This document summarizes research on the production of ferulic acid esterase by Streptomyces sp. Key findings include: - Streptomyces sp. isolate S10 was identified that produced ferulic acid esterase when grown in de-starched wheat bran medium. - Maximum ferulic acid esterase production of 2.0 mU/ml occurred in MBS medium containing 1.5% de-starched wheat bran at 30°C and pH 6.5 with agitation after 96 hours of fermentation. - Hydrolysis experiments confirmed the enzyme produced was ferulic acid esterase as it released ferulic acid from the de-starched wheat bran substrate.

1. Bioresource Technology 98 (2007) 211–213
0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.12.001
Short Communication
Ferulic acid esterase production by Streptomyces sp.
G. Mukherjee a
, R.K. Singh a
, A. Mitra b
, S.K. Sen a,¤
a
Microbiology Division, School of Life Science, Visva-Bharati, Santiniketan 731 235, India
b
Department of Agriculture and Food Engineering, Indian Institute of Technology, Kharagpur 721 302, India
Received 25 July 2005; received in revised form 22 November 2005; accepted 2 December 2005
Available online 19 January 2006
Abstract
Studies were carried out on ferulic acid esterase production using a culture of Streptomyces S10. In optimized condition, enzyme yield
was 2.0 mU/ml in MBS medium, containing 1.5% de-starched wheat bran at 30 °C and initial pH 6.5 under agitated submerged culture.
© 2005 Elsevier Ltd. All rights reserved.
Keywords: Ferulic acid esterase; Ferulic acid; Streptomyces; Submerged fermentation
1. Introduction
Sugar–phenolic acid ester linkages are hydrolysed by
ferulic acid esterase (FAE). Cell wall biodegradation is
strongly inXuenced by phenolic acids and commonly per-
formed by fungi and bacteria (Christov and Prior, 1993).
Certain microXora can well survive in presence of phenolic
acids by utilizing them as carbon source. FAE activity has
been reported in Streptomyces olivochromogenes (Faulds
and Williamson, 1993), Streptomyces avermitilis UAH30
(Garcia et al., 1998), Schizophyllum commune (Mackenzie
and Biolous, 1988), and also in other microbial sources
(Faulds and Williamson, 1993).
Ferulic acid (FA) and p-coumaric acid are common con-
stituents of forage and may represent up to 2.5% of cell
walls. FA is ester-linked to arabinose in various plant poly-
saccharides, such as arabinoxylans and pectins, and may
play a role in cell wall growth and stabilization (Ishii and
Hiroi, 1990). Wheat bran (WB), a common agro-residue,
contains 0.66% (w/w) alkali extractable FA. Considerable
interest has been shown in FAE to obtain FA from agro-
wastes (Faulds et al., 1997a). FA exhibits a number of
potential commercial applications, such as natural antioxi-
dant, food preservative agent, anti-inXammatory agent,
photoprotectant (Graf, 1992). Above all, FA is the most
promising substrate for natural vanillin production by bio-
transformation (Overhang et al., 2002). The aim of the pres-
ent investigation was to identify FAE production by a new
isolate of Streptomyces sp.
2. Methods
2.1. Screening for FAE producer
Soils samples were screened using dilution method on
arginine glycerol salt agar plates to isolate strains of Strep-
tomyces sp. Several colonies appeared, which were sub-
cultured and one of them (isolate S10) was picked up for
detailed characterization (results not shown). The selected
strain (isolate S10) was characterized following the methods
described in Bergey’s Manual of Systematic Bacteriology
(Williams et al., 1989) and was identiWed as Streptomyces
sp.
2.2. De-starching of wheat bran and fermentation
De-starched wheat bran (DSWB) was prepared follow-
ing the method of Johnson et al. (1988) with modiWcation.
WB was treated with 0.30% (w/v) potassium acetate at
95°C for 30 min followed by extensive washing with water
to remove starch. Fermentation was carried out using MBS
medium (Crawford, 1978), supplemented with 1% DSWB
*
Corresponding author. Tel.: +91 3463 261686; fax: +91 3463 261268.
