Biotechnol Lett DOI 10.1007/s10529-015-1834-0

ORIGINAL RESEARCH PAPER

Enzymatic synthesis of aroma acetoin fatty acid esters by immobilized Candida antarctica lipase B Zijun Xiao • Xiaoyuan Hou • Xin Lyu • Jing-yi Zhao • Lijun Xi • Jing Li • Jian R. Lu

Received: 17 February 2015 / Accepted: 2 April 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Objective To enzymatically synthesize aroma acetoin fatty acid esters, useful as flavor and fragrance ingredients in foods. Results Immobilized Candida antarctica lipase B (CALB), performed significantly better than lipases from Rhizopus niveus and Candida rugosa in carrying out the esterification of acetoin and fatty acids. C4–C12 straight chain fatty acids were suitable acyl donors and CALB had a strong preference for longer straight chains up to ten carbon atoms. Higher temperatures, 40–60 °C, and higher acetoin/fatty acid molar ratios favored the conversion. The maximum yield of acetoin octanoate obtained was (51 ± 1) % after 24 h reaction time in hexane with 0.25 M octanoic acid, 5:1 excess acetoin and an enzyme concentration of 6 g/mol fatty acid at 60 °C. The enzyme activity declined at a steady

Electronic supplementary material The online version of this article (doi:10.1007/s10529-015-1834-0) contains supplementary material, which is available to authorized users. Z. Xiao (&)  X. Hou  X. Lyu  J. Zhao  L. Xi  J. Li Center for Bioengineering and Biotechnology, China University of Petroleum, No. 66 Changjiang West Road, Huangdao District, Qingdao 266580, People’s Republic of China e-mail: [email protected] J. R. Lu Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Manchester M13 9PL, UK

rate during reuse at 60 °C and after the 10th cycle, 65 % of initial activity was still be retained. Conclusion This is the first report of acetoin fatty acid ester synthesis by biological method and CALB has been shown to be effective for the lipase-catalyzed esterification of acetion and C4–C12 straight chain fatty acids. Keywords Acetoin  Aroma esters  Enzymatic synthesis  Fatty acids  Lipase

Introduction Acetoin fatty acid esters (AFAEs), with their representative molecular structure shown in Fig. 1, generally have a pleasant tonality of fruity, buttery, fermented and sweet attributes and the odors have subtle differences according to the carbon number of the aliphatic acyl moieties (Shiota 1991). These rich flavors are desirable for a special aroma of certain foods. Currently, there are at least four types of natural AFAEs (acetoin acetate, acetoin butyrate, acetoin hexanoate, and acetoin octanoate) found in dietary materials. For example, acetoin acetate is sometimes present in wines (Izquierdo Can˜as et al. 2008) and vinegars (Chinnici et al. 2009). Some bananas contain not only acetoin acetate but also acetoin butyrate (Shiota 1993) and pawpaw fruits contain all the four AFAEs (Shiota 1991).

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Materials and methods Chemicals and reagents

Fig. 1 The lipase catalyzed synthesis reaction of acetoin fatty acid esters (up) and the electron ionization MS fragmentation pathway of the typical ions (down). R = C3–C11 alkanoic chains in this study

Three lipases from Candida antarctica, Rhizopus niveus and Candida rugosa were purchased from Sigma. Acetoin (98 %) and 11 fatty acids (acetic acid, 99 %; butyric acid, 99 %; 2-methylbutanoic acid, 98 %; 3-methylbutanoic acid, 99 %; pentanoic acid, 99 %; hexanoic acid, 99 %; heptanoic acid, 99 %; octanoic acid, 99 %; nonanoic acid, 97 %; decanoic acid, 98 %; dodecanoic acid, 99.8 %) were tested as the substrates. Other chemicals and reagents used were of analytical grade. Reaction procedure

