Enzyme and Microbial Technology 39 (2006) 612–620

Molecular cloning of a gene encoding the sucrose phosphorylasefrom Leuconostoc mesenteroides B-1149 and the

expression in Escherichia coli

Jin-Ha Lee a,∗, Seung-Heon Yoon b, Seung-Hee Nam c, Young-Hwan Moon d,You-Youn Moon e, Doman Kim a,f,∗

a Engineering Research Institute, Chonnam National University, Gwang-Ju 500-757, South Koreab Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA

c Laboratory of Functional Carbohydrate Enzymes and Microbial Genomics, Chonnam National University, Gwang-Ju 500-757, South Koread Department of Material and Chemical and Biochemical Engineering, Chonnam National University, Gwang-Ju 500-757, South Korea

e Department of Chemical Engineering, Iowa State University, Ames, IA 50011, USAf School of Biological Sciences and Technology, Research Institute for Catalysis and Institute of Bioindustrial Technology,

Chonnam National University, 300 Young-bong dong, Buk-Gu, Gwang-Ju 500-757, South Korea

Received 10 April 2005; accepted 11 November 2005

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Leuconostoc mesenteroides NRRL B-1149 sucrose phosphorylase (SPase) gene, 1149sp, was isolated and characterized. It is composed of 1479 bpucleotides and encodes a 1149SPase of 492 amino acid residues with a calculated molecular mass of 56.1 kDa. It has unique C-terminal amino acidequence (439DVETPSDTTIKITRKDKSGENVAVLVANAADKTFTITANGEEILANTEADKQQL492). 1149sp was expressed in Escherichia colind the purified 1149SPase specific activity was 1.49 U/mg for sucrose. The optimum temperature and pH for SPase activities were ranged broadetween 20 and 50 ◦C, between pH 6.0 and 7.5, respectively. The optimum temperature and pH were 37 ◦C at pH 6.7 and it showed Km of 6.3 mMnd kcat of 1.59 s−1 for sucrose. It had a broad range of acceptor specificity and transferred the glucosyl moiety of sucrose or glucose-1-phosphateo various acceptors.

2005 Elsevier Inc. All rights reserved.

eywords: Leuconostoc mesenteroides; Sucrose phosphorylase; Expression; Acceptor reaction

. Introduction

Sucrose phosphorylase (EC 2.4.1.7) (SPase) belongs to fam-ly 13 of the glycosyl hydrolases [1]. It catalyzes the syn-hesis of �-d-glucose-1-phosphate (Glc-1-P) and d-fructoserom sucrose and inorganic phosphate [2]. The presence ofPase was first reported from Leuconostoc mesenteroides ATCC2291 [3] and other SPases were also studied from vari-us bacteria, such as Pseudomonas saccharophila [4,5], Pseu-omonas puterfaciens [6], L. mesenteroides [7,8], Streptococ-us mutans [9] and Bifidobacterium adolescentis DSM2008310]. Corresponding SPase genes were cloned from L. mesen-

∗ Corresponding author. Tel.: +82 62 530 1844; fax: +82 62 530 1849.E-mail addresses: tre98@korea.com (J.-H. Lee), dmkim@chonnama.ac.kr

D. Kim).

teroides ATCC 12291 and No. 165 [11,12], S. mutans [13],Agrobacterium vitis [14] and B. adolescentis DSM20083[10].

SPase can be used for the determination of inorganic phos-phate in clinical analysis [15], for the specific and rapid deter-mination of sucrose concentrations in sugar transport by plants[16], for the flow-injection method to determine sucrose concen-tration without interference from glucose or fructose in sucroseelectrode [17,18] and for a one-pot enzymatic galactosyltrans-ferase reaction in which SPase scavenged for the accumulatedinorganic phosphate and utilized it with sucrose to generate insitu glucose-1-phosphate [19].

