Introduction

Dextransucrase (EC 2.4.1.5) is an extracellular glucosyltransferase that is usually produced by oral streptococci and various strains of Leuconostoc mesenteroides. Dextransucrase is induced when strain L. mesenteroides 0326 is grown on sucrose-rich media. It catalyses the transfer of d-glucosyl units from sucrose to acceptor molecules. Thus, two different products can be synthesized: α-dextran or α-oligosaccharides when efficient acceptors (like maltose) are present (Dols et al. 1997; Monchois et al. 1999). Dextran that is produced by dextransucrase of L. mesenteroides has 95% α(1 → 6) linkages in the main chains and 5% α(1 → 3) branch linkage (Brown et al. 1989). Dextran has important medical applications in the production of fine chemicals such as plasma substitutes and Sephadex. However, they can also be used in texture improvement in the food industry, e.g. in milk drinks, yoghurts and ice creams (Neubauer et al. 2003).

A gene coding for dextransucrase was isolated from L. mesenteroides NRRL B-512F and sequenced (Wilke-Douglas et al. 1989). The amino acid residues were identified, which are composed of two different functional domains: the N-terminal catalytic domain (about 900 aa) and the C-terminal domain (300–400 aa) (Monchois et al. 1999). The dextransucrase of L. mesenteroides NRRL B-512F was expressed in the presence of carbon sources other than sucrose but at low activities (Quirasco et al. 1999).

In this paper, we describe the cloning and sequencing of a dextransucrase gene (dexYG) of L. mesenteroides 0326, and the alignment of the deduced amino acid sequences with those expressed from other dextransucrase genes. Expression of the dextransucrase dexYG and its activity are also reported.

Materials and methods

Materials, bacterial strains, plasmid and medium

Restriction endonuclease, LA Taq DNA polymerase, phenol/chloroform and T4 DNA ligase were purchased from TaKaRa. E. coli DH5α was used as a host in standard cloning experiments, and was grown in Luria Bertani (LB) medium. E. coli BL21 (DE3) was used for dextransucrase expression and the strain bearing plasmid pET28a(+) were grown in LB supplemented with 50 μg kanamycin/ml.

Molecular techniques

Genomic DNA from L. mesenteroides 0326 was extracted according to the established protocols (Sambrook et al. 1989). Restriction endonucleases, LA Taq DNA polymerases were used as described by the suppliers (TaKaRa Inc.). Transformation of plasmids into E. coli, DNA purification, digestion and agarose gel electrophoresis were performed according to standard procedures (Sambrook et al. 1989). pUCm-T vector (Shanghai Sangon) was used for cloning of PCR products. DNA sequencing was carried out by Invitrogen biotechnology company (China).

Cloning of the dextransucrase gene

Based on the sequence of gene dsrD (AY017384, GenBank), two primers [sense (5′-ATTTATGCCATTTACAAAAAAGCT-3′) and antisense (5′-CTTATGCTGACACAGCATTTCC-3′] were designed to amplify the gene by PCR. The 50 μl reaction mixture contained 5 μg genomic DNA, 0.5 μM of each primers, 250 μM of each dNTPs, 5 U LA Taq DNA polymerase, 2.5 mM MgCl2 and 1 × LA Buffer (Ryu et al. 2000). The PCR amplification protocol consisted of a denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 47°C, for 30 s, extension at 72°C for 5 min, and a final hold for an extra 10 min at 72°C. The resulting PCR-amplified fragments were ligated into plasmid pUCm-T to generate plasmid pYGdex, and the plasmid pYGdex was transformed into the E. coli DH5α strain.

The nucleotide sequence of the dexYG gene has been submitted to the GenBank databases under accession no. DQ345760.

