Biochemical and Biophysical Research Communications 443 (2014) 194–199

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Biochemical and Biophysical Research Communications

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Purification and characterization of a carboxymethyl cellulase

from Artemia salina
Hyun Woo Zin a, Kwang-Hyun Park b, Tae Jin Choi a,⇑
a
Department of Microbiology, Pukyong National University, 45, Yongso-ro, Nam-Gu, Busan 608-737, Republic of Korea
b
Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cel-
Received 11 November 2013 lulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase
Available online 28 November 2013 activity was purified and the activity analyzed under different conditions. After initial identification of
cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted
Keywords: to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was
Artemia purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the
Cellulase
final purification, a 70-fold increase in specific enzyme activity was observed. SDS–PAGE results revealed
Halophilic
Bioethanol
that the cellulase enzyme had a molecular mass of 96 kDa. Temperature, pH, and salinity values were
Macroalgae found to be optimal at 55 °C, pH 8.0, and 600 mM NaCl, respectively. Specifically, the enzyme showed
a fivefold increase in enzyme activity in seawater compared to 600 mM NaCl in phosphate buffer. Further
analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indi-
cating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol
from marine macroalgae.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction and distillation. Sometimes saccharification and fermentation

(SSF) are conducted simultaneously [5]. The pretreatment that pro-
Biofuels produced from biomass are expected to be an alterna- duces polysaccharides from biomass is usually conducted by using
tive energy source in the future, which in liquid or gaseous form diluted acid (0.2–2.5%) and temperatures between 130 °C and
are usually produced from plant matter and residues, and believed 210 °C, but this complex and energy-intensive pretreatment is
to reduce harmful carbon emissions [1]. not necessary for macroalgae because they do not contain lignins
Algae include diverse species inhabiting freshwater and seawa- [3]. The use of extremely low acid conditions for the pretreatment
ter and 50% of global biomass is thought to be generated in the of macroalgae biomass can simplify downstream processing such
marine environment [2]. Compared to most productive land plants, as neutralization and waste treatment, and reduces equipment
algae are capable of producing high yields of carbohydrates, lipids, costs [6].
and proteins over a short period of time, which can be processed to Similar to the cellulosic biomass from other plant sources, that
generate biofuels. Algae are generally divided into microalgae and from algae can be enzymatically hydrolyzed using cellulase en-
macroalgae based on their morphology and size. Microalgae are zymes and converted to simple sugars that can be fermented to
usually single-celled and do not have to spend energy for distribu- ethanol [3]. Cellulose is a major polysaccharide component of
tion and transportation of storage molecules; thus, they are very plants and algae cell walls. Although cellulose is a common carbo-
effective at producing storage materials such as starch, glycogen, hydrate, only a few organisms have the ability to utilize it
and cellulose, which can be used for biofuel production [3]. Similar efficiently. Cellulase, an important inducible enzyme synthesized
to microalgae, macroalgae grow at a fast rate and yield large mostly by microorganisms during their growth on cellulosic mate-
amounts of biomass. In addition, macroalgae can be cultured on rials, is required to release glucose prior to the production of eth-
a large scale, even three-dimensionally, by seeding onto thin, anol. Most biotechnologically significant cellulases are derived
weighed strings suspended over a larger horizontal rope [4]. from bacteria and fungi [7,8]. Due to their origin, most cellulases
The production of ethanol from biomass is generally performed have limited activity under high-salt conditions such as hydrolysis
in four steps: pretreatment, enzymatic hydrolysis, fermentation, of polysaccharides derived from marine macroalgae, and the raw
macroalgal biomass needs to be desalted before processing [9].
Therefore, a cellulase that is active under a high-salt condition is
⇑ Corresponding author. Fax: +82 51 629 5619.
desirable for bioethanol production from marine macroalgae.
E-mail address: [email protected] (T.J. Choi).

