Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel

Article pubs.acs.org/JAFC

Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus Hao Dong,† Francesco Secundo,§ Changhu Xue,† and Xiangzhao Mao*,† †

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China Istituto di Chimica del Riconoscimento Molecolare, CNR, v. Mario Bianco 9, 20131 Milan, Italy

§

S Supporting Information *

ABSTRACT: Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h. KEYWORDS: cinnamyl acetate, whole-cell biocatalyst, transesterification, genomic DNA library, esterase, acetic esters

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obtained under optimal reaction conditions.8 Immobilized porcine pancreatic lipase was also used to synthesize cinnamyl acetate using acetic acid and cinnamyl alcohol as substrate. However, the yield of cinnamyl acetate was only up to 62.56%.14 These lipases were also reported to prepare another flavor compound. Sharma et al. used porcine pancreatic lipase to synthesize ethyl cinnamate, and the maximum conversion was only 55% after 27 h.15 Jakovetic et al. reported the synthesis of ethyl cinnamate using Novozyme 435. However, only 35.2% ethyl cinnamate was obtained at 96 h.16 Wang et al. enhanced the yield of ethyl cinnamate to a high level using Lipozyme TLIM, whereas it took 24 h to achieve the high level and the concentration of cinnamic acid was low.17 Lipases are important industrial biocatalysts and have been widely used in the preparation of flavor compounds. Analogously to lipases, esterases do not require cofactors, have high regio-, chemo-, and enantioselectivities, and are also stable in organic solvents, where they can catalyze the formation of ester bonds by transesterification and esterification. Esterases have been exploited in biotechnological applications, such as synthesis of biodiesel, flavor compounds, and resolution of racemic mixtures.18 It has to be emphasized that flavor and fragrance compounds produced by esterase are considered as “natural”, satisfying the demand for industries.9 Furthermore, considering that cinnamyl acetate has a short-chain acyl moiety, esterases can be better than lipases because they catalyze the hydrolysis and formation of short-chain acyl esters. However, despite these differences in substrate preference for these two groups of hydrolases, only in a few cases have esterases been used for the synthesis of cinnamyl acetate.

INTRODUCTION Acetic esters are one of the most important esters, and they are widely applied in the food industries as flavorings or fragrances.1 Cinnamyl acetate is a member of the acetic esters, and it naturally occurs in fresh bark of cinnamon.2,3 Cinnamyl acetate has beneficial biological properties, particularly the antioxidant activity, which is ascribed to the structural characteristics of cinnamyl acetate.4 It has been also reported that cinnamyl acetate can be used as precursor for the synthesis of chloramphenicol and butyrolactone lignans.5−7 Furthermore, cinnamyl acetate has a wide application in the flavor and fragrance industries because of its sweet, balsamic, floral odor,8 and it has already been approved by the U.S. Food and Drug Administration.2 Cinnamyl acetate can be extracted and purified from plants, but the low yield leads to a high production cost.9 Chemical methods might be an efficient strategy to produce cinnamyl acetate. Nevertheless, the use of hazardous chemicals pollutes the environment, reduces the security of products, and leads to the production of some unwanted byproducts under hightemperature and high-pressure conditions.10 Thus, enzymemediated catalysis is considered as a promising alternative for the mild reaction conditions and environmentally friendly process.11 Lipolytic enzymes, including esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), represent a family of hydrolases that catalyze, by the same mechanism, the cleavage or formation of ester bonds, and they are widely distributed in microorganisms, plants, and animals.12 The difference between esterases and lipases is that lipases can catalyze hydrolysis and synthesis of water-insoluble long-chain (≥10) acylglycerols, whereas esterases catalyze hydrolysis and synthesis of water-soluble short-chain (50% of the original activity was retained at 30−40 °C. However, the residual activity of EstK1 decreased sharply at high temperatures, and the residual activities were 28.3% (50 °C). D

DOI: 10.1021/acs.jafc.6b05799 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 3. Characterization of EstK1: (a) substrate specifity of EstK1 using pNP esters with different acyl chain lengths; (b) effect of pH on EstK1 activity in different buffers using pNPB as substrate; (c) optimal temperature and thermostability of EstK1 at temperatures ranging from 30 to 60 °C; (d) effect of detergents (0.5%) on EstK1 activity. The reaction without detergent addtion (control) was defined as 100%.

