Production of a novel glycerol-inducible lipase from thermophilic Geobacillus stearothermophilus strain-5

Abstract

In a screening program for isolation of thermophilic lipase-producing bacteria, a number of thermophilic bacteria were isolated
from desert soil from Baltim, Egypt. Among 55 isolates, a potent bacterial candidate (starin-5) was characterized and identified
by biochemical and PCR techniques, based on 16S rRNA sequencing. Phylogenetic analysis revealed its closeness to geobacilli
especially the thermophilic Geobacillusstearothermophilus with optimal growth and lipolytic enzyme activity at 60°C and pH 7.0. An inducible nature of lipolytic enzyme synthesis using
glycerol and glucose was demonstrated. Approximately, 94–100% of the original activity was retained due to thermal stability
of the crude enzyme after heat treatment for 15min at 30–60°C. The enzyme retained 84.84% of its original activity during
incubation at 70°C (pH 8.0) for 15min. Lipase enzyme from G.stearothermophilus strain-5 was immobilized on various carriers and the most suitable carrier was chitin that showed 73.03% of activity yield.

Full text

ORIGINAL PAPER
Production of a novel glycerol-inducible lipase from thermophilic
Geobacillus stearothermophilus strain-5
Mahmoud M. Berekaa ÆTaha I. Zaghloul Æ
Yasser R. Abdel-Fattah ÆHesham M. Saeed Æ
Mohamed Sifour
Received: 19 May 2008 / Accepted: 15 October 2008 / Published online: 6 November 2008
ÓSpringer Science+Business Media B.V. 2008
Abstract In a screening program for isolation of thermo-
philic lipase-producing bacteria, a number of thermophilic
bacteria were isolated from desert soil from Baltim, Egypt.
Among 55 isolates, a potent bacterial candidate (starin-5)
was characterized and identified by biochemical and PCR
techniques, based on 16S rRNA sequencing. Phylogenetic
analysis revealed its closeness to geobacilli especially the
thermophilic Geobacillus stearothermophilus with optimal
growth and lipolytic enzyme activity at 60°C and pH 7.0. An
inducible nature of lipolytic enzyme synthesis using glyc-
erol and glucose was demonstrated. Approximately, 94–
100% of the original activity was retained due to thermal
stability of the crude enzyme after heat treatment for 15 min
at 30–60°C. The enzyme retained 84.84% of its original
activity during incubation at 70°C (pH 8.0) for 15 min.
Lipase enzyme from G. stearothermophilus strain-5 was
immobilized on various carriers and the most suitable carrier
was chitin that showed 73.03% of activity yield.
Keywords Geobacillus stearothermophilus 
Immobilization Production of lipase Thermostable lipase
Introduction
The major share of industrial enzyme market is occupied
by hydrolytic enzyme such as proteases, amylases, ester-
ases and lipases (Gupta et al.2004a,b). Lipolytic enzymes
(lipases and esterases) are industrially interesting, and
they catalyse both hydrolysis and synthesis reactions
(Dominguez et al. 2005). Production of thermostable
lipolytic enzyme from thermophilic bacteria gained a lot of
interest in recent years. Several enzymes were isolated
from thermophilic microorganisms (Kambourova et al.
2003; Dominguez et al. 2005; Nawani et al. 2006; Soliman
et al. 2007). The advantages of application of thermophilic
enzymes as biocatalyst include increased substrate solu-
bility, the diffusion rate, mass transfer effect due to reduced
viscosity and reduced risk of contamination (Abdel-Fattah
2002; Haki and Rakshit 2003; Li and Zhang 2005).
The potential for industrial applications of lipases
comprises the industry of additives (flavor modification),
fine chemistry (synthesis of ester), detergents (hydrolysis
of fat), wastewater treatment, leather (removal of fat from
animal skin), pharmaceuticals and cosmetics (removal of
lipids, medicines and enzymes for diagnosis) (Eltaweel
et al. 2005).
In this study, we described the isolation and the char-
acterization of a new thermophilic Geobacillus
stearothermophilus strain, a potent producer of thermo-
stable lipolytic enzyme. The enzyme production in
presence of different carbon sources was investigated.
