- Email: [email protected]
A novel β-glucanase gene from Bacillus halodurans C-125
FEMS Microbiology Letters 248 (2005) 9–15 www.fems-microbiology.org
A novel b-glucanase gene from Bacillus halodurans C-125 Masatake Akita *, Kinya Kayatama, Yuji Hatada, Susumu Ito, Koki Horikoshi Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 215 Natsushima, Yokosuka 237 0061, Japan Received 15 February 2005; received in revised form 13 April 2005; accepted 9 May 2005 First published online 24 May 2005 Edited by M. Moracci
Abstract A novel endo-b-1,3(4)-D-glucanase gene was found in the complete genome sequence of Bacillus halodurans C-125. The gene was previously annotated as an ‘‘unknown’’ protein and assigned an incorrect open reading frame (ORF). However, determining the biochemical characteristics has elucidated the function and correct ORF of the gene. The gene encodes 231 amino acids, and its calculated molecular mass was estimated to be 26743.16 Da. The amino acid sequence alignment showed that the highest sequence identity was only 28% with that of the b-1,3-1,4-glucanase from Bacillus subtilis. Moreover, the nucleotide sequence did not match any other known Bacillus b-glucanase gene. The member of the gene cluster that includes this novel gene was apparently different from that of the gene cluster including the putative b-glucanase genes (bh3231 and bh3232) from B. halodurans C-125. Therefore, the novel gene is not a copy of either of these genes, and in B. halodurans cells, the putative role of the encoded protein may differ from that of bh3231 and bh3232. To examine the activity of the gene product, the gene was cloned as a His-tagged protein and expressed in Escherichia coli. The purified enzyme showed activity against lichenan, barley b-glucan, laminarin, and carboxymethyl curdlan. Thin-layer chromatography showed that the enzyme hydrolyzes substrates in an endo-type manner. When b-glucan was used as a substrate, the pH optimum was between 6 and 8, and the temperature optimum was 60 C. After 2 h incubation at 50 and 60 C, the residual activity remained 100% and 50%, respectively. The enzymatic activity was abolished after 30 min incubation at 70 C. Based on the results, the gene encodes an endo-type b-1,3(4)-D-glucanase (E.C. 3.2.1.6). 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: b-glucanase; b-1,3(4)-glucanase; Glycoside hydrolase family 16; Bacillus halodurans C-125; Post-genomic study
1. Introduction b-1,3-1,4-Glucans are linear polysaccharides of cell walls of higher plants that comprise a mixture of both b-1,3- and b-1,4-linked D-glucose [1]. The percentage of the b-1,3-linkage varies from 25% to 30%. The endo-type enzymes hydrolyzing b-1,3-1,4-glucans (b-1,3-1,4glucanases) are b-1,4-D-glucan 4-glucanohydrolase (EC. 3.2.1.4), b-1,3-D-glucan 3-glucanohydrolase (E.C. *
Corresponding author. Tel.: +81 46 867 9713; fax: +81 46 867 9645. E-mail address: [email protected] (M. Akita).
3.2.1.39), and b-1,3-1,4-D-4-glucanohydrolase (lichenase) (E.C. 3.2.1.73). The enzymes hydrolyzing both b-1,3-1,4-glucan and b-1,3-glucan are classified as a b-1,3(4)-glucanase (E.C. 3.2.1.6). The b-1,3-1,4glucanases from several bacilli, which are classified into the glycoside hydrolase family 16 (GH-16) (http:// afmb.cnrsmrs.fr/CAZY/index.html), share a similar amino acid sequence having a conserved ‘‘EIDIEF’’ motif [2]. The two glutamic acid residues in this motif are known to be involved in the hydrolytic activity [3,4]. b-1,3-1,4-glucanases in GH-16 from Bacillus subtilis [5–7], Bacillus amyloliquefaciens [8], Bacillus
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.05.009
10
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
macerans [9], Bacillus circulans [10,11], Bacillus polymyxa [12], Bacillus licheniformis [13], Bacillus brevis [14], and Bacillus sp. strain N137 [15] have been cloned and characterized. Bacillus halodurans C-125 is an alkaliphilic bacterium [16] that can grow well at pH 7–10.5 when sufficient sodium chloride is present in the medium. It is well characterized physiologically, biochemically, and genetically [16–19]. The chromosomal DNA of B. halodurans C-125 has been sequenced, revealing that the genome contains more than 4000 protein coding sequences (CDSs) [20]. In the CDSs, several lichenase-like genes have been found, and two of the genes are designated bh3231 and bh3232. The proteins translated from these two genes are now classified into GH-16. The CDS designated bh2115 was annotated as an ‘‘unknown’’ protein. However, upstream of the CDS, there is an amino acid sequence that is conserved among the enzymes of GH-16. An extended CDS (designated bgn2115) has been found at the position from 2242244 to 2242939 (693 base pairs, 231 amino acids) on the complete genome sequence. Although the gene product contains the conserved amino acid motif ‘‘EIDIEF’’, the results of a BLAST search revealed that the maximal identity of the gene product was very low. This suggests that bgn2115 is a novel protein gene. Here, we report sequence analyses, protein expression, and enzymatic properties of this novel gene and its product from B. halodurans C-125. The biochemical data elucidated the function of this gene product that was previously annotated as ‘‘unknown’’.
2. Materials and methods 2.1. Materials Unless otherwise stated, all chemicals used were from Wako Pure Chemical. Coomassie Brilliant Blue R-250 (CBB), SDS, and EDTA were products of Bio-Rad. Peptone and yeast extract were purchased from Difco. Lichenan from Cetraria islandica, laminarin from Laminaria digitata, b-glucan from barley, starch, j-carrageenan from Irish moss, xylan from birch wood, and p-nitrophenyl-b-D-glucopyranoside were purchased from Sigma. 2.2. Sequence analyses Database analyses were performed using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST). Amino acid sequence alignment was performed using the CLUSTAL program [21]. The amino acid sequences used were Bgn2115 in this work, lichenase from B. subtilis NCIB 8565 (SWISS-PROT Q45691), b-1,3-1,4endoglucanase from B. licheniformis (SWISS-PROT
