INTRODUCTION

Top Abstract Introduction Materials and methods Results and discussion References

Amylosucrase is a glucosyltransferase (EC 2.4.1.4) which catalyzes the transfer of the D-glucopyranosyl unit from sucrose onto a glucan primer and synthesizes an insoluble -glucan, without using -D-glucosylnucleoside-di-P as substrate (19-21, 28, 29, 42), unlike glycogen synthase. Indeed, the bacterial synthesis of glycogen is frequently based on the transfer of glucose from adenosine-5'-di-P-glucose (ADPG) onto an -glucan chain, the ADPG itself being formed by ADPG synthetase (ADPG--D-glucose-1-P-adenyltransferase) from -D-glucose-1-P (32).

Amylosucrase was first discovered in cultures of Neisseria perflava (7). In 1974, Neisseria polysaccharea was isolated from the throats of healthy children in Europe and Africa (34). This nonpathogenic strain was shown to possess an extracellular amylosucrase that uses sucrose to produce a linear polymer composed of -(14)glucopyranosyl residues having strong similarities with amylose (5, 33, 35). More recently, Büttcher et al. (5) cloned and sequenced the gene coding for amylosucrase from N. polysaccharea. They report that the deduced amino acid sequence of the enzyme has homology with the -amylase class of enzymes. However, regions known to be essential for the catalytic activity of -amylases were scarcely found in the amylosucrase sequence reported.

This study describes the subcloning and sequencing of the gene encoding amylosucrase from N. polysaccharea isolated by Remaud-Simeon et al. (33). The deduced amino acid sequence differs significantly from that published by Büttcher et al. (5). This allowed us to compare it more specifically with enzymes from the -amylase family. The recombinant enzyme was then overexpressed by Escherichia coli, purified, and characterized.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results and discussion References

Bacterial strains. N. polysaccharea ATCC 43768 was used as the source of chromosomal DNA. E. coli C-600 was used as the cloning host for direct expression screening of the gene library. E. coli DH1 was used as the host for the pUC19 and ptrc99a vectors; strain BL21 was used as the host for the expression plasmid pGEX-6-P-3.

Vectors. Phage EMBL3A was used to construct the genomic library. pUC19 (New England Biolabs) and ptrc99a (Amersham Pharmacia Biotech) were used for subcloning and overexpression, respectively. pGEX-6-P-3 (Amersham Pharmacia Biotech) was used for expression of amylosucrase fused to glutathione S-transferase (GST-AS).

Cloning of amylosucrase. The gene library was constructed by partially digesting DNA from N. polysaccharea with Sau3A and cloning the fragments into BamHI sites of EMBL3A as previously described (33). The recombinant E. coli C-600 bacteria in YT medium soft agar were plated on top of M9 medium containing nutritional supplements (MgSO₄, CaCl₂, thiamine, threonine, and leucine) and 0.7% sucrose. After 6 h of growth at 37°C, plaques appeared in the soft agar (37). When sucrase activity was expressed, the enzyme catalyzed the release of fructose and/or glucose which were metabolized by the bacteria and stimulated growth around the plaque, forming a halo. As sucrose cannot be utilized by E. coli C-600, the halo appeared only around the plaque where sucrase activity was expressed. Recombinant phage DNA purification was carried out as described by Sambrook et al. (37). A 15-kb fragment was purified (33) and digested with KpnI and HindIII to isolate a 6-kb fragment that was inserted between the KpnI and HindIII sites of pUC19. The plasmid obtained was named AS/pUC19. The fragment was shown to encode amylosucrase. Activity of the subclone was detected as described below.