E-mail address: sksenvb@rediVmail.com (S.K. Sen).

2. 212 G. Mukherjee et al. / Bioresource Technology 98 (2007) 211–213
as carbon source to 20ml medium in 100 ml conical Xask.
The whole set was sterilized under 15 lb/in.2
. The medium
was inoculated with 1ml (in 0.1% sterile NaCl) spore
suspension (1.5£ 106
spores/ml, counted using haemocyto-
meter) and initial pH, and temperature were 7.0 and 30°C
and fermentation carried out for 96 h and assay was done at
every 24h. Fermented broth was centrifuged (10,000 rpm,
15 min) and supernatant was used as enzyme source. To
optimize FAE production, several media (Table 1) were
tried substituting their carbon source with 1% DSWB. To
Wnd out the eVect of agitation during fermentation, the
experiment was carried out in still and shaking (120 rpm;
Orbitek, Scigenics, India) conditions in Xasks (100 ml). For
determination of optimum pH diVerent buVer system was
used.
2.3. Assay of FAE
For FAE assay, the reaction mixture contained 100 mg
DSWB and crude enzyme (2.5 ml, centrifuged supernatant
of fermented broth) in phosphate buVer (2.5 ml, 70 mM, pH
6.5) to a Wnal volume of 2.0 ml and incubated for 30 min at
50 °C. The reaction was stopped by putting the mixture in
boiling water for 3min. After centrifugation (10,000g,
15 min), the FA content of the supernatant was determined
through HPLC. FAE activity (1mU) was deWned as the
enzymes required to release 1 mol FA per min at 50 °C and
pH 6.5. Background FA levels were subtracted during cal-
culations. Protein content was estimated using bovine
serum albumin as standard (Lowry et al., 1951).
2.4. Detection of FA
The cell free supernatant was scanned through Spectro-
photometer (Jasco 7800, Japan) in a range of 240–400nm
and compared with authentic sample (Ferulic acid, Hi-
Media, India). HPLC analysis was performed in Torrence
(CA, USA) equipped with a UV detector, using C18 (RP-
HYDRO 4 m, 250 £4.6 mm) column. Feruloylated mate-
rial was analyzed (software version 3.20, Waters) using an
isocratic linear solvent gradient of methanol:water:triXuou-
roacetic acid (3:2:6) as eluant at a Xow rate of 1 ml/min for
25 min and monitored at 320 nm.
2.5. Hydrolysis of DSWB
Alkali extracted FA content of DSWB (10 mg) was mea-
sured following incubation of substrate with 1 M NaOH
(2 ml) for 2 h at 100 °C in dark. The FA content of the sam-
ple was determined by HPLC (Garcia et al., 1998). The
enzymatically hydrolysed product was separated by centri-
fugation (10,000 rpm, 10 min) and the hydrolysate was ana-
lyzed by TLC, using cellulose (Merck, India) along with
authentic sample. The chromatogram was developed using
aqueous formic acid (2%). The plate was observed under
254 nm (Uvitec, Genei, India).
3. Results
After 96 h of fermentation with DSWB as the substrate,
the cell free culture broth was analyzed for RA production
and out of 52 isolates, only six isolates viz; S6, S7, S8, S10, S35
and S39, showed notable FAE production (results not
shown). On the basis of FA production, the isolate S10 was
selected for further studies. The studies of morphological,
micromorphological, cultural, physiological and biochemi-
cal characteristics of S10 showed rectiXexible yellow spore
chain, smooth spore surface, production of diVusible pig-
ment and melanin on tyrosine agar, reduction of nitrate to
nitrite, utilization of phenylalanine and no growth at 45 °C
indicated it to belong to Streptomyces sp.
