Biotechnology Letters, Vol 19, No 9, September 1997, pp. 903-907 7826 · Cloning and characterization of a sucrase from Leuconostoc mesenteroides S.M. Holt* and G.L. Cote

1 Biopolymer Research Unit, National Center for Agricultural Utilization Research, ARS, USDA , 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 for sucrose phosphorylase include production of fructose Leuconostocs are heterofermentative lactic acid bacteria and glucose-I-phosphate from sucrose and synthesis of involved in the fermentation processes of numerous novel disaccharides (Vandamme et aI., 1987). Despite the vegetable, dairy, and industrial products (Holzapfel and research interest in sucrases, no sucrase enzyme that Schillinger, 1992). Many L. mesenteroides strains can possesses invertase activity has been cloned from L. mesen­ produce sucrases when grown in a medium containing teroides. In this study, a sucrase enzyme with invertase sucrose as the sole carbohydrate source, however, not activity was cloned from L. mesenteroides to better under­ much is known about the molecular biology of the stand the molecular biology of sucrose utilization by this enzymes involved. Dextransucrase is an extracellular industrially significant microorganism. 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 Materials and methods has many commercial applications (Cote and Alhgren, Bacterial strains, plasmids, and media 1995). Dextransucrase liberates fructose from sucrose and Leuconostoc mesenteroides NRRL B-21297 was recently transfers glucosyl residues to glucose or a nascent glucan isolated by Leathers et al. (1997). For DNA isolation, chain. Recently, Monchois et al. (1996) cloned a novel L. mesenteroides NRRL B-21297 was cultured in MRS dextransucrase from L. mesenteroides NRRL B-1299 that (de Man et aI., 1960) broth containing 2% (w/v) glucose synthesized a dextran composed of85% a(1-6) and 15% as the sole carbohydrate source. E. coli XLI-blue MRF' aO-3) linked glucose residues. A dextransucrase gene was the host strain for the Lambda ZAP II vector from NRRL B-512F was cloned by Wilke-Douglas et al. (Stratagene, Inc, Lajolla, CA) and was prepared for phage (989) that synthesized a dextran composed of 95 % infection according to manufacturers instructions. E. coli aO-6) and 5% a(1-3) links. The amino acid sequence SOLR strain was used to maintain the recombinant from this dextransucrase exhibited a high degree of pBluescript phagemid following in vivo excision from homology with the sucrase from NRRL B-1299 and with the lambda ZAPII vector. E. coli SOLR harboring pSH5 streptococcal glucosyltransferases (Monchois et aI., was maintained on Luria-Bertani (LB, Maniatis et al. 1996). A sucrose phosphorylase gene was cloned from 1982) agar plates with ampicillin (50 J.Lg/ml) and L. mesenteroides ATCC 12291 and the amino acid cultured in LB broth with the antibiotic. sequence showed 68% homology with the sucrose phosphorylase gene gtfA from Streptococcus mutans (Kitao Chemicals and enzymes and Nakano, 1992). Sucrose phosphorylase catalyzes the Tetrazolium Red was from Sigma. Restriction enzymes formation of glucose-I-phosphate and fructose in were from New England Biolabs (Beverly, MA) and the presence of phosphate. Biotechnological applications DNA mass standards were from Stratagene.

ames 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 Supplied by U.S. Dept. of Agriculture Biotechnology Letters· Vol 19 . No 9 . 1997 903 National Center for Agricultural Utilization Research, Peoria, Illinois S.M. Holt and C.L. Cote