Q8GMY0), lichenase from B. amyloliquefaciens (SWISS-PROT P04957), a b-1,3-1,4-glucanase precursor from Bacillus pumilus (SWISS-PROT Q8GB49), a b-1,31,4-glucanase precursor from Paenibacillus polymyxa (SWISS-PROT Q8GB48), lichenase M from Paenibacillus macerans (SWISS-PROT Q846Q0), a lichenase from a Bacillus sp. (GenBank A00896), a lichenase from B. brevis (SWISS-PROT P37073), BH3231 (SWISS-PROT Q9K7X6), and BH3232 (SWISS-PROT Q9K7X5) from B. halodurans C-125. 2.3. Cloning the bgn2115 gene Genomic DNA of B. halodurans C-125 was prepared as described by Saito and Miura [22]. The bgn2115 gene was amplified from the genomic DNA by polymerase chain reaction (PCR) using primers, 5 0 -GCATAAAATCATATGAAACAACTAG-3 0 (NdeI site underlined) and 5 0 -TACAAGTTCTCGAGGTTACAATATG-3 0 (XhoI site underlined). PCR was performed using Takara Pyrobest DNA polymerase (Takara) in accordance with the manufacturerÕs instructions. The amplified DNA was purified using a High Pure PCR Product Purification kit (Roche), and then digested using restriction enzymes, NdeI and XhoI (New England Biolabs). The resulting DNA was inserted into pET-15b (Novagen) using a DNA ligation kit ver. 2, Takara. The plasmid, which encodes an N-terminal Histag with six histidine residues, was introduced by transformation into Escherichia coli HB-101 (Takara) by the methods of Hanahan [23] and Chang and Cohen [24]. The plasmid DNA sequencing was performed using an ABI Prism Big Dye Terminator Cycle Sequencing kit and an ABI 377 Sequencer (Applied Biosystems). After the sequence was checked, the desired plasmids were selected (designated pBGN2115). 2.4. Protein expression and purification To produce the protein (Bgn2115) translated from bgn2115, the plasmid pBGN2115 was introduced by transformation into E. coli BL21 (DE3) (Novagen). Then, the transformed E. coli BL21 (DE3)-pBGN2115 was cultured in 2 · YT medium (1.6% (w/v) peptone, 1.0% (w/v) yeast extract, and 0.5% (w/v) NaCl) containing 100 lg/ml ampicillin at 37 C until the OD600 reached the level of 0.5. The target protein was induced by the addition of isopropyl-b-thiogalactopyranoside (IPTG) to the medium at a final concentration of 0.1 mM. After 2 h incubation, cells were collected by centrifugation at 8000 · g for 15 min. The pellets were suspended in 20 mM sodium phosphate buffer at pH 7.0, followed by sonication. The sonicate was centrifuged, and the supernatant was loaded onto a nickel nitrilotriacetic acid-agarose
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
column (5 ml; Novagen) equilibrated with a buffer (20 mM sodium phosphate, pH 7.2, 0.5 M NaCl, and 10 mM imidazole). The column was washed with 10 mM imidazole buffer, and then the histidine-tagged protein was eluted by a stepwise gradient of imidazole from 100 to 500 mM. The purity of the protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). SDS–PAGE was performed essentially as described by Laemmli [25] on slab gels [10% (w/v) acrylamide, 80 · 100 mm, 1.0-mm thickness], and the gels were stained for proteins with CBB. The molecular mass was estimated by SDS–PAGE with molecular mass standards (BioRad), which included phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). The resultant protein was dialyzed against a large volume of 20 mM sodium phosphate buffer at pH 7.0, followed by a centrifugation at 12000 · g for 10 min. The supernatant was used for the enzymatic assay. The protein concentration was measured using the DC protein assay (Bio-Rad). 2.5. Determination of substrate specificity The enzyme activity was assayed with various substrates at a final concentration of 1.0% (w/v). The substrates used were lichenan, laminarin, carboxymethyl curdlan (CM-curdlan), b-glucan, carboxymethyl cellulose (CM-cellulose), j-carrageenan, xylan, and agarose. The assay was carried out at 50 C for 30 min in a reaction mixture containing 1.0% (w/v) substrate solution, 50 mM buffer solution, and 6.5 lg of the protein. The buffers used were sodium acetate, pH 5.0, sodium phosphate, pH 7.0, and glycine-NaOH, pH 9.0. After the mixture was centrifuged, the enzymatic activity was measured using the dinitrosalicylic acid procedure [26] with D-glucose as the standard. One unit (U) of enzymatic activity was defined as the amount of protein that produced 1 lmol of reducing sugar as D-glucose per min under the conditions of the assay. Glucosidase activity was measured using p-nitrophenyl-b-D-glucopyranoside at a final concentration of 3 mM in a 50 mM buffer (sodium acetate, pH 5.0, sodium phosphate, pH 7.0, or glycine-NaOH, pH 9.0). 2.6. Enzyme assay of b-glucan The assay was carried out at 50 C for 30 min in a reaction mixture containing 1% (w/v) b-glucan in a buffer and 6.5 lg of the protein. The enzymatic activity was measured using the method described in Section 2.5. The pH optimum of the protein was determined in various buffers: sodium acetate, pH 4.0–6.0, sodium phosphate, pH 6.0–8.0, and glycine-NaOH, pH 8.0–11, at a final concentration of 50 mM. The pH stability was
11
examined by measuring the residual activity after the protein had been incubated with the same buffers at 50 C for 30 min. Determinations of the optimal temperature and thermal stability of the protein were performed using sodium phosphate, pH 7.0. Effects of metal salts and chemical reagents were examined by measuring the residual activity after the protein had been incubated with various chemicals in 50 mM sodium phosphate buffer, pH 7.0, at 50 C for 30 min. 2.7. Chromatographic analysis of products Thin-layer chromatography (TLC) was used to identify the products. Enzymatic hydrolysis of b-glucan was carried out at 50 C in 50 mM sodium phosphate, pH 7.0, containing a 1.0% (w/v) substrate. The reaction mixtures were treated with a 70% (v/v) ethanol solution to remove unreacted b-glucan, and then applied to silica gel 60 TLC plates (10 · 20 cm, Merck). The plates were developed using a solvent system composed of 1-butanol-acetic acid-H2O (2:1:1, v/v). The spots, which were oligosaccharides resulting from the hydrolysis of the substrate, were visualized by spraying with 10% (v/v) H2SO4 and then heating at 115 C for 5 min.
3. Results 3.1. Gene and translated amino acid sequence analyses The bgn2115 gene encodes a polypeptide comprised of 231 amino acids with a calculated molecular mass of 26743.16 Da. The gene contained a putative ribosomebinding site as a 5 0 -AAAGGA-3 0 sequence, a 10 and 35 promoter sequence upstream of the initiation codon (TTG) (Fig. 1). Database searches of the gene and deduced amino acid sequences were performed using BLAST. Although the nucleotide sequence did not match any significant sequences, the amino acid sequence matched several b-1,3-1,4-glucanases. The search result suggests that the encoded protein belongs to the GH-16 family. It also had partial homology to several b-glucanases: lichenase from B. subtilis NCIB 8565 (28% in a 174 aa overlap), b-1,3-1,4-endoglucanase from B. licheniformis (27.0% in a 174 aa overlap), lichenase from B. amyloliquefaciens (27% in a 174 aa overlap), a b-1,3-1,4-glucanase precursor from B. pumilus (26% in a 171 aa overlap), an endo-b-1,3-1,4-glucanase precursor from P. polymyxa (26% in a 176 aa overlap), lichenase M from P. macerans (27% in a 175 aa overlap), BH3231 from B. halodurans C-125 (20% in a 150 aa overlap), and BH3232 from B. halodurans C-125 (29% in a 202 aa overlap). The b-glucanases belonging to GH-16 have the conserved motif ‘‘EIDIEF’’, which is also conserved in the amino acid sequence of Bgn2115 (Fig. 2).
12
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
Fig. 1. Nucleotide sequence containing the bgn2115 gene. The nucleotide sequence is numbered throughout. Putative promoter sequences (35 and 10 regions) and the ribosome binding site (SD) are underlined and boxed, respectively. The bold characters refer to the initiation codon (TTG) and termination codon (TAA). Subjected to cloning, the initiation codon of the expressed protein was ATG (Met) instead of TTG (Leu). A conserved motif of GH-16 is doubleunderlined. The initiation codon and SD sequence of bh2115 (original CDS) are indicated by dotted underline and dotted box, respectively.
