Appl Microbiol Biotechnol
DOI 10.1007/s00253-010-2897-4
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Citrus peel influences the production of an extracellular
naringinase by Staphylococcus xylosus MAK2
in a stirred tank reactor
Munish Puri & Aneet Kaur & Colin J. Barrow &
Ram Sarup Singh
Received: 7 July 2010 / Revised: 17 September 2010 / Accepted: 18 September 2010
# Springer-Verlag 2010
Abstract Staphylococcus xylosus MAK2, Gram-positive
coccus, a nonpathogenic member of the coagulase-negative
Staphylococcus family was isolated from soil and used to
produce naringinase in a stirred tank reactor. An initial
medium at pH 5.5 and a cultivation temperature of 30°C
was found to be optimal for enzyme production. The
addition of Ca+2 caused stimulation of enzyme activity. The
effect of various physico-chemical parameters, such as pH,
temperature, agitation, and inducer concentration was
studied. The enzyme production was enhanced by the
addition of citrus peel powder (CPP) in the optimized
medium. A twofold increase in naringinase production was
achieved using different technological combinations. The
process optimization using technological combinations
allowed rapid optimization of large number of variables,
which significantly improved enzyme production in a 5-
l reactor in 34 h. An increase in sugar concentration (15 gl−1)
in the fermentation medium further increased naringinase
production (8.9 IUml−1) in the bioreactor. Thus, availability
of naringinase renders it attractive for potential biotechno-
logical applications in citrus processing industry.
Keywords Citrus peel . Rhamnosidase . Naringin .
Bioreactor . Flavonoid
M. Puri (*) : C. J. Barrow
Centre for Biotechnology and Interdisciplinary Sciences
(Biodeakin), Institute for Technology Research and Innovation
(ITRI), Deakin University,
Victoria 3217, Australia
e-mail: munish.puri@deakin.edu.au
M. Puri : A. Kaur : R. S. Singh
Fermentation and Protein Biotechnology Laboratory, Department
of Biotechnology, Punjabi University,
Punjab 147002, India
Introduction
Naringinase (EC 3.2.1.40), a hydrolytic enzyme (an
enzyme containing both α- L -rhamnosidase and β-
glucosidase activities) detected in different species of
microorganisms, hydrolyzes naringin (4,5,7-trihydroxy
flavonone 7-rhamnoglucoside) to release L-rhamnose
and naringenin (4,5,7-trihydroxy flavonone; Puri et al.
2005). The α-L-rhamnosidase activity of this enzyme is of
particular interest to the structure determination of poly-
saccharides, glycosides, and glycolipids (Kamiya et al.
1985). Naringin and its hydrolyzed product naringenin are
useful for inhibiting acyl-coA-cholesterol-o- acyltransferase
activity, which helps in preventing accumulation of macro-
phage–lipid complex that causes hepatic diseases (Bok et al.
2000). It has several important industrial applications, and
these are reviewed elsewhere (Puri and Banerjee 2000).
Therefore, α-L-rhamnosidase activity of naringinase is
important not only for elucidation of the structures of
bioactive compounds containing L-rhamnose, but also for
potential application in the food and beverage industry (Puri
et al. 2010a). Recently, a process has been reported for the
production of α-L-rhamnosidase employing Penicillium
ulaiense in a stirred-batch reactor using rhamnose as the
main carbon source (Rajal et al. 2009).
Naringinase produced by several fungi, especially
Aspergillus niger, is used for elimination of bitter flavor
from narigin in citrus fruit juice (Bram and Solomons 1965;
Puri et al. 2005). The characteristics of naringinase from
fungi and plants have been thoroughly documented,
although the production of this enzyme in bacteria has to
date received little attention. Few naringinases have been
reported from bacterial origin, partially because their
industrial utility has led to protection by industry as trade
secrets. However, recently a few reports on α-L-rhamnosi-

dase activity from bacteria have appeared. These reports have
documented purification of α-L-rhamnosidases from Bacillus
sp. (Hashimoto et al. 2003), Burkholderia cenocepacia
(Cardona et al. 2006), Clostridium stercorarium (Zverlov et
al. 2000), Lactobacillus acidophilus (Beekwilder et al.