Cloning tion and polymer formation consisted of 0.1 M sucrose Chromosomal DNA was isolated from L. mesenteroides in 480 IJ.J 50 mM potassium phosphate buffer, pH 6.8, NRRL B-21297 by using the method of Pitcher et at. and 20 j.LI enzyme extract. Assays were incubated for (1989). The chromosomal DNA was partially digested 1 h at 37°e. Sucrase activity was measured by using the with EcoR I and fractionated by using 0.8% agarose gel copper-bicinchoninate reducing sugar test (Waffen­ electrophoresis. DNA fragments between 5 and 10 kb schmidt and Jaenicke, 1987). Polymer formation was in size were sliced from the agarose gel and added to a determined by visual inspection of digests and by HPLC SPIN-X centrifuge filter unit (0.22 /-Lm, Costar, analysis. Sucrose phosphorylase activity was assayed by Cambridge, MA) containing 400 IJ.J TE (10 mM Tris­ the method of Silverstein et at. (1963). A commercial HCL, pH 8.0, and 1 mM EDTA) buffer. The gel was preparation of sucrose phosphorylase (Sigma) was used centrifuged at 16,000 X g for 10 min and the DNA in as a positive control for the assay. For HPLC detection the filtrate was concentrated by using a Centricon ultra­ of sucrase products, the enzyme assay consisted of 0.05 filtration unit (30,000 molecular weight cut off, M sucrose in 300 /-LI enzyme extract and 200 j.LI 50 mM Millipore, Bedford, MA). A partial EcoR I DNA library phosphate buffer. was prepared by ligating the EcoR I precut and dephos­ phorylated lambda ZAP II vector arms with the EcoR I digested and fractionated genomic DNA. Recombinants HPLC and GC-MS analyses were packaged into phage heads with a packaging HPLC separation of fructose, glucose, and sucrose was extract (Stratagene). The phage genomic library was carried out by using a 4.6 mm X 250 mm amino column incubated with the XL1-Blue MRF' E. coli cells for 15 with a 5 j.Lm particle size (Supelco, Supelcosil LC-NH2). min at 37°C in 8 ml 0.7% soft agar containing NZY Compounds were eluted with acetonitrile/water (85: 15 v/v) at 1.0 ml min-to For detection of the glucose-1­ medium (0.08 M NaCI, 0.02 M MgS04, 0.5% yeast extract, and 1% casein hydrolysate) and 0.1 % phosphate standard, the column was eluted with acetoni­ Tetrazolium Red. After infection, the soft agar was over­ trile/water (60:40, v/v). HPLC detection of polymer laid onto M9 medium (Maniatis et at., 1982) containing formation was performed by using a gel permeation 1% sucrose to give approximately 1,500 plaques per column (8 mm X 300 mm, Shodex Ohpak KB806M) 1 150-mm dish. The dishes were incubated for 4 days at eluted wi th water at 0.5 ml min- . Detection of HPLC 37°e. The genomic phage library was screened for eluted products was by refractive index. Samples to sucrase activity as described by Aoki et at. (1986). be analyzed by GC-MS chromatography were freeze Sucrase-positive clones were identified as plaques dried and acetylated according Seymour et at. (1975), surrounded by heavy E. coli growth and red halo forma­ except that hydroxylamine hydrochloride was not used. tion. Sucrase-positive plaques were removed from the GC-MS detection of acetylated derivatives of fructose, M9 medium with a Pasteur pipette and were suspended glucose and sucrose was performed with a Hewlett­ in 0.5 ml of SM buffer (0.05 M Tris HCI, pH 7.5, 0.1 Packard 5970B mass selective detector operating at 70 eV and using a methylsilicone column (25 m X 0.022 M NaCI, 0.01 M MgS04 X 7H20, and 0.01 % gelatin). The plaques were mixed by vortex action for 1 min and i.d. X 0.1 /-Lm thickness; Hewlett-Packard, Wilmington, cultured onto M9 medium as previously described to DE). The column temperature was held at 160°C for confirm purity. The phagemid from one purified sucrase­ 3 min then increased at 5°C per min to 1850e. positive plaque was subcloned into E. coli SOLR by in vivo excision from the lambda ZAPII vector with the Restriction enzyme mapping aid of a helper phage (Stratagene). The subcloned To determine the size of the cloned DNA fragment from pBluescript phagemid containing the sucrase gene from L. mesenteroides, CsCI purified pSH5 was digested with L. mesenteroides was designated pSH5. EcoR I, Not I, Sac I, and Xho I in a number of enzyme combinations and examined by 0.8% agarose gel elec­ trophoresis. To determine the restriction map of the Enzyme assays insert, single, double and triple digests were performed Enzyme extracts were prepared from E. coli cell lysates. with EcoR I, EcoR V, Hind III, Sac I, Sal I, and Xho I Cell pellets of E. coli cultures (10 ml) were harvested by in a number of enzyme combinations. The DNA centrifugation, resuspended in 1 ml of 50 mM potas­ banding profiles were examined by 0.8% agarose gel sium phosphate buffer, pH 6.8, and disrupted by using electrophoresis and the restriction map was deduced ultrasonication. The cell debris was removed by centrifu­ from the patterns. All DNA manipulations were gation and the supernatant was used as the enzyme performed according to the methods of Maniatis et at. source. A typical assay mixture for reducing sugar detec- (1982), unless otherwise indicated.