AFAEs are mainly used in food and beverage industries to enhance the flavors of products. Due to the moderate volatile ability, characteristic odor, and chemical structure, AFAEs have great potential as semi-synthetic chemicals (Sto¨kl et al. 2010) to be used as artificial lures for mass-trapping techniques of pests (Xiao and Lu 2014). AFAEs also have found utility as solvents, especially for epoxy and vinyl resins (Burton and Wiese 1968). At present, there are only a few chemical synthetic methods available for the preparation of AFAEs. These methods involve radical reactions (Burton and Wiese 1968; Zoeller 1990; Shiota 1991) and require multiple processing steps, imposing safety issues and causing environmental impact. On the other hand, consumers prefer natural products even though they are generally much more expensive than their synthetic counterparts, especially when the product is used in food, medicine, and cosmetics (Xiao et al. 2014). There are intense public appeals for green industry and more environmentally benign processes as well. However, direct extraction of AFAEs from natural materials is infeasible due to the shortage of proper raw materials. Biotechnology based methods such as fermentation and enzymatic transformation are unavailable to date. In this study, ten AFAEs were synthesized by the esterification of acetoin and fatty acids catalyzed by solvent-tolerant lipases. Operational conditions including enzymes, substrates and their ratios, reaction time, temperature, solvents, and enzyme reuse times were examined. This is the first report of a biosynthetic approach for the production of AFAEs.

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Reactions were carried out using 71 9 20.6 mm screw cap glass vials. Briefly, the desired amount of lipase and substrates was placed into a vial and solvent added to make 8 ml. Vials were sealed and shaken at 150 rpm at a specific temperature. All the tests were performed in triplicate. Purification of products The enzyme beads of Candida antarctica lipase B (CALB) were removed from the reaction mixture by filtration. Saturated Na2CO3 was then applied to neutralize the residual fatty acid. Most residual acetoin was removed from the mixture by repeatedly wash with pure water. The solvent was removed by evaporation and the residue was loaded onto a silica gel column (13.4 9 254 mm glass column packed with 200–300 mesh silica gel) and eluted with cyclohexane/ethyl acetate/acetic acid (90:9.5:0.5, by vol.). Fractions were analyzed by TLC with phosphomolybdic acid staining and by gas chromatography (GC). The fractions with the same composition were combined and the solvents were evaporated under vacuum. Analytical methods 60 ll of the reaction mixture was withdrawn at the desired time, diluted to 1 ml with ethanol and then quantitatively analyzed using an Agilent 7890A GC system equipped with a 30-m HP-5 (0.32 mm inside diam., 0.25 lm film thickness) capillary column (19091 J-413, Agilent) and a flame ionization detector. Isoamyl isovalerate was used as the internal

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standard. The column was kept at 50 °C for 2 min, and then programmed to 250 °C with an increase of 10 °C/ min and maintained at 250 °C for 8 min. The GC retention times (min) for each of the 10 synthesized AFAEs: acetoin butyrate (11.2), acetoin 2-methylbutanoate (11.9), acetoin 3-methylbutanoate (12.1), acetoin pentanoate (12.9), acetoin hexanoate (14.3), acetoin heptanoate (15.9), acetoin octanoate (17.3), acetoin nonanoate (18.9), acetoin decanoate (20.1), and acetoin dodecanoate (22.6). The percentage conversion was defined as the amount of AFAE produced from the initial substrate (mol ester/mol initial substrate 9100 %). Reaction products were also qualitatively analyzed using GC–MS: Agilent 7890A equipped with a 30-m HP-5MS (0.25 mm inside diam., 0.25 lm film thickness) capillary column (19091S-433, Agilent) and a 5975C MS detector. The column was programmed from 40 to 300 °C with an increase of 10 °C/min and maintained at 300 °C for 5 min. MS in the electron impact mode were generated at 70 eV and scan mode in the range of 30–600 amu. 1 H-NMR analysis was conducted on a Bruker Avance 400 spectrometer using deuterated chloroform as the solvent and the chemical shifts were calibrated against tetramethylsilane.

