters synthesis.
The esterification between acetic acid and butanol catalyzed by immobilized lipase B from Candida antarctica, commercially available as Novozym 435 has been recently optimized using the standard mechanical mixing system. Results were positive, but reaction rates and reuse of the biocatalyst were low to justify its use at industrial level, demanding further optimization of the process to meet these requirements. Optimal results were obtained using hexane as solvent, and a concentration of 0.3 M acetic acid to avoid enzyme inactivation [19].
Considering these aspects, in this work it was evaluated the influence of ultrasound energy on the esterification reaction between acetic acid and butanol in hexane catalyzed by Novozym 435 starting from the conditions previously reported as optimal for this reaction under standard mixing system. The reaction parameters temperature, substrate molar ratio, enzyme content, and added water were optimized using central composite design (CCD) and the response surface methodology (RSM). Finally, the enzyme stability on acetic acid and the recycling of the enzyme for multiple batches were analyzed and compared with the standard mechanical agitation technique.
Section snippets
Materials
Lipase B from C. antarctica immobilized in a macroporous resin (Novozym 435) was kindly donated by Novozymes (Spain). The substrates, solvents and other chemicals were purchased from Sigma–Aldrich (Sigma, St. Louis, USA) and were of analytical grade.
Esterification reaction
The substrates n-butanol and acetic acid (0.3 M based on a previous work [19]) were dissolved in n-hexane at different molar ratios into 50 mL. Erlenmeyer flasks (working volume of 10 mL), followed by the addition of various amounts of water and
Experimental design, model fitting and ANOVA
Response surface methodology is an empirical modeling technique used to evaluate the relationship between a set of controllable experimental factors and the observed results. In Table 2 are presented the results for the ultrasound-assisted butyl acetate synthesis using Novozym 435 as biocatalysts after 2.5 h of reaction. Highest yield of conversion (99.3%) was obtained in treatment 22 (45 °C, 3:1 alcohol:acid, enzyme content 10%, added water 0.5%), while the less effective treatment was the 21,
Conclusion
The ultrasound energy has shown to be a good technology to improve the enzymatic esterification reaction catalyzed by Novozym 435 for flavor ester synthesis in three ways: (a) higher biocatalyst operational stability, (b) biocatalyst stabilization in the presence of high concentrations of acetic acid in n-hexane, and (c) increased productivity of ester, when compared to the traditional method of mechanical agitation. The reaction parameters were optimized and the optimal conditions were similar
Acknowledgments
This work was supported by grants from Fundação de Amparo a Pesquisa do Rio Grande do Sul (FAPERGS – ARD/2011), from CNPq (Brazilian Bureau of Science and Technology), and CTQ2009-07568 from Spanish Ministerio de Ciencia e Innovación. The authors would like to thank Mr. Ramiro Martínez (Novozymes, Spain) for kindly supplying the enzymes used in this research. We also thank CNPq – Brazil for a fellowship to J.L.R. Friedrich and FAPERGS – Brazil for a fellowship to A.B. Martins.
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