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Cloning and characterization of a sucrase from Leuconostoc mesenteroides S.M. Holt* and G.L. Cote Biopolymer Research Unit, National Center for Agricultural Utilization Research, ARS, USDA1, Peoria, IL, USA A sucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The cloned enzyme did not show dextransucrase or sucrose phosphorylase activity. HPLC and GC-MS analyses of the sucrase products indicated the presence of fructose and glucose in equimolar amounts. IPTG induction did not increase sucrase activity in E. coli indicating that the cloned gene may be transcribed from its own promoter. To our knowledge, this is the first sucrase cloned from L. mesenteroides that has invertase activity.

Introduction Leuconostocs are heterofermentative lactic acid bacteria involved in the fermentation processes of numerous vegetable, dairy, and industrial products (Holzapfel and Schillinger, 1992). Many L. mesenteroides strains can produce sucrases when grown in a medium containing sucrose as the sole carbohydrate source, however, not much is known about the molecular biology of the enzymes involved. Dextransucrase is an extracellular enzyme secreted by L. mesenteroides and is responsible for dextran synthesis. Dextran is a high molecular weight homopolymer comprised of a-linked glucose units and has many commercial applications (Cote and Alhgren, 1995). Dextransucrase liberates fructose from sucrose and transfers glucosyl residues to glucose or a nascent glucan chain. Recently, Monchois et al. (1996) cloned a novel dextransucrase from L. mesenteroides NRRL B-1299 that synthesized a dextran composed of 85% a(1–6) and 15% a(1–3) linked glucose residues. A dextransucrase gene from NRRL B-512F was cloned by Wilke-Douglas et al. (1989) that synthesized a dextran composed of 95% a(1–6) and 5% a(1–3) links. The amino acid sequence from this dextransucrase exhibited a high degree of homology with the sucrase from NRRL B-1299 and with streptococcal glucosyltransferases (Monchois et al., 1996). A sucrose phosphorylase gene was cloned from L. mesenteroides ATCC 12291 and the amino acid sequence showed 68% homology with the sucrose phosphorylase gene gtfA from Streptococcus mutans (Kitao and Nakano, 1992). Sucrose phosphorylase catalyzes the formation of glucose-1-phosphate and fructose in the presence of phosphate. Biotechnological applications

for sucrose phosphorylase include production of fructose and glucose-1-phosphate from sucrose and synthesis of novel disaccharides (Vandamme et al., 1987). Despite the research interest in sucrases, no sucrase enzyme that possesses invertase activity has been cloned from L. mesenteroides. In this study, a sucrase enzyme with invertase activity was cloned from L. mesenteroides to better understand the molecular biology of sucrose utilization by this industrially significant microorganism. Materials and methods Bacterial strains, plasmids, and media Leuconostoc mesenteroides NRRL B-21297 was recently isolated by Leathers et al. (1997). For DNA isolation, L. mesenteroides NRRL B-21297 was cultured in MRS (de Man et al., 1960) broth containing 2% (w/v) glucose as the sole carbohydrate source. E. coli XL1-blue MRF′ was the host strain for the Lambda ZAP II vector (Stratagene, Inc, Lajolla, CA) and was prepared for phage infection according to manufacturers instructions. E. coli SOLR strain was used to maintain the recombinant pBluescript phagemid following in vivo excision from the lambda ZAPII vector. E. coli SOLR harboring pSH5 was maintained on Luria-Bertani (LB, Maniatis et al. 1982) agar plates with ampicillin (50 mg/ml) and cultured in LB broth with the antibiotic. Chemicals and enzymes Tetrazolium Red was from Sigma. Restriction enzymes were from New England Biolabs (Beverly, MA) and DNA mass standards were from Stratagene.

1

Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.