3.2. Expression of bgn2115 The bgn2115 gene was expressed in the transformant harboring pBGN2115 induced by IPTG. The protein was mostly produced in inclusion bodies. The amount of purified protein obtained from 500 ml culture was approximately 1.3 mg. When the purified protein was dialyzed, a large amount of the protein precipitated. The molecular mass of Bgn2115 determined by SDS– PAGE was approximately 31 kDa (Fig. 3). 3.3. Activity of Bgn2115 The sequence analysis of bgn2115 suggested that the gene might have a function similar to the enzymes in GH-16. The possible functions were as a b-1,3-D-glucanase, b-1,4-D-glucanase, b-1,3-1,4-D-glucanase, j-carrageenase, and agarase. The results of activity assays using various substrates showed that Bgn2115 had activity against lichenan, barley b-glucan, laminarin, and CM-curdlan. The specific activities (U/mg) toward bglucan, lichenan, laminarin, and CM-curdlan were 217
Fig. 2. Alignment of amino acid sequence of bgn2115 b-glucanase with enzymes from different Bacillus strains using CLUSTAL. Identical and similar amino acids among the proteins in GH-16 are marked by asterisks (*) and colons (:), respectively. A conserved motif is boxed. Abbreviations: Bgn2115, this work; Bsubt, a lichenase from B. subtilis NCIB 8565; Blich, a b-1,3-1,4-endoglucanase from B. licheniformis; Bamyl, a lichenase from B. amyloliquefaciens; Bpumi, a b-1,3-1,4glucanase precursor from B. pumilus; Bpoly, a b-1,3-1,4-glucanase precursor from Paenibacillus polymyxa; Bmace, lichenase M from Paenibacillus macerans; Bspp, a lichenase from Bacillus sp.; Bbrev, a lichenase from B. brevis, BH3231 and BH3232 from B. halodurans C125.
(standard error = 4), 64 (6), 57 (7), and 13.0 (0.1), respectively. In contrast, activity against agarose, jcarageenan, starch, xylan and CM-cellulose was not observed. Glucosidase activity was also not observed. When b-glucan was used as the substrate, the pH optimum was from 6 to 8, and the temperature optimum was 60 C (Fig. 4). The enzyme was stable between pH 6 and pH 9, having 85% of the original activity. It also retained approximately 80% of the original activity after treatment at pH 10. After 2 h incubation at 50 and 60 C, the residual activity was at 100% and 50%, respectively. The enzymatic activity was abolished after
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
13
Fig. 3. Coomassie Brilliant Blue R-250 (CBB)-stained SDS–PAGE gel. Lanes 1 and 3, marker; lane 2, purified Bgn2115. Protein mass markers (in kDa) are indicated on the left.
30 min incubation at 70 C. Based on the TLC analysis, the enzyme does not produce glucose, cellobiose, cellotriose, laminaribiose, laminaritriose, or laminaritetraose from b-glucan (Fig. 5). Metal ions, such as Ni2+, Cu2+, Zn2+, and Hg2+ (each at 1 mM), inhibited the enzymatic activity. In contrast, the activity was not affected by CaCl2 (up to 10 mM) or EDTA (up to 100 mM). It was also not inhibited by N-bromosuccinimide (0.1 mM).
4. Discussion The chromosomal DNA of B. halodurans C-125 contains approximately 4000 genes. Of these, the two b-glucanase-like genes designated bh3231 and bh3232 have been previously annotated [20]. Those two genes were classified into GH-16. In this study, we found a novel b-1,3(4)-D-glucanase gene designated bgn2115 in the chromosomal DNA by extending the CDS designated bh2115. Upstream of bgn2115, putative promoter sequences (35 and 10 regions) and a ribosome binding site (SD) were found (Fig. 1). The gene starts with TTG (Leu), and the gene product does not contain a signal sequence. Therefore, the resultant protein may not be secreted extracellularly. The gene product, Bgn2115, contains the ‘‘EIDIEF’’ motif that is conserved among b-1,3-1,4-D-glucanases in GH-16. The translated protein, Bgn2115, was expressed by transformed E. coli BL21 (DE3) cells. The molecular mass of Bgn2115 was approximately 31 kDa by SDS–
Fig. 4. Effect of pH and temperature on activity of the purified Bgn2115. (a) The pH–activity curve. Activities were determined at the pHs indicated. Values are expressed as percentages of the optimal pH, about pH 6.0, which is taken to be 100%. (b) The temperature–activity curve. The activities were determined at the temperature indicated. Values are expressed as percentages of the optimal temperature, about 60 C, which is taken to be 100%.
PAGE (Fig. 3). Bgn2115 had activity against lichenan, barley b-glucan, laminarin, and CM-curdlan. The ratio of the activity for b-glucan, lichenan, laminarin, and carboxymethyl curdlan was 1:0.30:0.26:0.06, respectively. The relative activity for b-glucan as a substrate was higher than that for lichenan, laminarin, and CMcurdlan. b-glucan and lichenan have both b-1,3 and b-1,4 linkages. However, the ratios of the b-1,3 and b1,4-linkages are 1:3 for b-glucan and 2:1 for lichenan, respectively. Laminarin and CM-curdlan consist of mainly b-1,3-linked glucose. The reason the activity of barley b-glucan was higher than the other substrates may depend on the number of b-1,3 and b-1,4 linkages. Bgn2115 does not cleave CM-cellulose, starch, j-carrageenan, agarose, xylan, or p-nitrophenyl-b-D-glucopyranoside. The TLC analysis showed that the hydrolytic pattern was apparently an endo type, and Bgn2115 did not produce glucose, cellobiose, cellotriose, laminaribiose, laminaritriose, or laminaritetraose from b-glucan (Fig. 5). The product would be a mixture of b-1,3- and b-1,4-linkages, and the smallest product would be a
14
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
Fig. 5. Thin-layer chromatography of the products of b-glucan hydrolysis by Bgn2115. Reactions were carried out at 50 C at pH 7.0 in 50 mM sodium phosphate with 0.65 lg enzyme and 1.0% (w/v) b-glucan. At intervals, aliquots from the reaction mixture were sampled and developed by TLC. Lanes 1 and 9: standard sugars for b-1,4-linked glucose (glucose: Glu, cellobiose: cel2, and cellotriose: cel3), lanes 2 and 8: standard sugars for b-1,3-linked glucose (Glu, laminaribiose: lam2, laminaritriose: lam3, and laminaritetraose: lam4), lanes 3–7: reaction mixtures at 0, 3, 6, 12, and 24 h.
3-O-b-D-cellobiosyl-D-glucose because lichenases and laminarinases generally yield mainly tri- and tetrasaccharides as the final product. Based on these results, the bgn2115 gene encodes an endo-type b-1,3(4)-D-glucanase (E.C. 3.2.1.6). The enzymes that hydrolyze both b-1,3-1,4-glucans (lichenan and barley b-glucan) and b-1,3-glucans (laminarin) are from Rhizopus arrhizus [1,27,28], Clostridium thermocellum [28], Cellvibrio mixtus [29], Cochilobolus carbonum [30], Phaffia rhodozyma [31], and Pyrococcus furiosus [32]. With respect to lichenan and b-glucan as a substrate, the substrate specificity of Bgn2115 was similar to the b-1,3(4)-glucanase from C. mixtus, which has low activity on lichenan compared to barley b-glucan [29]. The pH dependency and temperature profile were similar to the b-glucanase from B. subtilis [33], which showed 28% similarity with Bgn2115. Bgn2115 contains the ‘‘EIDIEF’’ motif that is conserved among b-1,3-1,4-D-glucanases in GH-16. However, it has low sequence similarity to known b-1,3-1,4-D-glucanases, even when compared to BH3232 and BH3231 of B. halodurans C-125. The genes coding BH3232 and BH3231 are sequentially aligned on the chromosome, followed by a transcriptional regulator protein (BH3230). The BH3232 has a signal peptide, and it may be secreted extracellularly. BH3232 and BH3231 might be produced to degrade extracellular b-glucan. In contrast, the genes upstream of bgn2115 produce an unidentified protein (BH2111), a transmembrane lipoprotein (BH2112), an ABC transporter (BH2113), and b-1,4-xylosidase (BH2114). According to this gene cluster, the putative role of the bgn2115
product would differ from that of bh3231 or bh3232. The reason why b-1,3(4)-glucanase activity is needed in this cluster is still unknown. Moreover, the nucleotide sequence of bgn2115 did not match that of bh3231 and bh3232. Based on these results, bgn2115 is not a copy of either of those two genes. The open reading frame (ORF) of bh2115 (the originally determined CDS) was apparently wrong. The annotation program picked up the ORFs and then analyzed the sequence with respect of the SD sequence and codon usage for a series of two amino acids [20]. The assignment of the wrong ORF may due to the SD-like sequence that is located upstream of the original CDS, bh2115. bh2115 starts from Met163, and an SD-like sequence (AAATGGAC) is found upstream of the initiation codon, (Fig. 1). In this case, because the two ORFs (bh2115 and bgn2115) are redundant, the propensity of codon usage is the same. Therefore, the annotation program compared the quality of the SD sequences and selected bh2115 as a protein coding sequence. This study indicates that there is a limitation to automatic annotation. The case of bgn2115 is just one of 4066 CDSs of B. halodurans C-125. To elucidate gene functions, biochemical data provide the most authoritative information. Therefore, it is worthwhile to continue performing biochemical experiments based on the genome sequence rather than to fix the annotation program.
