An unusual pectate lyase from a Bacillus sp. with high activity on pectin: cloning and characterization

Margarita Soriano1, Ana Blanco1, Pilar Díaz1 and F. I. Javier Pastor1

Department of Microbiology, Faculty of Biology, University of Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain1

Author for correspondence: F. I. Javier Pastor. Tel: +34 3 4029012. Fax: +34 3 4110592. e-mail: fpastor{at}bio.ub.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The gene pelA encoding a pectate lyase from the strain Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1214 bp DNA fragment containing pelA gene was determined, revealing an ORF of 666 nucleotides that encoded a protein of 23233 Da. The deduced amino acid sequence of the encoded enzyme showed homology to pectate lyases A, B, C and D from Fusarium solani, Pel-3 and PelB from Erwinia carotovora and PelI from Erwinia chrysanthemi. Homology was also found to the protein deduced from the Bacillus subtilis yvpA gene, the function of which is unknown. The heterologous expressed enzyme depolymerized polygalacturonate and pectins of methyl esterification degree from 22 to 89%, and exhibited similar activity on polygalacturonate and on 89% esterified citrus pectin. Optimum temperature and pH for enzymic activity were 50 °C and pH 10, respectively. Ca2+ was required for activity on pectic substrates, while the enzyme was strongly inhibited by Ba2+.

Keywords: pectate, pectin, lyase

The EMBL accession number for the nucleotide sequence determined in this work is AJ237980.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pectin is a heteropolysaccharide composed of 1,4-{alpha}-linked galacturonate chains with a high percentage of methyl esterification. Pectin degradation requires the combined action of several enzymes that can be classified into two main groups: methylesterases, which remove methoxyl groups from pectin, and depolymerases (hydrolases and lyases), which cleave the bonds between galacturonate units. Lyases cleave glycosidic bonds by ß-elimination, giving rise to unsaturated products. Among these enzymes, pectin lyases show specificity for methyl esterified substrates (pectin) while pectate lyases are specific for unesterified polygalacturonate (pectate). Pectate lyases are widely distributed among microbial plant pathogens and have been the focus of several studies to ascertain their role as virulence factors (Barras et al., 1994 ; Hugouvieux-Cotte-Pattat et al., 1996 ). They have also been found in saprophytic micro-organisms, including the genus Bacillus (Nasser et al., 1990 ), and in some thermophilic bacteria (Kozianowski et al., 1997 ). Multiple sequence alignment of pectate lyases has been used to classify these enzymes into several subfamiles and to identify consensus sequences of some of them (Henrissat et al., 1995 ; Hinton et al., 1989 ), although recent sequencing and characterization of novel enzymes has resulted in the proposal of new groups of pectate lyases (Shevchik et al., 1997 ; Kim & Beer 1998 ).

Pectin-degrading enzymes are widely used in the food industry for improving the yield and the clarification of fruit juices (Alkorta et al., 1998 ). At present, pectinolytic enzymes are being introduced into the textile industry to release fibres from flax stems, as an alternative to conventional retting (Henriksson et al., 1999 ). In these fields, finding new enzymes has special interest to improve the efficiency of the production systems.

The strain Bacillus sp. BP-23, previously isolated from a rice field (Blanco & Pastor, 1993 ), shows a multiple glycanase system, some enzymes of which have been cloned and characterized (Blanco et al., 1995 , 1998 , 1999 ). In this article, we describe the cloning and characterization of pectate lyase A from Bacillus sp. BP-23, an enzyme with some unusual features among pectin and pectate lyases.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
Bacillus sp. strain BP-23 was isolated from soil from a rice field in the Ebro river delta (Blanco & Pastor, 1993 ) and has been deposited in the Spanish Type Culture Collection as strain CECT 4592. The strain was grown as described previously (Blanco & Pastor, 1993 ). Escherichia coli 5K (Godessart et al., 1988 ) was used as the recipient strain for recombinant plasmids. E. coli 5K was cultivated at 37 °C in LB medium. Plasmid pBR322 (Boehringer Mannheim) was used as cloning vector.