0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2013.11.085
 H.W. Zin et al. / Biochemical and Biophysical Research Communications 443 (2014) 194–199 195

In this study, Artemia salina was used as the source of a new in 10% increments with constant stirring for 70 min. For each
salt-tolerant cellulase. A. salina is a primitive invertebrate belong- ammonium sulfate concentration, the mixture was allowed to
ing to a group of crustaceans in the kingdom Animalia [10]. This stand for 30 min and then centrifuged at 10,000g for 15 min. The
species can survive under extreme conditions including high pellet was dissolved in 10 mL of 50 mM phosphate buffer (pH
salinity due to its special adapting abilities against environmental 7.0) with 10 mM b-mercaptoethanol and 1 mM EDTA, and used
stresses. A. salina feeds on microalgae and uses cellulase for the for the cellulose activity test. The supernatant was used for the
digestion of the microalgae cell wall components. Additionally, next fractionation with a 10% increase in ammonium sulfate.
considering the high-salt habitat of this organism, the cellulase
was expected to be active under high-salt conditions, which was 2.4. Enzyme assay
confirmed by the purified enzyme in the present study.
Cellulase activity was measured by the 3,5-dinitrosalicylic acid
(DNS) method [13], which determines the amount of reducing sug-
2. Materials and methods
ars liberated by the cellulase from 1% CMC solubilized in 50 mM
phosphate buffer (pH 7.0). Purified cellulose (500 lL) was incu-
2.1. A. salina culture
bated with 500 lL of 1% CMC for 30 min at room temperature
and the reaction was stopped by adding 1 mL DNS solution.
Lyophilized cysts of A. salina (Inve Aquaculture, Salt Lake City,
Treated samples were boiled for 5 min and cooled at room temper-
UT, USA) coated with iron were washed using 70% ethanol for
ature, and then the optical density was measured at 550 nm. The
2 min in a 1.5 mL tube, and then the ethanol was removed by
cellulase activity was determined using a calibration curve for
standing the tube on a magnetic stand. The cysts were incubated
glucose (Sigma–Aldrich, Gillingham, Dorset, UK). One unit of activ-
in a 20 L plastic tank containing autoclaved seawater for 48 h at
ity was defined as the amount of enzyme that released 1 lM of
28 °C with fluorescent light and mild aeration. After visible signs
glucose equivalents from substrate per minute. The specific
of hatching, the volume was reduced using an autoclaved nylon
activity was expressed in lmol/min/mg.
mesh, and the empty cysts were removed using a magnetic stand.
Pure A. salina was collected on autoclaved nylon mesh and used for
2.5. Cellulase purification and amino acid sequence analysis
homogenization.