Acknowledgements We thank Dr. H. Takami of the Japan Agency for Marine-Earth Science and Technology for providing strains and genome sequences of Bacillus halodurans C-125 and useful advice.
References [1] Parrish, F.W., Perlin, A.S. and Reese, E.T. (1960) Selective enzymolysis of poly-b-D-glucans, and the structure of the polymers. Can. J. Chem. 38, 2094–2104. [2] Michel, G., Chantalat, L., Duee, E., Barbeyron, T., Henrissat, B., Kloareg, B. and Dideberg, O. (2001) The j-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of clan-B glycoside hydrolases. Structure 9, 513– 525. [3] Juncosa, M., Pons, J., Dot, T., Querol, E. and Planas, A. (1994) Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase by site-directed mutagenesis. J. Biol. Chem. 269, 14530–14535. [4] Hahn, M., Olsen, O., Politz, O., Borriss, R. and Heinemann, U. (1995) Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-b-glucanase. J. Biol. Chem. 270, 3081– 3088. [5] Cantwell, B.A. and McConnell, M.J. (1983) Molecular cloning and expression of a Bacillus subtilis b-glucanase gene in Escherichia coli. Gene 23, 211–219.
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15 [6] Murphy, N., McConnell, D.J. and Cantwell, B.A. (1984) The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme b-glucanase. Nucleic Acid Res. 12, 5355–5367. [7] Hinchliffe, E. (1984) Cloning and expression of a Bacillus subtilis endo-1,3-1,4-b-D-glucanase gene in Escherichia coli K12. J. Gen. Microbiol. 130, 1285–1291. [8] Hofemeister, J., Kurtz, A., Borriss, R. and Knowles, J. (1986) The b-glucanase gene from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis. Gene 49, 177–187. [9] Borriss, R., Buettner, K. and Maentsaelae, P. (1990) Structure of the b-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other b-glucanases. Mol. Gen. Genet. 222, 278–283. [10] Bueno, A., Vazquez de Aldana, C.R., Correa, J. and del Rey, F. (1990) Nucleotide sequence of a 1,3-1,4-b-glucanase-encoding gene in Bacillus circulans WL-12. Nucleic Acid Res. 18, 4248. [11] Bueno, A., Vazquez de Aldana, C.R., Correa, J., Villa, T.G. and del Rey, F. (1990) Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-b-D-glucanase in Escherichia coli. J. Bacteriol. 172, 2160–2167. [12] Gosalbes, M.J., Pe´rez-Gonza´lez, J.A., Gonzalez, R. and Navarro, A. (1991) Two b-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-b-(1,3)-(1,4)-glucanase. J. Bacteriol. 173, 7705–7710. [13] Lloberas, J., Perez-Pons, J.A. and Querol, E. (1991) Molecular cloning, expression and nucleotide sequence of the endo-b-1,3-1,4D-glucanase gene from Bacillus licheniformis. Predictive structural analyses of the encoded polypeptide. Eur. J. Biochem. 197, 337– 343. [14] Louw, M.E., Reid, S.J. and Watson, T.G. (1993) Characterization, cloning and sequencing of a thermostable endo-(1,3-1,4) bglucanase-encoding gene from an alkalophilic Bacillus brevis. Appl. Microbiol. Biotechnol. 38, 507–513. [15] Tabernero, C., Coll, P.M., Fernandez-Abalos, J.M., Pe´rez, P. and Santamarı´a, R.I. (1994) Cloning and DNA sequencing of bgaA, a gene encoding an endo-b-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137). Appl. Environ. Microbiol. 60, 1213–1220. [16] Takami, H. and Horikoshi, K. (1999) Reidentification of facultatively alkaliphilic Bacillus sp. C-125 to Bacillus halodurans. Biosci. Biotech. Biochem. 63, 943–945. [17] Horikoshi, K. (1999) Alkaliphiles: some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63, 735– 750. [18] Ikura, Y. and Horikoshi, K. (1979) Isolation and some properties of b-galactosidase producing bacteria. Agric. Biol. Chem. 43, 85–88. [19] Honda, H., Kudo, T., Ikura, Y. and Horikoshi, K. (1985) Two types of xylanases of alkalophilic Bacillus sp. no. C-125. Can. J. Microbiol. 31, 538–542.
15
[20] Takami, H., Nakasone, K., Takaki, Y., Maeno, G., Sasaki, R., Masui, N., Fuji, F., Hirama, C., Nakamura, Y., Ogasawara, N., Kuhara, S. and Horikoshi, K. (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acid Res. 28, 4317–4331. [21] Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G. and Gibson, T.J. (1998) Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23, 403–405. [22] Saito, H. and Miura, K. (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72, 619–629. [23] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580. [24] Chang, S. and Cohen, S.N. (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen. Genet. 168, 111–115. [25] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. [26] Anderson, M.A. and Stone, B.A. (1975) A new substrate for investigating the specificity of b-glucan hydrolases. FEBS Lett. 52, 202–207. [27] Clark, D.R., Johnson, J., Chung, K.H. and Kirkwood, S. (1978) Purification, characterization, and action-pattern studies on the endo-(1 linked to 3)b-D-glucanase from Rhizopus arrhizus. Carbohydr. Res. 61, 457–477. [28] Schwarz, W.H., Schimming, S. and Staudenbauer, W.L. (1988) Isolation of a Clostridium thermocellum gene encoding a thermostable b-1,3-glucanase (laminarinase). Biotechnol. Lett. 10, 225– 230. [29] Sakellaris, H., Pemberton, J.M. and Manners, J.M. (1993) Characterization of an endo-1,3(4)-b-D-glucanase gene from Cellvibrio mixtus. FEMS Microbiol. Lett. 109, 269–272. [30] Gorlach, J.M., Van Der Knaap, E. and Walton, J.D. (1998) Cloning and targeted disruption of MLG1, a gene encoding two of three extracellular mixed-linked glucanases of Cochliobolus carbonum. Appl. Environ. Microbiol. 64, 385–391. [31] Bang, M.L., Villadsen, I. and Sandal, T. (1999) Cloning and characterization of an endo-b-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938. Appl. Microbiol. Biotechnol. 51, 215–222. [32] Gueguen, Y., Voorhorst, W.G.B., van der Oost, J. and de Vos, W.M. (1997) Molecular and biochemical characterization of an endo-b-1,3-glucanase of the hyperthermophilic archaeon Pyrococcus furiosus. J. Biol. Chem. 272, 31258– 31264. [33] Moscatelli, E.A., Ham, E.A. and Rickes, E.L. (1961) Enzymatic properties of a b-glucanase from Bacillus subtilis. J. Biol. Chem. 236, 2858–2862.