L. mesenteroides SPase was reported to be producedconstitutively [20] and it transferred the glucosyl moiety ofGlc-1-P to various sugars, sugar alcohols [21,22], phenolicand alcoholic compounds [23–27] to synthesize novel structureacceptor products [28]. Acceptor products by SPase also

141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2005.11.008

J.-H. Lee et al. / Enzyme and Microbial Technology 39 (2006) 612–620 613

have various potential applications as anticarigenic mate-rials, probiotics, antibiotic substitutes and food ingredients[22,24–27]. Recently, we reported the optimum conditions forsimultaneous production of glucosyltranferase (GTase) andfructosyltranferase (FTase) in L. mesenteroides NRRL B-1149by changing substrate concentrations using response surfacemethodology based on the Box–Behnken design [29]. Duringpurification of GTase and FTase in B-1149 culture supernatant,L. mesenteroides SPase was also purified. The 1149SPaseshowed a molecular size of 56 kDa on SDS-PAGE and theoptimum pH and temperature of L. mesenteroides NRRLB-1149 SPase were 6.2–6.5 and 37 ◦C, respectively. It had anapparent Km of 6.0 mM and kcat of 1.62 s−1 for sucrose [29].

L. mesenteroides SPase has been studied in detail [30],although based on the information, the gene encoding sucrosephosphorylase in L. mesenteroides B-1149 (1149sp) could notbe obtained. Since different kinds of glucansucarases are pro-duced by different strains or species of Leuconostoc, it wouldbe different SPases in different Leuconostoc stains or species.

In this paper, we report the purification of sucrose phos-phorylase, isolation and characterization of corresponding gene(1149sp) from L. mesenteroides NRRL B-1149. There is aunique nucleotide sequence at the C-terminal ends of 1149SPaseand lower similarity compared to other Leuconostoc SPasegenes from L. mesenteroides No. 165 and L. mesenteroidesATCC 12291 [12]. We also constructed a sucrose phosphorylase-ootfhcp

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mleMCt[csb6TwtpDai(fif

(Bio-Rad, Hercules, USA) [34]. The protein band was excised and its aminoacid sequence was analyzed by Edman degradation method using an AppliedBiosystems model 476A Protein/Peptide Sequencer (Applied Biosystems Inc.,CA).

2.2. DNA isolation and manipulation

Genomic DNA from L. mesenteroides B-1149 was prepared as previouslydescribed [35], and routine DNA manipulations, including plasmid purificationand Escherichia coli transformation, were performed with the methods describedby Maniatis et al. [36]. Plasmid DNA was isolated using the alkaline lysis method[36]. Extraction of chromosomal DNA and plasmid DNA from agarose gels wereperformed with an AccuPrep® Gel Purification kit (Bioneer Co., Dae-Jeon,Korea), as suggested by the manufacturer. Restriction endonuclease, alkalinephosphatase and T4 DNA ligase were purchased from Boehringer Mannheim(Mannheim, Germany), DCC-Bionet (Seong-nam, Korea) and Takara (Shiga,Japan), respectively. Competent E. coli DH5� cells, prepared by the procedureof Cohen et al., were transformed with plasmid DNA using the CaCl2 method[37].

2.3. PCR cloning of the 1149sp gene

A pair of PCR primers for amplification of an internal sequence of1149sp gene were designed based on the N-terminal amino acid sequence,NH2 MEIQNKAM COOH (determined in this study) and the multiple align-ment of amino acid sequences conserved in other bacterial SPases (Gen-Bank accession numbers AF065394, AAKO04742, AB029313, AJ401152 andAJ303085): SP1F, 5′-ATGGAAATTCAAAACAAAGCAA-3′ and SP1R, 5′-TAGTAGATTTGTGGAATACC-3′. The first PCR was performed using chro-mosomal DNA from L. mesenteroides B-1149 as a template. The PCR mixturecde(faaGbcgSButibc5sa

2

AstldcwiTiCt

verproducing strain using a pRSETA vector. The informationf SPase genes from various characteristic SPases is usefulo analyze the molecular diversity among SPases, especiallyrom various L. mesenteroides, and to construct new SPaseaving unique transglucosylation characteristics to synthesizeompounds that have designed structure and physico-chemicalroperties.