Nucleic acid manipulation.
Chromosomal DNA from Bacillus sp. BP-23 was prepared as described by Dubnau & Davidoff-Abelson (1971) . Plasmid DNA was purified by the alkaline lysis procedure (Sambrook et al., 1989 ). Restriction nucleases and ligase were purchased from Boehringer Mannheim and used according to the manufacturer’s specifications. Chromosomal DNA was partially digested with Sau3AI; fragments of 4–7 kb were isolated from a 0·8% (w/v) agarose gel by electroelution and ligated to BamHI-digested pBR322. The resulting molecules were introduced into E. coli 5K cells by transformation according to the method of Hanahan (1983) and Apr Tcs colonies were selected. Southern hybridization analysis was performed as described by Sambrook et al. (1989) .

The sequence of both strands of pelA was determined by automated fluorescence sequencing with an ABI PRISM dye terminator cycle sequencing ready reaction mix (Perkin Elmer) in a 377 Perkin Elmer DNA sequencer.

Enzyme assays.
The screening of the gene library for recombinant bacteria producing pectinase was performed on LB agar plates supplemented with 0·4% (w/v) polygalacturonic acid (Sigma) as described by Keen et al. (1984) . After growth, plates were flooded with 1 M CaCl2 and pectinase-producing colonies were detected by the appearance of a halo around them.

Cells from cultures of E. coli 5K carrying the recombinant plasmid pP22 (see below) were disrupted by sonication and the lysates obtained were cleared by centrifugation, dialysed and tested for activity. Pectate lyase activity was assayed spectrophotometrically by measuring the formation of unsaturated products from polygalacturonic acid at 232 nm (Collmer et al., 1988 ). The standard assay mixture contained 0·2% polygalacturonic acid (Sigma) in a final volume of 3 ml 50 mM glycine buffer pH 10·0 containing 0·5 mM CaCl2. Assay mixtures were incubated for 2·5 min at 50 °C and the increase in absorbance at 232 nm was measured. To test activity on substrates with various degrees of methylation, polygalacturonic acid was replaced by 22, 64 and 89% esterified citrus pectin (Sigma). Apple pectin (68% esterification; Sigma) was also tested as a substrate. One unit of enzymic activity was defined as the amount of enzyme that produces 1 µmol unsaturated product min-1 under the assay conditions described. For inhibition studies, 1 mM different ions were added to the assay mixture and activity was determined as indicated.

Gel electrophoresis and zymograms.
SDS-PAGE was performed in 13% (w/v) gels, essentially as described by Laemmli (1970) . Samples were heated for 10 min at 45 °C in sample buffer before being applied to gels. After electrophoresis, gels were soaked in 2·5% (w/v) Triton X-100 for 30 min, washed in 100 mM glycine buffer pH 10·0, 1·5 mM CaCl2 for 30 min and overlaid with an 1% agarose gel containing 0·1% polygalacturonic acid in the same buffer as above. After 30 min incubation at 45 °C, gels were stained with 0·05% (w/v) Ruthenium red (Sigma) for 10 min and washed with water until pectate lyase bands became visible.