Table 1. Effect of Metal Ions on EstK1 Activity

Table 2. Effect of Organic Solvents on EstK1 Activity

relative activity (%) ion control Co2+ K+ Li+ Na2-EDTA Fe2+ Fe3+ Mn2+ Ca2+ Mg2+ Zn2+ Ni2+

1 mM 100 105.81 129.74 97.97 93.37 104.25 94.61 92.59 102.20 103.25 36.44 84.67

± ± ± ± ± ± ± ± ± ± ± ±

1.2 2.65 0.46 1.57 1.09 2.34 3.90 0.53 1.12 4.46 0.69 6.43

residual activity (%) at a concentration of 10 mM

100 127.38 152.58 83.52 75.73 234.97 70.16 81.20 93.87 94.53 31.79 53.75

± ± ± ± ± ± ± ± ± ± ± ±

organic solvent

0.5 0.01 0.25 3.17 2.93 6.06 4.52 4.16 3.87 2.82 0.89 0.27

control DMSO methanol ethanol acetone acetonitrile isopropanol n-propanol chloroform cyclohexane n-hexane isooctane

log P −1.3 −0.76 −0.24 −0.23 −0.15 0.1 0.28 2.0 3.2 3.5 4.5

25%, v/v 100 50.92 44.33 24.18 16.70 4.63 6.94 6.48 18.19 91.33 97.14 90.01

± ± ± ± ± ± ± ± ± ± ± ±

0.46 0.37 3.71 0.98 0.27 0.47 1.01 0.23 1.72 0.86 0.86 0.56

50%, v/v 100 28.61 5.72 2.11 1.72 1.56 5.47 5.32 14.63 59.58 92.69 78.08

± ± ± ± ± ± ± ± ± ± ± ±

2.34 0.33 0.17 0.05 0.11 0.17 0.13 0.69 0.56 0.57 0.81 0.49

Solvent is an important factor for the synthesis of cinnamyl acetate, because it could affect the conversion ratio and reaction speed by changing the three-dimensional conformation of proteins.9 It was also reported that hydrophilic organic solvents could denature the enzyme by stripping the essential water for esterase activity in the enzyme structure.45,46 Therefore, to estimate the effect of organic solvents, hydrophobic organic solvents were chosen to do further study. The reactions were carried out under similar conditions, and the result is shown in Figure 4b. Various organic solvents had significantly different effects on the transesterification activity. The conversion was

It can be seen from Figure 4a that acyl donors had a great influence on the transesterification activity of EstK1. In particular, with vinyl acetate the reaction rate was highest and it was slightly (about 20%) less with isopropenyl acetate. With the other acyl donors (isoamyl, methyl, ethyl, and butyl acetate), the reaction rate was no higher than 15% than that observed with vinyl acetate. The reason might be attributed to the irreversible process, as the product vinyl or isopropenyl alcohol could easily tautomerize to aldehydes or acetone, respectively.43,44 In the end, vinyl acetate was chosen as the optimal acyl donor. E


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Figure 4. Optimization of cinnamyl acetate synthesis in a nonaqueous system: (a) effect of acyl donor on EstK1 transesterification activity; (b) effect of organic solvents on EstK1 transesterification activity; (c) effect of molar ratio on EstK1 transesterification activity; (d) effect of temperature on EstK1 transesterification activity.

low with MTBE, whereas activity reached the highest level with isooctane. Isooctane was chosen as the optimal organic solvent. The effect of the molar ratio of cinnamyl alcohol and vinyl acetate was investigated (Figure 4c). The conversion of cinnamyl alcohol increased with the increase in molar ratio from 1:1 to 1:6. However, the conversion increased slightly after 1:4, so 1:4 was selected as the optimal molar ratio for the financial considerations. Temperature, as an important factor, not only influences the activity and thermostability of esterases but also affects the reaction equilibrium and properties of reaction media.9,17 From 25 to 40 °C, the conversion increased slightly and then decreased by 23% at 50 °C (Figure 4d). Therefore, 40 °C was considered the optimal temperature, under which the highest transesterification activity could be obtained and the loss of products could be reduced. Under the optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio = 1:4, temperature = 40 °C), the relationship between reaction time and the conversion ratio of cinnamyl alcohol was studied. As can be seen, the reaction speed increased rapidly with the increase of biocatalyst loading from 2.5 to 10 mg/mL (Figure 5). With a loading of 10 mg/mL biocatalyst, the conversion of cinnamyl alcohol reached 94.1% at 1.0 h and 97.1% at 2.0 h (Figure S3), which corresponds to a yield of cinnamyl acetate of 19.42 mM at 2.0 h. It is important to emphasize that the conversion obtained in the present study is higher than that reported in a previous

Figure 5. Time course of synthesis of cinnamyl acetate with different biocatalyst loadings. Reaction conditions: vinyl acetate as acyl donor; isooctane as solvent; molar ratio, 1:4; temperature, 40 °C.

study with Novozym 435 used to synthesize cinnamyl acetate.8 The reaction was carried out using cinnamyl alcohol as substrate and ethyl acetate as acyl donor, and only a 90.06% conversion was obtained under the optimal conditions.8 In another study, with porcine pancreatic lipase immobilized on metal ceramic powder and used for synthesizing cinnamyl acetate, the cinnamyl acetate yield was 62.56% after 10 h.14 Thus, it can be concluded that EstK1 is a far more efficient F


