Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm

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Two-step puri

Wcation of Bacillus circulans chitinase A1 expressed

in Escherichia coli periplasm

Chun-Ti Chen, Chien-Jui Huang, Yi-Huei Wang, and Chao-Ying Chen

¤

Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan, ROC

Received 16 December 2003, and in revised form 2 March 2004 Available online 24 June 2004

Abstract

A protein puriWcation procedure was developed to eYciently and eVectively purify the target enzyme, chitinase A1 of Bacillus

cir-culans WL-12, from Escherichia coli DH5 carrying the chiA gene with its natural promoter in the plasmid pNTU110. Chitinase A1

was puriWed to apparent homogeneity from E. coli periplasm with a Wnal recovery of 90.6%. Two main steps were included in this protein puriWcation procedure, ammonium sulfate precipitation (40% saturation) and anion-exchange chromatography at pH 6.0 using Q Ceramic HyperD column. The yield of chitinase A1 was estimated at 95g/L. A polyclonal antibody against chitinase A1 was raised by immunizing BALB/c mice with chitinase A1 puriWed from E. coli DH5(pNTU110). As indicated by Western blot analysis, a 3000-fold diluted antibody detected puriWed chitinase A1 from E. coli DH5(pNTU110) in an amount of at least 1 ng and speciWcally detected chitinase A1 produced by B. circulans WL-12.

Keywords: Chitinase A1; Low isoelectric point; PuriWcation; Anion-exchange chromatography

Chitin, a -1,4-linked homopolymer of N-acetylglu-cosamine (GlcNAc), is one of the most abundant poly-saccharides found in nature [1]. It is degraded to GlcNAc monomers by the cooperative interaction of two enzymes, chitinase (EC 3.2.1.14) and -N-acetylglu-cosaminidase (EC3.2.1.30; GlcNAcase) [2]. Chitinases are found in a wide range of organisms, including bacte-ria, fungi, higher plants, insects, crustaceans, and some vertebrates [1–4]. They play important physiological and ecological roles in chitin metabolism of microorganisms for the production of carbon and energy sources and the antagonism toward target fungi [1–3]. In higher plants which do not contain chitin constituent, production of chitinase is generally involved in the defense mechanisms against fungal pathogens [1,2].

Bacillus circulans WL-12, a Gram-positive bacterium, secretes chitinases that are encoded by three genes (chiA, chiC, and chiD) [5–8]. Chitinase A1, encoded by chiA, is a key enzyme in the chitinolytic system of B. circulans

WL-12 [9]. It has been studied from several aspects including gene structure [7] and catalytic mechanism [10–14]. In this study, we proposed a two-step procedure to purify chitinase A1 from Escherichia coli cells harbor-ing chiA gene based on the trait of low isoelectric point (pI) of this protein [7,9]. This puriWed chitinase A1 was used in raising polyclonal antibody speciWc for the detec-tion of corresponding chitinase produced by B. circulans WL-12 and could be useful for the study of possible function of chitinase A1 toward fungi.

Materials and methods

Bacterial strains and growth conditions

Escherichia coli DH5 was used as an E. coli host for transformation and protein expression. Luria–Bertani (LB) medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, and 0.5% NaCl, pH 7.0; 2% agar was added for solid medium) was used for general purposes and supple-mented with 50g/ml ampicillin if required. B. circulans

¤_{Corresponding author. Fax: +886-2-23636490.}

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WL-12 [15], a bacterial stock from Culture Collections and Research Center in Taiwan (originally from Ger-man Collection of Microorganisms and Cell Cultures, DSM596), was used as the source of chiA gene and cul-tured in LB medium supplemented with 0.2% colloidal chitin. Colloidal chitin was prepared from crab shell chi-tin (Sigma). Ten grams of crab shell chichi-tin was mixed with 300 ml of 85% phosphoric acid and stirred at 4 °C for two days. The resulting hydrolysate was neutralized by washing with distilled water and collected as colloidal chitin after centrifugation at 1500g for 10 min. All bacte-ria were cultured at 37 °C.