. Materials and methods

.1. Purification and determination of N-terminal amino acidequence of purified 1149SPase

L. mesenteroides NRRL B-1149 was prepared by culturing in glucoseedium as described before [31,32]. It was grown at 28 ◦C for 48 h in a 20-jar containing 18 l of LWG medium of 2% (w/v) glucose, 0.5% (w/v) yeastxtract and peptone, 2% (w/v) K2HPO4 and 1% (v/v) mineral solution (2%gSO4·7H2O, 0.1% NaCl, 0.1% FeSO4·7H2O, 0.1% MnSO4·H2O, 0.13%aCl2·2H2O) [31,32]. 1149SPase was purified by using the membrane filtra-

ion (30 K cut-off), DEAE-Toyopearl 650M and following DEAE-Sepharose33]. After removing cells by centrifugation, the culture supernatant was con-entrated using membrane filtration (30 K cut-off). The concentrated enzymeolution (672 mg) was dialyzed overnight against 8 l of 20 mM sodium phosphateuffer (pH 6.5). The dialyzate (672 mg) was loaded onto a DEAE-Toyopearl50M column (2.5 cm × 35 cm) that was equilibrated with the same buffer.he column was washed with 500 ml of this buffer and the adsorbed proteinas eluted with a linear gradient of NaCl (0–0.5 M) in the buffer. The frac-

ions exhibiting SPase activity were pooled and dialyzed against a sodiumhosphate buffer (pH 6.5). The resulting dialyzate (315 mg) was applied to aEAE-Sepharose column (2.5 cm × 35 cm) equilibrated with the same buffer

nd proteins were eluted with a linear NaCl gradient up to 1 M. The fractions hav-ng SPase activity were pooled and the re-applied to a DEAE-Sepharose column2.5 cm × 35 cm) and the protein was eluted again as described above. The puri-ed 1149SPase was electrophoretically transferred from a sodium dodecyl sul-ate (SDS)-polyacrylamide gel to a polyvinylidene difluoride (PVDF) membrane

ontained 10 mM Tris–HCl (pH 8.5), 50 mM KCl, 3 mM MgCl2, 2 mM each ofeoxyribonucleotide triphosphate (dNTP), 0.25 mg template DNA and 10 pmolach primers. After incubation for 5 min at 94 ◦C, 1 �l Taq DNA polymeraseTakara, Japan) was added and followed by 25 cycles of denaturation at 94 ◦Cor 30 s, annealing at 57 ◦C for 30 s and elongation at 72 ◦C for 90 s. Themplified fragment was ligated into pGEM-T Easy vector (Promega, USA)nd DNA sequence was analyzed by Korea Basic Science Institute (KBSI,wang-Ju) in Korea. Further 5′ and 3′ region of the 1149sp were amplifiedy the thermal asymmetric interlaced (TAIL) PCR method [38]. The spe-ific sense and antisense primers to amplify the 5′ and 3′ region of 1149spene were designed as follows: SP2F, 5′-GGATATCTACCAAATCAAC-3′ andP2R, 5′-TAATCAGAAGGTGCAAAACC-3′. Self-ligated L. mesenteroides-1149 chromosomal DNA after partial digestion with BamHI and PstI wassed as a template. The PCR reaction was performed as described above andhe amplified fragment was then inserted into pGEM-T Easy vector and thensert DNA was further sequenced. The whole 1149sp gene was then amplifiedy PCR using L. mesenteroides B-1149 chromosomal DNA and two oligonu-leotide primers (SP1F, 5′-ATGGAAATTCAAAACAAAGCAA-3′ and SP3R,′-TTACAATTGTTGCTTATCAGC-3′) obtained from the above nucleotideequence results. The amplified gene was then inserted into pGEM-T Easy vectornd transformed into E. coli DH5�.

.4. Overexpression of 1149sp and purification of 1149SPase

To express 1149sp, the sense primer SP-EF (5′-ACAGGATCCAT-ACTATGGAAATTCAAA-3′: the BamHI site is underlined) and the anti-

ense primer SP-ER (5′-TTTAAGAATTCATATATTTAAAATTACAATTG-3′:he KpnI site is underlined) were constructed. The PCR fragment was thenigated into the corresponding sites of pRSETA vector (Invitrogen, USA) pre-igested with BamHI and KpnI. The ligated vector was then transformed into E.oli BL21(DE3)pLysS (Invitrogen). E. coli BL21(DE3)pLysS carrying p1149spas grown in 50 ml LB liquid medium containing 50 �g/ml ampicillin and 1 mM

sopropyl-�-d-thiogalactopyranoside (Duchefa, Netherlands) at 28 ◦C for 6 h.he cells were harvested by centrifugation (15,000 × g, 10 min, 4 ◦C), suspended

n 200 �l of phosphate buffer (50 mM, pH 6.0) and disrupted by sonication.ell debris was removed by centrifugation (15,000 × g, 10 min, 4 ◦C). The N-

erminal, 6-histidyl tagged protein was purified by nickel-nitrilotriacetic acid

614 J.-H. Lee et al. / Enzyme and Microbial Technology 39 (2006) 612–620

(Ni-NTA) affinity chromatography (6xHis tag; Qiagen, Germany). The molecu-lar weight of recombinant 1149SPase was determined, using the Laemmli system(10% acrylamide gel) [39] and proteins were stained with Coomassie BrilliantBlue R250 [40].