All the tested media supported FAE production but
maximum production (1.6 mU/ml) was obtained with MBS
medium (Table 1). Studies on the eVect of agitation showed
better enzyme production under agitation (1.74 mU) than
under static (1.60 mU). To optimize the period of fermenta-
tion, experiment was carried out for 120 h. FAE production
started after 24 h but maximum production was at 96 h
(Fig. 1). The eVect of temperature on FAE was evaluated in
a range of 20–50 °C. The optimum production temperature
was 30 °C, with enzyme activity and speciWc activity of
1.74 mU/ml and 14.58 mU/mg, respectively (Fig. 1). To
Table 1
Suitability of fermentation medium for FAE production by the selected
isolate S10
Media FAE
(mU/ml)
SpeciWc activity
(mU/mg)
IAF medium (Ishaque and Kleupfel,
1980)
1.52 12.45
MBS medium (Crawford, 1978) 1.60 13.54
Minial medium (Donnelly and Crawford,
1988)
1.56 13.45
Basal medium (Christakopoulos et al.,
1996)
1.48 12.35
Mineral salt medium (Asther et al., 2002) 1.58 13.50
Mineral medium (Uchida et al., 2003) 1.50 12.38
Fig. 1. EVect of fermentation time period and temperature for FAE
production.
Temperature (°C)
Time (hr)
0 20 40 60 80 100 120 140
FAE(mU/ml)
0
1
2
3
Specificactivity(mU/ml)
0
2
4
6
8
10
12
14
16
15 20 25 30 35 40 45 50 55
FAE activity (Time)
Specific activity (Time)
FAE activity (Temp)
Specific activity (Temp)

3. G. Mukherjee et al. / Bioresource Technology 98 (2007) 211–213 213
optimize the pH of the medium, the fermentation was car-
ried out in a range of pH 5.0–9.0 (Fig. 2). The isolate S10
showed its adaptive quality to this varied range and pH 6.5
was optimum. EVect of substrate (DSWB) concentration of
the fermentation medium was tested and maximum yield
was at 1.5% DSWB (Fig. 2), showing 2.0 mU/ml enzyme
activity and 15.45 mU/mg of speciWc activity. Cochroma-
tography of FAE hydrolysed product and authentic sample
showed Rf value 0.28 that corresponded to FA (data not
shown).
4. Discussion
It has been reported that WB contained several ester-
linked dehydrodimers of FA, which was released by enzyme
activity (Ferreira et al., 1999). DSWB supplemented MBS
medium was most suitable for FAE fermentation with the
isolate S10. Higher FAE production as observed under agi-
tated condition in this study was similar to earlier reports,
which indicated oxygen tension during metabolism (John-
son et al., 1988; Donnelly and Crawford, 1988). The present
study showed optimum FAE production at 96 h. However,
in other Streptomyces spp., it was reported as 48 h (Ferreira
et al., 1999) and 72 h (Johnson et al., 1988).
Several authors reported DSWB as the main carbon
source for FAE production by Streptomyces (Faulds and
Williamson, 1993; Garcia et al., 1998; Ferreira et al., 1999).
The isolate S10 showed optimum FAE production with 1.5%
DSWB. The FAE production was triggered with oat spelt
xylan as carbon source in S. olivochromogenes (Johnson
et al., 1988), but the component responsible for FAE induc-
tion was not been identiWed (Ferreira et al., 1999). FAE of
S. avermitilis CECT 3339 could release 10% FA by hydroly-
sing DSWB (Ferreira et al., 1999). FAE activity could not be
correlated with the esteriWed FA of DSWB (Garcia et al.,
1998; Ferreira et al., 1999). In Aspergillus niger, induction of
FAE was possible only with the presence of free FA but not
with esteriWed FA (Faulds et al., 1997b).
Acknowledgements
We gratefully acknowledge the Wnancial support
received from DBT, Ministry of S&T, Government of
India.
References
Asther, M., Haon, M., Roussos, S., Record, E., Delattre, M., Lesage-Mees-
sen, L., Labat, M., Asther, M., 2002. Feruloyl esterase from Aspergillus
niger a comparison of the production in solid state and submerged
fermentation. Process Biochem. 38, 685–691.
Christakopoulos, P., Mamma, D., Nerinckx, B., Kekos, D., Macris, B.,
Claeyssens, M., 1996. Production and partial characterization of
xylanase from Fusarium oxysporum. Bioresour. Technol. 58, 115–
119.
Christov, L.P., Prior, B.A., 1993. Esterases of xylan-degrading microorgan-
isms: production properties and signiWcance. Enzyme Microb. Technol.