1997 Chapman & Hall

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Cloning Chromosomal DNA was isolated from L. mesenteroides NRRL B-21297 by using the method of Pitcher et al. (1989). The chromosomal DNA was partially digested with EcoR I and fractionated by using 0.8% agarose gel electrophoresis. DNA fragments between 5 and 10 kb in size were sliced from the agarose gel and added to a SPIN-X centrifuge filter unit (0.22 mm, Costar, Cambridge, MA) containing 400 ml TE (10 mM TrisHCL, pH 8.0, and 1 mM EDTA) buffer. The gel was centrifuged at 16,000 × g for 10 min and the DNA in the filtrate was concentrated by using a Centricon ultrafiltration unit (30,000 molecular weight cut off, Millipore, Bedford, MA). A partial EcoR I DNA library was prepared by ligating the EcoR I precut and dephosphorylated lambda ZAP II vector arms with the EcoR I digested and fractionated genomic DNA. Recombinants were packaged into phage heads with a packaging extract (Stratagene). The phage genomic library was incubated with the XL1-Blue MRF′ E. coli cells for 15 min at 37°C in 8 ml 0.7% soft agar containing NZY medium (0.08 M NaCl, 0.02 M MgSO4, 0.5% yeast extract, and 1% casein hydrolysate) and 0.1% Tetrazolium Red. After infection, the soft agar was overlaid onto M9 medium (Maniatis et al., 1982) containing 1% sucrose to give approximately 1,500 plaques per 150-mm dish. The dishes were incubated for 4 days at 37°C. The genomic phage library was screened for sucrase activity as described by Aoki et al. (1986). Sucrase-positive clones were identified as plaques surrounded by heavy E. coli growth and red halo formation. Sucrase-positive plaques were removed from the M9 medium with a Pasteur pipette and were suspended in 0.5 ml of SM buffer (0.05 M Tris HCl, pH 7.5, 0.1 M NaCl, 0.01 M MgSO4 × 7H20, and 0.01% gelatin). The plaques were mixed by vortex action for 1 min and cultured onto M9 medium as previously described to confirm purity. The phagemid from one purified sucrasepositive plaque was subcloned into E. coli SOLR by in vivo excision from the lambda ZAPII vector with the aid of a helper phage (Stratagene). The subcloned pBluescript phagemid containing the sucrase gene from L. mesenteroides was designated pSH5. Enzyme assays Enzyme extracts were prepared from E. coli cell lysates. Cell pellets of E. coli cultures (10 ml) were harvested by centrifugation, resuspended in 1 ml of 50 mM potassium phosphate buffer, pH 6.8, and disrupted by using ultrasonication. The cell debris was removed by centrifugation and the supernatant was used as the enzyme source. A typical assay mixture for reducing sugar detec-

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tion and polymer formation consisted of 0.1 M sucrose in 480 ml 50 mM potassium phosphate buffer, pH 6.8, and 20 ml enzyme extract. Assays were incubated for 1 h at 37°C. Sucrase activity was measured by using the copper-bicinchoninate reducing sugar test (Waffenschmidt and Jaenicke, 1987). Polymer formation was determined by visual inspection of digests and by HPLC analysis. Sucrose phosphorylase activity was assayed by the method of Silverstein et al. (1963). A commercial preparation of sucrose phosphorylase (Sigma) was used as a positive control for the assay. For HPLC detection of sucrase products, the enzyme assay consisted of 0.05 M sucrose in 300 ml enzyme extract and 200 ml 50 mM phosphate buffer. HPLC and GC-MS analyses HPLC separation of fructose, glucose, and sucrose was carried out by using a 4.6 mm × 250 mm amino column with a 5 mm particle size (Supelco, Supelcosil LC-NH2). Compounds were eluted with acetonitrile/water (85:15 v/v) at 1.0 ml min–1. For detection of the glucose-1phosphate standard, the column was eluted with acetonitrile/water (60:40, v/v). HPLC detection of polymer formation was performed by using a gel permeation column (8 mm × 300 mm, Shodex Ohpak KB806M) eluted with water at 0.5 ml min–1. Detection of HPLC eluted products was by refractive index. Samples to be analyzed by GC-MS chromatography were freeze dried and acetylated according Seymour et al. (1975), except that hydroxylamine hydrochloride was not used. GC-MS detection of acetylated derivatives of fructose, glucose and sucrose was performed with a HewlettPackard 5970B mass selective detector operating at 70 eV and using a methylsilicone column (25 m × 0.022 i.d. × 0.1 mm thickness; Hewlett-Packard, Wilmington, DE). The column temperature was held at 160°C for 3 min then increased at 5°C per min to 185°C. Restriction enzyme mapping To determine the size of the cloned DNA fragment from L. mesenteroides, CsCl purified pSH5 was digested with EcoR I, Not I, Sac I, and Xho I in a number of enzyme combinations and examined by 0.8% agarose gel electrophoresis. To determine the restriction map of the insert, single, double and triple digests were performed with EcoR I, EcoR V, Hind III, Sac I, Sal I, and Xho I in a number of enzyme combinations. The DNA banding profiles were examined by 0.8% agarose gel electrophoresis and the restriction map was deduced from the patterns. All DNA manipulations were performed according to the methods of Maniatis et al. (1982), unless otherwise indicated.