2009), Sphingomonas paucimobilis (Hashimoto and Murata
1998), and Thermomicrobium roseum (Jang and Kim 1996).
In this study, we have optimized medium and process
parameters to increase naringinase production from the
newly isolated strain Staphylococcus xylosus MAK2, in a
batch bioreactor. In addition, nariginase production has
been further improved by optimizing parameters in the
presence of CPP (comprised of peel, membranes, and juice
vesicles), a waste generated from the citrus processing
industry, through technological combination experiments.
CPP contains a high portion of polyphenols and has been
used for the production of fiber, ethanol, and D-rhamnose
(Puri et al. 2010b). These results show that production of
flavonoid-degrading enzymes from citrus peel powder
holds promises for by-product utilization.
Materials and methods
Chemicals
Naringin, soybean meal, and corn steep liquor (CSL) were
obtained from Sigma, St. Louis, MO, USA. Different
growth factors and organic and inorganic nitrogen sources
were obtained from Hi-Media Laboratories, Mumbai, India.
All other reagents used were of analytical grade. Dry
orange (Citrus reticulata) peel waste was collected from
local orange industry to prepare CPP as per little
modifications (Puri et al. 2010a).
Microorganism and maintenance of culture
S. xylosus MAK2, isolated and identified by 16S rRNA
analysis in our laboratory, was used in the present study
(Puri and Kaur 2010). The isolated strain (MAK2) was
deposited with International Depository Microbial Type
culture Collection and Gene Bank (MTCC, India) and
assigned no. 7443. The stock culture was maintained on
nutrient agar plates. The microorganism was initially grown
at 37°C for 12 h in a medium with the following
composition (gl−1): peptone, 10; beef extract, 5; and
sodium chloride, 5 by inoculating the medium with a
loopful of culture and incubating it in a temperature
controlled orbital shaker (25°C, 200 rpm). A 12-h-old, 5%
(v/v) seed culture was used to inoculate sterilized produc-
tion medium (100 ml in 500 ml Erlenmeyer flask) with the
following composition (gl−1): peptone, 10; beef extract, 5;
sodium chloride, 5; and citrus peel powder, 2. CPP
Appl Microbiol Biotechnol
containing maximum concentration of naringin (0.5%, w/v)
was used for the induction of naringinase in the fermentation
medium. The medium was sterilized for 15 min at 121°C,
15 psi (103,421.35 Pa) pressure, and the initial pH of the
medium was adjusted to 5.5. Flasks were incubated (30°C,
200 rpm) in a rotary shaker for 36 h. Samples were
withdrawn aseptically at regular time intervals and analyzed
for cell mass, naringinase activity, and residual substrate.
Optimization of process parameters
Using S. xylosus MAK2 for naringinase production, a range
of media constituents were optimized in shake flask for
enzyme production. To study the effect on enzyme
production, different carbon sources and their concentration
(1%, w/v) were added to the medium before autoclaving.
Different organic (1%, w/v) and inorganic (1%, w/v)
nitrogen sources and their concentrations were added to
the medium to determine their effect on naringinase
production. The chloride salts of different metal ions,
including Ca2+, Co2+, Cu2+, Fe3+, Mg2+, Ni2+, Mn2+, and
Zn2+ were used in the culture medium. The concentration
of CPP (0.5%, 1%, 1.5%, 2%, 2.5%, and 3%) was
optimized to test its effect on enzyme production. The
effect of temperature (25, 30, and 37°C), pH (4, 4.5, 5, 5.5,
6, 6.5, 7, 8, and 9) and agitation rate (150–350 rpm) on
enzyme production was tested in 500-ml Erlenmeyer flasks
containing 100 ml of medium. The effect of inoculum age
(4, 6, 8, 10, 12, 14, and 16 h) and inoculum volume (1–7%
v/v) was tested at the shake flask level. All experiments
were carried out in duplicate. Fermentation was carried out
in a 5-L bioreactor (In situ Bioage, India) with a working
volume of 3.5 L at 37 °C with aeration and agitation rates
of 0.5 vvm and 350 rpm, respectively. Samples were
withdrawn at regular time intervals and analyzed for cell
mass, naringinase activity, and residual substrate. Fermen-
tation was carried out at 30°C for 40 h, with aeration
(1 vvm) and agitation (300 rpm).