A novel b-glucanase gene from Bacillus halodurans C-125 Masatake Akita *, Kinya Kayatama, Yuji Hatada, Susumu Ito, Koki Horikoshi Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 215 Natsushima, Yokosuka 237 0061, Japan Received 15 February 2005; received in revised form 13 April 2005; accepted 9 May 2005 First published online 24 May 2005 Edited by M. Moracci
Abstract A novel endo-b-1,3(4)-D-glucanase gene was found in the complete genome sequence of Bacillus halodurans C-125. The gene was previously annotated as an ‘‘unknown’’ protein and assigned an incorrect open reading frame (ORF). However, determining the biochemical characteristics has elucidated the function and correct ORF of the gene. The gene encodes 231 amino acids, and its calculated molecular mass was estimated to be 26743.16 Da. The amino acid sequence alignment showed that the highest sequence identity was only 28% with that of the b-1,3-1,4-glucanase from Bacillus subtilis. Moreover, the nucleotide sequence did not match any other known Bacillus b-glucanase gene. The member of the gene cluster that includes this novel gene was apparently different from that of the gene cluster including the putative b-glucanase genes (bh3231 and bh3232) from B. halodurans C-125. Therefore, the novel gene is not a copy of either of these genes, and in B. halodurans cells, the putative role of the encoded protein may differ from that of bh3231 and bh3232. To examine the activity of the gene product, the gene was cloned as a His-tagged protein and expressed in Escherichia coli. The purified enzyme showed activity against lichenan, barley b-glucan, laminarin, and carboxymethyl curdlan. Thin-layer chromatography showed that the enzyme hydrolyzes substrates in an endo-type manner. When b-glucan was used as a substrate, the pH optimum was between 6 and 8, and the temperature optimum was 60 C. After 2 h incubation at 50 and 60 C, the residual activity remained 100% and 50%, respectively. The enzymatic activity was abolished after 30 min incubation at 70 C. Based on the results, the gene encodes an endo-type b-1,3(4)-D-glucanase (E.C. 3.2.1.6). 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: b-glucanase; b-1,3(4)-glucanase; Glycoside hydrolase family 16; Bacillus halodurans C-125; Post-genomic study
1. Introduction b-1,3-1,4-Glucans are linear polysaccharides of cell walls of higher plants that comprise a mixture of both b-1,3- and b-1,4-linked D-glucose [1]. The percentage of the b-1,3-linkage varies from 25% to 30%. The endo-type enzymes hydrolyzing b-1,3-1,4-glucans (b-1,3-1,4glucanases) are b-1,4-D-glucan 4-glucanohydrolase (EC. 3.2.1.4), b-1,3-D-glucan 3-glucanohydrolase (E.C. *
Corresponding author. Tel.: +81 46 867 9713; fax: +81 46 867 9645. E-mail address: [email protected] (M. Akita).
3.2.1.39), and b-1,3-1,4-D-4-glucanohydrolase (lichenase) (E.C. 3.2.1.73). The enzymes hydrolyzing both b-1,3-1,4-glucan and b-1,3-glucan are classified as a b-1,3(4)-glucanase (E.C. 3.2.1.6). The b-1,3-1,4glucanases from several bacilli, which are classified into the glycoside hydrolase family 16 (GH-16) (http:// afmb.cnrsmrs.fr/CAZY/index.html), share a similar amino acid sequence having a conserved ‘‘EIDIEF’’ motif [2]. The two glutamic acid residues in this motif are known to be involved in the hydrolytic activity [3,4]. b-1,3-1,4-glucanases in GH-16 from Bacillus subtilis [5–7], Bacillus amyloliquefaciens [8], Bacillus
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.05.009
10
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
macerans [9], Bacillus circulans [10,11], Bacillus polymyxa [12], Bacillus licheniformis [13], Bacillus brevis [14], and Bacillus sp. strain N137 [15] have been cloned and characterized. Bacillus halodurans C-125 is an alkaliphilic bacterium [16] that can grow well at pH 7–10.5 when sufficient sodium chloride is present in the medium. It is well characterized physiologically, biochemically, and genetically [16–19]. The chromosomal DNA of B. halodurans C-125 has been sequenced, revealing that the genome contains more than 4000 protein coding sequences (CDSs) [20]. In the CDSs, several lichenase-like genes have been found, and two of the genes are designated bh3231 and bh3232. The proteins translated from these two genes are now classified into GH-16. The CDS designated bh2115 was annotated as an ‘‘unknown’’ protein. However, upstream of the CDS, there is an amino acid sequence that is conserved among the enzymes of GH-16. An extended CDS (designated bgn2115) has been found at the position from 2242244 to 2242939 (693 base pairs, 231 amino acids) on the complete genome sequence. Although the gene product contains the conserved amino acid motif ‘‘EIDIEF’’, the results of a BLAST search revealed that the maximal identity of the gene product was very low. This suggests that bgn2115 is a novel protein gene. Here, we report sequence analyses, protein expression, and enzymatic properties of this novel gene and its product from B. halodurans C-125. The biochemical data elucidated the function of this gene product that was previously annotated as ‘‘unknown’’.
2. Materials and methods 2.1. Materials Unless otherwise stated, all chemicals used were from Wako Pure Chemical. Coomassie Brilliant Blue R-250 (CBB), SDS, and EDTA were products of Bio-Rad. Peptone and yeast extract were purchased from Difco. Lichenan from Cetraria islandica, laminarin from Laminaria digitata, b-glucan from barley, starch, j-carrageenan from Irish moss, xylan from birch wood, and p-nitrophenyl-b-D-glucopyranoside were purchased from Sigma. 2.2. Sequence analyses Database analyses were performed using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST). Amino acid sequence alignment was performed using the CLUSTAL program [21]. The amino acid sequences used were Bgn2115 in this work, lichenase from B. subtilis NCIB 8565 (SWISS-PROT Q45691), b-1,3-1,4endoglucanase from B. licheniformis (SWISS-PROT
Q8GMY0), lichenase from B. amyloliquefaciens (SWISS-PROT P04957), a b-1,3-1,4-glucanase precursor from Bacillus pumilus (SWISS-PROT Q8GB49), a b-1,31,4-glucanase precursor from Paenibacillus polymyxa (SWISS-PROT Q8GB48), lichenase M from Paenibacillus macerans (SWISS-PROT Q846Q0), a lichenase from a Bacillus sp. (GenBank A00896), a lichenase from B. brevis (SWISS-PROT P37073), BH3231 (SWISS-PROT Q9K7X6), and BH3232 (SWISS-PROT Q9K7X5) from B. halodurans C-125. 2.3. Cloning the bgn2115 gene Genomic DNA of B. halodurans C-125 was prepared as described by Saito and Miura [22]. The bgn2115 gene was amplified from the genomic DNA by polymerase chain reaction (PCR) using primers, 5 0 -GCATAAAATCATATGAAACAACTAG-3 0 (NdeI site underlined) and 5 0 -TACAAGTTCTCGAGGTTACAATATG-3 0 (XhoI site underlined). PCR was performed using Takara Pyrobest DNA polymerase (Takara) in accordance with the manufacturerÕs instructions. The amplified DNA was purified using a High Pure PCR Product Purification kit (Roche), and then digested using restriction enzymes, NdeI and XhoI (New England Biolabs). The resulting DNA was inserted into pET-15b (Novagen) using a DNA ligation kit ver. 2, Takara. The plasmid, which encodes an N-terminal Histag with six histidine residues, was introduced by transformation into Escherichia coli HB-101 (Takara) by the methods of Hanahan [23] and Chang and Cohen [24]. The plasmid DNA sequencing was performed using an ABI Prism Big Dye Terminator Cycle Sequencing kit and an ABI 377 Sequencer (Applied Biosystems). After the sequence was checked, the desired plasmids were selected (designated pBGN2115). 