Fractions from 50% to 70% ammonium sulfate fractionations

2.2. Preliminary test for cellulose activity and bacterial contamination were pooled and used for enzyme purification. The pooled sample
was first filtered through a 0.45 lm syringe filter and concentrated
Hatched A. salina was ground in liquid nitrogen and resus- using VIVASPIN 20 with a 10,000 MWCO (Sartorius Stedim Biotech,
pended in 50 mM phosphate buffer (pH 7.0 with 10 mM b-mercap- Aubagne, France) at 1700g for 45 min. The concentrated enzyme
toethanol, 5 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM was purified using gel filtration chromatography and ion exchange
EDTA). The homogenate was centrifuged at 8000g for 5 min and chromatography using the Fast Protein Liquid Chromatography
then the supernatant was filtered through a 0.2 lm syringe filter. (FPLC) system (Pharmacia Biotech, Piscataway, NJ, USA). For gel fil-
The crude homogenate, supernatant, and filtrate (20 lL) were tration chromatography, preparations were applied to a HiLoad 16/
placed on a 1.5% agar plate containing 1% carboxymethylcellulose 60 Superdex 75 (GE Healthcare Life Sciences, Piscataway, NJ, USA)
(CMC) and incubated for 30 min at room temperature. Cellulast equilibrated with 50 mM phosphate buffer (pH 7.0 with 10 mM
(Novozymes, Bagsvaerd, Denmark) of 70 endoglucase units (EGU) b-mercaptoethanol, 50 mM NaCl, and 5% glycerol) at a flow rate
in 20 lL 50 mM phosphate buffer was used as a positive control. of 1 mL/min and collected by an autofraction collector. Every frac-
The carboxymethyl cellulolytic activity was confirmed by the tion of 0.3 mL was collected and assayed for cellulase activity. The
Congo Red overlay method [11]. The crude homogenate superna- active fractions were dialyzed overnight with 50 mM Tris–HCl
tant and the filtrate were streaked on Luria Broth plates and Mar- buffer (pH 8.0 with 10 mM b-mercaptoethanol, 50 mM NaCl) and
ine 2216 agar (Difco, Franklin Lakes, NJ, USA), respectively, and further processed in a RESOURCE Q 6 mL column (GE Healthcare
incubated at 37 °C and 25 °C, respectively, overnight to observe Life Sciences), which was equilibrated with elution buffer
any bacterial growth. Total DNA was extracted from the crude (50 mM Tris–HCl, pH 8.0, with 50 mM NaCl). Elution was achieved
homogenate using phenol/chloroform and PCR was conducted with a linear gradient of 5–100 mM NaCl in equilibration buffer at
using two universal primers for the bacterial 16S rRNA gene a flow rate of 1 mL/min. Every 0.3 mL fraction was collected and
(27F: 50 -AGAGTTTGATGGCTCAG-30 , 1492R: 50 -TACGGYTACCTTGT- assayed for cellulase activity, and the active fractions were col-
TACGACTT-30 ) [12]. lected and concentrated using VIVASPIN 20 with a 10,000 MWCO
(Sartorius Stedim Biotech) at 1700g for 45 min and used for the
2.3. Ammonium sulfate fractionation analyses described below. The purified proteins were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–
All purification steps were performed at 4 °C except where PAGE) was performed on a 12% polyacrylamide gel with a protein
otherwise specified. Hatched A. salina was separated from the marker (GenDepot, Barker, TX, USA).
empty cyst as above, collected on nylon mesh, and washed with The amino acid sequence of the purified protein was deter-
sterile distilled water. The collected A. salina was ground in liquid mined by ultra- performance liquid chromatography (UPLC) and
nitrogen using a mortar and pestle, and resuspended in 50 mM mass spectrophotometry at Yonsei Protomics Research Center,
phosphate buffer (pH 7.0) containing 10 mM b-mercaptoethanol, Yonsei University, Seoul, Korea.
5 mM PMSF, 1 mM EDTA, and 10% sucrose in 100 mL buffer to a
10 g sample ratio. The homogenate was centrifuged at 10,000g 2.6. Optimum conditions for enzyme activity
for 20 min. The supernatant was saved and the pellet was resus-
pended with 100 mL of 50 mM phosphate buffer (pH 7.0) with The optimum temperature, pH, and salinity of the purified
10 mM b-mercaptoethanol and 1 mM EDTA, and centrifuged as cellulase were determined by incubating the 50 lL of purified
above. The two supernatants were pooled and subjected to ammo- cellulase with 50 lL of 1% CMC in 50 mM phosphate buffer (pH
nium sulfate fractionation. Solid ammonium sulfate was slowly 7.0) under different conditions. The reaction was conducted in a 96
added to the homogenate in a stepwise manner from 10% to 70% well ELISA plate. To determine the optimum temperature, the
196 H.W. Zin et al. / Biochemical and Biophysical Research Communications 443 (2014) 194–199