Sequencing. The recombinant plasmid AS/pUC19 was automatically sequenced over the entire length of the insert by Genome Express (Grenoble, France), using the method of Sanger et al. (38). The sequencing reaction was performed by PCR amplification in a final volume of 20 µl, using 100 ng of PCR products, 5 pmol of primer, and 9.5 µl of BigDyeTerminators premix, in accordance with the Applied Biosystems protocol. After heating of the mixture to 94°C for 2 min, the reaction was cycled as follows: 25 cycles of 30 s at 94°C, 30 s at 55°C, and 4 min at 60°C (Perkin-Elmer 9600 thermal cycler). Removal of excess of BigDyeTerminators was performed by using Quick Spin columns (Boehringer Mannheim). The samples were dried in a vacuum centrifuge and dissolved with 2 µl of deionized formamide-EDTA (pH 8.0) (5/1). The samples were loaded onto an Applied Biosystems 373A sequencer and run for 12 h on a 4.5% denaturing acrylamide gel. Each reaction was repeated three times to verify the sequence obtained.

Enzyme extraction methods. Cultured recombinant bacteria were centrifuged (8,000 × g, 10 min, 4°C), and the culture supernatant was stored at 4°C for assay. Depending on the intended use the bacterial pellet underwent one of two treatments.

(i) Osmotic shock. To extract enzyme secreted into the periplasmic space, the pellet was washed with 0.01 M Tris-HCl-0.03 M NaCl (pH 7.3) and then centrifuged (5,000 × g, 5 min, 4°C). It was resuspended, concentrated to 1/20 the initial culture volume with hypertonic solution (0.03 M Tris, 20% [wt/vol] methyl--D-glucopyranoside, 1 mM EDTA [pH 7.3]), gently mixed for 10 min at room temperature, and then centrifuged (5,000 × g, 5 min, 4°C); cells were resuspended and concentrated to 1/20 the initial culture volume with hypotonic solution (water). Then the pellet was centrifuged again (7,500 × g, 10 min, 4°C), and the supernatant was stored at 4°C.

(ii) Sonication. To extract the intracellular enzyme, the bacterial pellet was resuspended and concentrated at an optical density at 600 nm of 80 in 50 mM maleate buffer (pH 6.4) or, for strain BL21, in phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ [pH 7.3]). After sonication, 1% (vol/vol) Triton X-100 was added to the extract. After 30 min at 4°C and centrifugation (10,000 × g, 10 min, 4°C), the supernatant was stored at 4°C.

Amylosucrase assay. The amylosucrase assay was carried out at 30°C in 50 mM maleate buffer (pH 6.4) supplemented with sucrose (50 g/liter) and glycogen (0.1 g/liter) (G-8751; Sigma Chemical Co.) as a primer. Activities of the GST-AS amylosucrase fusion protein and the purification fractions were measured at 30°C in sample buffer (pH 7.0) in the presence of sucrose (50 g/liter) and glycogen (0.1 g/liter).

Overexpression of amylosucrase. For high-level expression, the gene encoding amylosucrase was first amplified by PCR and cloned into the expression vector ptrc99a, to obtain plasmid AS/ptrc99a. PCR was carried out with DNA from recombinant EMBL3A with a 15-kb insert fragment as the matrix. The PCR primer oligonucleotides were synthesized by Isoprim (Toulouse, France).

Purification of amylosucrase. Affinity chromatography between the GST-AS fusion protein and the glutathione-Sepharose 4B support was performed to purify the amylosucrase.

(i) Cloning of the GST-AS fusion protein. To clone GST-AS, we used the expression vector pGEX-6-P-3, which includes the GST protein just downstream of the strong promoter P_tac and upstream of the cloning site. Cloning was carried out between the EcoRI and NotI restriction sites. The amylosucrase gene was amplified by PCR from AS/pUC19, using the direct primer 5'-CAGCAAGTCGGTTTGAATTCACAGTACCTCAAAACACGC-3' and reverse primer 5'-TCCGGTTCGGCGCAGCGGCCGCCTGAAACGGTTCAGA-3', including the EcoRI and NotI restriction sites, respectively. These primers are localized between bases 151 and 189 and bases 2067 and 2103, respectively, of the amylosucrase gene nucleotide sequence. PCR was carried out in the presence of 10% (vol/vol) dimethyl sulfoxide so as to avoid nonspecific hybridization of the primers and thus the amplification of interfering fragments. The PCR fragment was cloned into the EcoRI and NotI sites of the vector pGEX-6-P-3. This cloning results in a mutated amylosucrase in positions 11 and 12 (IN and LS), with deletions of amino acids 1 to 10, corresponding to the first part of the putative signal sequence described by Büttcher et al. (5). The plasmid obtained was used to transform a protease-negative strain E. coli BL21.