2.4. Protein expression and purification To produce the protein (Bgn2115) translated from bgn2115, the plasmid pBGN2115 was introduced by transformation into E. coli BL21 (DE3) (Novagen). Then, the transformed E. coli BL21 (DE3)-pBGN2115 was cultured in 2 · YT medium (1.6% (w/v) peptone, 1.0% (w/v) yeast extract, and 0.5% (w/v) NaCl) containing 100 lg/ml ampicillin at 37 C until the OD600 reached the level of 0.5. The target protein was induced by the addition of isopropyl-b-thiogalactopyranoside (IPTG) to the medium at a final concentration of 0.1 mM. After 2 h incubation, cells were collected by centrifugation at 8000 · g for 15 min. The pellets were suspended in 20 mM sodium phosphate buffer at pH 7.0, followed by sonication. The sonicate was centrifuged, and the supernatant was loaded onto a nickel nitrilotriacetic acid-agarose
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
column (5 ml; Novagen) equilibrated with a buffer (20 mM sodium phosphate, pH 7.2, 0.5 M NaCl, and 10 mM imidazole). The column was washed with 10 mM imidazole buffer, and then the histidine-tagged protein was eluted by a stepwise gradient of imidazole from 100 to 500 mM. The purity of the protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). SDS–PAGE was performed essentially as described by Laemmli [25] on slab gels [10% (w/v) acrylamide, 80 · 100 mm, 1.0-mm thickness], and the gels were stained for proteins with CBB. The molecular mass was estimated by SDS–PAGE with molecular mass standards (BioRad), which included phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). The resultant protein was dialyzed against a large volume of 20 mM sodium phosphate buffer at pH 7.0, followed by a centrifugation at 12000 · g for 10 min. The supernatant was used for the enzymatic assay. The protein concentration was measured using the DC protein assay (Bio-Rad). 2.5. Determination of substrate specificity The enzyme activity was assayed with various substrates at a final concentration of 1.0% (w/v). The substrates used were lichenan, laminarin, carboxymethyl curdlan (CM-curdlan), b-glucan, carboxymethyl cellulose (CM-cellulose), j-carrageenan, xylan, and agarose. The assay was carried out at 50 C for 30 min in a reaction mixture containing 1.0% (w/v) substrate solution, 50 mM buffer solution, and 6.5 lg of the protein. The buffers used were sodium acetate, pH 5.0, sodium phosphate, pH 7.0, and glycine-NaOH, pH 9.0. After the mixture was centrifuged, the enzymatic activity was measured using the dinitrosalicylic acid procedure [26] with D-glucose as the standard. One unit (U) of enzymatic activity was defined as the amount of protein that produced 1 lmol of reducing sugar as D-glucose per min under the conditions of the assay. Glucosidase activity was measured using p-nitrophenyl-b-D-glucopyranoside at a final concentration of 3 mM in a 50 mM buffer (sodium acetate, pH 5.0, sodium phosphate, pH 7.0, or glycine-NaOH, pH 9.0). 2.6. Enzyme assay of b-glucan The assay was carried out at 50 C for 30 min in a reaction mixture containing 1% (w/v) b-glucan in a buffer and 6.5 lg of the protein. The enzymatic activity was measured using the method described in Section 2.5. The pH optimum of the protein was determined in various buffers: sodium acetate, pH 4.0–6.0, sodium phosphate, pH 6.0–8.0, and glycine-NaOH, pH 8.0–11, at a final concentration of 50 mM. The pH stability was
11
examined by measuring the residual activity after the protein had been incubated with the same buffers at 50 C for 30 min. Determinations of the optimal temperature and thermal stability of the protein were performed using sodium phosphate, pH 7.0. Effects of metal salts and chemical reagents were examined by measuring the residual activity after the protein had been incubated with various chemicals in 50 mM sodium phosphate buffer, pH 7.0, at 50 C for 30 min. 2.7. Chromatographic analysis of products Thin-layer chromatography (TLC) was used to identify the products. Enzymatic hydrolysis of b-glucan was carried out at 50 C in 50 mM sodium phosphate, pH 7.0, containing a 1.0% (w/v) substrate. The reaction mixtures were treated with a 70% (v/v) ethanol solution to remove unreacted b-glucan, and then applied to silica gel 60 TLC plates (10 · 20 cm, Merck). The plates were developed using a solvent system composed of 1-butanol-acetic acid-H2O (2:1:1, v/v). The spots, which were oligosaccharides resulting from the hydrolysis of the substrate, were visualized by spraying with 10% (v/v) H2SO4 and then heating at 115 C for 5 min.
3. Results 3.1. Gene and translated amino acid sequence analyses The bgn2115 gene encodes a polypeptide comprised of 231 amino acids with a calculated molecular mass of 26743.16 Da. The gene contained a putative ribosomebinding site as a 5 0 -AAAGGA-3 0 sequence, a 10 and 35 promoter sequence upstream of the initiation codon (TTG) (Fig. 1). Database searches of the gene and deduced amino acid sequences were performed using BLAST. Although the nucleotide sequence did not match any significant sequences, the amino acid sequence matched several b-1,3-1,4-glucanases. The search result suggests that the encoded protein belongs to the GH-16 family. It also had partial homology to several b-glucanases: lichenase from B. subtilis NCIB 8565 (28% in a 174 aa overlap), b-1,3-1,4-endoglucanase from B. licheniformis (27.0% in a 174 aa overlap), lichenase from B. amyloliquefaciens (27% in a 174 aa overlap), a b-1,3-1,4-glucanase precursor from B. pumilus (26% in a 171 aa overlap), an endo-b-1,3-1,4-glucanase precursor from P. polymyxa (26% in a 176 aa overlap), lichenase M from P. macerans (27% in a 175 aa overlap), BH3231 from B. halodurans C-125 (20% in a 150 aa overlap), and BH3232 from B. halodurans C-125 (29% in a 202 aa overlap). The b-glucanases belonging to GH-16 have the conserved motif ‘‘EIDIEF’’, which is also conserved in the amino acid sequence of Bgn2115 (Fig. 2).
12
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
Fig. 1. Nucleotide sequence containing the bgn2115 gene. The nucleotide sequence is numbered throughout. Putative promoter sequences (35 and 10 regions) and the ribosome binding site (SD) are underlined and boxed, respectively. The bold characters refer to the initiation codon (TTG) and termination codon (TAA). Subjected to cloning, the initiation codon of the expressed protein was ATG (Met) instead of TTG (Leu). A conserved motif of GH-16 is doubleunderlined. The initiation codon and SD sequence of bh2115 (original CDS) are indicated by dotted underline and dotted box, respectively.