Construction of recombinant plasmid for the expression of chitinase A1

Genomic DNA of B. circulans WL-12 was isolated according to a procedure described elsewhere [16] and used as the template for ampliWcation of chiA-contain-ing fragment with oligonucleotide primers, 5⬘-AGC GGCTGGAGGCGGCTATACGGC-3⬘ and 5⬘-CTAA ACTAAGCTCGCCAACACTGC-3⬘, by polymerase chain reaction (PCR). PCR thermal proWle consisted of 30 cycles of 95 °C for 1 min, 50 °C for 2 min, and 72 °C for 2 min, followed by a Wnal extension step at 72 °C for 10 min. The ampliWed DNA fragment of 2.4 kb was cloned into pCR2.1-TOPO (Invitrogen). The sequence of insert DNA was determined by an ABI-310 autose-quencer (Applied Biosystems) and compared with the sequence in GenBank Accession No. M57601. Production of chitinase of E. coli DH5 cells carrying recombinant plasmid was conWrmed by the enzyme assay using pro-teins from the periplasmic fraction of transformants [17]. Preparation of crude enzyme extract from E. coli cells for puriWcation of chitinase A1

A 3 ml overnight culture of E. coli DH5 carrying the recombinant plasmid was added to 200 ml fresh LB medium supplemented with ampicillin and cultured for 20–24 h to express chitinase. Crude enzyme extract was prepared from E. coli periplasm by an osmotic shock method [17]. Bacterial cells were harvested by centrifuga-tion at 8000g at 4 °C for 10 min and suspended in 10 ml cold (4 °C) spheroplast buVer [100 mM Tris–HCl, pH 8.0, 0.5 mM EDTA, 0.5 mM sucrose, and 20g/ml phen-ylmethylsulfonyl Xuoride (PMSF)]. After incubation for 5 min on ice, bacterial cells were collected by centrifuga-tion at 8000g at 4 °C for 10 min and re-suspended in 6.67 ml cold sterile water supplemented with a proteinase inhibitor cocktail (Roche). This bacterial suspension was incubated for 45 s on ice and subsequently mixed with 334l of 20 mM MgCl₂ (a Wnal concentration of 1 mM). The supernatant of nearly 7 ml was collected by centrifu-gation at 8000g at 4 °C for 10 min as the periplasmic fraction.

PuriWcation of chitinase A1

Twenty milliliters of pooled periplasmic fractions was prepared from 600 ml culture of E. coli DH5 carrying the recombinant plasmid. All subsequent puriWcation steps were performed at 4 °C. Proteins in the periplasmic fractions were fractionated by adding solid ammonium sulfate to 40% saturation. The precipitate was dissolved in 1 ml of 25 mM potassium phosphate buVer, pH 6.0, containing 1 mM PMSF and dialyzed overnight against the same buVer. The dialysate was applied to a 5 ml anion-exchange (Q Ceramic HyperD) column (Sigma) equilibrated with the same buVer. Proteins were eluted with NaCl of 0.1, 0.3, and 0.5 M in 25 mM potassium phosphate buVer, pH 6.0. Aliquot fractions (1 ml each) were collected. Proteins obtained from each step were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and in-gel chitinase activ-ity assay.

Protein concentration determination

Protein concentration was determined by the method described by Bradford [18] using a protein assay dye reagent concentrate (Bio-Rad). Bovine serum albumin was used as standard.

Enzyme assay

The enzymatic activity by using assay with Xuoro-genic 4-methylumbelliferyl-N,N⬘,N⬙-triacetylchitotriose (4-MU-(GlcNAc)₃) [10] was measured by a Xuorescence spectrophotometer (F-4500, Hitachi). One unit of chiti-nase activity was deWned as the amount of enzyme required to release 1mol of 4-methylumbelliferone (4-MU) per minute.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis SDS–PAGE in 10% separating gel containing 0.01% glycol chitin was carried out as described by Ames [19] using the buVer system of Laemmli [20]. Duplicate gels were run. After electrophoresis, one gel was soaked in 0.1 M sodium acetate buVer (pH 5.0) containing 1% Tri-ton X-100 for protein renaturation, then the gel was stained with 0.01% CalcoXuor White M2R in 0.5 M Tris–HCl (pH 8.9). Protein bands exhibiting chitinase activity were visualized under UV transilluminator [21]. In addition, proteins in another gel were stained with Coomassie brilliant blue G-250.

Preparation of antibody

Polyclonal antibody against puriWed chitinase A1 was raised in 4-week-old BALB/c mice. Chitinase of 100g was applied as immunogen in the initial immunization of

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BALB/c mice and succeeding injections 2 and 4 weeks thereafter.

Preparation of crude chitinases from the culture supernatant of B. circulans WL-12

Three-day culture of B. circulans WL-12 was centri-fuged at 10,000g at 4 °C for 15 min. Proteins in the culture supernatant were precipitated with 10% trichloroacetic acid at 4 °C for 30 min. The precipitated proteins were subsequently collected by centrifugation at 10,000g at 4 °C for 15 min and suspended in 0.1 M Tris–HCl buVer (pH 8.0).