(ii) Purification. Purification was carried out at 4°C except during the elution step. A 2-ml column of glutathione-Sepharose 4B (Amersham Pharmacia Biotech) was equilibrated with 30 ml of PBS and then with 6 ml of PBS-1% (vol/vol) Triton X-100. A volume of 1.5 ml of enzyme extract from the sonication supernatant (corresponding to 12.3 mg of protein and 12.2 U of amylosucrase activity) was injected into the column, which was then washed with 70 ml of PBS. The fusion protein was eluted with 2 ml of elution buffer (20 mM reduced glutathione-G-4251 [Sigma], 100 mM Tris-HCl [pH 8.0], 120 mM NaCl) after 10 min of contact at room temperature. The procedure was carried out three times. The column was regenerated to remove residual noneluted fusion proteins by using 30 ml of PBS-3 M NaCl and then 10 ml of 70% (vol/vol) ethanol and was then equilibrated with 30 ml of PreScission buffer (50 mM Tris-HCl [pH 7.0], 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol). The fraction containing the eluted fusion protein was dialyzed against PreScission buffer at 4°C overnight. The fusion protein was then subjected to proteolysis at 4°C for 4 h by adding 2 U of PreScission protease (Amersham Pharmacia Biotech) per 100 µg of fusion protein. The mixture was then injected into the glutathione-Sepharose 4B column, the purified amylosucrase being eluted in the PreScission buffer. The column was eluted and regenerated as described above and then equilibrated and stored in PBS at 4°C.

Electrophoresis. Electrophoresis was carried out with the PHAST system (Amersham Pharmacia Biotech). Under denaturing conditions, the sample was diluted with denaturing buffer (10 mM Tris-HCl, 1 mM EDTA, 2.5% [wt/vol] sodium dodecyl sulfate [SDS], 5% [wt/vol] -mercaptoethanol, 0.01% [wt/vol] bromophenol blue) and heated to 90°C for 1 min before being placed on a polyacrylamide PhastGel gradient 8-25 (Amersham Pharmacia Biotech). The buffer system in PhastGel SDS buffer strips was 0.2 M tricine (trailing ion)-0.2 M Tris-0.55% SDS (pH 8.1).

Production and characterization of the polymer synthesized. The synthesis reaction was performed at 30°C for 24 h in PreScission buffer, supplemented with sucrose (50 g/liter) and oyster glycogen (1 g/liter) with 19.5 mg of pure amylosucrase per liter. The reaction was stopped by adding 60% (vol/vol) dimethyl sulfoxide (which solubilized the water-insoluble polymer) and heating to 100°C for 1 h. The polymer was then precipitated with ethanol at 4°C and dried by solvent exchange (ethanol-acetone).

(i) -Amylolysis and deramification experiments. Glucans (0.8%) were solubilized in dimethyl sulfoxide. After dilution in 50 mM morpholinepropanesulfonic acid (MOPS) buffer (pH 7.0) to a final concentration of 2 mg/ml, 10 µl of -amylase from Bacillus sp. (2000 U/mg; Megazyme) was added, and the reaction was run for 3 h at 40°C. The determination of reducing ends was performed as described by Nelson (27).

(ii) Sample preparation for high-performance size-exclusion chromatography (HPSEC)-MALLS. To prepare -glucan solutions in 0.1 M KOH, amylose powder was dispersed in 1 M KOH, gently stirred overnight at 4°C, and then diluted to 0.1 M KOH. For water solutions, -glucan powder was dispersed by boiling for 5 to 30 min, until the aqueous buffer used as the eluent was clarified. -Glucan solutions were then cooled to room temperature.

