Introduction

Trehalose (α-d-glucopyranosyl α-d-glucopyranoside), a non-reducing disaccharide, is widely distributed in nature (Elbein et al. 2003; Richards et al. 2002). It has been reported that this heat- and acid-stable sugar protects tissues of some plants and animals against environmental stresses such as freezing, desiccation, heat shock, and osmotic stress (Elbein et al. 2003; Richards et al. 2002). As an additive or stabilizer, trehalose can be used in foods, cosmetics, and medicines (Richards et al. 2002; Schiraldi et al. 2002).

Trehalose synthase (TreS) catalyzes the reversible inter-conversion of trehalose and maltose (Koh et al. 2003). It would provide a prospective biochemical method for the trehalose production. Recently, the complete genome of Arthrobacter aurescens TC1 was sequenced (Mongodin et al. 2006), but the gene noted as “treS” has not been identified yet. In this study, we cloned the treS gene from A. aurescens CGMCC 1.1892, identified its biochemical function, and also characterized its main enzyme features. This is the first TreS report from Arthrobacter genus, and it could afford some useful information for the further study on TreS.

Materials and methods

Bacterial strains and plasmids

Bacterial strains and plasmids used in this study are listed in Table 1.

Table 1 Bacterial strains and plasmids used in this work
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Growth media and conditions

Escherichia coli strains were routinely grown at 37 °C in LB media. Final concentrations of 50 μg/ml kanamycin (Kan), 50 μg/ml ampicillin (Amp), and 34 μg/ml chloramphenicol (Cam) were used for the selection of transformed bacteria in LB plates. A. aurescens was grown in nutrient broth medium (pepton 1%, beef extract 0.3%, NaCl 0.5%) at 30 °C.

Gene cloning and expression vector construction

Genomic DNA from A. aurescens CGMCC 1.1892 was prepared as described (Sambrook et al. 1989). Based on the ORF (gi = 119951278) of treS in GenBank, we designed the sense primer AaTreSF 5′-AGATCTGAGTTTTTCTCCGCAGAACC-3′ and antisense primer AaTreSR 5′-CTCGAGTCATCCTTTGGAGGTCAC-3′ (the Bgl II and Xho I restriction sites are underlined, respectively). The PCR products were ligated with pGEM-T cloning vector. After sequence analysis, the treS gene was digested by Bgl II and Xho I and inserted into pET-30a (+) vector to generate the recombinant vector pET-30a-treS. A His (6)-tag was added to the N-terminus of the recombinant protein to facilitate a one-step purification. The purified pET-30a-treS plasmid was transformed into BL21(DE3) pLysS for expression.

Protein expression and purification

The recombinant strain was cultured in 1 l LB medium containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol with vigorous shaking until the OD600 reached 0.6. To maximize the amount of soluble recombinant TreS, cultures were incubated at 25 °C for 6 h with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation at 4,000 ×g for 15 min at 4 °C and suspended in 250 ml of 100 mM potassium phosphate buffer (pH 7.5). The cells were disrupted by sonication and insoluble cell debris were removed by centrifugation at 10,000 ×g for 20 min at 4 °C. The supernatant was filtered through a 0.45-μm filter and loaded onto a Ni–NTA column according to the manufacturer’s purification protocol manual (Novagen, Ni–NTA His•Bind Resins). The purified enzyme was analyzed by 12% SDS-PAGE and protein concentration was determined by the method of Bradford using BSA as a standard.

Function identification and enzyme activity assay

The function of the recombinant protein was identified by TLC, using maltose and trehalose as a substrate, respectively. A reaction mixture containing the enzyme solution and 90 mM maltose or trehalose in 100 mM phosphate buffer (pH 7.5) was incubated at 35 °C for 4 h. Then the reaction mixture was heated in boiling water for 10 min to stop the reaction. After removal of the insoluble fraction, the product mixtures were separated on silica gel plates (Merck, Darmstadt, Germany) using a solvent system of 1-butanol/pyridine/water (4:5:1, v/v). Products were visualized by heating TLC plates at 100 °C for 5 min after spraying with 50% (v/v) sulfuric acid in ethanol. The recombinant trehalose synthase activity was measured by the amount of trehalose produced from maltose. One unit (U) of trehalose synthase was defined as the amount of enzyme required to produce 1 mmol trehalose per minute under the specified conditions. The amount of trehalose was quantified by IC using a Dionex 2500 system equipped with a CarboPacPA-2 column.

Nucleotide sequence accession number

The nucleotide and amino acid sequences of TreS in A. aurescens CGMCC 1.1892 are deposited in GenBank with accession number FJ545264.