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Journal of Agricultural and Food Chemistry biocatalyst than lipase for the synthesis of cinnamyl acetate. Because of the preference of EstK1 for short-chain acyl esters, we tested and found that whole-cell biocatalyst of EstK1 could be used as an efficient alternative to synthesize cinnamyl acetate. There were also some studies on the enzyme-mediated synthesis of ethyl esters. In a recent study, the synthesis reaction of ethyl cinnamate was carried out using isooctane as solvent and Lipozyme TLIM as biocatalyst. It is worth pointing out that the reaction system was pre-equilibrated for 3 days to obtain the initial water activity (aw) in this study.17 However, our reaction was carried out using a nonaqueous system without the addition of additional water, which can significantly simplify the procedures and reduce the time. In another paper, the conversion yield of ethyl cinnamate was not higher than 35.2% (at 96 h), which indicated that it was needed to obtain an alternative to Novozyme 435 in the synthesis of ethyl cinnamate.47 Porcine pancreatic lipase was also used to synthesize ethyl cinnamate, which allowed a lower yield level (55% at 27 h) in a remarkably longer reaction time.15 Furthermore, the use of DMSO as solvent is not desirable, which may complicate the procedures for postprocessing.15 Synthesis of Other Acetic Esters. The dried whole-cell biocatalyst was used to synthesize various acetic esters under similar conditions (Scheme 1). EstK1 showed substrate

Figure 6. Synthesis of other acetic esters. Cinnamyl acetate was used as control. The conversion ratio of cinnamyl alcohol was defined as 100%.

was used as biocatalyst to synthesize cinnamyl acetate. Through optimizing reaction conditions, the conversion of cinnamyl alcohol could be up to 94.1% at 1 h. Furthermore, most of the cinnamyl alcohol (97.1%) was converted to cinnamyl acetate at 2 h, which indicated whole-cell EstK1 was an efficient biocatalyst. EstK1 could also be used to prepare other acetic esters, making it a promising alternative biocatalyst in the industries. This study also demonstrated that esterase might be used as a better biocatalyst to prepare cinnamyl acetate because of its preference for short-chain acylglycerols.

Scheme 1. EstK1-Catalyzed Transesterification of Acetic Esters

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05799. Figure S1: partial digestion of genomic DNA with Sau3AI. Figure S2: SDS-PAGE analysis of the expression level of EstK1. Figure S3: GC analysis of cinnamyl alcohol and cinnamyl acetate. Table S1: Gradient elution of acetic esters (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(X.M.) Phone: +86-532-82031360. Fax: +86-532-82032272. E-mail: [email protected].

specifity toward different alcohols. In particular, n-octyl alcohol, 2-phenylethanol, citronellol, and geraniol could be used as substrates to prepare corresponding acetic esters (Figure 6). The reaction rate of 2-phenethyl acetate (110.03%) was higher than that of cinnamyl acetate (100%), whereas the reaction rates of other acetic esters were lower (n-octyl acetate, 14.97%; citronellol acetate, 13.11%; geraniol acetate, 22.06%) than that of cinnamyl acetate. The reason might be that the optimal conditions were for the synthesis of cinnamyl acetate. After optimization respectively, the conversion of n-octyl alcohol, citronellol, and geraniol could be improved further. EstK1 showed transesterification activities toward different alcohols in the nonaqueous system, which indicated that EstK1 might be an efficient candidate for the industrial process. In this work, we identified a novel esterase (EstK1) gene from the genomic DNA library of A. hemolyticus. Whole-cell catalysis has significant advantages, such as simplifying the process and reducing the production cost. Therefore, after overexpression in BL21 (DE3), the dried whole cell of EstK1

ORCID

Xiangzhao Mao: 0000-0002-6315-1338 Funding

This work was supported by the Fundamental Research Funds for the Central Universities (201564018), Qingdao Shinan District Science and Technology Development Funds (2014− 14-002-SW), Major Special Science and Technology Projects in Shandong Province (2016YYSP016), and the National Natural Science Foundation of China−Shandong Joint Fund for Marine Science Research Centers (U1406402). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED CIP, alkaline phosphatase calf intestinal; pNP, p-nitrophenol; pNPA, p-nitrophenyl acetate; pNPB, p-nitrophenyl butyrate; pNPC, p-nitrophenyl caprylate; pNPD, p-nitrophenyl decanoate; pNPL, p-nitrophenyl laurate; pNPM, p-nitrophenyl G


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myristate; pNPP, p-nitrophenyl palmitate; ORF, open reading frame; MTBE, methyl tert-butyl ether; IPTG, isopropyl-β-D-1thiogalactopyranoside

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Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel

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