Special emphasis was given to characterization of the crude
enzyme especially temperature and pH as well as its
M. M. Berekaa (&)
Department of Environmental Science, Faculty of Science
(Moharam Bay), University of Alexandria, Alexandria, Egypt
e-mail: Berekaa2005@yahoo.com
T. I. Zaghloul H. M. Saeed
Department of Biotechnology, Institute of Graduate Studies
and Research, University of Alexandria, Alexandria, Egypt
Y. R. Abdel-Fattah
Mubarak City for Scientific Research and Technology
Applications, Alexandria, Egypt
M. Sifour
Department of Molecular and Cell Biology, Faculty of Science,
University of Jijel, Jijel, Algeria
123
World J Microbiol Biotechnol (2009) 25:287–294
DOI 10.1007/s11274-008-9891-3

immobilization on different carriers namely; Amberlite
IRC 50 for ionic binding, silica gel for physical adsorption
and chitin and chitosane for covalent binding.
Materials and methods
Bacterial strains
In a screening program for isolation of thermophilic bac-
teria with a potent lipolytic activity, soil samples from
Baltim city, Egypt, were preliminary suspended in PY
medium (Bernhard et al. 1978)(gl
-1
: peptone: 10, yeast
extract: 5 and NaCl: 5) in Erlenmeyer flasks and incubated
at 65°C for 3 h in reciprocal shaker. An aliquot of 1% was
used as inoculum for successive enrichment cultures on the
same medium. At the end of incubation period, sample of
1 ml was used for inoculation of solid PY medium. Isolates
able to grow only over 40°C were considered as true
thermophiles. Separate colonies were streaked on PA
medium (PY medium supplemented with 15 g l
-1
agar) to
ensure purity. Pure isolates were qualitatively screened for
lipolytic activity by plating on PA plates supplemented
with 1% tributyrin, Tween 20, or Tween 80 in presence of
0.01% CaCl
2
. The positive isolates were subjected to
quantitative estimation of lipolytic activity using p-nitro-
phenyl palmitate (pNPP) method (Vorderwuelbecke et al.
1992). This was done by inoculating 50 ml of PY medium
supplemented with 1% Tween 20, Tween 80 or olive oil
and 0.02% CaCl
2
in 250 ml Erlenmeyer flask with 4%
bacterial suspension from 2 days-old freshly prepared slant
of each tested isolate. Incubation was carried out at
60 ±2°C for 48 h using reciprocal shaker (180 rpm). At
the end of incubation period, cells were harvested by
centrifugation and the cell-free supernatant was used for
the determination of lipolytic activity.
Lipase assay
Lipase activity was routinely determined colorimetrically by
p-nitrophenyl palmitate (pNPP) method (Vorderwuelbecke
et al. 1992). The assay mixture contained 900 ll of the assay
reagent and 100 ll of enzyme solution. The assay reagent
was prepared by adding 1 ml of solution (1) to 9 ml of
solution (2) dropwise to get an emulsion that remained stable
for 2 h. The solutions were prepared as follows; solution (1)
contained 90 mg p-nitrophenyl palmitate dissolved in 30 ml
2-propanol, solution (2) contained 2 g Triton-X 100 and
0.5 g gum arabic dissolved in 450 ml buffer (Tris/HCl,
50 mM, pH 8). After incubation of the enzyme solution with
substrate for 20 min at 60°C, the liberated p-nitrophenol was
measured at 410 nm. One unit of enzyme was defined as the
amount of enzyme that releases 1 lmol p-nitrophenol from
the substrate per minute. The protein content was determined
according to Bradford method (Bradford 1976).
DNA extraction and PCR sequencing of 16S rDNA
DNA was isolated from the isolate strain-5 according to
(Zaghloul et al. 1985). Amplification of the 16S rDNA
gene was carried out by polymerase chain reaction (PCR)
using DNA thermal cycler, Progene. The forward primer
was 50AGAGTTTGATCMTGGCTCAG30and the reverse
primer was 50TACGGYTACCCTGTTACGACTT30. Ther-
mocycling consisted of an initial denaturation of 2.5 min
at 95°C and of 30 cycles of 1 min at 94°C, 1 min at
55°C, and 1 min at 72°C. The PCR product was purified
using QIAquick PCR purification reagents (QIAgen).
