Enzyme and Microbial Technology 27 (2000) 95–99 www.elsevier.

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Electroelution as a simple and fast protein purification method:

isolation of an extracellular xylanase from Bacillus sp. CCMI 966
Paula Sá–Pereira*, José Duarte, Maria Costa–Ferreira
National Institute of Industrial Engineering and Technology, Department of Biotechnology, Bioengineering and Bioprocessing Unit,
Estrada do Paço do Lumiar, 22, 1649 – 038 Lisboa, Portugal

Received 21 April 1999; received in revised form 31 January 2000; accepted 15 February 2000.

Abstract

An efficient and simple modified method of electroelution is described that can be used as a time-saving method for eluting multiple
protein bands. Provided that the proteins are highly expressed, they can be purified rapidly and without requiring any prior knowledge of
the protein characteristics. A xylanase excreted by Bacillus sp. CCMI 966 was purified directly from the polyacrylamide gel. Some of the
properties of this enzyme are presented. It had an unusually apparent high molecular mass of 340kDa, as determined by native PAGE. The
specific activity of the purified xylanase was 137 U/mg. © 2000 Elsevier Science Inc. All rights reserved.

Keywords: Electroelution; High molecular weight xylanase; Purification

1. Introduction
In recent years interest in new xylanolytic enzymes from
microbial sources has grown due to their numerous appli-
cations in different industrial sectors [1– 4]. Hemicellulases
are typically produced as a mixture of different hydrolytic
enzymes that act on xylan, degrading its backbone into
small oligomers. Among these, the best known are endo-
xylanases (endo-1,4-␤-d-xylan xylanohydrolase; EC
3.2.1.8.).
Xylanases are typically purified using two or more chro-
matographic steps [5–13]. These protocols often involve a
precipitation step, followed by an ion exchange and/or gel
filtration. Another approach is based on molecular biology
tools, which involves cloning the codifying gene (s) in order
to produce the recombinant enzymes in an adequate expres-
sion vector (reviewed in [14]).
In this article, a simple method involving electroelution
for the purification of a high molecular weight xylanase
excreted by the strain CCMI 966 of Bacillus sp. is de-
scribed. Although electroelution is a well-described method,
it usually requires appropriate apparatus [15]. Furthermore,
* Corresponding author.Tel.: ⫹00-351-21-7165141; fax: ⫹00-351-21-
7163636.
E-mail address: paula.pereira@mail.ineti.pt (P. Sa–Pereira).
this approach has not been previously reported for xylanase
purification.
2. Materials and methods
2.1. Bacterial strain
The Bacillus strain was isolated from a hot spring in the
Azores, Portugal. The strain was deposited in a certified
laboratory (The Laboratory of Industrial Microbiology-
INETI) as Bacillus sp. CCMI 966.
2.2. Medium and culture conditions
The strain was maintained in a medium based on that
previously described [16,17] but further optimized for xy-
lanase overproduction while minimizing protease excretion
(results not shown). It contained K2HPO4 (1.0 g/liter), NaCl
(1.0 g/liter), soybean (CIPAN, S.A., Portugal) (2.0 g/liter),
oat spelts xylan (Sigma, St. Louis, MO, USA) (10 g/liter),
MgSO4 䡠 7H2O (0.5 g/liter), CaCO3 (2.0 g/liter) and 2%
(w/v) agar. Calcium carbonate was sterilized separately and
added to the medium after cooling. The pH was adjusted to
pH 6.0 with HCl. For long-term storage, 1 ml aliquots of
Bacillus grown in the liquid medium were kept in 25%
glycerol at ⫺70°C under sterile connotations.