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Sequencing The partial nucleotide sequence for the cloned DNA fragment from L. mesenteroides was determined from the five prime and three prime ends by the dideoxy-chain termination method of Sanger et al. (1980), using T3/T7 promotor primers and a dye-terminator kit (Perkin Elmer, ABI Prism, Foster City, CA). Labeled nucleotides were detected with an ABI Prism 377 DNA sequencer (Perkin Elmer). Results and discussion Cloning A partial EcoR I gene library was prepared from L. mesenteroides B-21297 genomic DNA by using the insertion vector lZAP II. Fourteen of 140,000 plaques (0.01%) that were screened on M9-sucrose medium displayed sucrase activity. All fourteen of the sucrase-positive plaques showed the same reaction on the M9-sucrose medium with the NZY-0.1% Tetrazolium Red overlay. E. coli XL1-blue cells normally cannot grow on sucrose. Sucrases released from the recombinant plaques, however, split sucrose into products that E. coli can use for growth such as glucose and fructose. The sucrase products initiate heavy E. coli growth and Tetrazolium Red reduction (red halo formation) surrounding the recombinant plaques on the M9-sucrose medium. Incorporation of Tetrazolium Red into the NZY soft agar overlay enhanced the detection of sucrase-positive plaques. Figure 1 shows the typical reaction of a purified sucrase-positive plaque when cultured on M9sucrose medium and overlaid with NZY top agar containing 0.1% Tetrazolium Red. After purification of the sucrase-positive plaque, the recombinant pBluescript phagemid was excised from the lambda ZAPII vector and subcloned into E. coli SOLR strain. The subcloned plasmid harboring the sucrase gene was designated pSH5 and was further characterized for sucrase expression and restriction mapping. Enzyme assays The sonicated cell lysates from E. coli (pSH5) showed sucrase activity when assayed on sucrose as determined by a reducing sugar test. Sonicated cell lysates from the control strains E. coli SOLR harboring pBluescript vector without an insert and from E. coli SOLR without plasmid did not exhibit sucrase activity. CsCl purified pSH5 was electrotransformed into E. coli SOLR and cell lysates were sucrase-positive. The E. coli (pSH5) enzyme lysate did not exhibit polymer-forming (dextransucrase) activity and also did not show sucrose phophorylase activity. Analysis of the sucrase products by using HPLC indicated the presence of fructose and glucose in equimolar proportions (data not shown). GC-MS analysis

Figure 1 Reaction of sucrase-positive recombinant plaques on an M9-sucrose plate with an NZY soft agar overlay containing 0.1% Tetrazolium Red. The plate was incubated for two days at 37°C. The plaques were isolated from a L. mesenteroides DNA library.

of the sucrase products also confirmed the presence of fructose and glucose (data not shown). Based on the enzyme assay data, the cloned sucrase from L. mesenteroides has invertase activity. Due to interfering enzyme activities (a-glucosidase) in the native E. coli cell lysate, further characterization of the cloned enzyme was hampered. Purification of the cloned enzyme is therefore necessary to distinguish it as a b-fructosidase or an a-D-glucosidase. The influence of the inducer IPTG on sucrase activity from E. coli (pSH5) cell lysates was examined since the lacZ promotor in pSH5 might drive expression of cloned genes. IPTG induction, however, did not alter the sucrase activity from E. coli (pSH5) cell lysates indicating that the cloned enzyme might be transcribed from its own promoter. Restriction mapping Restriction endonuclease analysis of CsCl purified pSH5 indicated that the subcloned plasmid was about 9.0 kb in size and the cloned DNA fragment was about 6.0 kb in size (Fig. 2, lanes 6 and 8). The host pBluescript vector was about 3.0 kb in size (Stratagene). Although the gene library was constructed with EcoR I, subsequent digestion of pSH5 with the endonuclease did not release the insert (Fig. 2, lane 3). Restriction endonuclease analysis showed that only one EcoR I site near the Sac I site was valid (Fig. 3). Nucleotide sequence data Biotechnology Letters · Vol 19 · No 9 · 1997

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still intact. Star activity usually results from digestion under extreme nonstandard enzyme conditions (Polisky et al., 1975), however, standard enzyme conditions were used. The reason for the star activity is unknown but it is interesting to speculate that the sucrase gene from L. mesenteroides may not have been cloned without it. Single, double and triple enzyme digests were used to generate the insert restriction map shown in figure 3. There are two EcoR V sites, one Hind III site, and one Sal I site within the 6.0 kb cloned fragment. The insert was also digested by Hinc II, Pvu II, and Sau 3A1 but the patterns were too complex to accurately assess indicating multiple recognition sites for each enzyme. The insert DNA was not digested by numerous other restriction endonucleases commonly used.