reaction mixtures were incubated for 30 min at different tempera- cellulase activity on a 1.5% agar plate containing 1% CMC and were
tures ranging from 4 °C to 60 °C. For the optimum pH, the purified stained with Congo Red. When the three samples were spread on
cellulase was mixed with 1% of CMC in 50 mM phosphate buffer that LB and Marine 2216 agar plates, no bacterial colony growth was
ranged from pH 3.0 to pH 12.0 and incubated at room temperature. observed. Furthermore, no PCR products for the 16s rRNA gene
For optimum salinity, the purified cellulase was mixed with 1% of was amplified from the samples.
CMC in 50 mM phosphate buffer (pH 7.0) with salinity ranging from
0.1 M to 1.0 M for 30 min at room temperature. The reactions were
stopped by adding 100 lL DNS solution, and enzyme activity was 3.2. Purification and molecular characterization of cellulase
measured using the enzyme assay procedures described above.
The values represent the percentages of the enzyme activity as The crude enzyme obtained from A. salina was precipitated
compared to the observed maximum activities under optimum using ammonium sulfate up to 80% saturation in 10% increments.
conditions. When each fraction was tested for cellulase activity, most of the
cellulase activity was observed in the protein fractions precipitated
2.7. Effect of metal ions on cellulase activity with 60–70% ammonium sulfate. The proteins pooled from these
two fractions showed cellulase of 1.1 U/mg (Table 1).
The effects of various metal ions on purified cellulase activity These two pooled fractions were used for further purification by
were determined by preincubating the enzyme with individual me- gel filtration chromatography and ion exchange chromatography.
tal ions of 1 mM (CaCl2, CoCl2, CuCl2, FeSO4, KCl, MgCl2, MnCl2) in In the gel filtration chromatography, the protein peak was ob-
50 mM phosphate buffer (pH 7.0) at room temperature for 30 min. served in fractions numbered 11–13 after the initial flowoff
The enzyme activity was measured as described above. The activity (Fig. 2). In the cellulase activity test, these fractions also showed
assayed in the absence of metal ions was recorded as 100%. cellulase activity. When the fractions were pooled and cellulase
activity was tested, the specific enzyme activity was 11.6 U/mg
(Table 1). Notably, these protein contaminating fractions also
3. Results showed a high concentrations of NaCl, which are shown in the dot-
ted line in Fig. 2. The active fractions 11–13 of the gel filtration
3.1. Verification of cellulase from A. salina chromatography were pooled, concentrated, and further purified
using ion exchange chromatography. When the purified protein
The cellulase activity from the crude extract of hatched A. salina, was subjected to SDS–PAGE analysis, a major protein with an esti-
the supernatant after centrifugation, and the filtrate were tested mated molecular mass of 96 kDa was observed (Fig. 3). The partial
for the presence of cellulase. As shown in Fig. 1, all samples showed amino acid sequence of the purified protein was analyzed using a
mass spectrometer after trypsin digestion. Although peptide
sequences with a diverse molecular weight were verified from
the analysis, none of the peptides showed any sequence similarity
to known proteins (Table 2).

Fig. 1. Pretest for the presence of cellulase activity from A. salina extract by the Fig. 2. Enzyme purification by gel filtration chromatography. Fractions (300 lL)
Congo Red overlay method. Samples (20 lL) were tested with 1% CMC for 30 min were collected and assayed for cellulase activity and the NaCl concentration was
and stained with Congo Red. (1) Positive control, 70 EGU of cellulast; (2) crude determined. Protein concentration is indicated by the black line, and the dash
homogenate; (3) filtrate from 0.2 lm syringe filter; and (4) supernatant after 5 min represents the NaCl concentration. The boxed area denotes the fractions showing
centrifugation at 8000g. cellulase activity.

Table 1
Purification steps of the cellulase enzyme isolated from A. salina.

Purification steps Protein (mg) Total activity (U) Specific activity (U mg1) Purification fold Yield
Crude enzyme 2680 1420.4 0.5 – 100
Ammonium sulfate precipitation 552 590.6 1.1 2.2 41.6
Superdex 75 3 34.8 11.6 23.2 2.5
RESOURCE Q6 0.5 18.1 35.1 70.2 1.3
 H.W. Zin et al. / Biochemical and Biophysical Research Communications 443 (2014) 194–199 197

Fig. 3. SDS–PAGE analysis of the purified cellulase. The purified protein after ion
exchange chromatography was analyzed on 12% SDS–PAGE (lane 1) with a
molecular weight marker (lane M). The arrow points to the band used for mass
spectrophotometry.

Table 2
Amino sequence of peptide from trypsin digestion of cellulase enzyme isolated from
A. salina.

Peptide sequence Molecular weight (Da)

IKDDR 645.34
SRTLLPR 841.51
RDIELDR 915.48
LSSSFDFSR 1044.5
VTISSTSSTSR 1124.57
RGPSLAHYFR 1202.63
GNTEYKLAVDR 1264.64
MACMLVQDKR 1282.58
MLQQAIQENFR 1376.69
AKSWLAASPCLPK 1427.76
AWIFTNITDQDR 1488.72
SQETIGELLSGGFR 1492.75
LLIMDTMMVQDR 1496.7
ELAILMGSLLDEVTR 1674.89
ENIDALKEVLPSFPR 1726.93
NNINLAIVAMVNYTAIR 1889.02
ISLADWTIASVWAFKSAK 1993.07
ISLADWTIASVWAFKSAK 1993.07
FFKNNFFSNSTESVTSESQR 2355.06