2.5. Enzyme activity assay

To prepare the crude enzyme extracts for measurement of SPase activity,E. coli pellets (5 ml culture) were resuspended in 100 �l of 50 mM potassiumphosphate, pH 6.8, and disrupted by ultrasonication and centrifuged (15,000 × g,10 min, 4 ◦C). A sucrose phosphorylase activity was assayed by the methodof Silverstein et al. [5,7]. The glucose-1-phosphate from sucrose and Pi weredetermined by coupling the reduction of NADP+ in the presence of phos-phoglucomutase (Sigma-P3397, USA) and glucose-6-phosphate dehydrogenase(Sigma-G5760, USA). The standard assay medium contained 50 mM potas-sium phosphate buffer (pH 6.8), 140 mM sucrose, 0.09 mM EDTA-2Na, 15 mMMgCl2, 0.36 mM of NADP+ (Sigma-N0505, USA), 0.0015 mM of glucose-1,6-bisphosphate (Sigma-G7137, USA), 20 U of phosphoglucomutase, 20 U ofglucose-6-phosphate dehydrogenase and the 1149SPase (20 �l) in a final vol-ume of 3.3 ml. Increase in absorbance of NADPH at 340 nm was measured at25 ◦C. One unit of sucrose phosphorylase activity was defined as the amount ofenzyme that caused the reduction of 1 �mol of NADP+ per minute under theabove assay conditions [11,30]. An extinction coefficient of 6.22 × 103 M/cmwas used for the calculation of enzyme activity. Protein concentration was mea-sured according to the method of Bradford [40], with bovine serum albumin(Sigma) as a standard.

2.6. Thermostability

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tc6m

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plates was analyzed with a NIH image program by using the standard materials[42–44].

3. Results

3.1. Analysis of N-terminal amino acid sequence

The 1149SPase was purified with a specific activityof 2.18 U/mg [29]. The N-terminal amino acid sequenceof 1149SP was MEIQNKAM. The amino acid sequencewas applied to a database homology search using BLAST(http://www.ncbi.nlm.nih.gov). The 1149SP of N-terminalamino acid sequence was same as SPase of L. mesenteroidesNo. 165 and ATCC 12291 [11,12]. We designed a degeneratedprimer, SP1F, based on this amino acid sequence.

3.2. Cloning

The PCR amplification of 1149SPase by using a sense primer(SP1F) and an antisense primer (SP1R) resulted a fragment of1100 bp. By TAIL-PCR method [45], 300 and 550 bp fragmentswere amplified from 5′ and 3′ region of 1149sp gene, respec-tively. Then, the whole 1149sp gene, 1500 bp, was amplified byPCR using chromosomal DNA and two oligonucleotide primersof the 5′ and 3′ terminal sequences. The recombinant plasmidcontaining a 4.7 kb DNA insert was designated p1149sp.

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1aEP(s[l[

For the thermostability determination, the purified 1149SPase (1.88 mg/ml)as incubated at different temperatures between 20 and 70 ◦C for 30 min in0 mM potassium phosphate buffer (pH 6.8) and the residual activity was mea-ured as described above. Relative activity is expressed as a percentage of theaximum activity left under the experimental conditions.

.7. pH stability

To measure the pH stability, the enzyme (1.88 mg/ml) was incubated at roomemperature for 30 min in different buffers. The residual activity was then mea-ured. The buffer solutions used were 20 mM citric acid–Na2HPO4 buffer (pH–7), 20 mM Na2HPO4–NaH2PO4 buffer (pH 7–8) and 20 mM glycine–NaOHuffer (pH 8–10).

.8. Reaction kinetics

The kinetic constants, Km and kcat, were determined from measurements ofhe initial reaction rates, using Lineweaver–Burk plots [41]. The reaction mixtureontaining sucrose (2.3–37.4 mM) in 50 mM potassium phosphate buffer (pH.8) was incubated with 1149SPase (1.88 mg/ml) at 25 ◦C for the designatedinutes. Then reaction was stopped by heating.

