Food Chemistry 124 (2011) 1096–1098

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Food Chemistry

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Short communication

Enzymatic hydrolysis of capsaicins for the production of vanillylamine usingECB deacylase from Actinoplanes utahensis

Diego Romano a, Raffaella Gandolfi b, Simone Guglielmetti a, Francesco Molinari a,*

a Department of Food Science and Microbiology, University of Milan, Via Mangiagalli 25, 20133 Milan, Italyb Department of Molecular Science Applied to Biosystems, University of Milan, Via Venezian 21, 20133 Milan, Italy

a r t i c l e i n f o

Article history:Received 9 February 2010Received in revised form 23 April 2010Accepted 17 June 2010

Keywords:AcylaseCapsaicinVanillylamineBiotransformationActinoplanes utahensis

0308-8146/$ - see front matter � 2010 Published bydoi:10.1016/j.foodchem.2010.06.070

* Corresponding author. Fax: +39 0250319191.E-mail address: francesco.molinari@unimi.it (F. M

a b s t r a c t

Echinocandin acylase from Actinoplanes utahensis NRRL 12052 (ECB deacylase) was used for the hydroly-sis of capsaicins (trans-8-methyl-N-vanillyl-6-nonenamide and 8-methyl-N-vanillyl-6-nonanamide) intovanillylamine. Caspaicins were extracted from Capsicum annuum var. Red Cayenne. The enzyme was par-tially purified and used for improving molar conversion and reaction rate of the biotransformation. Underoptimised conditions, the preparative biotransformation of 8.2 mM capsaicins on 200 ml scale allowedfor 92% molar conversions and 59% of recovered vanillylamine.

� 2010 Published by Elsevier Ltd.

1. Introduction

Chily peppers and related plants of the Capsicum family producea mixture of capsaicins (trans-8-methyl-N-vanillyl-6-nonenamideand 8-methyl-N-vanillyl-6-nonanamide), which are the mostimportant molecules in terms of abundance and pungency (Suzuki& Iwai, 1984). Deacylation of these amides furnishes vanillylamine(4-hydroxy-3-methoxybenzylamine) which can be furtherenzymatically converted into vanillin using specific microbial orplant oxidase (Sudhakar Johnson, Ravishankar, & Venkataraman,1996; Yoshida et al., 1997;). This two-step enzymatic conversionof natural capsaicins into vanillin (Scheme 1) is attractive sincethe product obtained can be classified as a natural flavour. Indeed,US and European legislations state that ‘natural’ flavour substancescan only be prepared either by extraction from natural sources orby enzymatic or microbial processes, which involve precursors iso-lated from nature (Serra, Fuganti, & Brenna, 2005).

Candida antarctica lipase B (CALB) (Reyes Duarte, Castillo,Barzana, & Lopez-Munguia, 2000; Torres-Gavilan, Castillo, & Lo-pez-Munguia, 2006) and with an extracellular enzyme fromStreptomyces mobaraensis (Koreishi et al., 2006) with best yieldsaround 2.3 mM vanillylamine after 72 h. In this work, we havescreened twenty actinomycetes known as producers of acylaseactivity (Gandolfi, Jovetic, Marinelli, & Molinari, 2007) for selectingenzymes able to catalyse the hydrolysis of capsaicins with high

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olinari).

molar conversions. An acylase produced from Actinoplanes utahen-sis NRRL 12052 (Boeck, Fukuda, Abbott, & Debono, 1988; Kreuzmanet al., 2000) was selected and the enzymatic transformation wasoptimised.

2. Materials and methods

2.1. Chemicals

Unless otherwise stated, all reagents were of analytical gradefrom Sigma (St. Louis, MO).

2.2. Microorganisms

The actinomycetes used in this study (Gandolfi et al., 2007)were from our collection and from commercial collections (NRRL,Northern Utilisation Research Branch, USDA, Peoria, Illinois, USAand ATCC-LGC Promochem, American Type Culture Collection,LGC Promochem, Sesto San Giovanni, Milano, Italy). Spores of acti-nomycetes were maintained in Nutrient Glycerol (Nutrient brothDifco 8 g l�1, glycerol 150 ml l�1) in 96-well microtitre plates at�20 �C.

2.3. Culture conditions

Actinomycetes were inoculated into a vegetative mediumcomposed with glucose 20 g, yeast extract 2 g, soybean meal 8 g,NaCl g and CaCO3 1 g in 1 l tap water at pH 7. After incubation

NH

R

OMe

HO

O

R =

R =

NH2

OMe

HO

trans-8-methyl-N-vanillyl-6-nonenamide (capsaicin)

trans-8-methyl-N-vanillyl-6-nonanamide(dihydrocapsaicin)

vanillylamine

CHO

OMe

HO vanillin

acylase

amino oxidase

Scheme 1. Two-step enzymatic conversion of capsaicins into vanillin.