3.3. Effect of temperature on the cellulase activity

The effect of temperature on the purified enzyme was deter-

mined after 30 min of incubation at temperatures ranging from
4 °C to 60 °C. The enzyme activity increased as the temperature
increased up to 55 °C, and then declined (Fig. 4A). The purified
enzyme exhibited the highest activity at 55 °C and the relative
activity of the purified enzyme at 4 °C, 10 °C, 20 °C, 25 °C, 30 °C,
40 °C, 50 °C, 55 °C, and 60 °C were 38 ± 6.2, 56 ± 4.0, 62 ± 2.9, Fig. 4. Effect of temperature (A), pH (B) and salinity (C) on cellulase activity. The
67 ± 4.2, 70 ± 4.1, 77 ± 3.3, 84 ± 3.8, 90 ± 2.8, 100 ± 2.0, and activity of purified cellulase was measured at different temperatures, pH and NaCl
84 ± 6.3, respectively. concentrations using a phosphate buffer (pH 7.0).

3.4. Effect of pH on the cellulase activity The enzyme activity increased as the pH increased to pH 8.0 at
which point the activity was the highest and the activity decreased
The effect of pH on the cellulase activity of the purified protein rapidly afterward (Fig. 4B). The relative enzyme activity at pH val-
was examined at various pH values ranging from pH 3.0 to pH 12.0. ues of 3, 4, 5, 6, 7, 8, 9, 10, and 12 were 72 ± 4.9, 83 ± 3.8, 90 ± 5.0,
198 H.W. Zin et al. / Biochemical and Biophysical Research Communications 443 (2014) 194–199