Western blot analysis

Western blot analysis was performed to analyze polyclonal antibody against chitinases from E. coli cells expressing the chiA gene and the culture supernatant of B. circulans WL-12. Immunoreactive proteins were detected by an enzyme immunoassay using peroxidase-conjugated goat anti-mouse immunoglobulin (Amersham Biosciences) as secondary antibody and diaminobenzi-dine as a substrate of peroxidase.

Results

PuriWcation of chitinase A1 from E. coli DH5 harboring pNTU110

The PCR-ampliWed DNA containing chiA gene and its promoter region was ligated into pCR2.1-TOPO to generate plasmid pNTU110. The E. coli DH5 cells car-rying pNTU110, exhibiting chitinase activity, were used for the production of chitinase A1. Chitinase A1 in the periplasmic fraction of E. coli DH5(pNTU110) was puriWed to apparent homogeneity by a two-step proce-dure that included ammonium sulfate precipitation and anion-exchange chromatography. Approximately 33.7-fold puriWcation was achieved with 90.6% yield. Overall yield of puriWed chitinase A1 was estimated at 95 g/L. The speciWc activity of puriWed chitinase A1 was deter-mined to be 2.193 U/mg. The degree of puriWcation and yield at individual steps are given in Table 1. As shown in Fig. 1, chitinase was mostly eluted with NaCl at a

concentration of 0.3 M in 25 mM potassium phosphate buVer, pH 6.0, in anion-exchange chromatography, as indicated by the assay of chitinase activity. Smaller amounts of chitinase bound to anion exchanger were eluted with NaCl at a concentration of 0.5 M in 25 mM potassium phosphate buVer, pH 6.0. In addition, at least two peaks without chitinase activity appeared before elution of chitinase protein, which were proteins of higher pI values than that of chitinase A1 (pI 4.7). Electrophoresis and in-gel chitinase activity assay

In SDS–PAGE and in-gel chitinase activity assay, two major protein bands appeared in ammonium sul-fate-precipitated periplasmic protein sample of E. coli DH5 harboring pNTU110 (Fig. 2A). One of the bands corresponded to chitinase A1 as shown by in-gel activity assay. Smaller proteins exhibiting chitinase activity appeared in the gel, similar to that shown in the sample of periplasmic fraction (Fig. 2B). However, the other major band did not exhibit chitinase activity. After anion-exchange chromatography, only a major protein band was visualized in the protein samples eluted with 0.3 M NaCl in 25 mM potassium phosphate buVer, pH 6.0, which showed chitinase activity as indicated by Xuo-rometric assay (Fig. 2A). This puriWed protein exhibited chitinase activity in the in-gel activity assay and smaller proteins with chitinase activity became scarce (Fig. 2B). Table 1

PuriWcation of chitinase A1 from E. coli DH5 harboring pNTU110

a_{1 U of activity D 1}_{mol of 4-MU released per minute.}

PuriWcation step Total activity (U)a _{Total protein (mg)} _{SpeciWc activity (U/mg)} _{PuriWcation fold} _{Yield (%)}

Periplasmic fraction 0.138 2.130 0.065 1 100 Ammonium sulfate precipitation 0.109 0.145 0.752 11.6 79.0 Anion-exchange chromatography 0.125 0.057 2.193 33.7 90.6

Fig. 1. ProWle of chitinase A1 puriWcation by anion-exchange chroma-tography. The proteins were eluted stepwise with NaCl of 0.1, 0.3, 0.5, and 2 M in 25 mM potassium phosphate buVer, pH 6.0. Most of the target protein, chitinase A1, was eluted by 0.3 M NaCl.

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SpeciWcity assay of polyclonal antibody against chitinase A1

The polyclonal antibody against chitinase A1, pre-pared in BALB/c mice, was examined for its sensitivity and speciWcity by Western blot analysis. A 3000-fold diluted antibody detected puriWed chitinase A1 from E. coli DH5(pNTU110) at an amount of at least 1 ng and speciWcally detected chitinase A1 produced by B. circulans WL-12 (Fig. 3). A degradation product of chitinase A1, present in the sample of periplasmic frac-tion of E. coli DH5(pNTU110), was detected by poly-clonal antibody against chitinase A1. However, this degradation product of chitinase A1 did not hydrolyze glycol chitin and exhibited chitinase activity in the in-gel activity assay (Fig. 3).