Growth and lipolytic activity
Growth and lipolytic activity were monitored in PY med-
ium supplemented with 1% olive oil. About 50 ml of the
medium in 250 ml Erlenmeyer flask were inoculated with
4% of preculture. The inoculated flasks were incubated in a
reciprocal shaker at 60°C for 24 h. Growth (measured at
420 nm) and lipolytic activity were monitored during dif-
ferent time intervals. In order to study the inducible or
constitutive nature of the enzyme, lipolytic activity was
monitored during growth of Geobacillus strain-5 on three
different media namely; minimal medium (composed of
gl
-1
: glucose, 10; (NH
4
)
2
SO
4
,1;K
2
HPO
4
, 7; MgSO
4
.
7H
2
O, 0.1; NaCl, 2 and pH 7.0.), complex medium (PY)
and PY supplemented with 1% olive oil. The inoculated
flasks were incubated under shaked conditions at 60°C for
48 h and growth at 420 nm and lipolytic activity were
monitored.
Effect of cultivation conditions
A preliminary investigation to study the effect of some
culture conditions on lipase enzyme production by Geo-
bacillus strain-5 was carried out. The effect of two
different environmental factors namely; temperature (50,
60 and 70°C) and pH (5, 6, 7, 7.5, 8 and 9) on lipase
enzyme production was monitored. On the other hand, the
most important chemical factors namely; carbon sources
(glucose, galactose, glycerol and gum Arabic), were clo-
sely investigated. Measurements were taken in duplicate.
Characterization of the crude lipase
The enzyme activity assay was carried out at different
temperatures (30–100°C) to determine its optimum tem-
perature of activity. The effect of temperature on
thermostability of the crude lipase was also investigated.
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123

The crude enzyme was incubated at the indicated temper-
atures for 15 min. The residual activity was determined.
The effect of organic solvents on lipolytic activity was
obtained by measuring the residual activity after pre-
incubation of the crude enzyme with organic solvents for
30 min at 60°C. The final concentration of the organic
solvents was 50%.
Enzyme immobilization
For immobilization of the partially purified lipase enzyme,
the following compounds were used as carriers; Amberlite
IRC 50 (Fisher) for ionic binding, Silica gel (Merck) for
physical adsorption and chitin (WinlaB) and chitosane
(Sigma) for covalent binding. The carriers were washed
with 20 mM Tris-buffer (pH 8.0). For preparation of the
crude enzyme preparation, the cell-free supernatant was
passed through a 10 kDa membrane and one-tenth of the
retentate was collected. One milliliter of the ultrafiltrate
was incubated overnight with 100 mg of the corresponding
carrier at room temperature. At the end of incubation
period, the unbound enzyme was removed and the carriers
were washed with 20 mM Tris-buffer (pH 8.0). Lipase
activity was subsequently estimated in the unbound and
immobilized enzyme at 60°C. The activity yield and
immobilized yield were determined according to the fol-
lowing equations:
Activity yield %ðÞ¼C=A100
Immobilized yield %ðÞ¼ABðÞ=A½100;
where A is the activity of the enzyme added to the
immobilization solution, B the activity of the unbound
enzyme and C the activity of the immobilized enzyme
(Kambourova et al. 2003).
Results and discussion
Identification and phylogenetic analysis of a potent
thermostable lipase producer
In a screening program for isolation of thermophilic bac-
terial candidate with thermostable lipase activity, 55
thermophilic bacterial strains isolated from different soil
samples (see Materials and methods) were investigated.
Among them, bacterial candidate strain-5 was chosen as a
potent lipase producer as determined by qualitatively and
quantitatively assays during growth on different media.