Assay methods
Naringin hydrolysis was measured using a modification of
the Davis method (Puri et al. 2005). A typical assay mixture
comprised of 0.9 ml naringin (0.05%, w/v), dissolved in
sodium acetate buffer (0.1 M, pH 4.0) and 100 μl of culture
filtrate. The assay mixture was incubated at 50°C for
60 min after which 100 μl was added to 5 ml 90%
diethylene glycol followed by the addition of 100 μl 4 N
NaOH. Samples were kept at room temperature (28°C) for
10 min, and intensity of the resultant yellow colour was
determined at 420 nm. One unit of naringinase activity was
defined as 1 μmol of naringin hydrolyzed under the above
assay conditions. Naringinase (i.e. α-L-rhamnosidase com-
Appl Microbiol Biotechnol
ponent) activity was also confirmed using modification of
the Romero method (Romero et al. 1985). The reaction
mixture contained 35 μl p-nitrophenyl-α-L-rhamnopyrano-
side, 5 μl enzyme solution, and 10 μl 0.5 M KH2PO4/
K2HPO4 buffer, pH 7.5 for 60 min. Reactions were stopped
by the addition of 100 μl of 1 M Na2CO3. The absorbance
was measured at 405 nm. One unit of enzyme activity was
defined as the amount of the enzyme that released 1 μmol
of p-nitrophenol per minute. Extracellular protein was
measured by the method of Lowry et al. (1951). Total
sugars were determined with anthrone method (Updegraff
1969).
Estimation of growth
The growth of the organism was estimated by taking 1-ml
aliquot periodically and measuring the optical density (OD) at
600 nm in a UV–visible spectrophotometer (Shimadzu1601,
Japan) with medium as a blank. The OD value was converted
into cell mass concentration (mgL−1) with the help of a
standard curve.
Designing technological combinations (24) on optimized
media components concentration
To reduce the cost of production of S. xylosus MAK 2 cells
with high naringinase activity, technological combinations
were applied in designing few set of experiments. Critical
components of the medium were selected on the basis of
the optimized conditions, which were determined using
classical variable methodology. Two levels of four factors
were considered leading to a set of 22 combinations of
culture conditions. Different combinations of optimum and
one lower level of optimized media concentration were
used under optimized conditions, rather than following a
variation of single parameter approach. The naringinase
activity and growth were then estimated.
Results
Media optimization for growth and naringinase production
S. xylosus MAK2 was initially grown on a nutrient medium
with citrus peel powder as an inducer. This organism
produces significant amount of extracellular naringinase in
the medium. The medium composition (detailed below)
was optimized in shake flasks to improve naringinase yield.
Effect of carbon source
Various carbon compounds were added at a concentration
of 10 gl−1 to the fermentation medium containing citrus
peel powder. Sucrose and molasses (1%, w/v) exhibited the
highest naringinase production (5.7 IUml−1) after 36 h of
fermentation. Other carbon sources, for example, lactose
(4.9 IUml−1) and maltose (4.9 IUml−1) also supported
naringinase production, but the yield was lower when
compared to that obtained with sucrose. However,
naringinase production was very low using either glucose
(2.25 IUml−1) or fructose (2.7 IUml−1). Suppression of
naringinase activity by rhamnose, citrate, and some other
carbon sources has been reported, although these carbon
sources supported excellent growth (Tsen et al. 1989).
Although molasses as a carbon source resulted in high
naringinase production, it was not selected as the main
carbon source, as it also produced a thick brown color,
which hindered the estimation of microbial growth during
fermentation runs. For all subsequent experiments, sucrose
was used as the primary carbon source. In order to determine
the optimum concentration of sucrose for enzyme production,
different concentrations (5–30 gl−1) of sucrose were used in
the medium. With increased sucrose concentration, naringi-
nase production increased (5.7 IUml−1) up to 15 gl−1 sucrose
in the medium and thereafter declined.