Results

Gene and amino acid sequence analysis of TreS

We used the software Clustalx1.83 to analyze all the TreS sequences published online and found several highly conserved amino acid motifs (Table 2). These conserved motifs were identified in all TreS proteins reported so far (Pan et al. 2004; Wei et al. 2004; Lee et al. 2005; Chen et al. 2006). According to the published genome sequence of A. aurescens TC1 (gi = 119947346), there are two assumed treS genes in it. One is 1,797 bp (gi = 119951278), which has all the conserved motifs, but the other (2,136 bp, gi = 119947556) has not. In this study, we cloned the first treS gene from A. aurescens CGMCC 1.1892, which is more similar to the other known trehalose synthases, into E. coli to identify its biochemical function and for enzyme characterization. Using the primer pairs AaTreSF and AaTreSR, we got one 1,797 bp gene, which encoded 598 amino acids. The sequencing result showed that its amino sequence was 98.16% identical to the TreS online (gi = 119951278), which was higher than the corresponding nucleotide sequence homology of 90.14%. The result of amino acid sequence analysis by SignalP (http://www.cbs.dtu.dk/services/SignalP/) showed that there was no obvious signal peptide, which indicated that the TreS of A. aurescens CGMCC 1.1892 was an intracellular enzyme.

Table 2 Comparisons of conserved amino acid sequences in the TreS
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Expression and purification of recombinant protein

The recombinant expression vector pET-30a-treS was transformed into the BL21 (DE3) pLysS strain, and then TreS was produced by induction of T7 polymerase. The SDS-PAGE result (Fig. 1) showed that a recombinant protein about 68 kDa, which was in accordance with the predicted value, was expressed. Expression studies indicated that the induction with 0.4 mM IPTG at 25 °C for 5 h was the best condition for the expression of soluble TreS. Higher temperature or IPTG concentrations lead to a higher rate of inclusion bodies, whereas a temperature below 20 °C and lower IPTG concentrations decreased the total amount of TreS (data not shown).

Fig. 1
figure 1

SDS-PAGE analysis of the recombinant protein. All the protein samples were analyzed on a 12.5% polyacrylamide gel under denaturing conditions. The gel was stained with Coomassie Brilliant Blue R-250. The arrow indicates recombinant TreS. Lane 1 purified recombinant TreS. Lane 2 soluble fraction of BL21(DE3)pLysS transformed with pET-30a-TreS. Lane 3 total cell lysate of BL21(DE3)pLysS transformed with pET-30a-TreS. Lane M, molecular weight standards (97.4, 66.2, 43.0, 31.0 kDa)

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To determine whether the gene encoded a functional TreS, we purified the recombinant protein from the supernatant of cell lysate as shown by the single band in Fig. 1, lane A. Crude protein concentration in the supernatant of the cell lysate was 3.04 mg/ml, and the purified TreS concentration was 295 μg/ml.

Identification of the recombinant protein

To identify the function of the recombinant protein, we incubated the purified enzyme with trehalose and maltose, respectively. The reaction mixture was analyzed by standard TLC. TLC results (data not shown) showed that the enzyme could catalyze the conversion of maltose and trehalose, which meant this recombinant protein was TreS.

Effects of pH and temperature on the activity and stability of TreS

The pH dependence of TreS was studied at various pH values ranging from 4.5 to 10.0. To determine the pH stability, the recombinant enzymes were preincubated at various pH values (pH 4.5–10.0) for 20 min before the residual activity was measured at pH 6.5, immediately. The optimum pH for TreS was 6.5, but it remained highly stable within a pH range from 5.5 to 7.5 (Fig. 2).

Fig. 2
figure 2

Effects of pH on the activity and stability of the recombinant TreS. The enzyme activity of TreS at various pH was studied at 35 °C in 100 mM phosphate buffer (pH 4.5–10.0) for 30 min, using 90 mM maltose as a substrate. To examine the stability of TreS, the enzymes were preincubated at various pH values (pH 4.5–10.0) for 20 min at 35 °C. The residual activities were measured at pH 6.5. The square (■) represents effects of pH on the activity of TreS; the triangle (▲) represents effects of pH on the stability of TreS

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The effects of temperature on TreS activity were determined at various temperatures ranging from 20 to 60 °C. To determine the stability against thermal denaturation, the recombinant enzymes were preincubated at various temperatures (20–60 °C) for 20 min and then cooled immediately to assay the residual activity at 35 °C. The optimum temperature was 35 °C, and the enzyme remained stable up to 40 °C (Fig. 3).

Fig. 3
figure 3

Effects of temperature on the activity and stability of the recombinant TreS. The enzyme activity of TreS at various temperatures was studied at pH 6.5 in 100 mM phosphate buffers (pH 4.5–10.0) for 30 min, using 90 mM maltose as a substrate. To examine the thermal stability of TreS, the enzymes were preincubated at various temperatures (20–60 °C) for 20 min at pH 6.5. The residual activities were measured at 35 °C. The square (■) represents effects of temperature on the activity of TreS; the triangle (▲) represents effects of temperature on the stability of TreS

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Conversion profile of maltose to trehalose by TreS

The purified enzyme was incubated in 100 mM phosphate buffer (pH 6.5) at 35 °C for 0–10 h, using 90 mM maltose as a substrate. All the reactions were stopped by boiling them for 10 min before the samples were analyzed by IC. The retention time for trehalose, glucose, and maltose were 1.4, 2.1, and 4.6 min, respectively (data not shown). After 8 h of reaction, the conversion rates of trehalose and glucose were 59.5% and 13.2%, respectively (Fig. 4).