D. Romano et al. / Food Chemistry 124 (2011) 1096–1098 1097

for 72–96 h at 28 �C, the mycelia suspension (4%) was transferredinto the AUR/M liquid medium composed with maltose 20 g, dex-trin 10 g, yeast extract 2 g, meat extract 4 g, peptone 4 g, soybeanmeal 15 g and CaCO3 2 g, in 1 l tap water at pH 7. A. utahensis NRRL12052 was also grown in AUR/M medium in a 20-l Chemap fer-menter (Model CF 3000-DCU, Type SG) at 28 �C, stirrer speed at600 rpm and aeration rate 0.5 vvm. The mycelia were harvestedafter 96 h by centrifugation, washed with 0.05 M KH2PO4 pH 6and used for enzyme purification.

2.4. Partial purification of the enzyme

Mycelia of A. utahensis were suspended in 0.05 M KH2PO4 pH 6in the presence of KCl (0.7 M) and 15% dimethylsulphoxide andmaintained at 4 �C for 24 h; the suspension was then treated at60 �C for 1 h and centrifuged; the supernatant (containing mostof the acylase activity) was dia-filtered through a 10,000 MWultrafiltration (UF) membrane using a UF stirred cell (Model8050, capacity 50 ml) using 0.05 M KH2PO4 pH 6 solution. Theultrafiltrate fraction containing 0.38–0.42 mg/ml of total proteinswas used for bioconversion optimisation. Total protein contentwas determined according to a standard Bradford assay (Bradford,1976).

2.5. Extraction of capsaicins from Capsicum annuum var. Red Cayenne

The extraction of capsaicins was performed using the followingoptimised protocol: dry C. annuum var. Red Cayenne (200 g) washomogenised by grinding in a mortar in acetone (500 ml) and ex-tracted with 500 ml of acetone twice; the organic extracts werecollected and evaporated, furnishing 10.1–10.3 g of crude extract.The crude product was purified by silica gel 60 (70–230 mesh,Merck) chromatography (ethyl acetate/hexane 1/1) yielding1.47 g of pure capsaicins, having a relative composition of capsai-cin 65/dihydrocapsaicin 35.

2.6. Biotransformations

The screening of whole microbial cells was carried out by usingthe mycelium and the supernatant independently. The myceliumwas recovered by centrifugation at 12,000 rpm for 20 min, washedwith physiological solution and suspended in 0.1 M phosphate buf-fer pH 7.0 at the same volume used for the growth; substrate(0.4 g l�1) was added as dimethylformamide (DMF) solution(1% vol vol�1). The supernatant was directly used for biotransfor-mation by adding the substrate (0.4 g l�1) in the presence of DMF

(1% vol vol�1). After incubation on a rotary shaker (200 rpm) at28 �C for 24 h, samples (200 ll) were withdrawn, microfiltered(0.45 lm), diluted with an equal amount of CH3CN and analysedby HPLC.

Biotransformations, with partially purified acylase from A. utah-ensis, were performed by adding the substrate dissolved in differ-ent co-solvents (acetone, acetonitrile, DMF, dimethylsulphoxide,ethanol and mixtures of these solvents) to the enzymatic solution(0.038–0.042 mg ml�1 of total protein in different buffers). Theconditions of the sequential experimental trials were selectedemploying the Multisimplex� 2.0 software (Walters, Parker, Mor-gan, & Deming, 1991). The control variables were temperature,pH, type of buffer and the two response variables were initial reac-tion rate and the molar conversion after 24 h. Initial reaction ratewas defined as the lmol of vanillylamine produced in one minuteper mg of total protein.

A preparative biotransformation was carried out using 200 mlof enzymatic extract added with 0.5 g of capsaicins in the presenceof 2.5% (vol/vol) of DMF. The reaction mixture was incubated at60 �C for 24 h on a reciprocal shaker (120 spm) and stopped byultrafiltration. The ultrafiltrate was treated with a solution ofNaOH (0.2 M) until pH 9.0 and extracted with diethyl ether(100 ml, three times); the organic extracts were washed withwater (100 ml), dried on Na2SO4 and concentrated by evaporationunder reduced pressure; the residue was treated with 1 ml of asolution of HCl (0.1 N) and crystallised by adding 4 ml of ethanol.Vanillylamine hydrochloride was recrystallised from 95% ethanol,filtered off, washed with acetone and dried at 50 �C and allowedfor the obtainment of light yellow crystals of pure vanillylaminehydrochloride (191 mg, 59%). Purity was checked by melting pointand 1H: mp: 221–223 �C. 1H NMR (CDCl3/DMSO-d6) d: 9.26 (s, 1H),8.41 (s, 2H), 7.19 (s, 1H), 6.89 (d, 1H), 6.80 (d, 1H), 3.94 (s, 2H), 3.78(s, 3H).