Construction of expression plasmid pETdex

Using the plasmid pYGdex as template, a pair of specific primers were designed as follows: Sense 5′-CCGTAGATCTTCCATGCCATTTACAGAAAAAGT-3′ with a BglII site (underlined) and Antisense 5′-CCGCTCGAGTTATGCTGACACAGCATTT-3′ with a XhoI site (underlined). The PCR product obtained was cleaved with BglII and XhoI and ligated into the vector pET28a(+) previously digested with BamHI and XhoI to construct an expression plasmid pETdex. Plasmid pETdex was then transformed into E. coli BL21 (DE3) to obtain recombinant E. coli BL21 (DE3)/pETdex.

Expression and SDS-PAGE of dextransucrase

Cells of BL21 (DE3)/pETdex were grown at 37°C in LB medium containing 50 μg kanamycin/ml, and the expression of dextransucrase was induced by adding IPTG to 1 mM when the OD600 reached 0.6. The cultivation was continued for 5 h and the cells were harvested by centrifugation at 12,000 × g for 5 min (Malten et al. 2005). Bacteria pellet was disrupted by pulse sonication in lysis buffer as crude enzyme (Funane et al. 2001).

Proteins from the lysed cells were separated by 8% (w/v) SDS-PAGE (Sambrook et al. 1989) and were stained with Coomassie Brilliant Blue. After SDS-PAGE, the gel was washed three times with 20 mM sodium acetate buffer, pH 5.4, containing 0.05 g CaCl2/l to eliminate the SDS.

Activity assay and enzymatic biotransformation

Dextransucrase activity was determined spectrophotometrically by measuring the initial rate of fructose production using the dinitrosalicylic acid method (Monchois et al. 1997). The enzymatic reaction was carried out at 30°C with magnetic stirring in 20 mM sodium acetate buffer (pH 5.4) containing 100 g sucrose/l and 0.05 g CaCl2/l. Samples were centrifuged for 8 min at 9,000 × g before measuring the absorbance. A calibration curve was obtained with a 1 g fructose/l solution. One unit of dextransucrase activity was defined as that catalyzing the formation of 0.1 mg fructose/h under the above-mentioned conditions.

Results and discussion

Sequence analysis of the cloned dexYG and comparison of dexYG with others

The nucleotide sequence of the dexYG gene was cloned and sequenced. The nucleotide sequence of the dexYG gene was 4,584 bp in length (see Supplementary Table 1) and encodes a protein of 1,527 aa (Fig. 1) with a molecular mass of 170 kDa. The highly conserved residues identified in the N-terminal domain (Neubauer et al. 2003) were also conserved in dexYG. Analysis of the dexYG protein sequence revealed that it consists of a hydrophilic core domain with a hydrophobic N-terminal region. This N-terminal region displayed the sequence characteristics of a typical signal peptide sequence for secretion. The signal peptidase cleavage site at residues between position 42 and 43 (MPFTEKVMRKKLYKVGKSWV VGGVCA FALTASFALATPSVLG-DSSVPD) was predicted using SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP). Dextransucrase can be secreted into the culture medium of L. mesenteroides strain. However, the recombinant protein (dextransucrase) cannot be secreted expressed into the culture medium of recombinant E. coli (DE3)/pETdex since it does not have a signal sequence of the correspondent E. coli host.

Fig. 1
figure 1

Deduced amino acid sequence of the L. mesenteroides 0326 dextransucrase. The N-terminal amino acids of the secreted dextransucrase are shown in bold and marked by an arrow

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The obtained results were analysed by a BLAST similarity search of the GenBank database (http://www.ncbi.nlm.nih.gov.cn/BLAST). Comparison of the deduced amino acid sequence of dexYG with the sequences of other proteins revealed similarities corresponding to some conserved regions of dextransucrases. The highest level of homology was found with DsrD (GenBank AY017384) from L. mesenteroides Lcc4 (98.8% identity). The amino acid sequence is also highly similar to DEX (GenBank LMU81374; 97.4% identity). dexYG also shows significant similarities to other dextransucrases such as DsrB (66.7% identity) (Monchois et al. 1998) and DsrC (GenBank LME250172, 65.5% identity).

Expression of dexYG in E. coli

Dextransucrase can be efficiently expressed in E. coli BL21 (DE3) by IPTG induction at 25°C. SDS-PAGE analysis clearly showed a band after 3 h induction, corresponding to a Mr 170 kDa. This value is consistent with the theoretical molecular mass (Fig. 2).