Effect of nitrogen source
Eight different nitrogen sources were used at a concentra-
tion of 10 gl−1 in a medium containing sucrose (15 gl−1).
Among all the organic nitrogen sources tested, tryptone and
soybean meal were the most effective for naringinase
production, producing 6.2 and 6.1 IUml−1, respectively. In
order to determine the optimum concentration of soybean
meal for naringinase production, different concentrations
(2.5–20 gl−1) of soybean meal were used in the medium
containing sucrose. Naringinase production was found to be
highest (6.1 IUml−1) with the optimum concentration of
soybean meal being 7.5 gl−1. Higher concentrations of
soybean meal in the fermentation medium did not signif-
icantly increase enzyme yield. For further experiments,
soybean meal (7.5 gl−1) was used in the cultivation of S.
xylosus for the production of naringinase.
Effect of CPP concentration
The concentration of inducer and its source plays an
important role in the enzyme synthesis. In this study, CPP
prepared from dried citrus peel waste has been used to
improve naringinase production. The highest naringinase
production yield of 6.1 IUml−1 was observed at 2% (w/v) of
citrus peel, and it decreased further on increasing inducer
concentration (Table 1). Among other inducers tested,
naringenin and rhamnose gave the lowest enzyme yield.
In the absence of an inducer, growth of microorganism was
supported but enzyme biosynthesis was low.
 Appl Microbiol Biotechnol

Table 1 Effect of citrus peel powder (CPP) in addition to sugar Table 2 Effect of metal ions on the production of nariginase by S.
(10 gl−1) on naringinase production by S. xylosus MAK 2 xylosus MAK 2

Inducer concentration Final Enzyme activity Metal ions Concentration (mM) Enzyme activity (IUml−1)
(gl−1) citrus peel powder medium pH (IUml−1)
Control 6.0
Control 5.5 2.0 Fe2+ 5 4.4
0.5 5.4 4.2 Ca2+ 5 6.5
1 5.3 4.8 10 6.84
1.5 5.3 5.4 30 5.2
2.0 5.2 6.0 Mg2+ 5 6.2
2.5 5.3 2.4 10 5.1
3.0 5.3 1.8 30 4.4
Naringenin (from Sigma) 5.5 4.5 Cu2+ 5 3.6
Rhamnose (from Sigma) 5.5 4.5 Mn2+ 5 4.4
Zn2+ 5 4.0
Co2+ 5 3.8
Effect of inoculum age and size Ni2+ 5 3.9

A 10-h-old seed culture when used as inoculum, gave

maximum naringinase production in the fermentation
medium (data not shown). The inoculum level of 1–7%
(v/v) was used in the cultivation medium to establish the
effect of inoculum size on the enzyme production by S.
xylosus MAK2. A 5% (v/v) inoculum was optimal for
growth and naringinase production, and the lag phase was
also minimal (data not shown). With 2–4% (v/v) inoculum
size, the lag phase did not reduce significantly, and
maximal enzyme production was obtained at a longer
incubation time of 60 h.
soybean meal, and citrus peel powder) at different initial
conditions. Varying the initial pH of the fermentation
medium between pH 4 and 9 with intervals of 0.5 U had
an effect on enzyme production. Naringinase production was
found to be highest (6 IUml−1) when the pH of the medium
was set at pH 5.5 (data not shown). Varying temperature at
5°C intervals between 25 and 35°C had little effect on
enzyme production, whereas at 37°C naringinase production
was significantly reduced (3 IUml−1). The temperature of
30°C was used for further studies.

Effect of metal ions Technological combinations on optimized media

Different metal ions were used in the cultivation medium to
determine the effects of metal ions on growth and
naringinase production by S. xylosus MAK2. Fe3+, Cu2+,
Mn2+, Zn2+, Co2+, and Ni2+ were found to be inhibitory to
naringinase production at a concentration of 5 mM,
whereas Ca2+ (5–10 mM) and Mg2+ (5 mM) stimulated
the naringinase synthesis (Table 2). In the case of Ca2+, the
maximum naringinase yield (6.84 IUml−1) was observed at
10 mM, which corresponded to a 13% increase in enzyme
production. Above 10 mM, enzyme production decreased
significantly. Mg2+ at 5 mM showed maximal production
(6.2 IUml−1) of naringinase, whereas at higher metal ion
concentration (30 mM), a decrease in enzyme production
(4.4 IUml−1) was observed.
