Electroelution as a simple and fast protein purification method:isolation of an extracellular xylanase fromBacillus sp. CCMI 966

Paula Sa´–Pereira*, Jose´ Duarte, Maria Costa–FerreiraNational Institute of Industrial Engineering and Technology, Department of Biotechnology, Bioengineering and Bioprocessing Unit,

Estrada do Pac¸o 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 multipleprotein bands. Provided that the proteins are highly expressed, they can be purified rapidly and without requiring any prior knowledge ofthe protein characteristics. A xylanase excreted byBacillussp. CCMI 966 was purified directly from the polyacrylamide gel. Some of theproperties of this enzyme are presented. It had an unusually apparent high molecular mass of 340kDa, as determined by native PAGE. Thespecific 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 frommicrobial sources has grown due to their numerous appli-cations in different industrial sectors [1–4]. Hemicellulasesare typically produced as a mixture of different hydrolyticenzymes that act on xylan, degrading its backbone intosmall oligomers. Among these, the best known are endo-xylanases (endo-1,4-b-d-xylan xylanohydrolase; EC3.2.1.8.).

Xylanases are typically purified using two or more chro-matographic steps [5–13]. These protocols often involve aprecipitation step, followed by an ion exchange and/or gelfiltration. Another approach is based on molecular biologytools, which involves cloning the codifying gene (s) in orderto produce the recombinant enzymes in an adequate expres-sion vector (reviewed in [14]).

In this article, a simple method involving electroelutionfor the purification of a high molecular weight xylanaseexcreted by the strain CCMI 966 ofBacillus sp. is de-scribed. Although electroelution is a well-described method,it usually requires appropriate apparatus [15]. Furthermore,

this approach has not been previously reported for xylanasepurification.

2. Materials and methods

2.1. Bacterial strain

TheBacillusstrain was isolated from a hot spring in theAzores, Portugal. The strain was deposited in a certifiedlaboratory (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 thatpreviously 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 z 7H2O (0.5 g/liter), CaCO3 (2.0 g/liter) and 2%(w/v) agar. Calcium carbonate was sterilized separately andadded to the medium after cooling. The pH was adjusted topH 6.0 with HCl. For long-term storage, 1 ml aliquots ofBacillus grown in the liquid medium were kept in 25%glycerol at270°C under sterile connotations.

* Corresponding author.Tel.:100-351-21-7165141; fax:100-351-21-7163636.

E-mail address:paula.pereira@mail.ineti.pt (P. Sa–Pereira).

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0141-0229/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.PII: S0141-0229(00)00185-X

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 withBacilli grown overnight. Cultiva-tions were carried in 1-liter Erlenmeyer flasks containing200 ml of the above medium. These were incubated for 18 hat 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 0003 g for 30 min at 4°C, ina Sorval RC5 (Dupont) centrifuge.

Ammonium sulfate was added slowly with agitation at65% saturation. The pellet was obtained by centrifugation(10 000 3 g) and was resuspended in 50 mM phosphatebuffer, pH 6.0.

2.4. Activity assays

Xylanolytic activity was determined by using soluble oatspelt 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 axylose standard curve. The assay was carried out at 60°C.

For preliminary identification of xylanase activity ofelectrically eluted proteins from polyacrylamide gel strips(see below), samples (10ml) 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 for24 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 themanufacturer’s instructions (Biorad, Richmond, CA, USA),according to Laemmli buffer system [21].

Preparative electrophoresis was performed in a verticalslab gel unit (HSI SG 600 Series, Hoefer Scientific Instru-ments). The crude enzyme preparation was run in a 10 mlstacking gel at 4% acrylamide concentration (stock solution30% T, 2,67% C) and 32 ml separating gel with acrylamideconcentration (5%). The gel was run for about 12 h at 50 V.

Analytical electrophoresis was done in a vertical slabunit (Mini Protean II, Biorad). The purity of eluted sampleswas ascertained by running the proteins for 30 min at 150 Vusing a stacking gel (4% acrylamide concentration) over aseparating 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 aswell 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 determinedaccording to the method of Bradford [23]. The optical den-sity was read at 590 nm and the amount of protein wascalculated 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 andexpressed, usually inE. coli [14,24]. In the present work, adifferent methodology was used in order to simplify thepurification 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 endoglucanasefrom Cellvibrio mixtuschromosomal DNA [25].

Fig. 1 depicts the various steps used in the present workto 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). Afterrunning the gel, a slice (about 2 cm) was cut vertically alongthe gel and proteins were silver stained (Fig. 1B). Thestained slice was aligned with the remainder of the gel andthen each band corresponding to the stained proteins wasexcised horizontally (Fig. 1C). Each slice was subsequentlyintroduced 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 ahorizontal gel electrophoresis apparatus (GNA-200 Pharma-cia) filled with glycine buffer (Fig. 1E). The proteins wereeluted from the gel slices at 100 V. An electroelution timeof 30 min was found to be sufficient for xylanase transferinto the buffer. The contents of each dialysis tube wascentrifuged and the gels discarded (Fig. 1F). Xylanolyticactivity was detected in the supernatant (Fig. 1G) accordingto 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 ammoniumsulfate precipitation step, this value is within the rangereported in the literature [6,11]. PEG 8000 (Sigma) was alsoused to concentrate the culture supernatant, but this did notimprove the recovery (results not shown).

Analytical electrophoresis of the electroeluted proteinsstained with silver nitrate is shown in Fig. 2. The nativePAGE shows that the xylanase migrated as a single bandconfirming its purity to homogeneity. It was found to havean unusually high apparent molecular mass of about 340kDa. Other authors have described xylanases fromBacillussp. that have much lower molecular weights [6,11,14]. Thisaspect is being further studied using complementary tech-niques.

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Fig. 1. A schematic representation of the purification process of a xylanase excreted by theBacillus sp. CCMI 966 (For details see Section 3).

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The activity of the purified xylanase was determinedwithin a pH range of 4 to 10 (Fig. 3). The enzyme exhibitedhigh activity from pH 5.0 to 6.5, with the optimum at pH6.0. Similar values have been reported for otherBacilli sp.such asB. licheniformis[26], B. stearothermophilus[27],and B. thermoalkalophilus[28], assayed at a similar tem-perature (60°C) as that used in the present work.

4. Conclusion

This method is efficient and reproducible enough to beused 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 parametervaries according to the particular protein studied. Clearly,when a certain protein is excreted in very small quantities,its visual identification could be difficult despite high activ-

ity levels. On the other hand, for proteins with high expres-sion levels this method offers varies advantages. It is sim-ple, fast, cost-effective, and does not require priorknowledge of the protein characteristics.

Fig. 2. Native PAGE of a purified xylanase fromBacillus 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, purifiedxylanase (from preparative electrophoresis with acrylamide 5%). Lanes 6 and 7, partially purified xylanase (from preparative electrophoresis withacrylamide10%).

Fig. 3. Effect of assay pH on activity of purified xylanase fromBacillussp.CCMI 966. The xylanase activity was measured at 60°C for 10 min. Theassay pH values were adjusted with the following buffer systems (50 mM):(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 7Units.

Table 1Details of enzyme purification

Purification step Total activity(Units)

Total protein(mg)

Specific activity(U z mg21)

Culture supernatant 65.6 1.2832 51.1(NH4)2SO4 precipitation 21.3 0.2341 91.0Electroelution 0.9 0.0066 137.0

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