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96 P. Sá–Pereira et al. / Enzyme and Microbial Technology 27 (2000) 95–99
Before inoculation, colonies were tested for xylan hy-
drolysis by the appearance of digestion halos on agar plates,
as revealed by Congo Red staining [18]. Cultivation me-
dium was inoculated with Bacilli grown overnight. Cultiva-
tions were carried in 1-liter Erlenmeyer flasks containing
200 ml of the above medium. These were incubated for 18 h
at 50°C under rotary agitation (200 rpm).
2.3. Preparation of culture supernatant
The extracellular enzymes were obtained by centrifuga-
tion of the culture broth at 10 000 ⫻ g for 30 min at 4°C, in
a Sorval RC5 (Dupont) centrifuge.
Ammonium sulfate was added slowly with agitation at
65% saturation. The pellet was obtained by centrifugation
(10 000 ⫻ g) and was resuspended in 50 mM phosphate
buffer, pH 6.0.
2.4. Activity assays
Xylanolytic activity was determined by using soluble oat
spelt xylan (0.5%, w/v) in 50 mM phosphate buffer, pH 6.0,
prepared according to the method described by Bailey [19].
Activity was determined using the DNS reagent [20] and a
xylose standard curve. The assay was carried out at 60°C.
For preliminary identification of xylanase activity of
electrically eluted proteins from polyacrylamide gel strips
(see below), samples (10 ␮l) were placed onto plates con-
taining 3% (w/v) oat spelt xylan in 50 mM glycine buffer,
pH 7. 0 and 1% (w/v) agarose. These were incubated for
24 h at 50°C and the halos revealed by Congo-Red staining.
2.5. Gel electrophoresis
Reagent and gel preparation for PAGE were based on the
manufacturer’s instructions (Biorad, Richmond, CA, USA),
according to Laemmli buffer system [21].
Preparative electrophoresis was performed in a vertical
slab gel unit (HSI SG 600 Series, Hoefer Scientific Instru-
ments). The crude enzyme preparation was run in a 10 ml
stacking gel at 4% acrylamide concentration (stock solution
30% T, 2,67% C) and 32 ml separating gel with acrylamide
concentration (5%). The gel was run for about 12 h at 50 V.
Analytical electrophoresis was done in a vertical slab
unit (Mini Protean II, Biorad). The purity of eluted samples
was ascertained by running the proteins for 30 min at 150 V
using a stacking gel (4% acrylamide concentration) over a
separating gel (5% acrylamide concentration). The elec-
trode-running buffer was Tris base (15 g/liter), glycine
(72 g/liter), pH 8.3 for both electrophoretic processes as
well as for electroelution. The method described by Merril
(1990) [22] was used for silver staining of the gels.
2.6. Protein estimation
The concentration of soluble proteins was determined
according to the method of Bradford [23]. The optical den-
sity was read at 590 nm and the amount of protein was
calculated by using BSA (Fraction V, Sigma, St. Louis,
MO, USA) as standard.
3. Results and discussion
Xylanases are typically purified using two or more chro-
matographic steps, or the xylanase genes are cloned and
expressed, usually in E. coli [14,24]. In the present work, a
different methodology was used in order to simplify the
purification of xylanase excreted by Bacillus. This ap-
proach, based on electrophoretic elution, was used in pre-
vious work used in relation to the cloning of endoglucanase
from Cellvibrio mixtus chromosomal DNA [25].
Fig. 1 depicts the various steps used in the present work
to purify the xylanase to homogeneity. Preparative electro-
phoresis of the appropriately diluted crude enzyme prepa-
ration was run for about 12 h at 50 V (Fig. 1A). After
running the gel, a slice (about 2 cm) was cut vertically along
the gel and proteins were silver stained (Fig. 1B). The
stained slice was aligned with the remainder of the gel and
then each band corresponding to the stained proteins was
excised horizontally (Fig. 1C). Each slice was subsequently
introduced into dialysis tubing (visking size 1– 8/32", Medi-
cell International, Ltd. London, UK) about 30 cm long,
previously filled with 50 mM phosphate buffer, pH 6.0 (Fig.
1D). The appropriately identified tubing were placed in a
horizontal gel electrophoresis apparatus (GNA-200 Pharma-
cia) filled with glycine buffer (Fig. 1E). The proteins were
eluted from the gel slices at 100 V. An electroelution time
of 30 min was found to be sufficient for xylanase transfer
into the buffer. The contents of each dialysis tube was
centrifuged and the gels discarded (Fig. 1F). Xylanolytic
activity was detected in the supernatant (Fig. 1G) according
to the assay described under Materials and methods.
The details of enzyme purification are given in Table 1.
The specific activity of the purified xylanase was 137 U/mg.
Although considerable activity was lost in the ammonium
sulfate precipitation step, this value is within the range
reported in the literature [6,11]. PEG 8000 (Sigma) was also
used to concentrate the culture supernatant, but this did not
improve the recovery (results not shown).
Analytical electrophoresis of the electroeluted proteins
stained with silver nitrate is shown in Fig. 2. The native
PAGE shows that the xylanase migrated as a single band
confirming its purity to homogeneity. It was found to have
an unusually high apparent molecular mass of about 340
kDa. Other authors have described xylanases from Bacillus
sp. that have much lower molecular weights [6,11,14]. This
aspect is being further studied using complementary tech-
niques.
 P. Sá–Pereira et al. / Enzyme and Microbial Technology 27 (2000) 95–99 97