Table 3 Ammonium sulfate fractionation, gel filtration, and ion

Effects of various metal ions on cellulase activity from A. salina. exchange chromatography were performed to purify the novel
Metal ions (1 mM) Residual activity (%) cellulase enzyme. Notably, during purification, the protein frac-
None 100 tions that contained the cellulase activity showed a high NaCl con-
EDTA 83 centration (Fig. 2). These results are in agreement with the effects
CaCl2 124 of salt concentration on enzyme activity as discussed below. After
CoCl2 420 the last purification, specific activity was 35.1 U/mg, which was a
CuCl2 236
FeSO4 88
70-fold increase compared to the crude enzyme before purification
KCl 94 (Table 1). The specific activity after final purification was compara-
MgCl2 138 ble to activity purified from various microorganisms, which ranged
MnCl2 188 from 3.8 U/mg to 71 U/mg [7,8,15].
The SDS–PAGE showed a major protein band with a molecular
93 ± 5.2, 97 ± 4.3, 100 ± 3.1, 99 ± 2.0, 80 ± 5.3, and 45 ± 6.4, respec- mass of 96 kDa and a small protein band with smaller molecular
tively. As shown in Fig. 4B, the enzyme showed over 60% activity weight, which we ignored considering the relative amount of the
compared to the highest activity at pH 8.0 at values ranging from major band (Fig. 3). Cellulolytic enzymes include diverse enzymes
pH 3.0 to pH 10.0. such as endo-b-1,4-glucanase (EG), exo-b-1,4-cellobiohydrase
(CBH), and b-glucosidase that are classified into 78 families in 10
3.5. Effect of salinity on the cellulase activity clans [16]. Therefore, the molecular weights of these cellulases
are diverse. For example, a thermostable cellulase isolated from
The effect of salinity on the cellulase activity of the purified pro- blue mussel has a molecular weight of 20 kDa [17], yet another
tein was measured at various NaCl concentrations ranging from thermophilic cellulase from Panenibacillus sp. strain B39 has a
0.1 M to 1 M for 30 min at room temperature. The enzyme activity molecular weight of 148 kDa [15]. Further confirmation of the
increased in accordance with the increase in NaCl concentration up molecular weight of the cellulase purified in this study by amino
to 600 mM and sharply declined afterward (Fig. 4C). The relative acid sequencing, or the cloning of the gene encoding this protein,
enzyme activity at the NaCl concentration of 100 mM, 300 mM, is necessary.
400 mM, 500 mM, 600 mM, 700 mM, 800 mM, and 1000 mM was As the molecular weight of cellulases differ, the optimum tem-
62 ± 5.6, 74 ± 4.4, 80 ± 3.6, 80 ± 4.5, 100 ± 2.2, 92 ± 3.4, 72 ± 4.5, perature, pH, and effects of metal ions are also different for each
and 50 ± 6.4, respectively. When the purified enzyme was tested cellulase and usually show a broad range of optimum conditions.
with seawater, the enzyme showed 500% increased activity com- When considering cellulases from marine invertebrates, the cellu-
pared to its 100% activity at 600 mM. lase isolated from blue mussel showed an optimum pH of 4.6 with
over 80% activity in the pH range of 4.0–5.5 [17]. Additionally, the
enzyme showed a broad optimum activity in the temperature
3.6. Effect of metal ions on the cellulase activity
range of 30–50 °C but its activity dropped rapidly above 50 °C. In
another example, the cellulase isolated from abalone showed an
The cellulase activity in the presence of various metal ions of
optimal temperature and pH of 38 °C and 6.3, respectively [18].
the same concentration (1 mM) was compared in phosphate buffer,
The cellulase purified in this study also showed a broad optimum
and the results are shown in Table 3. The enzyme showed the high-
activity in pH and temperature but with a higher pH (pH 8.0)
est activity in the presence of CoCl2 followed by CuCl2, MnCl2,
and temperature (55 °C) at the highest enzyme activity (Fig. 4A
MgCl2, and CaCl2. In contrast, the presence of KCl, FeSO4, and EDTA
and B). Therefore, the broad optimal range of temperature and
showed inhibition of the enzyme activity although the inhibition
pH of this cellulase is not unusual.
was less than 20%.
The effect of NaCl concentration on the enzyme activity is an
interesting characteristic. As shown in Fig. 4C, the activity in-
4. Discussion creased up to 600 mM NaCl and decreased afterward. When this
enzyme was tested in seawater, the activity increased fivefold,
Microalgae and macroalgae are regarded as new and promising compared to the activity at 600 mM NaCl. The NaCl concentration
bioenergy sources that can solve the problems such as the scarcity in seawater is typically 3.5%, equivalent to 598 mM. Therefore, the
of food and high cost for processing when using the first- and sec- highest activity at 600 mM NaCl concentration is consistent with
ond-generation biofuels [3]. Bioethanol production from algae re- the NaCl concentration in seawater. The activity increase in seawa-
quires four processing steps: pretreatment, enzymatic hydrolysis, ter compared to pure NaCl can be explained by the presence of
fermentation, and distillation. Of these four steps, we focused on other metal ions in seawater. As shown in Table 2, the presence
the hydrolysis of cellulosic polymers. Specifically, we looked for a of metal ions such as CoCl2, CuCl2, MnCl2, MgCl2, and CaCl2 in-
cellulase that can be active under high-salt conditions such as sea- creased the enzyme activity. Compared to the metal concentration
water in which macroalgae are cultivated so that a laborious used in this experiment (1 mM), the concentrations of metal ions
desalting process can be avoided. are much lower. For example, the concentration of Co in the North
Although cellulase activity has been detected from many inver- Pacific was 4–50 pM on the surface and 10–20 pM in deep water
tebrates including marine vertebrates, most are believed to be and that of Cu was 0.5–1.3 nM on the surface and 4.5 nM in deep
produced from symbiotic microorganisms living in the gut of these water [19]. Although the effect of each metal ion at different con-
animals [6,14]. After initial confirmation of the cellulase activity centrations was not determined, the higher enzyme activity in sea-
from the homogenate of A. salina (Fig. 1), we tested for possible water could have resulted from the additional effects of NaCl and
bacterial contamination. As the initial step, we surface-sterilized metal ions present in seawater. The high activity of this enzyme
the cyst before hatching, and sterile seawater was used for hatch- in seawater is an important characteristic because this enzyme
ing. No colony appeared on the LB and Marine agar plates. For can be used for the hydrolysis of biomass from marine cultures
further confirmation, PCR amplification of the bacterial 16S rRNA without the desalting process.
gene was performed and no PCR product was detected. Although For the confirmation of the purified protein as a cellulase and
other microorganisms may not have been detected, the data sug- future application of the information for cloning this gene for the
gested that the e cellulase activity originated from A. salina. production of recombinant protein, we analyzed the purified
 H.W. Zin et al. / Biochemical and Biophysical Research Communications 443 (2014) 194–199 199

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