904 Biotechnology Letters· Vol 19 . No 9 . 1997 Cloning and characterization ofa sucrase from Leuconostoc mesenteroides

Sequencing The partial nucleotide sequence for the cloned DA fragment from L. mesenteroides was determined from the five prime and three prime ends by the dideoxy-chain termination method of Sanger et at. (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. mesen­ teroides B-21297 genomic DA by using the insertion vector ",ZAP 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, Figure Reaction of sucrase-positive recombinant plaques on an M9-sucrose plate with an NZY soft agar however, split sucrose into products that E. coli can use overlay containing 0.1 % Tetrazolium Red. The plate was for growth such as glucose and fructose. The sucrase incubated for two days at 37°C. The plaques were isolated products initiate heavy E. coli growth and Tetrazolium from a L. mesenteroides DNA library. Red reduction (red halo formation) surrounding the recombinant plaques on the M9-sucrose medium. Incorporation of Tetrazolium Red into the ZY soft of the sucrase products also confirmed the presence of agar overlay enhanced the detection of sucrase-positive fructose and glucose (data not shown). Based on the plaques. Figure 1 shows the typical reaction of a puri­ enzyme assay data, the cloned sucrase from L. mesen­ fied sucrase-positive plaque when cultured on M9­ teroides has invertase activity. Due to interfering enzyme sucrose medium and overlaid with ZY top agar activities (a-glucosidase) in the native E. coli cell lysate, containing 0.1 % Tetrazolium Red. After purification of further characterization of the cloned enzyme was the sucrase-positive plaque, the recombinant pBluescript hampered. Purification of the cloned enzyme is there­ phagemid was excised from the lambda ZAPII vector fore necessary to distinguish it as a ~-fructosidase or an and subcloned into E. coli SOLR strain. The subcloned a-D-glucosidase. The influence of the inducer IPTG on plasmid harboring the sucrase gene was designated sucrase activity from E. coli (pSHS) celilysates was exam­ pSHS and was further characterized for sucrase expres­ ined since the lacZ promotor in pSHS might drive sion and restriction mapping. expression of cloned genes. IPTG induction, however, did not alter the sucrase activity from E. coli (pSHS) cell Enzyme assays lysates indicating that the cloned enzyme might be tran­ The sonicated cell lysates from E. coli (pSHS) showed scribed from its own promoter. sucrase activity when assayed on sucrose as determined by a reducing sugar test. Sonicated cell lysates from the Restriction mapping control strains E. coli SOLR harboring pBluescript vector Restriction endonuclease analysis of CsCI purified pSHS without an insert and from E. coli SOLR without indicated that the subcloned plasmid was about 9.0 kb plasmid did not exhibit sucrase activity. CsCI purified in size and the cloned DA fragment was about 6.0 kb pSHS was electrotransformed into E. coli SOLR and cell in size (Fig. 2, lanes 6 and 8). The host pBluescript lysates were sucrase-positive. The E. coli (pSHS) enzyme vector was about 3.0 kb in size (Stratagene). Although lysate did not exhibit polymer-forming (dextransucrase) the gene library was constructed with EcoR I, subse­ activity and also did not show sucrose phophorylase quent digestion of pSHS with the endonuclease did not activity. Analysis of the sucrase products by using HPLC release the insert (Fig. 2, lane 3). Restriction endonu­ indicated the presence of fructose and glucose in clease analysis showed that only one EcoR I site near the equimolar proportions (data not shown). GC-MS analysis Sac I site was valid (Fig. 3). Nucleotide sequence data