3.2. Expression of bgn2115 The bgn2115 gene was expressed in the transformant harboring pBGN2115 induced by IPTG. The protein was mostly produced in inclusion bodies. The amount of purified protein obtained from 500 ml culture was approximately 1.3 mg. When the purified protein was dialyzed, a large amount of the protein precipitated. The molecular mass of Bgn2115 determined by SDS– PAGE was approximately 31 kDa (Fig. 3). 3.3. Activity of Bgn2115 The sequence analysis of bgn2115 suggested that the gene might have a function similar to the enzymes in GH-16. The possible functions were as a b-1,3-D-glucanase, b-1,4-D-glucanase, b-1,3-1,4-D-glucanase, j-carrageenase, and agarase. The results of activity assays using various substrates showed that Bgn2115 had activity against lichenan, barley b-glucan, laminarin, and CM-curdlan. The specific activities (U/mg) toward bglucan, lichenan, laminarin, and CM-curdlan were 217
Fig. 2. Alignment of amino acid sequence of bgn2115 b-glucanase with enzymes from different Bacillus strains using CLUSTAL. Identical and similar amino acids among the proteins in GH-16 are marked by asterisks (*) and colons (:), respectively. A conserved motif is boxed. Abbreviations: Bgn2115, this work; Bsubt, a lichenase from B. subtilis NCIB 8565; Blich, a b-1,3-1,4-endoglucanase from B. licheniformis; Bamyl, a lichenase from B. amyloliquefaciens; Bpumi, a b-1,3-1,4glucanase precursor from B. pumilus; Bpoly, a b-1,3-1,4-glucanase precursor from Paenibacillus polymyxa; Bmace, lichenase M from Paenibacillus macerans; Bspp, a lichenase from Bacillus sp.; Bbrev, a lichenase from B. brevis, BH3231 and BH3232 from B. halodurans C125.
(standard error = 4), 64 (6), 57 (7), and 13.0 (0.1), respectively. In contrast, activity against agarose, jcarageenan, starch, xylan and CM-cellulose was not observed. Glucosidase activity was also not observed. When b-glucan was used as the substrate, the pH optimum was from 6 to 8, and the temperature optimum was 60 C (Fig. 4). The enzyme was stable between pH 6 and pH 9, having 85% of the original activity. It also retained approximately 80% of the original activity after treatment at pH 10. After 2 h incubation at 50 and 60 C, the residual activity was at 100% and 50%, respectively. The enzymatic activity was abolished after
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
13
Fig. 3. Coomassie Brilliant Blue R-250 (CBB)-stained SDS–PAGE gel. Lanes 1 and 3, marker; lane 2, purified Bgn2115. Protein mass markers (in kDa) are indicated on the left.
30 min incubation at 70 C. Based on the TLC analysis, the enzyme does not produce glucose, cellobiose, cellotriose, laminaribiose, laminaritriose, or laminaritetraose from b-glucan (Fig. 5). Metal ions, such as Ni2+, Cu2+, Zn2+, and Hg2+ (each at 1 mM), inhibited the enzymatic activity. In contrast, the activity was not affected by CaCl2 (up to 10 mM) or EDTA (up to 100 mM). It was also not inhibited by N-bromosuccinimide (0.1 mM).
4. Discussion The chromosomal DNA of B. halodurans C-125 contains approximately 4000 genes. Of these, the two b-glucanase-like genes designated bh3231 and bh3232 have been previously annotated [20]. Those two genes were classified into GH-16. In this study, we found a novel b-1,3(4)-D-glucanase gene designated bgn2115 in the chromosomal DNA by extending the CDS designated bh2115. Upstream of bgn2115, putative promoter sequences (35 and 10 regions) and a ribosome binding site (SD) were found (Fig. 1). The gene starts with TTG (Leu), and the gene product does not contain a signal sequence. Therefore, the resultant protein may not be secreted extracellularly. The gene product, Bgn2115, contains the ‘‘EIDIEF’’ motif that is conserved among b-1,3-1,4-D-glucanases in GH-16. The translated protein, Bgn2115, was expressed by transformed E. coli BL21 (DE3) cells. The molecular mass of Bgn2115 was approximately 31 kDa by SDS–
Fig. 4. Effect of pH and temperature on activity of the purified Bgn2115. (a) The pH–activity curve. Activities were determined at the pHs indicated. Values are expressed as percentages of the optimal pH, about pH 6.0, which is taken to be 100%. (b) The temperature–activity curve. The activities were determined at the temperature indicated. Values are expressed as percentages of the optimal temperature, about 60 C, which is taken to be 100%.
PAGE (Fig. 3). Bgn2115 had activity against lichenan, barley b-glucan, laminarin, and CM-curdlan. The ratio of the activity for b-glucan, lichenan, laminarin, and carboxymethyl curdlan was 1:0.30:0.26:0.06, respectively. The relative activity for b-glucan as a substrate was higher than that for lichenan, laminarin, and CMcurdlan. b-glucan and lichenan have both b-1,3 and b-1,4 linkages. However, the ratios of the b-1,3 and b1,4-linkages are 1:3 for b-glucan and 2:1 for lichenan, respectively. Laminarin and CM-curdlan consist of mainly b-1,3-linked glucose. The reason the activity of barley b-glucan was higher than the other substrates may depend on the number of b-1,3 and b-1,4 linkages. Bgn2115 does not cleave CM-cellulose, starch, j-carrageenan, agarose, xylan, or p-nitrophenyl-b-D-glucopyranoside. The TLC analysis showed that the hydrolytic pattern was apparently an endo type, and Bgn2115 did not produce glucose, cellobiose, cellotriose, laminaribiose, laminaritriose, or laminaritetraose from b-glucan (Fig. 5). The product would be a mixture of b-1,3- and b-1,4-linkages, and the smallest product would be a
14
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15
Fig. 5. Thin-layer chromatography of the products of b-glucan hydrolysis by Bgn2115. Reactions were carried out at 50 C at pH 7.0 in 50 mM sodium phosphate with 0.65 lg enzyme and 1.0% (w/v) b-glucan. At intervals, aliquots from the reaction mixture were sampled and developed by TLC. Lanes 1 and 9: standard sugars for b-1,4-linked glucose (glucose: Glu, cellobiose: cel2, and cellotriose: cel3), lanes 2 and 8: standard sugars for b-1,3-linked glucose (Glu, laminaribiose: lam2, laminaritriose: lam3, and laminaritetraose: lam4), lanes 3–7: reaction mixtures at 0, 3, 6, 12, and 24 h.
3-O-b-D-cellobiosyl-D-glucose because lichenases and laminarinases generally yield mainly tri- and tetrasaccharides as the final product. Based on these results, the bgn2115 gene encodes an endo-type b-1,3(4)-D-glucanase (E.C. 3.2.1.6). The enzymes that hydrolyze both b-1,3-1,4-glucans (lichenan and barley b-glucan) and b-1,3-glucans (laminarin) are from Rhizopus arrhizus [1,27,28], Clostridium thermocellum [28], Cellvibrio mixtus [29], Cochilobolus carbonum [30], Phaffia rhodozyma [31], and Pyrococcus furiosus [32]. With respect to lichenan and b-glucan as a substrate, the substrate specificity of Bgn2115 was similar to the b-1,3(4)-glucanase from C. mixtus, which has low activity on lichenan compared to barley b-glucan [29]. The pH dependency and temperature profile were similar to the b-glucanase from B. subtilis [33], which showed 28% similarity with Bgn2115. Bgn2115 contains the ‘‘EIDIEF’’ motif that is conserved among b-1,3-1,4-D-glucanases in GH-16. However, it has low sequence similarity to known b-1,3-1,4-D-glucanases, even when compared to BH3232 and BH3231 of B. halodurans C-125. The genes coding BH3232 and BH3231 are sequentially aligned on the chromosome, followed by a transcriptional regulator protein (BH3230). The BH3232 has a signal peptide, and it may be secreted extracellularly. BH3232 and BH3231 might be produced to degrade extracellular b-glucan. In contrast, the genes upstream of bgn2115 produce an unidentified protein (BH2111), a transmembrane lipoprotein (BH2112), an ABC transporter (BH2113), and b-1,4-xylosidase (BH2114). According to this gene cluster, the putative role of the bgn2115
product would differ from that of bh3231 or bh3232. The reason why b-1,3(4)-glucanase activity is needed in this cluster is still unknown. Moreover, the nucleotide sequence of bgn2115 did not match that of bh3231 and bh3232. Based on these results, bgn2115 is not a copy of either of those two genes. The open reading frame (ORF) of bh2115 (the originally determined CDS) was apparently wrong. The annotation program picked up the ORFs and then analyzed the sequence with respect of the SD sequence and codon usage for a series of two amino acids [20]. The assignment of the wrong ORF may due to the SD-like sequence that is located upstream of the original CDS, bh2115. bh2115 starts from Met163, and an SD-like sequence (AAATGGAC) is found upstream of the initiation codon, (Fig. 1). In this case, because the two ORFs (bh2115 and bgn2115) are redundant, the propensity of codon usage is the same. Therefore, the annotation program compared the quality of the SD sequences and selected bh2115 as a protein coding sequence. This study indicates that there is a limitation to automatic annotation. The case of bgn2115 is just one of 4066 CDSs of B. halodurans C-125. To elucidate gene functions, biochemical data provide the most authoritative information. Therefore, it is worthwhile to continue performing biochemical experiments based on the genome sequence rather than to fix the annotation program.