Discussion

Chitinase A1 has been identiWed as a key enzyme in hydrolyzing chitin by B. circulans WL-12 and its encod-ing gene, chiA, was cloned and sequenced [7,9]. The chiA-transformed Bacillus subtilis F29-3 expressed higher antifungal activity than wild-type bacterium against Botrytis elliptica, a fungal pathogen of lily leaf and Xower blight [22]. Presumably, chitinase encoded by the chiA gene may increase the function of antifungal metabolites produced by B. subtilis F29-3.

To study the possible function of chitinase A1 toward fungi, pQE vectors (Qiagen) were Wrst used for the expression of chitinase A1 in E. coli. However, this approach was not a success in our study. Therefore, E. coli cells harboring plasmid pNTU110 were used to moderately express chitinase A1 for protein puriWcation. For the puriWcation of chitinase A1 from E. coli cells harboring pNTU110, chitin aYnity chromatography was Wrst followed [7,13], but the yield of chitinase after chitin adsorption step in our procedure was very low. The low eYciency was possibly due to the diYculty in manipulating chitin aYnity chromatography or the quality of the regenerated chitin. To prepare a suYcient amount of protein for our study on chitinase A1, an easy and rapid puriWcation procedure was therefore developed.

The major viewpoint of protein puriWcation proce-dure developed in this study is based on the fact that chi-tinase A1 has a low pI, thus making it negatively charged at pH 6.0. After ammonium sulfate precipitation, the protein pellet was dissolved in potassium phosphate buVer, pH 6.0. In this condition, the negative charge of most E. coli proteins [23,24] would be much weaker than the chitinase charge. Since the strong anion exchanger, Q Ceramic HyperD ion exchanger, could maintain a high ability of anion exchange at pH 6.0 to absorb negatively charged proteins such as chitinase A1, this protein could be subsequently eluted by NaCl-containing buVer at cer-tain concentration. The result veriWed that our proposed procedure could purify chitinase A1 from E. coli peri-plasm to near homogeneity. The Xow-through fraction in anion-exchange chromatography should include E. coli proteins positively charged at pH 6.0 [23,24]. The procedure to purify B. circulans chitinase A1 from E. coli harboring chiA gene using ion exchange at acidic pH is our own invention. PuriWed chitinase A1 was employed to prepare polyclonal antibody which could speciWcally detect chitinase A1 without cross reactivity to other unrelated chitinases produced by B. circulans WL-12 [7,9] and might be useful as an immunological tool in further studies related to chitinase A1.

The concept to develop an eVective and eYcient pro-cedure of chitinase A1 puriWcation in this study is based on the low pI of the target protein and the characteristic of the strong ion exchanger, which was Wrst applied to Fig. 2. SDS–PAGE analysis in each step of chitinase A1 puriWcation.

Periplasmic fraction of E. coli DH5(pNTU110) (lane 1), partially puriWed proteins from ammonium sulfate precipitation and overnight dialysis with 25 mM potassium phosphate buVer, pH 6.0 (lane 2), and the chitinase-containing fraction eluted with 0.3 M NaCl in 25 mM potassium phosphate buVer, pH 6.0, by anion-exchange chromatogra-phy (lane 3) were analyzed. Proteins in the polyacrylamide gels con-taining 0.01% glycol chitin were stained with Coomassie brilliant blue G-250 (A) and the presence of chitinase was detected by in-gel activity assay (B). The bands corresponding to chitinase A1 are indicated by arrows. M, low molecular weight standards (Amersham Biosciences).

Fig. 3. SpeciWcity assay of polyclonal antibody against chitinase A1. Periplasmic fraction of E. coli DH5(pNTU110) (lane 1), chitinase A1 puriWed by anion-exchange chromatography (lane 2), and the proteins in 3-day culture supernatant of B. circulans WL-12 (lane 3) were ana-lyzed by SDS–PAGE in polyacrylamide gel containing 0.01% glycol chitin. The gels were stained with Coomassie brilliant blue G-250 (A) or subjected to in-gel activity assay (B). Western blot analysis showed speciWcity of the polyclonal antibody against chitinase A1 produced by B. circulans WL-12 (C). The bands corresponding to chitinase A1 are indicated by arrows. M, low molecular weight standards.

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purify B. circulans chitinases. A similar strategy may be employed to purify proteins with low pI value as chitinase A1.

References

[1] J. Flach, P.E. Pilet, P. Jollès, What’s new in chitinase research? Experientia 48 (1992) 701–716.

[2] R. Cohen-Kupiec, I. Chet, The molecular biology of chitin diges-tion, Curr. Opin. Biotechnol. 9 (1998) 270–277.

[3] G.W. Gooday, Physiology of microbial degradation of chitin and chitosan, Biodegradation 1 (1990) 177–190.