IEF was performed in a Pharmacia PhastSystem unit. Gels with a pH range from 3·0 to 9·0 (Pharmacia) were used. For zymogram analysis, IEF gels were overlaid with polygalacturonic-acid-containing agarose gels and developed as described above.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of pectate-lyase-A-encoding gene
The strain Bacillus sp. BP-23, previously isolated from an enrichment culture on rice straw, secretes a complex system of carbohydrate-degrading enzymes including pectinases (Blanco & Pastor, 1993 ). The influence of carbon sources on pectate lyase production by the strain was evaluated. The highest activity was obtained in pectin-supplemented media (252 U ml-1). Other carbohydrates tested, such as polygalacturonate and rice straw, did not notably affect pectate lyase production, whilst activity was undetectable in glucose-supplemented media. A gene library from Bacillus sp. BP-23 was constructed in E. coli 5K and screened for pectinase activity on polygalacturonic-acid-containing agar plates. One clone out of 4000 tested showed pectin degradation around the bacterial growth and was selected. This strain carried the recombinant plasmid pP22, which contained a DNA insert of 1·2 kb. Hybridization analysis confirmed that the DNA insert in pP22 belonged to Bacillus sp. BP-23. A 0·7 kb PvuII–SphI fragment from pP22, used as a probe, hybridized to identically sized DNA bands from Bacillus sp. BP-23 and pP22 doubly digested with PvuII/SphI, whilst no hybridization was detected to E. coli 5K chromosomal DNA or pBR322.

Substrate specificity
Cell extracts from E. coli 5K/pP22 showed high lyase activity on pectic substrates, whilst no hydrolytic activity was found. Xylans, carboxymethylcellulose or other carbohydrates tested were not degraded by the enzyme. Among pectic substrates, maximum activity was found on polygalacturonic acid, although the cloned enzyme also showed high activity on pectin. The influence of substrate methylation on enzymic activity was tested by evaluating the activity of the enzyme on pectins of increasing degree of methyl esterification. The enzyme exhibited on all the pectins tested more than 60% of the activity found on polygalacturonic acid (Fig. 1a). On highly methylated pectins (89% esterification) the activity found was more than 90% of that detected on polygalacturonic acid. The results show that the cloned enzyme exhibits very similar activity on pectins and polygalacturonic acid, showing high levels of lyase activity on pectic substrates irrespective of the degree of methyl esterification.



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Fig. 1. Some enzymic properties of PelA. (a) Influence of the degree of pectin methylation on PelA activity. Activity of PelA on polygalacturonic acid (PGA), citrus pectins (CP) with degree of methyl esterification from 22 to 89% and apple pectin (AP) (68% esterification) was determined by the standard assay. Activity is expressed relative to that detected on non-methylated substrate. One hundred per cent pectate lyase activity was equivalent to 142 units (mg protein)-1. (b) Influence of Ca2+ concentration on PelA activity. Activity on polygalacturonic acid ({bullet}) and citrus pectin ({square}) was determined at 50 °C and pH 10·0 in the presence of different concentrations of CaCl2. One hundred per cent PelA activity was equivalent to 140 and 132 units (mg protein)-1 on polygalacturonic acid and on pectin, respectively.

 
Characterization of the cloned enzyme
The effect of pH and temperature on the activity of the cloned enzyme on polygalacturonic acid was determined. The enzyme was most active in a narrow alkaline pH range, showing the highest activity at pH 10·0. Optimum temperature for pectate lyase activity was 50 °C, whilst more than 50% of maximum activity was found in the temperature range from 40 to 60 °C. Stability assays showed that the enzyme was stable for 1 h at 40 °C in the pH range from 4·0 to 8·0. At pH 10·0, the enzyme retained more than 50% of the initial activity after 4 h incubation at 40 °C, whilst at 50 °C only 1% of the initial activity was found after the same treatment. At pH 7·0, the enzyme remained stable for at least 24 h at 40 °C.

The influence of Ca2+ ions on enzymic activity was tested by performing assays at different CaCl2 concentrations (Fig. 1b). The results showed that the cloned enzyme has an absolute requirement for Ca2+ ions for polygalacturonic acid degradation. Activity was undetectable without this ion. Maximum activity was found at 0·5–0·75 mM CaCl2, whilst activity decreased at higher concentrations and only 9% of maximum activity was found at 10 mM CaCl2. Conversely, the degradation of pectin was observed even without Ca2+ addition, although the activity was enhanced in the presence of added Ca2+ and reached a maximum at 0·75 mM CaCl2 (Fig. 1b). The activity found without exogenous Ca2+ could result from the presence of trace amounts of contaminant Ca2+ ions in the pectins used as substrates. In fact, when 2 mM EDTA was added to the assay mixture, no activity was detected on pectins or polygalacturonic acid. These results indicate a requirement for Ca2+ for enzymic activity on both substrates. According to this and to the substrate specificity results, we concluded that the cloned enzyme is a pectate lyase. We named it PelA.