Effect of environment factors on growth and naringinase
production
The effect of initial pH and temperature on growth and
naringinase production by S. xylosus was observed in 500-ml
Erlenmeyer flasks containing 100 ml medium (sucrose,
components concentration
The process optimization using technological combinations
allowed quick optimization of large number of variables,
which helps in designing the suitable fermentation conditions
for the production of naringinase by S. xylosus MAK 2. The
maximum naringinase production (8.23 IU ml−1) was
obtained when the production medium contained the
following (gl−1): sucrose, 10; soybean meal, 10; CPP,
0.5; and pH 5.5 (data not shown). There was a twofold
increase in naringinase production even in the absence of
metal ions.
Bioreactor studies
The effect of initial sugar concentration on growth and
enzyme production was studied in the bioreactor with all
the medium components optimized with the exception of
sugar concentration. The initial sugar concentration was
varied from 10 to 20 gl−1, and the bioreactor was operated
at constant aeration (1 vvm) and agitation (300 rpm) at 30°C.
The production profile of naringinase was recorded for up to
Appl Microbiol Biotechnol
40 h with an initial sugar concentration in the bioreactor of
15 gl−1. The profile for this fermentation is shown in Fig. 1a.
Enzyme production is growth associated in nature, and on
attaining the log phase of growth, enzyme biosynthesis
begins. Maximal enzyme production (8.25 IUml−1) was
obtained after 34 h. Dissolved oxygen (DO) concentration in
the bioreactor decreased initially, whereas it stabilized from
the late exponential phase to the stationary phase of the
growth until the end of fermentation (data not shown). The
pH of the medium was initially adjusted to the optimized
value of 5.5 after which (14 h) it was monitored but not
controlled. On the onset of fermentation, the pH decreased to
4.9, but after 12 h, it remained stable at 5.2 until harvesting.
It was observed that naringinase production increases with
increased cell mass upto 40 h and pH 5.2 of the medium,
during the course of fermentation when the sugar concentar-
Fig. 1 Fermentation profile for the production of nariginase by S.
xylosus MAK 2 in a stirred tank reactor (initial sugar concentrations, a
10 gl−1 and b 15 gl−1, pH 5.5 and DO 100%)
tion of the medium was 10 gl−1 (Fig. 1a). On increasing
sugar concentration (15 gl−1) in the medium, an increase in
the enzyme production (8.9 IUml−1) was observed in the
bioreactor. An increase in initial sugar concentration from 10
to 15 gl−1 in the fermentation medium resulted in increased
bacterial biomass. On attaining log phase of growth, the
enzyme biosynthesis began. The maximum growth
(5.88 gl−1) of S. xylosus was obtained in 30 h and remained
stable until the end of fermentation. The maximum naringi-
nase production was obtained in 34 h at 30°C (Fig. 1b).
On doubling the sucrose concentration (20 gl−1) in the
medium, increased cell mass formation was observed in
the bioreactor, but enzyme production was repressed
(7.5 IUml−1). A slight decrease in enzyme production at
higher sucrose concentration is attributed to substrate
inhibition. In the initial phase of fermentation, active cell
growth continued for upto 40 h of cultivation, and the
sugar concentration of the medium also decreased. It was
observed during the biorector run that the highest
consumption of sugar occurred within 24 h of fermenta-
tion. At higher sugar concentration in the medium, dense
growth of bacterial cell mass resulted in flocculation in the
reactor (results not shown). The repression of the enzyme
production is due to higher sugar concentration. On
extending the fermentation time up to 40 h, no significant
improvement in enzyme yield was observed, and a steady
state was attained.
Higher productivity of naringinase (about twofold) and a
reduction in fermentation time of up to 2 h was obtained in
a bioreactor. Higher sugar concentrations in the medium
(20 gl−1) did not improve enzyme yield in the present
study. A sugar concentration of 15 gl−1 was found to result
in the highest level of naringinase production.
