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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.
1. Introduction this approach has not been previously reported for xylanase
purification.
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 2. Materials and methods
are typically produced as a mixture of different hydrolytic
enzymes that act on xylan, degrading its backbone into 2.1. Bacterial strain
small oligomers. Among these, the best known are endo-
xylanases (endo-1,4--d-xylan xylanohydrolase; EC The Bacillus strain was isolated from a hot spring in the
3.2.1.8.). Azores, Portugal. The strain was deposited in a certified
Xylanases are typically purified using two or more chro- laboratory (The Laboratory of Industrial Microbiology-
matographic steps [5–13]. These protocols often involve a INETI) as Bacillus sp. CCMI 966.
precipitation step, followed by an ion exchange and/or gel
filtration. Another approach is based on molecular biology 2.2. Medium and culture conditions
tools, which involves cloning the codifying gene (s) in order
to produce the recombinant enzymes in an adequate expres- The strain was maintained in a medium based on that
sion vector (reviewed in [14]). previously described [16,17] but further optimized for xy-
In this article, a simple method involving electroelution lanase overproduction while minimizing protease excretion
for the purification of a high molecular weight xylanase (results not shown). It contained K2HPO4 (1.0 g/liter), NaCl
excreted by the strain CCMI 966 of Bacillus sp. is de- (1.0 g/liter), soybean (CIPAN, S.A., Portugal) (2.0 g/liter),
scribed. Although electroelution is a well-described method, oat spelts xylan (Sigma, St. Louis, MO, USA) (10 g/liter),
it usually requires appropriate apparatus [15]. Furthermore, 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
* Corresponding author.Tel.: ⫹00-351-21-7165141; fax: ⫹00-351-21- pH 6.0 with HCl. For long-term storage, 1 ml aliquots of
7163636. Bacillus grown in the liquid medium were kept in 25%
E-mail address: paula.pereira@mail.ineti.pt (P. Sa–Pereira). glycerol at ⫺70°C under sterile connotations.
0141-0229/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 0 0 ) 0 0 1 8 5 - X
96 P. Sá–Pereira et al. / Enzyme and Microbial Technology 27 (2000) 95–99
Before inoculation, colonies were tested for xylan hy- 2.6. Protein estimation
drolysis by the appearance of digestion halos on agar plates,
as revealed by Congo Red staining [18]. Cultivation me- The concentration of soluble proteins was determined
dium was inoculated with Bacilli grown overnight. Cultiva- according to the method of Bradford [23]. The optical den-
tions were carried in 1-liter Erlenmeyer flasks containing sity was read at 590 nm and the amount of protein was
200 ml of the above medium. These were incubated for 18 h calculated by using BSA (Fraction V, Sigma, St. Louis,
at 50°C under rotary agitation (200 rpm). MO, USA) as standard.
The extracellular enzymes were obtained by centrifuga- Xylanases are typically purified using two or more chro-
tion of the culture broth at 10 000 ⫻ g for 30 min at 4°C, in matographic steps, or the xylanase genes are cloned and
a Sorval RC5 (Dupont) centrifuge. expressed, usually in E. coli [14,24]. In the present work, a
Ammonium sulfate was added slowly with agitation at different methodology was used in order to simplify the
65% saturation. The pellet was obtained by centrifugation purification of xylanase excreted by Bacillus. This ap-
(10 000 ⫻ g) and was resuspended in 50 mM phosphate proach, based on electrophoretic elution, was used in pre-
buffer, pH 6.0. 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
2.4. Activity assays to purify the xylanase to homogeneity. Preparative electro-
phoresis of the appropriately diluted crude enzyme prepa-
Xylanolytic activity was determined by using soluble oat ration was run for about 12 h at 50 V (Fig. 1A). After
spelt xylan (0.5%, w/v) in 50 mM phosphate buffer, pH 6.0, running the gel, a slice (about 2 cm) was cut vertically along
prepared according to the method described by Bailey [19]. the gel and proteins were silver stained (Fig. 1B). The
Activity was determined using the DNS reagent [20] and a stained slice was aligned with the remainder of the gel and
xylose standard curve. The assay was carried out at 60°C. then each band corresponding to the stained proteins was
For preliminary identification of xylanase activity of excised horizontally (Fig. 1C). Each slice was subsequently
electrically eluted proteins from polyacrylamide gel strips introduced into dialysis tubing (visking size 1– 8/32", Medi-
(see below), samples (10 l) were placed onto plates con- cell International, Ltd. London, UK) about 30 cm long,
previously filled with 50 mM phosphate buffer, pH 6.0 (Fig.
taining 3% (w/v) oat spelt xylan in 50 mM glycine buffer,
1D). The appropriately identified tubing were placed in a
pH 7. 0 and 1% (w/v) agarose. These were incubated for
horizontal gel electrophoresis apparatus (GNA-200 Pharma-
24 h at 50°C and the halos revealed by Congo-Red staining.
cia) filled with glycine buffer (Fig. 1E). The proteins were
eluted from the gel slices at 100 V. An electroelution time
2.5. Gel electrophoresis of 30 min was found to be sufficient for xylanase transfer
into the buffer. The contents of each dialysis tube was
Reagent and gel preparation for PAGE were based on the centrifuged and the gels discarded (Fig. 1F). Xylanolytic
manufacturer’s instructions (Biorad, Richmond, CA, USA), activity was detected in the supernatant (Fig. 1G) according
according to Laemmli buffer system [21]. to the assay described under Materials and methods.
The details of enzyme purification are given in Table 1.
Preparative electrophoresis was performed in a vertical
The specific activity of the purified xylanase was 137 U/mg.
slab gel unit (HSI SG 600 Series, Hoefer Scientific Instru-
Although considerable activity was lost in the ammonium
ments). The crude enzyme preparation was run in a 10 ml
sulfate precipitation step, this value is within the range
stacking gel at 4% acrylamide concentration (stock solution
reported in the literature [6,11]. PEG 8000 (Sigma) was also
30% T, 2,67% C) and 32 ml separating gel with acrylamide used to concentrate the culture supernatant, but this did not
concentration (5%). The gel was run for about 12 h at 50 V. improve the recovery (results not shown).
Analytical electrophoresis was done in a vertical slab Analytical electrophoresis of the electroeluted proteins
unit (Mini Protean II, Biorad). The purity of eluted samples stained with silver nitrate is shown in Fig. 2. The native
was ascertained by running the proteins for 30 min at 150 V PAGE shows that the xylanase migrated as a single band
using a stacking gel (4% acrylamide concentration) over a confirming its purity to homogeneity. It was found to have
separating gel (5% acrylamide concentration). The elec- an unusually high apparent molecular mass of about 340
trode-running buffer was Tris base (15 g/liter), glycine kDa. Other authors have described xylanases from Bacillus
(72 g/liter), pH 8.3 for both electrophoretic processes as sp. that have much lower molecular weights [6,11,14]. This
well as for electroelution. The method described by Merril aspect is being further studied using complementary tech-
(1990) [22] was used for silver staining of the gels. 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
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 99