The effect of metal ions other than Ca2+ on pectate lyase activity was determined by performing enzymic assays on polygalacturonic acid in the presence of metal ions at 1 mM concentration, without added Ca2+. Activity was undetectable in all the assays, indicating that none of the ions tested could replace Ca2+. When the effect of metal ions was tested in the presence of 0·5 mM CaCl2, we found that the enzyme was strongly inhibited by Ba2+ (9% residual activity). Sn2+, Mg2+ and Ag+ also caused notable inhibition of the enzyme (36, 42 and 53% residual activity, respectively), whilst Zn2+, Ni2+, Hg2+ and Co2+ caused only low levels of inhibition (71, 75, 77 and 88% residual activity, respectively).

SDS-PAGE analysis of cell extracts from E. coli 5K/pP22 showed a prominent 25 kDa protein band not found in extracts from E. coli 5K/pBR322 (Fig. 2). The band showed pectate lyase activity in zymographic analysis, whilst no pectate lyase bands were detected in control samples. Analysis of E. coli 5K/pP22 extracts by IEF showed an intense pectate lyase band that migrated in the upper pH limit of the gel, indicating a pI of 9·0 or higher (data not shown).



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Fig. 2. Electrophoretic analysis of pectate lyase A. (a) Coomassie-blue-stained SDS-polyacrylamide gel. (b) Zymogram of an SDS-polyacrylamide gel. Lanes: 1, cell extracts from E. coli 5K/pBR322; 2, cell extracts from E. coli 5K/pP22. The positions of molecular mass marker proteins are indicated on the left.

 
Sequencing of the encoding gene
The nucleotide sequence of the 1214 bp DNA insert contained in pP22 was determined. It contains an ORF of 666 nucleotides that encodes a protein of 222 amino acids. The deduced protein sequence shows an N-terminal region of 25 amino acids with the features of a typical Bacillus signal peptide (Nagarajan, 1993 ). It contains two positively charged residues at its N-terminal end, followed by a hydrophobic span of 18 amino acids. This hydrophobic core is followed by a ß-turn-favouring residue (Pro) and an amino acid stretch with the sequence AAAA, together resembling the sequence recognized by bacterial signal peptidase (von Heijne, 1986 ), which would cleave the bond between the two last alanine residues. Eight nucleotides upstream of the ATG start codon there is a AAGGGAGGA sequence, resembling that of a ribosome-binding site (Shine–Dalgarno sequence). A putative promoter sequence, similar to those recognized by the {sigma}A subunit of Bacillus RNA polymerase, showing a -35 (TAGACA) and -10 (TCAAAT) region was found upstream of the pelA structural gene. An inverted repeat of 20 bp, which could act as a transcriptional terminator, was found in the 3' region of the pectate lyase gene.