Fig. 2
figure 2

SDS-PAGE analysis of the dexYG gene product, E. coli BL21 (DE3) cells bearing pET28a (+) plasmids were cultured at 25°C in LB medium containing 0.1 M Tris/HCl buffer, pH 5.4, and 50 μg kanamycin/ml. They were induced with 1 mM IPTG. 1 ml cultures were collected at different times of incubation (2, 3 and 4 h). Proteins were extracted by boiling bacterial pellets in a denaturation buffer and analyzed by 8% (w/v) SDS-PAGE and stained with Coomassie Brilliant Blue. Lane M: molecular weight markers; Lane1: 2 h after induction; Lane2: 3 h after induction; Lane3: 4 h after induction; Lane4: 0 h after induction. Arrow indicates an induced protein band with an Mr = 170 kDa

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Effect of pH on the enzymatic activity

The dextransucrase activity of dexYG was detected by measuring the release of reducing sugars in the presence of sucrose (Fig. 3). The dextransucrase activity was reached 5.4 U/ml after 4 h induction at 25°C. The degradation of activity was observed in a 5 h culture after induction. The pH value was increasing from 7.4 to 8.1 during the period of induction. Data showed that the loss of activity at 5 h post induction might be due to the increase in culture pH.

Fig. 3
figure 3

Time course of pH and dextransucrase activity after induction. The dextransucrase activity was measured by the dinitrosalicylic acid method. They were induced with 1 mM IPTG, and samples were taken at various time points after induction (1, 2, 3, 4, 5 and 6 h). The enzyme reaction is described in ‘Materials and methods’. (□) pH; (●) dextransucrase activity

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The effect of pH value on dextransucrase activity produced by engineered strain were studied (Fig. 4). The highest activity was reached 35.6 U/ml under the optimal culture conditions after 5 h induction in medium with pH 6.0 buffer, which was 3.5 times as that of in LB medium without pH-adjustment. The data demonstrated that the pH value was one of the main reasons which caused the degradation of enzyme activity in the later stage of induction. These results showed that dextransucrase could be efficiently heterologous expressed in E. coli and a strong dextransucrase activity had been detected and activity was shown to be pH dependent.

Fig. 4
figure 4

The effect of different pH value on dextransucrase activity produced by engineered strain. They were induced with 1 mM IPTG, and samples were taken at various time points after induction (3 ,4, 5 and 6 h). The enzyme reaction are described in ‘Materials and methods’. Different pH value in experiment: (■) pH 6.0 sodium phosphate buffer, (●) initial pH (not buffered), (Δ) pH 8.0 sodium phosphate buffer

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Enzymatic biotransformation

In the biotransformation test of dextransucrase, the substrate, sucrose, was transformed to dextran and fructose within 2 h by crude enzyme and the molecular weight of dextran was greater than 68 kDa (Fig. 5). Biotransformation yield of the enzyme reached 65% by measuring production of fructose using DNS method. These data demonstrated that the dextransucrase gene dexYG of L. mesenteroides 0326 was heterologously expressed in E. coli BL21 (DE3) and was able to drive dextran synthesis. Our results on the expression of this enzyme in E. coli BL21 (DE3) fermentations may help to optimize the industrial process of dextransucrase production. These may bring new opportunities for industrial applications of dextransucrases.

Fig. 5
figure 5

HPLC analysis of the product of dextransucrase biotransformation. In the biotransformation of dextransucrase, the substrate sucrose was transformed to dextran and fructose within 2 h by crude enzyme and the molecular weight of dextran was bigger than 68 kDa. Biotransformation yield of the enzyme reached 65% by measuring production of fructose using DNS method. The enzyme reaction is described in ‘Materials and methods’. HPLC conditions: GPC column, mobile phase: ultrapure water, flow rate: 0.6 ml/min, injection volume: 10 μl, detector temperature: 40°C, column temperature 60°C

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