15, 460–475.
Crawford, D.L., 1978. Lignocellulose decomposition by selected Strepto-
myces strains. Appl. Environ. Microbiol. 35, 1041–1045.
Donnelly, P., Crawford, D.L., 1988. Production by Streptomycess virido-
sporus T7A of an enzyme which cleaves aromatic acids from lignocellu-
lose. Appl. Environ. Microbiol. 54, 2237–2244.
Faulds, C.B., Williamson, G., 1993. Release of Ferulic acid from plant
polysaccharides by ferulic acid esterase from Streptomyces olivochrom-
ogenes. Carbohydr. Polym. 21, 153–155.
Faulds, C.B., Bartolome, B., Williamson, G., 1997a. Novel biotransforma-
tion of agro-industrial cereal waste by ferulic acid esterases. Ind. Crops
Prod. 6, 367–374.
Faulds, C.B., de Vries, R.P., Kroon, P.A., Visser, J., Williamson, G., 1997b.
InXuence of ferulic acid on the production of feruloyl esterases by
Aspergillus niger. FEMS Microbiol. Lett. 157, 239–244.
Ferreira, P., Diez, N., Gutierrez, C., Soliveri, J., Copa-Patino, J.L., 1999.
Streptomyces avermitilis CECT 3339 produces a ferulic acid esterase
able to release ferulic acid from sugar beet pulp soluble feruloylated
oligosaccharides. J. Sci. Food Agric. 79, 440–442.
Garcia, B.L., Ball, A.S., Rodriguez, J., Perez-Leblic, M.I., Arias, M.E.,
Copa-Patina, J.L., 1998. Induction of ferulic acid esterase and xylanase
activities in Streptomyces avermitilis UAH30. FEMS Microbiol. Lett.
158, 95–99.
Graf, E., 1992. Antioxidant potential of ferulic acid. Free Radical Biol.
Med. 13, 435–448.
Ishaque, M., Kleupfel, D., 1980. Cellulase complex of a mesophilic Strepto-
myces strain. Can. J. Microbiol. 26, 183–189.
Ishii, T., Hiroi, T., 1990. Linkage of phenolic acids to cell-wall polysaccha-
rides of bamboo shoot. Carbohydr. Res. 206, 297–310.
Johnson, K.G., Harrison, B.A., Schneider, H., Mackenzie, C.R., Fontana,
J.D., 1988. Xylan-hydrolysing enzymes from Streptomyces spp.
Enzyme Microbiol. Technol. 10, 403–409.
Lowry, O.H., Rosenbrough, J.N., Farr, A.L., Randall, R.J., 1951.
Protein measurement with folin–phenol reagent. J. Biol. Chem. 193,
265–275.
Mackenzie, C.R., Biolous, D., 1988. Ferulic acid esterase activity
from Schizophyllum commune. Appl. Environ. Microbiol. 54, 1170–
1173.
Overhang, J., Steinbüchel, A., Priefert, H., 2002. Biotransformation of
eugenol to ferulic acid by a recombinant strain of Ralstonia eutropha
H16. Appl. Environ. Microbiol. 68, 4315–4321.
Uchida, H., Kurakata, Y., Sawamura, H., Inamura, N., Kotani, T., Uwaj-
ima, T., 2003. PuriWcation and properties of an esterase from Asper-
gillus nomius HS-1 degrading ethylene glycol dibenzoate. FEMS
Microbiol. Lett. 223, 123–127.
Williams, S.T., Dharpeand, M.E., Holt, J.G., 1989. Bergey’s Manual of
Systematic Bacteriology (vol. 4). Williams and Wilkins Company,
Baltimore, USA.
Fig. 2. EVect of pH and wheat bran for FAE production.
Wheat bran (%)
pH
4 9 10
FAE(mU/ml)
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
2.1
0
Specificactivity(mU/ml)
14.4
14.6
14.8
15.0
15.2
15.4
15.6
FAE activity (pH)
Specific activity (pH)
FAE activity (WB)
Specific activity (WB)
5 6 7 8
1 2 3 4