The deduced amino acid sequence of PelA was compared to enzyme sequences contained in the SWISS-PROT and EMBL databases. Homology was found to enzymes belonging to class III pectate lyases (Shevchik et al., 1997 ; Kim & Beer 1998 ), whilst no homology was found to pectate lyases of other groups (Fig. 3). The highest homology was found to pectate lyases from phytopathogenic fungi. PelD from Fusarium solani (Guo et al., 1996 ) showed the highest homology (43% identity), whilst Pl1 from Fusarium oxysporum (TREMBL G3764095) and PelC, PelB and PelA from F. solani (Guo et al., 1995a , b ; González-Candelas & Kolattukudy, 1992 ) showed 41, 40, 39 and 37% identity, respectively, to Bacillus sp. BP-23 PelA. The enzyme also showed homology to pectate lyases from phytopathogenic bacteria. In this regard, 33% identity was found to Pel-3 and PelB from Erwinia carotovora (Liu et al., 1994 ; Heikinheimo et al., 1995 ), and 31% identity to PelI from Erwinia chrysanthemi (Shevchik et al., 1997 ). Sequence comparison also showed that PelA is highly homologous (54% identity) to the protein deduced from the yvpA gene from Bacillus subtilis, the function of which is unknown (Kunst et al., 1997 ).



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Fig. 3. Amino acid alignment of several pectate lyases belonging to class III. Alignment was performed with the CLUSTAL W program. Numbering of the amino acids starts at the N termini of the proteins. Gaps are indicated by dashes. Asterisks show identical amino acids and dots show conserved residues. Amino acids identical in at least eight of the sequences aligned are shaded. Blocks of conserved residues of class III pectate lyases are boxed. The sequences shown are: BspPelA, PelA from Bacillus sp. BP-23; BsuYvpA, YvpA from B. subtilis, FoxyPl1, Pl1 from F. oxysporum; FsoPelA, PelA from F. solani; FsoPelB, PelB from F. solani; FsoPelC, PelC from F. solani; FsoPelD, PelD from F. solani; EcaPelB, PelB from Erwinia carotovora; EcaPel3, Pel-3 from Erwinia carotovora; EchPelI, PelI from Erwinia chrysanthemi; EamHrpW, HrpW from Erwinia amylovora; PsyHrpW, HrpW from P. syringae.

 
The amino acid sequence of the cloned enzyme contains three blocks of conserved residues proposed as signatures of pectate lyases of class III (Shevchik et al., 1997 ) (Fig. 3). However, a fourth conserved region present in the enzymes of this class is missing in Bacillus sp. BP-23 PelA. Analysis for the occurrence of protein domains with ProDom at NCBI (Altschul et al., 1997 ) indicated a two-domain structure in PelA. An N-terminal pectate lyase domain (amino acids 33–160), shared with class III pectate lyases, comprises the three conserved regions of class III enzymes contained in PelA. The second domain, placed at the C-terminal region of PelA (amino acids 161–222) is only shared with the protein deduced from the yvpA gene from B. subtilis.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have identified and characterized a pectate lyase from the strain Bacillus sp. BP-23. Sequence comparison with proteins contained in databases revealed that the enzyme characterized, PelA, showed homology to enzymes belonging to class III pectate lyases, whilst no significant homology was found to pectate lyases of other groups, including pectate lyases from saprophytic micro-organisms such as Pel from Amycolata sp. (Brühlmann & Keen, 1997 ). It has recently been shown that Hrp proteins from Erwinia amylovora (Kim & Beer 1998 ) and Pseudomonas syringae (Charkowski et al., 1998 ) contain domains homologous to pectate lyases from class III although they do not show pectate lyase activity. This indicates that class III proteins are probably distributed in a wide range of micro-organisms. Our results indicate that the cloned enzyme belongs to class III pectate lyases. PelA from Bacillus sp. BP-23 is to our knowledge the only pectate lyase in this class reported from a non-pathogenic micro-organism.