The bacterial strain-5 was gram positive, endospore
forming rods. The bacterium is aerobic, unable to grow
bellow 37°C, with an optimum growth temperature of
60°C, catalase and amylase positive. Bacterial isolate-5
grows optimally at pH 7.0 and in the presence of 1–1.5%
NaCl. According to these morphological and physiological
characteristic, isolate-5 was found to belong to thermo-
philic Geobacilli sp. Furthermore, molecular identification
of bacterial isolate using 16S rRNA method was carried
out. Phylogenetic analysis based on 16SrDNA sequence
revealed its close relationship (99%) to G. stearothermo-
philus. The nucleotide sequence was deposited in the
GenBank sequence database, and given the accession
number DQ923400. Phylogenetic relation of G. stearo-
thermophilus strain-5 sequence with 16S rDNA of other 16
thermophilic Bacilli and Geobacilli and in relation to
B. subtilis DM-1 and B. licheniformis is shown in Fig. 1.
Production of lipolytic enzyme
by G. stearothermophilus strain-5
Monitoring of growth and extracellular lipolytic activity of
G. stearothermophilus strain-5 was carried in PY medium
supplemented with 1% olive oil. Results presented in Fig. 2
showed that the production of extracellular lipolytic activity
started at late log phase of the bacterial growth (*6 h after
inoculation) and increased gradually with bacterial growth,
it reached its maximal rate after 24 h. Results indicated that
G. stearothermophilus strain-5 has a moderate specific
growth rate (l=0.25 h
-1
). For enzyme production, the
strain showed a rate of production of 10 U l
-1
h
-1
. More-
over, the results collectively indicated that the specific
growth rate of G. stearothermophilus strain-5 was less than
that of other thermophilic bacilli such as; Bacillus sp. IHI-
91 (Becker et al. 1997), B. thermoleovorans ID-1 (Lee at al.
1999) and B. thermoleovorans YN (Abdel-Fattah et al.
2002), that showed growth rates of 1, 2.5 and 2.3 h
-1
,
respectively. Furthermore, thermophilic Bacillus sp. IHI-91
showed 34-fold increase in lipase production as compared
with G. stearothermophilus strain-5 (Becker et al. 1997).
Monitoring lipolytic activity of G. stearothermophilus
strain-5 on different growth media
To examine the nature of lipolytic enzyme produced by
G. stearothermophilus strain-5, the growth and enzyme
activity were closely monitored on three different media
namely; PY medium, PY supplemented with olive oil and
minimal medium without additives (see Materials and
methods). Results represented in Fig. 3showed that the
level of extracellular enzyme production started at late log
phase, about 6 h after inoculation, during growth on PY or
PY supplemented with olive oil and increased gradually
with bacterial growth to reach its maximal level (48.51 and
47.04 U ml
-1
) after 30 and 24 h of inoculation on PY and
PY medium supplemented with 1% olive oil, respectively.
These results indicated that olive oil may accelerate
World J Microbiol Biotechnol (2009) 25:287–294 289
123

enzyme production by G. stearothermophilus strain-5.
However, minimal medium devoid of olive oil support
neither growth nor enzyme production by G. stearother-
mophilus strain-5. In concurrence with other findings,
G. stearothermophilus strain-5 was found to produce lipase
enzyme in the late exponential phase and to be growth-
associated (Lee et al. 1999; Gupta et al. 2004a,b).
Effect of carbon source
One of the key factors affecting lipase enzyme production by
bacteria is oils and carbon source. Therefore, effect of oils
namely; olive oil, Tween 20, Tween 80 and Triton X-100 as
well as carbon sources namely; glucose, galactose, gum
Fig. 1 Phylogenetic
dendogram obtained by distance
matrix analysis showing the
position of
G. stearothermophilus strain-5
(isolate 5) among 16S rDNA of
the highest 16 similar
thermophilic Bacilli and
Geobacilli and in relation to
B. subtilis DM-1 and
B. licheniformis. The
dendogram was generated by
the neighbor-joining method
using BioEdit software
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0 5 10 15 20 25 30 35 40 45
incubation time (h)
absorbance at 420 nm
0
10
20
30
40
50
60
li
p
ol
y
tic activit
y
(U ml-1)
Fig. 2 Monitoring of lipase enzyme activity of G. stearothermophi-
lus strain-5 growing on PY medium supplemented with 1% olive oil.
Symbols: lipolytic activity (j), bacterial growth (h)
0
5
10
15
20
25
30
35
40
45
50
0 6 12 18 24 30 36
incubation (h)
lipolytic activity (U ml
-1
)
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
g
rowth (absorbance 420nm)
Fig. 3 Monitoring growth and extracellular lipolytic activity of
G. stearothermophilus strain-5 on different media. Symbols e,hand
Drepresent PY supplemented with olive oil, PY and minimal
medium, respectively. Open symbols, bacterial growth and closed
symbols, lipolytic activity (U ml
-1
)
290 World J Microbiol Biotechnol (2009) 25:287–294
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Arabic and glycerol on production of lipase from G. ste-
arothermophilus strain-5 was investigated. Results shown in
Table 1indicated that lipase enzyme was poorly induced by
oils. This observation is in accordance with results obtained
by Gupta et al. (2004a,b), they found that oils acts as poor
inducer of lipase production (Gupta et al. 2004a,b). Fur-
thermore, lipase from B. thermocatenulatus is reported to be
repressed in the presence of Tween 80 and Triton-X 100
(Schmidt-Dannert et al. 1994). However, other reports sta-
ted that lipases are generally induced by oils (Lee et al.