Fig. 4
figure 4

Conversion profile of maltose to trehalose by TreS. The conversion profile was obtained by incubating the enzyme (0.5 mg/ml) at 30 °C, pH 6.5 for 0–10 h, using 90 mM maltose as a substrate. Then, the reaction mixture was analyzed by IC, as described in the ‘Materials and methods’ section. Square (■): trehalose; triangle(▲): glucose

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Effects of metal ions and reagents on the activity of TreS

The effects of metal ions and reagents were determined by examining enzyme activity in the presence of either 1 or 5 mM of these substances under optimum reaction conditions (Table 3). The results showed that the enzyme activity was inhibited strongly by both 1 mM Cu2+ and SDS. However, when the concentration reached 5 mM, Tris could also inhibit most of the TreS activity.

Table 3 Effects of metal ions and reagents on the activity of TreS
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Discussion

In this paper, we confirmed that the gene (gi = 119951278) from A. aurescens CGMCC 1.1892 encoded a functional enzyme, trehalose synthase, which could catalyze the conversion of maltose to trehalose and vice versa. This enzyme showed its best activity at pH 6.5 and 35 °C. It could convert about 60% maltose to trehalose, accompanied by about 13% glucose as a byproduct. As reported in other studies, most TreS enzymes produce glucose as a byproduct, except the one from Pseudomonas stutzeri CJ38 (Lee et al. 2005). Glucose normally inhibits the enzyme activity (Chen et al. 2006) and lowers the conversion rate from maltose to trehalose (Wei et al. 2004). Those trehalose synthases producing less or no glucose have a relatively higher production rate for trehalose of about 70% to 80% (Lee et al. 2005; Chen et al. 2006; Nishimoto et al. 1996a, b). We found no obvious reasons why the enzyme derived from A. aurescens CGMCC 1.1892 produced a relatively high amount of glucose.

Till now, five pathways for trehalose synthesis have been identified: (a) TPS–TPP pathway, the most widely reported and best characterized pathway, involving the trehalose-phosphate synthase (TPS) and trehalose-phosphate phosphorylase (TPP) (Tzvetkov et al. 2003; Bell et al. 1998; Avonce et al. 2006). UDP-glucose + glucose-6-phosphate \(\underrightarrow {\operatorname{TPS} }\) trehalose-6-phosphate \(\underrightarrow {\operatorname{TPP} }\) trehalose + phosphate. (b) TreP pathway, existing in fungi (Saito et al. 1998a, b; Schwarz et al. 2007); it produces trehalose from α-d-glucose-1-phosphate and d-glucose by trehalose phosphorylase (TreP). (c) TreT pathway, found in extremophilic archaea (Tzvetkov et al. 2003); it converts ADP-glucose and glucose into trehalose by trehalose glycosyltransferring synthase (TreT). However, all these pathways are not suitable in trehalose industrial production due to their expensive substrates. (d) TreY–TreZ pathway; TreY mainly catalyses the formation of trehalosyl dextrins from dextrins by converting the α-1,4-glucosidic linkage at the reducing end to an α-1,1-glucosidic linkage. Then, TreZ cleaves the α-1,4-glucosidic linkage next to the α-1,1-glucosidic linkage of trehalosyl dextrins to produce trehalose and dextrins with lower molecular weight (Mukai et al. 1997; Fang et al. 2004). Trehalose has mainly been manufactured through this pathway since it was discovered in 1994. (e) The last pathway involves the TreS, which could produce trehalose from maltose in one step. Maltose is relatively cheap, and this pathway can be an alternative method for industrial trehalose production. So far, a number of TreS proteins from several bacterial strains (Ma et al. 2006; Lee et al. 2005; Pan et al. 2004; Nishimoto et al. 1996a, b; Chen et al. 2006; Zdzieblo and Synowiecki 2006; Wei et al. 2004) have been identified and characterized. All of these TreS proteins are encoded by uninterrupted prokaryotic genes, and the TreS from Mycobacterium smegmatis (Pan et al. 2004) proved to be a hexamer of six identical subunits by gel filtration, whereas all others suggested to be monomers by SDS-PAGE. This study provided the characteristics of the TreS from A. aurescens CGMCC 1.1892 for the trehalose catalysis metabolism.

There are other kinds of gene sequences, noted as “treS”, which do not have the five conserved motifs, and their amino acid sequences are not similar to each other. Till now, none of these sequences have been isolated as an enzyme. In the genome of A. aurescens TC1, a treS gene like that also exists. It may be a novel trehalose synthase with particular biochemical properties and is worthy of further study.