2.7. Analytical methods

Analysis of the biotransformations was performed at 30 �C onHPLC Merck Hitachi 655A, with UV detector Merck Hitachi L-4000 and a 5 lm particle size Supelco Hypersil ODS (C18) column(4.6 � 250 mm). The solvent system consisted of a phosphate buf-fer pH 8.0 (0.01 M) and acetonitrile (1/1). The flow-rate was1.0 ml min�1. Injection volume was 20 ll and UV detector(254 nm). Retention times were: vanillylamine 5.2 min, capsaicin8.5 min and dihydrocapsaicin 10.5 min. Samples of authentic cap-saicins and vanilyllamine hydrochloride (Sigma, St. Louis, USA)were used for comparison.

Table 1Mycelium-bound and extracellular activity of A. utahensis on a mixture of capsaicin/dihydrocapsaicin 65/35. The biotransfor-mation is expressed as molar conversion of the substrate. Biotransformation was performed by adding capsaicins (1 g l�1) in thepresence of N,N-dimethylformamide (DMF, 1% vol vol�1). Less than 3% hydrolysis was observed in the absence of enzymes.

1 h 24 h

Capsaicin Dihydrocapsaicin Capsaicin Dihydrocapsaicin

Mycelium-bound

6 7 42 44

Extracellular 0 0 5 5

1098 D. Romano et al. / Food Chemistry 124 (2011) 1096–1098

3. Results

A screening for the hydrolysis of capsaicins was performedamongst 20 actinomycetes previously assayed for acylase activity(Gandolfi et al., 2007), showing that A. utahensis NRRL 12052 wasthe only strain able to furnish molar conversion above 15%. Thebiotransformations were carried out using the mixture of capsaic-ins (capsaicin/dihydrocapsaicin 65/35) extracted from C. annuum(Table 1).

The activity was mostly mycelium-bound and both the capsaic-ins were hydrolysed with similar reaction rates and degrees of con-version. The hydrolysis of capsaicins occurred with transientformation of vanillylamine which was further degraded.

The activity towards capsaicins was mostly found in the salt-sol-ubilised extract (>95% of the total activity). A membrane-boundacylase from A. utahensis NRRL 12052 (ECB deacylase) has been al-ready used for the hydrolysis of different antibiotics, such as echi-nocandin B, A21978C and teicoplanin (Boeck et al., 1988;Kreuzman et al., 2000). The enzyme was solubilised from myceliumby treatment with KCl, heat-treated at 60 �C for 1 h and the solubleextract filtered through a 10,000 MW UF membrane until a totalprotein concentration of 0.39 mg ml�1. Biotransformation was per-formed by adding capsaicins (1 g l�1) in the presence of DMF(1% vol vol�1) observing an initial rate of 0.012 lmol mg�1

prot min�1.Under these conditions, vanillylamine could be accumulated inthe biotransformation medium with a molar conversion of 68%.

Capsaicins are poorly water soluble, therefore, different co-sol-vents were investigated for improving the solubility of the substrate:acetone, acetonitrile, dimethylformamide, dimethylsulphoxide,ethanol and mixtures of these solvents. The use of DMF(2.5% vol vol�1) allowed the highest conversion. The optimisationof the biotransformation was carried out by simultaneously evaluat-ing different parameters of the biotransformation (temperature, pH,type of buffer) using the Multisimplex experimental design. Citrate,phosphate and Tris–HCl buffers were used for achieving different pHvalues. The best conditions for the biotransformation were 10 mMphosphate buffer at pH 7.2, 60 �C in the presence of 2.5% DMF. Theeffect of the substrate concentration was studied under optimisedconditions with initial concentrations of capsaicins ranging from0.4 to 4 g l�1 (Fig. 1).

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5Substrate concentration (mg/ml)

Initi

al ra

te ( µ

mol

/mg

min

)

0

20

40

60

80

100

Mol

ar c

onve

rsio

n (%

)

initial rate molar conversion

Fig. 1. Effect of capsaicins concentration on initial rate and molar conversion after24 h.

The highest rates were observed using 2.5–3.0 g l�1, while thehighest yields after 24 h with initial substrate concentration of2.5 g l�1 (91–93%). Preparative biotransformation was carried outon 200 ml scale with an enzymatic extract containing 78–82 mgof total protein starting from 500 mg of substrate: after 24 h a90–92% molar conversion was determined by analytical means.Pure vanillylamine hydrochloride (191 mg) was recovered afterpurification.

4. Conclusions

In conclusion, we have described a novel method for the effi-cient bioconversion of capsaicins into vanillylamine using themembrane-bound ECB deacylase from A. utahensis. The enzymeshows better results in terms of rates and conversions than whatobserved with commercial CALB lipase (Torres-Gavilan et al.,2006) and an acylase from Streptomyces mobaraensis (Koreishiet al., 2006); moreover, the set-up of preparative biotransforma-tion using ECB deacylase as partially purified enzymatic prepara-tion is simple.

Acknowledgements

This work has been supported by Ministry of University and Re-search (MIUR) funding PRIN: ‘‘Production and application of plantand microbial enzymes for the synthesis of vanillin”.

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