Results Product confirmation Acetoin is a reactive molecule because the hydroxyl and carbonyl groups in the molecule are adjacent to each other (Xiao and Lu 2014). Figure 1 (top) shows the general esterification process for the fatty acids with different acyl chain lengths. Throughout the experiments in this work, however, no GC peaks indicative of by-products were observed because of the mild reaction conditions. The MS data of the ten enzymatically synthesized AFAEs are shown in Supplementary Figures. These AFAE molecules are rather fragile in electron ionization MS and their molecular ion peaks exist in very low abundances, or are even totally undetectable. As a conclusion, these 10 homologs share a similar fragmentation pathway with 3 typical ions being present in each of the measured MS spectrum: M-43, M-87, and 43 (Fig. 1, lower). Pure chemical standards of all the ten AFAEs

are currently unavailable and their reference spectra do not exist yet in GC–MS workstations or public MS databases. Of the ten AFAEs, only the MS data of acetoin butyrate, acetoin hexanoate, and acetoin octanoate have been reported previously (Shiota 1991). The results obtained from these three AFAEs in our study were consistent with the reported spectra. To bring this part of work forward, representative ones of the 10 AFAEs have been purified by silica gel column chromatography. The purities, as determined by the GC equipped with a flame ionization detector, were found to be 95, 98, 97, 96, and 96 % for acetoin hexanoate, acetoin heptanoate, acetoin octanoate, acetoin nonanoate, and acetoin decanoate, respectively. 1H-NMR data (in Supplementary Figures) thus reconfirmed the chemical structures and high purities. Selection of the lipases Reactions were carried out at 50 °C using hexane as the solvent. Acetoin and each of the 4 representative fatty acids (butyric acid, 2-methylbutanoic acid, 3-methylbutanoic acid, and octanoic acid) were dissolved in the solvent (0.25 M acetoin, 0.5 M fatty acid). 12 mg of immobilized C. antarctica lipase B (CALB or Novozym 435, activity C5 U/mg), with diam. 0.3–0.9 mm, 40 mg lipase from Rhizopus niveus (activity C1.5 U/mg), and 10 mg lipase from Candida rugosa Type VII (activity C700 U/mg) were individually used for the catalysis of the esterification. After following the reactions for 47 h, the product yields were checked and the results are shown in Fig. 2. The reaction systems with the lipase from Candida rugosa Type VII as the catalyst, despite the high doses added, did not yield any detectable products (results not shown). The lipase from Rhizopus niveus also showed no detectable activity when 2-methylbutanoic acid or 3-methylbutanoic acid was supplied as the substrate whilst CALB did generate some products even though the yield was low for 2-methylbutanic acid. For the two straight chain fatty acids of butyric acid and octanoic acid, CALB again exhibited much higher catalytic activities. Therefore, CALB was applied in all the further experiments undertaken in this work. Substrate specificity To extend the observations obtained from the work carried out above, more reactions were carried out at

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Fig. 2 Comparison of catalytic activities of CALB with the lipase from Rhizopus niveus. Reaction conditions: hexane as the solvent, 0.25 M acetoin, 0.5 M fatty acid, 12 mg of immobilized Candida antarctica lipase B (CALB) or 40 mg of lipase from Rhizopus niveus, 50 °C, 47 h. b, acetoin butyrate; c, acetoin 2-methylbutanoate; d, acetoin 3-methylbutanoate; h, acetoin octanoate

50 °C in hexane with acetoin and 11 single fatty acids as the substrates keeping the molar ratio of acetoin to fatty acid fixed at 1: 2 (0.25 M acetoin, 0.5 M fatty acid). As shown in Fig. 3, CALB preferred the longer straight chain fatty acids up to ten carbon atoms. It could not accept acetic acid as a substrate to form acetoin acetate under these conditions. Branched chain fatty acids were also poor substrates for the esterification reactions, with the yield of acetoin 2-methylbutanoate being significantly lower than acetoin 3-methylbutanoate. The yields from both branched acids were also lower than that obtained from the straight pentanoic acid, consistent with the observation from the results shown in Fig. 2. Typical time-dependent process of ester formation and the effect of reaction temperature The time-dependent formation processes of acetoin hexanoate, acetoin heptanoate, acetoin octanoate, acetoin nonanoate, and acetoin decanoate were recorded. As shown in Fig. 4 (top), all the processes followed the same pattern and the reactions came close to the equilibrium point at about 24 h. According to the product information of Novozym 435, CALB is a heat-tolerant lipase with a maximum activity in the range of 70–80 °C. However due to the