Figure 2 Restriction endonuclease analysis of pSH5. After enzymatic digestion of plasmids, the DNA patterns were examined by using 0.8% agarose gel electrophoresis. Lane 1, standards; lane 2, pBluescript-EcoR I digest; lanes 3–8, pSH5 digests. Lane 3, EcoR I; 4, Sac I; 5, Xho I; 6, Sac IXho I; 7, EcoR I-Sac I; and 8, EcoR I-Xho I.

Many L. mesenteroides strains can produce sucrases, however, few of the enzymes have been cloned and characterized. Most of the sucrases that have been characterized from L. mesenteroides strains were dextransucrases (Monchois et al., 1996; Wilke-Douglas et al., 1989) or a sucrose phosphorylase (Kitao and Nakano, 1992). To our knowledge, this is the first report of a sucrase with invertase activity that was cloned from L. mesenteroides and will contribute to our knowledge of sucrose utilization by this industrially significant microorganism. Acknowledgements We thank Drs. Jim Nicholson, Timothy Leathers, Robert Anderson, Jeff Ahlgren and Ms. Kathy PayneWahl for their contributions. References

Figure 3 Restriction map of the cloned DNA fragment from L. mesenteroides that contains the sucrase gene. E, EcoR I; E*, invalid EcoR I; H, Hind III; K, Kpn I, S, Sal I; SA, Sac I; V, EcoR V; and X, Xho I.

confirmed that the EcoR I site near the Xho I site did not contain the complete EcoR I recognition site. The Sac I and Xho I recognition sites originated from the multiple cloning site of the host vector pBluescript. The Sac I and Xho I sites bound the EcoR I site used to generate the clone (Fig. 3). The usual EcoR I recognition site is 5′-GAATTC-3′, however, sequence data for the invalid EcoR I site indicated a sequence containing 5′-TAATTC-3′. The inactivated EcoR I sequence probably resulted from star activity during digestion of the L. mesenteroides DNA. The resulting DNA products could still be ligated to the lambda vector arms since the central tetranucleotide sequence (5′-AATT-3′) was

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Aoki, H., Shiroza, T., Hayakawa, M., Sato, S., and Kuramitsu, H. K. (1986). Infect. Immun. 53:587–594. Cote, G. L., and Alhgren, J. A. (1995). Microbial polysaccharides. In: Kirk-Othmer Encyclopedia of Chemical Technology, 4th Ed. Vol. 16. pp578–611, John Wiley & Sons, Inc. deMan, J. C., Rogosa, M., and Sharpe, M. (1960). J. Appl. Bacteriol. 23:130–135. Holzapfel, W. H., and Schillinger, U. (1992). The genus Leuconostoc. In: The Prokaryotes, A. Balows, H. Truper, M. Dworkin, W. Harder, and K-H. Schleifer, eds 2nd Ed. pp1500–1535, NY, Springer-Verlag. Kitao, S., and Nakano, E. (1992). J. Ferm. Bioeng. 73:179–184. Leathers, T. D., Ahlgren, J. A., and Cote, G. L. (1997). J. Ind. Microbiol. Biotechnol. 18:278–283. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular cloning. A Laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Monchois, V., Willemot, R-M., Remaud-Simeon, M., Croux, C., and Monsan, P. (1996). Gene. 182:23–32. Pitcher, D. G., Saunders, N. A., and Owen, R. J. (1989). Lett. Appl. Microbiol. 8:151–156. Polisky, B., Greene, P., Garfin, D. E., McCarthy, B. J., Goodman, H. M., and Boyer, H. W. (1975). Proc. Nat. Acad. Sci. USA 72:3310–3314.

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Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., and Roe, B.A. (1980). J. Mol. Biol. 143:161–178. Seymour, F. R., Plattner, R. D., and Slodki, M. E. (1975). Carbohydr. Res. 44:181–198. Silverstein, R., Voet, J. Reed, D., and Abeles, H. (1963). J. Biol. Chem. 242:1338–1346. Vandamme, E. J., Van Loo, J., and DeLaporte, A. (1987).

Biotechnol. Bioeng. 29:8–15. Waffenschmidt, S., and Jaenicke, L. (1987). Anal. Biochem. 165:337–340. Wilke-Douglas, M., Perchorowicz, J. T., Houck, C. M., and Thomas, B. R. (1989). Methods and compositions for altering physical characteristics of fruit and fruit products. PCT Patent WO 89/12386.

Received 20 June 1997; Revisions requested 26 June 1997; Revisions received 15 July 1997; Accepted 15 July 1997

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