1999; Abdel-Fattah 2002). Results showed also that the
presence of certain carbon sources such as; glucose,
galactose and especially glycerol in cultures of G. stearo-
thermophilus strain-5 can enhance the level of lipase
production, whereas gum Arabic has no effect on the pro-
duction. Interestingly, Gupta et al. (2004a,b) reported that
the presence of sugars and sugar alcohols (glycerol and
mannitol), induced the production of lipase from a thermo-
philic Bacillus sp. (Gupta et al. 2004a,b).
Effect of growth temperature and pH on production
of lipase enzyme
To test the effect of growth temperature on lipase pro-
duction, G. stearothermophilus strain-5 cells were used for
inoculation of the production medium and incubated at
different temperatures (50, 60 or 70°C). Results in Fig. 4
illustrated that G. stearothermophilus strain-5 produced
lipase more efficiently at 60°C. Generally, the optimum
temperature for production of lipases from thermophilic
bacteria ranged from 50 to 70°C. The best temperature for
lipase production from B. thermoleovorans (Lee et al.
1999) was 65°C. However, the optimum temperature for
lipases production from a G. thermoleovorans (Abdel-
Fattah 2002) and Thermus sp. (Dominguez et al. 2004) was
70°C. On the other hand, investigations on the optimum pH
for lipase production indicated that pH of 7.0 was the most
suitable for production of lipolytic activity from G. ste-
arothermophilus strain-5 (Fig. 5). Interestingly, lipases
from thermophilic bacteria such as B. thermocatenelatus
(Schmidt-Dannert et al. 1994), T. thermophilus (Domin-
guez et al. 2005) and B. thermoleovorans (Lee et al. 1999)
are generally produced on a medium with initial pH around
7.0. However, production of lipases in a medium with
initial pH higher than 7.0 (pH 8.0–9.0) have been reported
(Abdel-Fattah 2002; Sharma et al. 2002).
Table 1 Lipolytic activity of G. stearothermophilus strain-5 grown
on different media
Medium Activity
a
(U ml
-1
)
Specific activity
(U mg
-1
)
1/2 PY 43.51 310.8
PY 97.02 433.13
PY?1% olive oil 81.31 361.37
PY?1% Tween 20 75.1 208.61
PY?1% Tween 80 77.35 161.14
PY?1% Triton X-100 64.86 91.35
PY?0.2% glucose 176.6 588.81
PY?0.2% gum
Arabic
133.35 493.92
PY?0.2% galactose 129.68 563.85
PY?0.2% glycerol 160.83 643.33
a
Activity was measured at 60°C after 24 h incubation
0
100
200
300
400
500
50 60 70
Temperature °C
lipolytic specific activity (U mg-1)
Fig. 4 Effect of temperature on the production of lipolytic enzyme
activity from G. stearothermophilus strain-5
0
50
100
150
200
250
300
350
400
450
500
4567891011
p
H
lipolytic specific activity (U mg-1)
Fig. 5 Effect of pH on the production of lipolytic enzyme activity
from G. stearothermophilus strain-5
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123

Characterization of the crude lipase enzyme produced
by G. stearothermophilus strain-5
Effect of temperature and pH
The crude lipase of G. stearothermophilus strain-5 was
active at different temperatures ranging from 30 to 100°C.
Results in Fig. 6a showed that the crude enzyme was most
active in the temperature range 55–70°C with maximal
activity at 60–65°C. However, the lipolytic activity was
strongly inhibited after increasing the reaction incubation
temperature to 90–100°C for 15 min. Generally, tempera-
tures optima of thermophilic enzymes were reported to be
in the range 50–80°C (Lee et al. 1999; Abdel-Fattah 2002;
Kambourova et al. 2003). The enzyme was active in the pH
range 7–9 with optimal pH at 8.0 in presence of 50 mM
Tris–HCl buffer (data not shown).
Enzyme stability
One of the key factors that determine the applicability of
lipase enzyme for industrial processes is its thermal sta-
bility. For this reason, the stability of lipase produced by
G. stearothermophilus was tested by determination of the
residual enzyme activity after heat treatment of the crude
enzyme preparations at different temperatures. Result in
Fig. 6b showed that the crude enzyme retained about
94–100% of the original activity after heat treatment for
15 min at 30–60°C. Interestingly, the enzyme retained
84.84% of its original activity during incubation at 70°C
(pH 8.0) for 15 min. Moreover, results showed that
96–100% of the original activity was retained after incu-
bation at pH 6–9 at 60°C for 30 min (Data not shown).