(iii) HPSEC. The HPSEC system was used as described previously (3). Dual detection of solutes was carried out with a detector Dawn DSP-F MALLS (Wyatt Technology Corporation, Santa Barbara, Calif.) and a differential refractive index detector (Erma ERC-7510) in series.

(iv) Data analysis. For each sampling time of the elution pattern corresponding to one elution volume (V_i), a concentration (c_i) is calculated from the differential refractive index response. Following this determination, measurement of the light scattered by the 15 Dawn-F photodiodes allows the determination of molecular weight (M_i) and radius of gyration (R_i = <r_g² >i^1/2) of -glucans by ASTRA version 1.4, based on the equation where K is the optical constant, R_q is the excess Rayleigh ratio of the solute,  is the wavelength (632.8 nm) of the incident laser beam, and c_i is calculated from the differential refractive index response (dn/dc being the refractive index increment with a value of 0.146 ml/g for -glucans) (30).

Nucleotide sequence accession number. The amylosucrase gene sequence has been submitted to the EMBL nucleotide sequence database and assigned accession no. AJ011781.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and methods Results and discussion References

Sequencing of the N. polysaccharea gene coding for amylosucrase. The sonicated cell extracts of E. coli DH1 clones transformed by plasmid AS/pUC19 tested positive for amylosucrase activity in the amylosucrase assay. Plasmid AS/pUC19 was sequenced on the full length of the 6-kb insert. An ORF search in the six possible reading frames revealed the presence of seven ORFs of 500 nucleotides (nt) or more. The deduced amino acid sequences from the seven ORFs were subjected to protein sequence comparison using Pfam. Only one of the seven ORFs belongs to a known enzyme family; it corresponds to the amylosucrase gene. This ORF consists of 1,911 nucleotides nt encoding a protein of 636 amino acids. Also, no ORF of more than 500 bases was present in the same reading frame as that of the gene encoding amylosucrase. No protein capable of interfering in the enzymatic reaction catalyzed by amylosucrase is present near it. Therefore, the amylosucrase from N. polysaccharea is not expressed via a multigenic system.

Sequence comparison of amylosucrase with related enzymes. A characteristic (/)₈-barrel fold was predicted in the protein structure by using Pfam. In addition, the eight-stranded / barrel is interrupted by the presence of a separate folding module (70 amino acids) homologous to a calcium-binding domain, protruding between  strand 3 and helix 3 (17). This domain is included in the part of the sequence that could correspond to domain B of amylolytic enzymes (G-184 to F-298 [amylosucrase numbering]) (11). The protein may also contain a C-terminal  barrel (17). These results suggest that amylosucrase is a member of the -amylase family.

View larger version (47K): [in this window] [in a new window]   FIG. 1.   Conserved sequence stretches (I to VIII) in amylosucrase, in the -amylase superfamily (A), and in glucosyltransferases (B) (12). The second line denotes the elements of secondary structure, as determined for pig pancreatic -amylase. Enzymes are numbered from the N-terminal end; invariable residues are in boldface. (A) AS, amylosucrase (N. polysaccharea); AMY, -amylase (pig pancreas); OGL, oligo-1,6-glucosidase (Bacillus cereus); AGL, -glucosidase (Saccharomyces cerevisiae); PUL, pullulanase (B. stearothermophilus); APU, amylopullulanase (Clostridium thermohydrosulfuricum); CMD, cyclomaltodextrinase (B. sphaericus); MTH, maltotetraohydrolase (Pseudomonas saccharophila); ISA, isoamylase (P. amyloderamosa); DGL, dextran-glucosidase (Streptococcus mutans); MHH, maltohexaohydrolase (Bacillus sp. strain 707); NPU, neopullulanase (B. stearothermophilus); BRE, branching enzyme (E. coli); CGT, cyclodextrin-glycosyltransferase (B. circulans); GDE, glycogen-debranching enzyme (human muscle); TAK, -amylase (A. oryzae). (B) AS, amylosucrase (N. polysaccharea); DSRB (Leuconostoc mesenteroides NRRL B-1299); DSRA (L. mesenteroides NRRL B-1299); GTFD (S. mutans GS5); GTFK (S. salivarius ATCC 25975); GTFS (S. downei Mfe 28); GFTI (S. sobrinus OMZ176 serotype D); GTFC (S. mutans GS5); GTFB (S. mutans GS5); DSRS (L. mesenteroides NRRL B-512F).