Fig. 1. A schematic representation of the purification process of a xylanase excreted by the Bacillus sp. CCMI 966 (For details see Section 3).
98 P. Sá–Pereira et al. / Enzyme and Microbial Technology 27 (2000) 95–99

Table 1
Details of enzyme purification

Purification step Total activity Total protein Specific activity

(Units) (mg) (U 䡠 mg⫺1)
Culture supernatant 65.6 1.2832 51.1
(NH4)2SO4 precipitation 21.3 0.2341 91.0
Electroelution 0.9 0.0066 137.0

The activity of the purified xylanase was determined

within a pH range of 4 to 10 (Fig. 3). The enzyme exhibited
high activity from pH 5.0 to 6.5, with the optimum at pH
6.0. Similar values have been reported for other Bacilli sp.
such as B. licheniformis [26], B. stearothermophilus [27],
and B. thermoalkalophilus [28], assayed at a similar tem-
perature (60°C) as that used in the present work.
Fig. 3. Effect of assay pH on activity of purified xylanase from Bacillus sp.
CCMI 966. The xylanase activity was measured at 60°C for 10 min. The
assay pH values were adjusted with the following buffer systems (50 mM):
4. Conclusion (f) Na2HPO4 – NaOH (pH 4.0 –7.0), (F) Tris –HCl (pH 7.0 – 8.5) and (Œ)
NaH2PO4. H2O – HCl (pH 8.0 –10.0). The 100% activity corresponds to 7
This method is efficient and reproducible enough to be Units.
used as a routine method for eluting multiple protein bands,
allowing considerable time saving. However, it is not pos-
sible to predict protein recovery, because this parameter ity levels. On the other hand, for proteins with high expres-
varies according to the particular protein studied. Clearly, sion levels this method offers varies advantages. It is sim-
when a certain protein is excreted in very small quantities, ple, fast, cost-effective, and does not require prior
its visual identification could be difficult despite high activ- knowledge of the protein characteristics.

Fig. 2. Native PAGE of a purified xylanase from Bacillus sp. CCMI 966 gel. Lane 1, standard molecular weight markers thyroglobulin (669 kDa); ferritin
(440 kDa); catalase (232 kDa); lactate dehydrogenase (140 kDa); albumin (67 kDa). Lanes 2,5 and 8, crude culture supernatant. Lanes 3 and 4, purified
xylanase (from preparative electrophoresis with acrylamide 5%). Lanes 6 and 7, partially purified xylanase (from preparative electrophoresis with acrylamide
10%).
 P. Sá–Pereira et al. / Enzyme and Microbial Technology 27 (2000) 95–99
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