Enzyme yield in terms of sugar utilized were higher at
lower concentrations of sugar (Table 3). Maximum specific
enzyme activity decreased at higher sugar concentrations.
Higher sugar concentrations in the medium (20 gl−1) did not
improve enzyme yield in the present study. With 15 gl−1
sugar, the maximum specific enzyme activity was two times
more than that at 20 gl−1. A sugar concentration of 15 gL−1
gave the highest level of extracellular naringinase
production.
Table 3 Effect of initial sugar concentration on the growth and
enzyme production of S. xylosus MAK 2 in a bioreactor
Initial Maximum Maximum Sugar Maximum
sugar cell mass EA (IUml−1) consumption specific EA
(gl−1) (gl−1) (%) (IU/g dw)
10 3.95 7.25 92 1,672
15 5.2 9.0 94 1,730
20 7.98 7.13 94 877

Discussion
Citrus peel (CP) accounts for about 40% of the total wet
weight of citrus used in the citrus processing industry. It is a
potential source for the fermentation industry but is not
widely utilized (Puri et al. 2010a). Polyphenols and sugar
resulting from CP can be used for the production of
ethanol, fiber, and rhamnose. We studied the naringinase
production from a new strain of S. xylosus, which we have
designated as MAK2. S. xylosus MAK2, a nonpathogenic
bacterium, belongs to coagulase-negative staphylococci
(CNS) family and has not been investigated for naringinase
production. The strain produced a high level of naringinase
(~8.9 IUml−1) in the presence of citrus peel powder,
sucrose, and soybean meal (Fig. 1b). The use of an inducer
is essential for inducing enzyme biosynthesis. Many work-
ers have used a variety of inducers for producing
extracellular enzymes (Singh et al. 2007; Dheeman et al.
2010), and orange peel has been used as an inducer for the
production of polygalacturonase (Nighojkar et al. 2006). In
our earlier studies (Puri et al. 2005), narigin was shown to
be an inducer for naringinase production by A. niger
MTCC 1344. Martins et al. (2002) also investigated the
use of orange bagasse and wheat bran for the production of
pectic enzymes. In some cases, continuous or stepwise
addition of inducer increases naringinase production by
Aspergillus sp. (Mateles et al. 1965). Higher naringinase
production was observed in the presence of CP as an
inducer, which is a rich source of naringin (Yanai and Sato
2000). Similarly, the use of hesperidin as an inducer along
with soybean residue and CSL for naringinase production
was reported in a Penicillium sp. (Fukumoto and Okada
1973). Mazzaferro et al. (2008) observed the highest
specific activity of α-L-rhamnosidase on using rhamnose
as substrate from Pseudoalteromonas, whereas glucose
decreased the activity levels.
The addition of different carbon sources to the basal
medium had a positive effect on naringinase production by
S. xylosus MAK2. Among the sugars, sucrose and molasses
showed the highest increase in naringinase production from
S. xylosus MAK2. Rhamnose and maltose also supported
naringinase production. Significant decrease in naringinase
levels were obtained with glucose and fructose. In one of
the studies, rhamnose has been used as the sole carbon
source in the induction of microbial rhamnosidase by
Burkholderia sp. (Cardona et al. 2006).
Nitrogen sources play an important role in the biosyn-
thesis of naringinase by microorganism. Among various
organic nitrogen tested, a significant increase in naringinase
production was observed with soybean meal and tryptone.
Soybean meal was selected as the best nitrogen source due
to its cost effectiveness during scale-up, and perhaps, it may
contain an inducer substance for α-L-rhamnosidase activity.
Appl Microbiol Biotechnol
Similar report of enhanced naringinase production was
observed from Phanopsis citri, Rhizoctonia solani, and
Cochiobolus miyabeanus using soybean meal (Ito and
Takiguchi 1970). Ammonium sulphate and ammonium
nitrate were inhibitory, presumably because of the release
of ammonium ions.