Wang et al. (1995) reported that lipase of the thermophilic
Bacillus, strain A30-1 retained 100% of its activity after
heating at 70°C for 30 min. Kambourova et al. (2003)
reported that heating at 70°C for 30 min half-inactivated
pure enzyme of B. stearothermophilus MC7, while crude
lipase had a half life of 3 h at 70°C.
Moreover, crude enzyme of G. stearothermophilus
strain-5 was stable in diethylether with residual activity of
92.24%. The enzyme showed good stability in butanol and
hexane with residual activity of 67.24% and 63.49%,
respectively. Eltaweel et al. (2005) reported that hexane
slightly enhanced the crude lipase activity of Bacillus sp.
strain 42. In contrast with these results, lipase of B. ste-
arothermophilus MC7 was completely inhibited by butanol
and diethylether (Kambourova et al. 2003). Propanol,
acetone and chloroform had a strong effect on lipolytic
activity of the crude enzyme of G. stearothermophilus
strain-5 (Fig. 7). Glycerol slightly enhanced activity of the
0
100
200
300
400
500
600
700
800
30 40 50 60 70 80 90 100
Temperature
Lipolytic activity (U/ml)
0
20
40
60
80
100
120
30 40 50 60 70 80 90 100
Tem
p
erature
Residual activity (%)
(a)
(b)
Fig. 6 Effect of temperature on the activity of lipolytic enzyme. a
Activity (measured at pH 8 and 60°C), bthermal stability (determined
by incubation of the enzyme for 15 min at different temperatures),
residual activity was expressed as percentage of original activity at
pH 8.0, 60°C
0
20
40
60
80
100
120
Ethanol
Methanol
Acetone
Propanol
Isopropanol
Butanol
Chloro form
diethyl ether
Hexane
Toluen
Glycerol
Solvent
Residual activity (%)
Fig. 7 Effect of some organic solvents on the lipolytic activity of the
crude enzyme. The enzyme was incubated with these organic solvents
for 30 min at 60°C. The final concentration of the organic solvents
was 50%. Activity was determined at pH 8, 60°C
292 World J Microbiol Biotechnol (2009) 25:287–294
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crude lipase of G. stearothermophilus strain-5, while
glycerol showed slight inhibition on lipase from B. ste-
arothermophilus MC7 (Kambourova et al. 2003).
Enzyme immobilization
It is known that preparation of immobilized enzyme is
useful for developing industrial processes of organic syn-
thesis. Immobilization may serve two main objectives;
improvement of enzyme stability and decrease in enzyme
consumption by repeated use of enzyme preparation for
many reaction cycles (Dosanjh and Kaur 2002). Therefore,
lipolytic enzyme from G. stearothermophilus strain-5 was
immobilized on various carriers and the activity was
evaluated. Results in Table 2indicated that the most suit-
able carrier was chitin with 73.03% of activity yield and
the lowest activity yield was recorded with Amberlite
RC50 (11.61% activity yield). Interestingly, natural mac-
romolecules, including chitosane, cellulose, agarose and
carrageenan, with excellent biocompatibility and hydro-
philicity, are non-toxic, biodegradable and inexpensive (Ye
et al. 2005). In addition, it was reported that B. stearo-
thermophilus MC7 lipase was efficiently immobilized on
DEAE cellulose (Kambourova et al. 2003). On the other
hand, thermophilic Bacillus sp. J33 lipase was immobilized
on phenyl Sepharose, where immobilization increased the
thermal stability (Nawani and Kaur 2000).
Acknowledgment M. Sifour is very grateful to the ‘‘Ministry of
Higher Education and Scientific Research of Algeria’’ for its financial
support.
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Carrier Enzyme activity (U ml
-1
) Activity yield (%) (C/A 9100) Immobilized (%) [(A–B)/A 9100]
Added (A) Unbound (B) Immobilized (C)
Chitin 56 12.06 40.9 73.03 78.4
Chitosane 56 22.17 33.62 60.03 60.41
Silica gel 56 16.25 18.25 32.6 70.98
Amberlite RC50 56 12.98 6.5 11.61 78.82
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