.9. Transglucosylation reaction using various acceptors

The transglucosylation reaction was performed by incubation of the purified149SPase (1.88 U/mg), sucrose [2% (w/v)] and various acceptors [monosac-harides (glucose, fructose and galactose), disaccharides (maltose, isomaltose,actose, cellobiose, turanose and gentiobiose), trisaccharides (panose, raffinose,altotriose and isomaltotriose), sugar alcohols (palatinose, sorbitol, xylitol and

rythritol), acarbose, rhamnose, arabinose, xylose, salicin and phosphate, 5%w/v)] in potassium phosphate buffer (50 mM, pH 6.8) for 30 min at 37 ◦C. TheLC plate used was a Whatman K5F plate (Whatman Inc., New Jersey) with two

o three ascents of 2:5:1.5 (v/v/v) nitromethane/1-propanol/water. The carbohy-rates were visualized by dipping the plates into 5% (v/v) H2SO4 in methanolontaining 0.3% (w/v) N-1-naphthylethylenediamine, followed by drying andeating for 10 min at 121 ◦C [42]. The quantity of each carbohydrate on the TLC

.3. Nucleotide sequence and deduced amino acidequence of 1149sp

Based on computer analysis one major open reading framef 1479 bp, which encodes a polypeptide of 492 amino acidesidues (Fig. 1) was identified. The sequence has been sub-itted to GenBank under accession number (AY795566). Theolecular mass of the 492 amino acid protein was corresponded

o 56.1 kDa.The deduced amino acid sequence of 1149sp was compared

ith other SPases gene sequences in GenBank using the Clustalprogram [46]. 1149SPase shared some degrees of iden-

ity with reported SPases: 89% with L. mesenteroides ATCC2291 SPase (GenBank accession number D90314), 66% with. mutans UA159 SPase (AE014929), 66% with Streptococcusneumoniae TIGR4 SPase (AE007480) and 66% with Lacto-occus acidophilus SPase (AY172020) (Fig. 2).

.4. Purification of 1149SPase

In order to characterize the enzymatic properties of149SPase, His-tagged 1149SPase was purified using Ni-NTAffinity column chromatography. The purified 1149SPase from. coli lysate showed more than 95% homogeneity on SDS-AGE as a band of 60 kDa (including His tags of 4 kDa)Fig. 3). In the respect of molecular mass, it was of similarize to the SPases of L. mesenteroides ATCC 12291 (55 kDa)7], S. mutans (55.7 kDa) [13] and A. vitis (53.9 kDa) [14], butarger than the SPase of Clostridium pasteurianum (36.5 kDa)47].

J.-H. Lee et al. / Enzyme and Microbial Technology 39 (2006) 612–620 615

Fig. 1. Nucleotide and deduced amino acid sequences of 1149sp. The N-terminal amino acid sequence of the purified Leuconostoc mesenteroides NRRL B-1149is boxed. Conserved residues with other bacterial sucrose phosphorylases of L. mesenteroides SPase (D90314), S. pneumoniae TIGR4 SPase (AE007480) and L.acidophilus SPase (AY172020) and primers are printed in boldface and underlined, respectively: double underlines are SPlF and SP1R primer sites, single underlinesare SP2F and SP2R primer sites.

3.5. Enzymatic properties of 1149SPase

The enzymatic characterizations, including the optimum pH,optimum temperature, thermostability and pH stability werestudied. The expressed and purified 1149SPase showed similaractivity behaviors for temperature and pH with other Leuconos-toc spp. SPase [8]. The optimum temperature and pH for SPaseactivities were ranged broad between 20 and 50 ◦C, betweenpH 6.0 and 7.5, respectively. The optimum pH and temperatureof the native 1149SPase were 6.2–6.5 and 37 ◦C, respectively[29]. The thermo-stability of 1149SPase showed at near up to40 ◦C and up to 50 ◦C, the enzyme was keeping over 70% oforiginal enzyme activity after 30 min. And, pH stability showedbetween pH 6.0 and 7.5 (Fig. 4). And the native and recom-

binant SPase showed similarity enzyme properties. 1149SPasedisplayed classical Michaelis–Menten kinetics. The Km and kcatfor sucrose were 6.3 mM and 1.59 s−1, respectively (Fig. 5). Kmvalue is smaller than the reported recombinant SPase originatedfrom Leuconostoc spp. (Kikkoman, Japan) having Km value of39 mM for sucrose.