It is important to provide an optimum inocula level in
fermentation processes. At a suitable incoulum size, the
nutrient and oxygen level are enough for sufficient growth
of bacteria and therefore enhance nariginase production. If
the incoulum size is too small, insufficient biomass will
lead to reduced levels of secreted nariginase. High
inoculums size can result in the lack of oxygen and nutrient
depletion in the culture media resulting in poor product
yield (Mudgetti 1986). There was a significant effect of size
of inoculum on naringinase production. Increased naringinase
production was observed with increase in inoculums size
(5%, v/v). Further increase in inoculum size resulted decrease
in enzyme synthesis, probably due to nutrient limitation.
The effect of temperature on naringinase production was
investigated for three temperatures. At the end of fermen-
tation, the naringinase yields obtained at 30°C were higher
than those obtained at 25 and 37°C, respectively (data not
shown). Various groups have reported an optimum temper-
ature of about 30°C for culturing S. xylosus (Ravyts et al.
2009). It appeared that 30°C was suitable for both S.
xylosus growth and enzyme activity. The maximum
naringinase activity of S. xylosus was obtained when the
initial medium pH was 5.5. At alkaline pH, there was a
decrease in enzyme activity. This observation was contrary
for bacterial α-L-rhamnosidases, for which neutral and
alkaline pH optima have been found (Hashimoto et al.
2003; Jang and Kim 1996; Miake et al. 2000; Zverlov et al.
2000).
Different metal ions play an important role in naringinase
production. For example, Ca2+ and Mg2+ had a stimulatory
effect, whereas Cu2+ and Ni2+ had an inhibitory effect on
naringinase production (Table 2). This suggests that Mg2+
and Ca2+ ions are required for the production of naringinase
by S. xylosus. Miake et al. (2000) reported the acceleration of
enzyme activity by Ca2+ in the case of Pseudomonas
paucimobilis FP2001.
The technological combination experiment is a statis-
tical approach that combines numerous combinations of
independent variables at appropriate levels in a single set
of experiment. Maximum enzyme production was ob-
served in a technological combinations containing lower
amount of CPP concentration than the optimum value
obtained during shake flask experiments (data not
shown). Minimum experimentation that leads to opti-
mized enzyme production by balancing nutrient concen-
tration is desirable in microbial metabolism (Prakasham
et al. 2007).
Appl Microbiol Biotechnol
In the bioreactor, naringinase production increased with
increasing biomass concentration. Two reasons accounted
for the increase in enzyme productivity. Firstly, increasing
sugar concentration from 10 to 15 (gl−1) gave a twofold
improvement in naringinase productivity. Enrichment with
maltose as the carbon source was found to increase protease
and lipase production by 6.3- and 1.6-fold, respectively
(Mahanta et al. 2008; Dheeman et al. 2010). Secondly,
better control of environmental factors in the vessel resulted
in increased enzyme production. A slight decrease in pH
and dissolved oxygen (DO) was observed in the fermenta-
tion medium, which otherwise remained almost unaltered
during the entire course of fermentation.
Maximum naringinase production was observed at 34 h,
which decreased on further incubation (Fig. 1b). This loss
could be due to secretion of other proteins at the late
logarithmic phase, leading to an apparent decrease in
naringinase activity. The biomass showed a substantial
increase after 14 h and was highest at 30 h before showing
a decrease. Thus, the present study identified reduction in
time period for naringinase production by 4 h when
fermentations were carried in a bioreactor. Similar trends
of reduction in fermentation time during the production of
nitrilase were observed in a stirred tank bioreactor (Naik et
al. 2008).
In conclusion, S. xylosus MAK 2 was identified as
producing extracelluar naringinase. A significant increase
in naringinase yield from strain was achieved by improving
the culture conditions. Continuous or step wise addition of
an inducer (CPP) increased naringinase production. Some
metals ions such as (Ca2+) were also shown to increase
enzyme production. In the present study, it was found that
citrus peel powder, a waste generated from citrus process-
ing industry, was important for enzyme production, and its
presence in the optimized medium significantly increased
the enzyme yield. On technological optimizing of fermen-
tation medium, further improvement in naringinase produc-
tion was observed. This new method for producing
extracellular naringinase could be potential cost effective
production method.
Acknowledgements This work was supported by a project grant
CSIR 38(1133)/07/EMR-II. Aneet Kaur gratefully acknowledges
CSIR, New Delhi for the award of Senior Research Fellowship.
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