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Evolutionary tree of amylolytic enzymes, glucosyltransferases, and amylosucrase, based on stretches I, II, IV, V, VII, and VIII. Abbreviations are given in the legend to Fig. 1; branch lengths are proportional to the sequence divergency. A similar tree can be found in reference 12.

Overexpression of the amylosucrase gene. As the amylosucrase gene was previously shown to contain a putative signal peptide sequence (5), the recombinant enzyme was tested for extracellular, periplasmic, or intracellular activity. E. coli DH1 transformation with plasmid AS/ptrc99a and induction by 2.5 mM isopropyl--D-thiogalactopyranoside, allowed determination of the initial activity in the enzyme extract: 30 U/liter of culture after osmotic shock, 3 U/liter of culture in the culture supernatant, and 658 U/liter of culture with sonication; 95% of the enzyme is intracellular, which confirms that E. coli cannot recognize or process the previously described putative signal peptide (5).

Purification of amylosucrase obtained from the GST-AS fusion protein. The yields obtained at various steps of the purification process and specific activities are presented in Table 1. The glutathione-eluted sample consists of pure fusion protein (see Fig. 3A). The purification process enabled a pure amylosucrase fraction to be obtained, as shown by the electrophoresis revealed with silver nitrate (Fig. 3A), with a specific activity of 9,565 U/g. The total yield of the purification was 38%; this value would have been increased to 43% if the fusion proteins contained in the washing solution of the first affinity chromatography had been recycled. The low yield is due mainly to the 50% loss in protein during the proteolytic cleavage step; a white precipitate that seemed to represent partial aggregation of proteins appeared during the incubation. The total purification factor was 9.6. 

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Purification of amylosucrase. (A) Expression of GST-AS fusion protein in pGEX-6-P-3 and purification of amylosucrase by using glutathione-Sepharose 4B. Electrophoresis was in denaturing conditions. Lanes: 1, low-molecular-weight electrophoresis calibration kit (Amersham Pharmacia Biotech); 2, sonicate supernatant of E. coli BL21 cells transformed with GST-AS expression plasmid pGEX-6-P-3 (142 ng); 3, eluate following purification of sonicate on glutathione-Sepharose 4B and elution with elution buffer (purified GST-AS) (117 ng); 4, flowthrough following PreScission protease digestion of GST-AS (purified amylosucrase) (111 ng); 5, low-molecular-weight electrophoresis calibration kit (Amersham Pharmacia Biotech). (B) Electrophoresis in native conditions. Lanes: 1, high-molecular-weight electrophoresis calibration kit (Amersham Pharmacia Biotech); 2, purified amylosucrase (148 ng); 3, low-molecular-weight electrophoresis calibration kit (Amersham Pharmacia Biotech). (C) Isoelectric focusing. Lanes: 1, isoelectric focusing calibration kit (Pharmacia); 2, purified amylosucrase (98 ng).

Characterization of the glucan synthesized by pure amylosucrase. Amylosucrase catalyzes the synthesis of -(14)-glucan from sucrose without any primer (5). Nevertheless, the polymer-synthesizing rate has been shown to be greatly increased in the presence of a glucan primer, and particularly of glycogen (33). To determine the role of the primer in the activated reaction, the polymer synthesized by pure amylosucrase from sucrose, with glycogen as a primer, was analyzed.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   HPSEC profiles of starches from native and modified glycogens.

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Ri versus Mi for amylose and for native and modified glycogens.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Three main hypotheses for the enzymatic action of amylosucrase on glycogen.