The 1149SPase produced various acceptor reaction productsusing sucrose as glucosyl donors (Table 1). It had a broad rangeof acceptor specificity and transferred the glucosyl moiety ofsucrose or glucose-1-phosphate to various acceptors such asgalactose, maltose, glucose, palatinose, sorbitol or beta linkagecompounds such as cellobiose and gentiobiose with the accep-tor reaction efficiency of 67.2, 44.9, 34.9, 24.1, 23.4, 13.6 and5.6%, respectively.

616 J.-H. Lee et al. / Enzyme and Microbial Technology 39 (2006) 612–620

Fig. 2. Amino acid sequence alignment of SPases. SP, S. pneumoniae TIGR4 SPase (AE007480); SM, S. mutans UA159 SPase (AE014929); LA, L. acidophilusSPase (AY172020); 1149SP, L. mesenteroides B-1149 SPase (AY795566); LM, L. mesenteroides SPase (D90314). Sequences were aligned using Clustal W [46].The fully conserved residues, conservation of strong group and conservation of weak groups are marked as star, semicolon and colon, respectively. The catalyticamino acid residues are bolded.

4. Discussion

Based on the amino acid sequence similarity, 1149SPase isbelonging to glucoside hydrolases family 13 having a (�/�)8-barrel comprising the catalytic domain, namely domain A [1].Recently, Sprogøe et al. reported the first crystal structure ofSPase from B. adolescentis, and they suggested that the catalyticactive-site residues (Asp192 and Glu232) of B. adolescentis

SPase are located at the tips of �-sheets 4 and 5 in the (�/�)8-barrel [48]. 1149SPase also has the potential catalytic active siteresidues (Asp196 and Glu237) similar to the sites of B. adoles-centis SPase.

Interestingly, the C-terminal sequence of 1149sp gene wasvery different from other bacteria SPases. The nucleotidesequence of the sucrose phosphorylase of L. mesenteroides No.165 gene showed 96.3% similarity to that of L. mesenteroides

J.-H. Lee et al. / Enzyme and Microbial Technology 39 (2006) 612–620 617

Fig. 3. SDS-PAGE and molecular weight determination of purified 1149SPase.The following molecular weight markers (Bio-Rad) were used: myosin(200 kDa), �-galactosidase (116.2 kDa), phosphorylase b (97.4 kDa), serumalbumin (66.2 kDa) and ovalbumin (45 kDa).

Table 1Transglucosylation products of 1149SPase

Acceptor Yielda (%)

Galactose 67.2Maltose 44.9d-Glucose 34.9Palatinose 24.1Sorbitol 23.4Salicin 19.5Xylitol 18.4d-Arabinose 18.0Maltotriose 17.1Isomaltotriose 16.6Lactose 14.1Cellobiose 13.6Erythritol 13.4Xylose 13.3Acarbose 13.0d-Fructose 12.3Rhamnose 11.5Panose 9.2Turanose 7.7Melizitose 7.6Phosphate 7.4�-Gentiobiose 5.6

a The yield of acceptor reaction was calculated based on the previous reportedmethod [44].

ATCC 12291 [12], however, L. mesenteroides B-1149 sucrosephosphorylase (SPase) gene (1149sp), showed only 89% identitywith that of L. mesenteroides ATCC 12291 (GenBank numberD90314) [11] and L. mesenteroides No. 165 [12]. In addition,the region of C-terminal from 448 Ile to 492 Leu (45 aminoacids) showed only 48.6% identity and two more amino acidswere existed. Using 1149sp nucleotide sequence from this study,we tried to amplify other SPase genes from various L. mesen-teroides that synthesize different structure dextrans or glucans.However, no PCR product was obtained (data not shown) and itis probably because of unique C-terminal nucleotide sequencesamong different L. mesenteroides. There are several report forglucansucrases from those strains and found there differencesare from 33 to 99%, and the difference gave significant dif-ferent polymer synthesizing differences [35,49–51]. We foundmost L. mesenteroides synthesize sucrose phosphorylase andalso found their gene sequences are different among variousstrains making different structure dextrans or glucans. Elevenpercent difference in total nucleotide sequences and only 48%identity of amino acids in C-terminal end may differentiatevarious Leuconostoc sp. making different structure glucans. L.mesenteroides strains have different genes and produce glucanshaving different structures [49–52]. Thus, the information of var-ious SPase genes are useful to analyze the molecular diversityamong SPases, especially from various L. mesenteroides, and toconstruct new SPase having unique transglucosylation charac-tp

woctSlmidpafistpsnauuag1mAsra

eristics to synthesize compounds having designed structure andhysico-chemical properties.