Acknowledgements We thank Dr. H. Takami of the Japan Agency for Marine-Earth Science and Technology for providing strains and genome sequences of Bacillus halodurans C-125 and useful advice.
References [1] Parrish, F.W., Perlin, A.S. and Reese, E.T. (1960) Selective enzymolysis of poly-b-D-glucans, and the structure of the polymers. Can. J. Chem. 38, 2094–2104. [2] Michel, G., Chantalat, L., Duee, E., Barbeyron, T., Henrissat, B., Kloareg, B. and Dideberg, O. (2001) The j-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of clan-B glycoside hydrolases. Structure 9, 513– 525. [3] Juncosa, M., Pons, J., Dot, T., Querol, E. and Planas, A. (1994) Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase by site-directed mutagenesis. J. Biol. Chem. 269, 14530–14535. [4] Hahn, M., Olsen, O., Politz, O., Borriss, R. and Heinemann, U. (1995) Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-b-glucanase. J. Biol. Chem. 270, 3081– 3088. [5] Cantwell, B.A. and McConnell, M.J. (1983) Molecular cloning and expression of a Bacillus subtilis b-glucanase gene in Escherichia coli. Gene 23, 211–219.
M. Akita et al. / FEMS Microbiology Letters 248 (2005) 9–15 [6] Murphy, N., McConnell, D.J. and Cantwell, B.A. (1984) The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme b-glucanase. Nucleic Acid Res. 12, 5355–5367. [7] Hinchliffe, E. (1984) Cloning and expression of a Bacillus subtilis endo-1,3-1,4-b-D-glucanase gene in Escherichia coli K12. J. Gen. Microbiol. 130, 1285–1291. [8] Hofemeister, J., Kurtz, A., Borriss, R. and Knowles, J. (1986) The b-glucanase gene from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis. Gene 49, 177–187. [9] Borriss, R., Buettner, K. and Maentsaelae, P. (1990) Structure of the b-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other b-glucanases. Mol. Gen. Genet. 222, 278–283. [10] Bueno, A., Vazquez de Aldana, C.R., Correa, J. and del Rey, F. (1990) Nucleotide sequence of a 1,3-1,4-b-glucanase-encoding gene in Bacillus circulans WL-12. Nucleic Acid Res. 18, 4248. [11] Bueno, A., Vazquez de Aldana, C.R., Correa, J., Villa, T.G. and del Rey, F. (1990) Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-b-D-glucanase in Escherichia coli. J. Bacteriol. 172, 2160–2167. [12] Gosalbes, M.J., Pe´rez-Gonza´lez, J.A., Gonzalez, R. and Navarro, A. (1991) Two b-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-b-(1,3)-(1,4)-glucanase. J. Bacteriol. 173, 7705–7710. [13] Lloberas, J., Perez-Pons, J.A. and Querol, E. (1991) Molecular cloning, expression and nucleotide sequence of the endo-b-1,3-1,4D-glucanase gene from Bacillus licheniformis. Predictive structural analyses of the encoded polypeptide. Eur. J. Biochem. 197, 337– 343. [14] Louw, M.E., Reid, S.J. and Watson, T.G. (1993) Characterization, cloning and sequencing of a thermostable endo-(1,3-1,4) bglucanase-encoding gene from an alkalophilic Bacillus brevis. Appl. Microbiol. Biotechnol. 38, 507–513. [15] Tabernero, C., Coll, P.M., Fernandez-Abalos, J.M., Pe´rez, P. and Santamarı´a, R.I. (1994) Cloning and DNA sequencing of bgaA, a gene encoding an endo-b-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137). Appl. Environ. Microbiol. 60, 1213–1220. [16] Takami, H. and Horikoshi, K. (1999) Reidentification of facultatively alkaliphilic Bacillus sp. C-125 to Bacillus halodurans. Biosci. Biotech. Biochem. 63, 943–945. [17] Horikoshi, K. (1999) Alkaliphiles: some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63, 735– 750. [18] Ikura, Y. and Horikoshi, K. (1979) Isolation and some properties of b-galactosidase producing bacteria. Agric. Biol. Chem. 43, 85–88. [19] Honda, H., Kudo, T., Ikura, Y. and Horikoshi, K. (1985) Two types of xylanases of alkalophilic Bacillus sp. no. C-125. Can. J. Microbiol. 31, 538–542.
15
[20] Takami, H., Nakasone, K., Takaki, Y., Maeno, G., Sasaki, R., Masui, N., Fuji, F., Hirama, C., Nakamura, Y., Ogasawara, N., Kuhara, S. and Horikoshi, K. (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acid Res. 28, 4317–4331. [21] Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G. and Gibson, T.J. (1998) Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23, 403–405. [22] Saito, H. and Miura, K. (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72, 619–629. [23] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580. [24] Chang, S. and Cohen, S.N. (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen. Genet. 168, 111–115. [25] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. [26] Anderson, M.A. and Stone, B.A. (1975) A new substrate for investigating the specificity of b-glucan hydrolases. FEBS Lett. 52, 202–207. [27] Clark, D.R., Johnson, J., Chung, K.H. and Kirkwood, S. (1978) Purification, characterization, and action-pattern studies on the endo-(1 linked to 3)b-D-glucanase from Rhizopus arrhizus. Carbohydr. Res. 61, 457–477. [28] Schwarz, W.H., Schimming, S. and Staudenbauer, W.L. (1988) Isolation of a Clostridium thermocellum gene encoding a thermostable b-1,3-glucanase (laminarinase). Biotechnol. Lett. 10, 225– 230. [29] Sakellaris, H., Pemberton, J.M. and Manners, J.M. (1993) Characterization of an endo-1,3(4)-b-D-glucanase gene from Cellvibrio mixtus. FEMS Microbiol. Lett. 109, 269–272. [30] Gorlach, J.M., Van Der Knaap, E. and Walton, J.D. (1998) Cloning and targeted disruption of MLG1, a gene encoding two of three extracellular mixed-linked glucanases of Cochliobolus carbonum. Appl. Environ. Microbiol. 64, 385–391. [31] Bang, M.L., Villadsen, I. and Sandal, T. (1999) Cloning and characterization of an endo-b-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938. Appl. Microbiol. Biotechnol. 51, 215–222. [32] Gueguen, Y., Voorhorst, W.G.B., van der Oost, J. and de Vos, W.M. (1997) Molecular and biochemical characterization of an endo-b-1,3-glucanase of the hyperthermophilic archaeon Pyrococcus furiosus. J. Biol. Chem. 272, 31258– 31264. [33] Moscatelli, E.A., Ham, E.A. and Rickes, E.L. (1961) Enzymatic properties of a b-glucanase from Bacillus subtilis. J. Biol. Chem. 236, 2858–2862.