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Fig. 3 The effects of fatty acid type and chain length. Reaction conditions: hexane as the solvent, 0.25 M acetoin, 0.5 M fatty acid, 12 mg CALB, 50 °C, 47 h. a, acetoin acetate; b, acetoin butyrate; c, acetoin 2-methylbutanoate; d, acetoin 3-methylbutanoate; e, acetoin pentanoate; f, acetoin hexanoate; g, acetoin heptanoate; h, acetoin octanoate; i, acetoin nonanoate; j, acetoin decanoate; k, acetoin dodecanoate

thermal inactivation at elevated temperatures, CALB is recommended to operate from 40 to 60 °C for optimum productivity. Under the recommended range, the temperature-dependent formation processes of acetoin octanoate were recorded as shown in Fig. 4 (lower). As higher conversion rates were achieved at 60 °C, further experiments were carried out at this temperature. Effect of the molar ratio of substrates As shown in Fig. 5 (top), when acetoin was excessively supplied (high acetoin/fatty acid ratio), conversion rates were enhanced, indicating acetoin had no or minor substrate inhibition effect on the lipase CALB. However, because the commercial prices of acetoin are usually much higher than fatty acids, this method appears not to be economical unless the residual acetoin can be easily recovered and reused. Fatty acids are relatively cheap, but increasing the fatty acid/acetoin ratio cannot help much to enhance the conversion. As shown in Fig. 5 (lower), the best conversion was achieved when the molar ratio of octanoic acid/acetoin =2. Increasing the amount of octanoic acid could help to push the equilibrium point towards the higher conversion, but it seems that this action will simultaneously impose serious substrate inhibition on the lipase.

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Fig. 4 The time courses of ester formation (up, at 50 °C) and the effect of reaction temperature (down, octanoic acid as the substrate). Reaction conditions: hexane as the solvent, 0.25 M acetoin, 0.5 M fatty acid, 12 mg CALB

Effect of organic solvent Choice of solvents is very important for a successful industrial process. As shown in Fig. 6, CALB works better in non-polar solvents than in polar solvents during AFAE synthesis in this study. This is consistent with the stated characteristics of this commercial lipase, i.e. Novozym 435 prefers inert solvents such as petroleum ether or hexane.

Fig. 5 Effect of the molar ratio of substrates. Top: acetoin was excessively supplied when octanoic acid was kept at 0.25 M; lower: octanoic acid was excessively supplied when acetoin was kept at 0.25 M. Reaction conditions: hexane as the solvent, 12 mg CALB, 60 °C, 24 h

inactivation of the long-time exposure at 60 °C, three control experiments were performed by storage of the lipase at high temperature before use. After 50 h of storage at 60, 50, and 40 °C in sealed vials, 94, 96, 98 % of the initial activity was retained, respectively. These results indicated some influence from temperature, but the greater extent of loss of catalytic activity might arise from other factors.

Enzyme reuse As shown in Fig. 7, the immobilized CALB can be reused many times even at 60 °C to synthesize AFAEs. Enzyme activity, however, declined at a steady rate and at the 10th cycle, 65 % of its initial activity was retained. To investigate whether this activity loss was mainly caused by the thermal

Discussion The three commercial enzymes used in this study represent the lipases from three sub-groups: CALB is a lipase with a funnel-like binding site; the lipase from Rhizopus niveus has a hydrophobic, crevice-like binding

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Fig. 6 Effect of solvents on the esterification reactions. Reaction conditions: 0.25 M acetoin, 0.5 M octanoic acid, 12 mg CALB, 60 °C, 24 h