The reaction kinetics of SPase was found to be consistentith a double displacement mechanism involving the formationf a glucose–enzyme complex and a subsequent reaction of thisomplex with acceptor to form the reaction products [5]. Accep-or reaction specificity was shown based on the source of SPase.Pase of P. saccharophila transferred glucosyl unit of Glc-1-P to-arabinose, d-fructose, or l-sorbose as acceptors having a com-on hydroxyl group at a cis position of glucosidic bond [53], and

t did not form the acceptor product from d-glucose, d-galactose,-xylose, d-mannose, d-ribose and l-fucose [31]. However, P.utrifaciens SPase was not able to use l-sorbose and d-xyluloses acceptors [6], although the hydroxyl group had the right con-guration. Sucrose phosphorylases from L. mesenteroides alsohowed broad acceptor specificities independent on the posi-ion of the hydroxyl group. Transglycosylation products fromentitols, d- and l-arabitol, d-fructose and xylitol were synthe-ized using Glc-1-P as glucosyl donor [22] and kojibiose andigerose were formed by using Glc-1-P and sucrose as a donornd an acceptor, respectively [23]. Recently, d-arabinose wassed to synthesize �-glcp(1 → 1)�-araf transglucosylation prod-ct by B. adolescentis SPase [10]. Recombinant SPase from B.dolescentis also showed that the cis configuration of hydroxylroup to the glucosidic bond in acceptor is not essential. The149SPase shows different acceptor specificity compared to L.esenteroides ATCC 12291 SPase [22]. Unlike L. mesenteroidesTCC 12291 SPase, galactose, maltose, glucose, palatinose andorbitol were good acceptors for 1149Spase transglucosylationeaction: the acceptor reaction yields were 67.2, 44.9, 34.9, 24.1nd 23.4% of total transglucosylation reaction products, respec-

618 J.-H. Lee et al. / Enzyme and Microbial Technology 39 (2006) 612–620

Fig. 4. Biological properties of 1149SPase: (a) optimum temperature, (b) optimum pH, (c) thermostability and (d) pH stability.

tively. Beta linkage compounds, cellobiose (13.6%) and gen-tiobiose (5.6%), were also effective acceptors, and xylitol anderythritol were also used to make transglucosylated products.Thus, different transglycosylation yield and acceptor efficiencyare possibly caused by the different protein structure. Thus, theamino acid sequences of C-terminal end in L. mesenteroidesSPase possibly have unique role for its own properties and therole of the 3′ end in 1149SPase is progress.

Sugar alcohols and their derivatives are potential antiplaqueagent ingredients and substitutes for other sweeteners in food[54]. Glucosylated maltooligosaccharides, fructooligosaccha-rides and �-linkages of carbohydrates can give resistant tomammalian digestive enzyme hydrolysis, resistances to disac-charidases (sucrase, maltase, isomaltase or lactase) of intestinalmucosa and to �-amylase of pancreatic homogenates [55]. The

glucosylated acarbose can also be used as novel inhibitors of �-amylases and �-glucosidases, and as pre-drugs working in thespecific intestinal targets, respectively. The modifying and con-trolling �-amylase action have medical applications, such as theinfluence in blood glucose, serum insulin and starch loading testsin animals and man [56]. 1149SPase also synthesized glucosyl-palatinose (24.1%), glucosyl-sorbitol (23.4%), glucosyl-xylitol(18.4%) and glucosyl-erythritol (13.4%) that can be used in sim-ilar applications. Studies for physical and biologically functionalcharacteristics of 1149Spase and its acceptor reaction productsare in progress.

Acknowledgement

This work was supported by the Korea Research FoundationGrant funded by the Korean Government (MOEHRD) (R08-2004-000-10256-0).

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