Biotechnology Letters· Vol 19 . No 9 . 1997 905 S.M. Holt and C.L. Cote

12345678 still intact. Star activity usually results from digestion under extreme nonstandard enzyme conditions (Polisky et aI., 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 ~ Insert 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. ~ Vector Many L. mesenteroides strains can produce sucrases, however, few of the enzymes have been cloned and char­ acterized. Most of the sucrases that have been charac­ terized from L. mesenteroides strains were dextransucrases Figure 2 Restriction endonuclease analysis of pSH5. After (Monchois et aI., 1996; Wilke-Douglas et aI., 1989) or enzymatic digestion of plasmids, the DNA patterns were a sucrose phosphorylase (Kitao and Nakano, 1992). To examined by using 0.8% agarose gel electrophoresis. Lane 1, standards; lane 2, pBluescript-EcoR I digest; lanes 3-8, our knowledge, this is the first report of a sucrase with pSH5 digests. Lane 3, EcoR I; 4, Sac I; 5, Xho I; 6, Sac 1­ invertase activity that was cloned from L. mesenteroides Xho I; 7, EcoR I-Sac I; and 8, EcoR I-Xho I. and will contribute to our knowledge of sucrose utiliza­ tion by this industrially significant microorganism.

Acknowledgements E S v H v X We thank Drs. Jim Nicholson, Timothy Leathers, SA K Robert Anderson, Jeff Ahlgren and Ms. Kathy Payne­ Wahl for their contributions. 1.0 kb E* L---J References Aoki, H., Shiroza, T., Hayakawa, M., Sato, S., and Kuramitsu, Figure 3 Restriction map of the cloned DNA fragment from H. K. (1986). Infect. Immun. 53:587-594. L. mesenteroides that contains the sucrase gene. E, EcoR Cote, G. 1., and Alhgren, ). A. (1995). Microbial polysaccha­ I; E*, invalid EcoR I; H, Hind III; K, Kpn I, S, SaIl; SA, Sac rides. In: Kirk-Othmer Encyclopedia of Chemical Technology, I; V, EcoR V; and X, Xho I. 4th Ed. Vol. 16. pp578-611, John Wiley & Sons, Inc. deMan, ). c., Rogosa, M., and Sharpe, M. (1960). J. App!. Bacteriol. 23:130-135. Holzapfel, W. H., and Schillinger, U. (1992). The genus Leuconostoc. In: The Prokaryotes, A. Balows, H. Truper, M. confirmed that the EcoR I site near the Xho I site did Dworkin, W. Harder, and K-H. Schleifer, eds 2nd Ed. not contain the complete EcoR I recognition site. The pp1500-1535, Y, Springer-Verlag. Sac I and Xho I recognition sites originated from the Kitao, S., and Nakano, E. (1992). J. Ferm. Bioeng. 73:179-184. multiple cloning site of the host vector pBluescript. The Leathers, T. D., Ahlgren, ). A., and Cote, G. 1. (1997). J. Ind. Microbiol. Biotechnol. 18:278-283. Sac I and Xho I sites bound the EcoR I site used to Maniatis, T., Fritsch, E. F., and Sambrook, ). (1982). Molecular generate the clone (Fig. 3). The usual EcoR I recogni­ cloning. A Laboratory manual. Cold Spring Harbor tion site is 5'-GAATTC-3', however, sequence data for Laboratory, Cold Spring Harbor, NY. the invalid EcoR I site indicated a sequence containing Monchois, V., Willemot, R-M., Remaud-Simeon, M., Croux, c., 5'-TAATTC-3'. The inactivated EcoR I sequence prob­ and Monsan, P. (1996). Gene. 182:23-32. Pitcher, D. G., Saunders, N. A., and Owen, R. ). (1989). Lett. ably resulted from star activity during digestion of the App!. Microbiol. 8:151-156. L. mesenteroides DNA. The resulting DNA products Polisky, B., Greene, P., Garfin, D. E., McCarthy, B.)., Goodman, could still be ligated to the lambda vector arms since H. M., and Boyer, H. W. (1975). Proc. Nat. Acad. Sci. USA the central tetranucleotide sequence (5'-AATT-3') was 72:3310-3314.

906 Biotechnology Letters· Vol 19 . No 9 . 1997 Cloning and characterization ofa sucrase from Leuconostoc mesenteroides

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

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