Fig. 7 Effect of lipase reuse times. Reaction conditions: hexane as the solvent, 0.75 M acetoin, 0.25 M octanoic acid, 12 mg CALB, 60 °C. In every cycle, after 5 h of reaction, the enzyme beads were removed from the reaction mixture by filtration and transferred into fresh substrates to start a new cycle

site located near the protein surface; the lipase from Candida rugosa Type VII has a tunnel-like binding site (Pleiss et al. 1998). The results in this study indicate that the lipases with funnel-like binding sites are comparatively better for the synthesis of AFAEs. The conversion rate was strongly influenced by the size and structure of the aliphatic chain from the acyl donor. The yield of acetoin pentanoate is 2.8- and 18.7-fold of the yield of acetoin 3-methylbutanoate and acetoin 2-methylbutanoate, respectively, indicating that steric hindrance is an important factor governing the activity

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of CALB with carboxylic acids leading to reduced conversion rates (Hollmann et al. 2009). The acyl and alcohol moieties are brought close in space during catalysis, which could explain CALB’s sensitivity to the length and structure of the acyl chain (Ottosson and Hult 2001). Our findings are in accordance with the shape and properties of the respective scissile fatty acid binding sites (Pleiss et al. 1998). But other structural elements may also play a role in mediating chain length and structure specificity, e.g., the binding site of acetoin. It is difficult to predict the impact of temperature on ester yield and rate because it may affect reaction efficiency in opposite ways (Romero et al. 2005). First, a temperature rise would have a positive effect on the kinetic constant as defined by the transition state theory. Conversely, the treatment at high temperatures may disrupt enzyme tertiary structure, causing their catalytic activity loss. As can be seen in Fig. 4 (lower), the initial reaction rates continuously increased at elevated temperatures in the range of 40–60 °C. As CALB is a heat-tolerant lipase, it appears that at these temperatures the kinetic effect was predominant. The choice of organic solvents is significant for lipase-catalyzed reactions. A relationship was suggested between solvent polarity and reaction conversion (Longo and Sanroma´n 2006). Highly hydrophobic solvents such as hexane are preferred because they do not penetrate the water layer surrounding the enzyme surface, thus favoring the maintenance of enzyme conformation. The amphiphilic nature of the organic phase increases while changing acetoin or fatty acid concentrations. Fatty acids may perform as competitive inhibitors and cause enzyme deactivation by dissolution in the micro-aqueous phase, which causes a pH drop. No indication of acetoin inhibition was observed in the tested range. On the contrary, excess acetoin enhanced conversion and the equilibrium of the reaction was pushed toward to product formation as the nucleophile (acetoin) concentration raised. Similar benefit from such an excess nucleophile was also observed for the enzymatic synthesis of esters by several other authors (for example, Gubicza et al. 2001).

Conclusion We present here the first report of AFAE synthesis by a biological route, i.e. by the lipase-catalyzed esterification of acetion and fatty acids. CALB, a lipase with a

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funnel-like binding site, was effective for the acceptance of C4–C12 straight chain saturated fatty acids. As acetoin and fatty acids can be commercially obtained in large quantities from renewable resources, the enzymatically synthesized AFAEs from the natural feedstock lead to natural products. This study will thus form a useful basis for further development of the enzymatic synthesis of natural AFAEs. Acknowledgments We thank the National Science Foundation of China (Grant No. 21376264) for funding support. Supporting information Supplementary Figure 1: – Electron ionization MS spectra (70 eV) of acetoin butyrate, acetoin 2-methylbutanoate, acetoin 3-methylbutanoate, acetoin pentanoate, acetoin hexanoate, acetoin heptanoate, acetoin octanoate, acetoin nonanoate, acetoin decanoate, acetoin dodecanoate, and 1H NMR spectra (CDCl3, 400 MHz) of acetoin hexanoate, acetoin heptanoate, acetoin octanoate, acetoin nonanoate, acetoin decanoate.

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Enzymatic synthesis of aroma acetoin fatty acid esters by immobilized Candida antarctica lipase B.

To enzymatically synthesize aroma acetoin fatty acid esters, useful as flavor and fragrance ingredients in foods...
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