[4] P.A. Felse, T. Panda, Regulation and cloning of microbial chiti-nase genes, Appl. Microbiol. Biotechnol. 51 (1999) 141–151. [5] M.M. Alam, T. Mizutani, M. Isono, N. Nikaidou, T. Watanabe,

Three chitinase genes (chiA, chiC and chiD) comprise the chitinase system of Bacillus circulans WL-12, J. Ferment. Bioeng. 82 (1996) 28–36.

[6] M.M. Alam, N. Nikaidou, H. Tanaka, T. Watanabe, Cloning and sequencing of chiC gene of Bacillus circulans WL-12 and relation-ship of its product to some other chitinases and chitinase-like pro-teins, J. Ferment. Bioeng. 80 (1995) 454–461.

[7] T. Watanabe, K. Suzuki, W. Oyanagi, K. Ohnishi, H. Tanaka, Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of Wbronectin, J. Biol. Chem. 265 (1990) 15659–15665.

[8] T. Watanabe, W. Oyanagi, K. Suzuki, K. Ohnishi, H. Tanaka, Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases, J. Bacteriol. 174 (1992) 408–414.

[9] T. Watanabe, W. Oyanagi, K. Suzuki, H. Tanaka, Chitinase sys-tem of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation, J. Bacteriol. 172 (1990) 4017–4022.

[10] T. Watanabe, K. Kobori, K. Miyashita, T. Fujii, H. Sakai, M. Uchida, H. Tanaka, IdentiWcation of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential resi-dues for chitinase activity, J. Biol. Chem. 268 (1993) 18567–18572. [11] T. Watanabe, M. Uchida, K. Kobori, H. Tanaka, Site-directed

mutagenesis of the Asp-197 and Asp-202 residues in chitinase A1

of Bacillus circulans WL-12, Biosci. Biotech. Biochem. 58 (1994) 2283–2285.

[12] M. Hashimoto, Y. Honda, N. Nikaidou, T. Fukamizo, T. Wata-nabe, Site-directed mutagenesis of Asp280_suggests

substrate-assisted catalysis of chitinase A1 from Bacillus circulans WL-12, J. Biosci. Bioeng. 89 (2000) 100–102.

[13] S. Armand, H. Tomita, A. Heyraud, C. Gey, T. Watanabe, B. Hen-rissat, Stereochemical course of the hydrolysis reaction catalyzed by chitinases A1 and D from Bacillus circulans WL-12, FEBS Lett. 343 (1994) 177–180.

[14] T. Imai, T. Watanabe, T. Yui, J. Sugiyama, Directional degrada-tion of -chitin by chitinase A1 revealed by a novel reducing end labelling technique, FEBS Lett. 510 (2002) 201–205.

[15] H. Tanaka, H.J. PhaV, Enzymatic hydrolysis of yeast cell walls. I. Isolation of wall-decomposing organisms and separation and puriWcation of lytic enzymes, J. Bacteriol. 89 (1965) 1570–1580. [16] S. Cutting, P.B. Vander Horn, in: C.R. Harwood, S.M. Cutting

(Eds.), Molecular Biological Methods for Bacillus, Wiley, Chiches-ter, England, 1990, pp. 27–74.

[17] C. Manoil, J. Beckwith, A genetic approach to analyzing mem-brane protein topology, Science 233 (1986) 1403–1408.

[18] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of pro-tein-dye binding, Anal. Biochem. 72 (1976) 248–254.

[19] G.F.L. Ames, Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs, J. Biol. Chem. 249 (1974) 634–644. [20] U.K. Laemmli, Cleavage of structural proteins during the

assem-bly of the head of bacterialphage T4, Nature (London) 227 (1970) 680–685.

[21] J. Trudel, A. Asselin, Detection of chitinase activity after poly-acrylamide gel electrophoresis, Anal. Biochem. 178 (1989) 362– 366.

[22] C.Y. Chen, Y.H. Wang, C.J. Huang, Enhancement of the anti-fungal activity of Bacillus subtilis F29-3 by the chitinase encoded by Bacillus circulans chiA gene, Can. J. Microbiol. (2004) (submit-ted and revised).

[23] B. Franzén, S. Becker, R. Mikkola, K. Tidblad, A. Tjernberg, S. Birnbaum, Characterization of periplasmic Escherichia coli pro-tein expression at high cell densities, Electrophoresis 20 (1999) 790–797.

[24] J.C. Nishihara, K.M. Champion, Quantitative evaluation of pro-teins in one- and two-dimensional polyacrylamide gels using a Xuorescent stain, Electrophoresis 23 (2002) 2203–2215.