The pectate lyase characterized differs in many aspects from known pectate lyases. One of the most notable features of the cloned enzyme is its activity on both polygalacturonic acid and highly methylated pectin. Most pectate lyases, besides degrading polygalacturonic acid, retain activity on pectins of low degree of methylation but are not active on highly methylated pectins (Burns, 1991 ; Hugouvieux-Cotte-Pattat et al., 1996 ). In some instances, as for Erwinia chrysanthemi PelB and PelC, pectate lyases are most active on substrates with a low degree of methylation (7–22%) and do not show activity on highly esterified pectin (Tardy et al., 1997 ). By contrast, Bacillus sp. BP-23 PelA, shows high activity on pectins of any degree of esterification. The activity on highly methylated pectins could indicate that the cloned enzyme is a pectin lyase. However, known pectin lyases show a decreasing activity as the methoxyl content of the substrate decreases and do not show noticeable activity on polygalacturonic acid. Additionally, pectin lyases usually do not require Ca2+ for activity, whilst the cloned enzyme does not show activity in the absence of this ion. From these results we conclude that Bacillus sp. BP-23 PelA is a pectate lyase showing a remarkable activity on highly methylated pectin.

Pectate lyases belonging to class III are usually more active on pectins than on polygalacturonate. Thus, PelI from Erwinia chrysanthemi and PelB from Erwinia carotovora show maximum activity on 45 and 68% esterified pectin, respectively (Shevchik et al., 1998 ; Heikinheimo et al., 1995 ), exhibiting low or undetectable activity on polygalacturonic acid. The cloned enzyme differs from these pectate lyases in showing a similar activity on polygalacturonic acid and pectins of any degree of methylation. Pectin methyl esterification seems not to affect greatly the activity of Bacillus sp. BP-23 PelA. This makes PelA a unique enzyme among known pectate and pectin lyases.

The pectate lyases grouped in class III show optimum activity at high Ca2+ concentrations and have a higher cysteine content (10–14 residues) than pectate lyases of other groups. PelA from Bacillus sp. BP-23 also shows optimum activity at elevated CaCl2 concentrations (0·75 mM), but it contains only two cysteine residues. In a similar way, Hrp proteins, despite their homology to class III pectate lyases, have no cysteine residues.

A pectate and a pectin lyase from other members of the genus Bacillus have been cloned and characterized (Nasser et al., 1990 , 1993 ; Sakamoto et al., 1994 , 1996 ). The pectate lyase, from B. subtilis, shows molecular size, pI and enzymic properties different from those of Bacillus sp. BP-23 PelA (Nasser et al., 1990 ). Sequence comparison of Bacillus sp. BP-23 PelA with Bacillus pectin-degrading enzymes did not show significant homology. However, the sequence of the cloned enzyme shows high homology (54% identity) to the protein deduced from the yvpA gene from B. subtilis (Kunst et al., 1997 ). Recently, a pectate lyase from an alkaliphilic strain of Bacillus has been characterized (Kobayashi et al., 1999 ). The enzyme shows similar molecular mass and pI (20 kDa and 10·3, respectively) to PelA, although it exhibits little activity on pectins. The sequence of its N-terminal region (28 amino acids) and that of an internal peptide of 12 amino acids has been determined and shows 75% identity with the corresponding regions of Bacillus sp. BP-23 PelA. This high homology suggests that this enzyme also belongs to class III and is similar to Bacillus sp. BP-23 PelA, although showing different substrate specificity. The sequence of the whole pectate lyase will be necessary to analyse the degree of homology between the two enzymes.

The results shown indicate that Bacillus sp. BP-23 PelA is a novel enzyme with unusual features that make it distinct from other known pectate lyases, and suggest that the cloned enzyme, together with YvpA, belongs to a new type of pectate lyases from the genus Bacillus. The high activity of the enzyme on pectins makes it a good candidate for biotechnological applications requiring the removal of pectin from natural substrates. At present, the activity of the enzyme on textile raw materials is being tested to evaluate the potential application of PelA for retting.


   ACKNOWLEDGEMENTS
 
We thank Serveis Científico-Tècnics of the University of Barcelona for their support in nucleotide sequencing. We thank X. Fontana for technical aid. This work was partially supported by the Scientific and Technological Research Council (CICYT, Spain), grant no. QUI98-0413-CO2-02. The experiments described in this article have been performed complying with current Spanish law.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 13 July 1999; revised 1 October 1999; accepted 5 October 1999.



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