Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei

Background Trehalose synthase (TreS) which converts maltose to trehalose is considered to be a potential biocatalyst for trehalose production. This enzymatic process has the advantage of simple reaction and employs an inexpensive substrate. Therefore, new TreS producing bacteria with suitable enzyme properties are expected to be isolated from extreme environment. Results Six TreS producing strains were isolated from a specimen obtained from soil of the Tibetan Plateau using degenerate PCR. A novel treS gene from Enterobacter hormaechei was amplified using thermal asymmetric interlaced PCR. The gene contained a 1626 bp open reading frame encoding 541 amino acids. The gene was expressed in Escherichia coli, and the recombinant TreS was purified and characterized. The purified TreS had a molecular mass of 65 kDa and an activity of 18.5 U/mg. The optimum temperature and pH for the converting reaction were 37°C and 6, respectively. Hg2+, Zn2+, Cu2+and SDS inhibited the enzyme activity at different levels whereas Mn2+ showed an enhancing effect by 10%. Conclusion In this study, several TreS producing strains were screened from a source of soil bacteria. The characterization of the recombinant TreS of Enterobacter hormaechei suggested its potential application. Consequently, a strategy for isolation of TreS producing strains and cloning of novel treS genes from natural sources was demonstrated.


Background
Trehalose, a non-reducing disaccharide with two glucoses linked by a 1, 1-glycosidic linkage, is widespread throughout the biology world. In some lower orders of plants and fungi, this disaccharide is mainly stored as a source for carbon and energy [1]. In the animal kingdom it is abundant and e.g. insects use it as a source for glucose to provide sufficient energy during flight [2]. In yeast and bacteria, it was reported that trehalose could protect cells from a variety of physical and chemical stresses, such as freezing, heat, desiccation, acidic conditions, and osmotic and oxidative stress [3,4]. Due to its desirable characteristics, it has been also applied as an additive, stabilizer and preservative to food, cosmetics, as well as medicinal and biological reagents [5].
The Tibetan plateau is a high-altitude region where it can be assumed that bacteria are frequently exposed to stress conditions, such as cold, hypoxia, nutritional stress and high ultraviolet radiation. The microorganisms in this environment have received much interest because of their special properties [22]. Therefore, it is highly desirable to develop an effective strategy for isolation and subsequent screening of new TreS from the extreme environment In this study, several TreS producing strains were screened from soil bacteria derived from the plateau soil sample by degenerate PCR based on the analysis of conserved domains. The full-length of the novel treS gene of Enterobacter hormaechei was obtained by thermal asymmetric interlaced PCR (TAIL-PCR). Subsequently, the gene was expressed in Escherichia coli (E. coli), and the recombinant TreS was purified and characterized.

Results and discussion
Gene sequence analysis and strain screenings By multiple alignment analysis, the four highly conserved motifs HE/QPDLN, NHDELD/TLE, GIRRRLAP and YGDEIGMGD were found among protein sequences of TreS published at NCBI (Figure 1). The NHDELD/TLE domain (the DF2 primer region) had the feature of glycoside hydrolase (GH) family 16 site, suggesting that the identified treS gene encoding products possibly had hydrolytic activity. In addition, there were some other motifs identified by sequence alignment with relatively lower homologies, including N/QHTSDQ/AH, GFRL/ ADA, VRTPMQW and GGFS ( Figure 1).
Among the 86 bacterial strains isolated from the plateau soil sample, 6 different bacterial species were identified to be treS positive. Amplified core regions were sequenced and showed different similarity to relevant genes as summarized in Table 1. This result provided evidence for the distribution of TreS producing bacteria in extreme environment.

Cloning of Enterobacter hormaechei treS gene and analysis
A 228 bp core region of Enterobacter hormaechei treS gene was amplified by degenerate PCR. Its 3' and 5' flanking sequences were amplified by TAIL-PCR. By this a 1626 bp open reading frame (ORF) sequence was obtained, encod-  [20]. It is also reported that a glycosidase of Thermobifida fusca had TreS activity [21]. Therefore, it was suggested that TreS might employ a hydrolysis mechanism.

Expression and purification of recombinant TreS
The pET30a(+)-treS plasmid was transformed into the E. coli BL21 (DE3) plysS expression host, and cells were induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). When compared to the sample without induction, only the induced cells containing the recombinant vector expressed an extra 65 kDa protein ( Figure 2). The recombinant protein was about 3 kDa heavier than the predicted M r of 61.8 kDa, which was due to the additional 46 amino acids including the 6-His tag at the N' terminus.

Activity assay of recombinant TreS and products assay of catalytic reaction
TreS activity was detected with purified TreS in reactions of the conversion between maltose and trehalose. It was confirmed that the cloned fragment was the intrinsically coding sequence of active TreS. The highest activity was calculated to be 18.5 U/mg. The activity value was lower than that of the recombinant TreS of Pseudomonas stutzeri CJ38 [13], but close to that of the recombinant TreS of Mycobacterium smegmatis [15].
The amount of glucose was also detected by ion chromatography (IC) ( Figure 3) and shown to be consistent with the features of glycoside hydrolase as discussed above. Commonly, glucose is released in most of the conversion reactions of bacterial-derived TreS [14][15][16][17][18][19][20][21]. The small amount of glucose is generated under entry of a water molecule into the catalytic pocket to induce hydrolysis prior to formation of the glycosidic linkage [19]. Individually, TreS from Pseudomonas stutzeri CJ38 was the only one reported without generation of glucose as a byproduct [13]. We found relatively low homology between the two TreS from Enterobacter hormaechei and Pseudomonas stutzeri CJ38 (data not shown), indicating that there exists a structural difference in the functional role. Hence, in order to improve the efficiency of the major trehalose formation, the strategy to screen the enzyme without byproducts still remains to be further exploited.

The effect of pH and temperature on recombinant TreS activity
The conversion rate of recombinant TreS was constantly above 30% (equivalent to 65% of the maximal activity) at a broad pH range of 4 to 9, and reached the highest activity at pH 6 ( Figure 4A). The maximal conversion rate of 48% was observed at the optimum temperature of 37°C ( Figure 4B). The optimum temperature was similar to that of Pseudomonas stutzeri CJ38 [13], Mycobacterium smegmatis [15], Arthrobacter aurescens [16] and Thermus caldophilus [23]. Although the conversion from maltose to trehalose could be accelerated at high temperatures (data not shown), TreS could produce more byproduct glucose when the temperature was increased [21,23]. Thus, it is probably more efficient to carry out conversions at moderate temperatures. In this report, the recombinant TreS showed a stable performance under the wide working conditions (pH 4-9 and 20-55°C, Figure 4). It suggested that the enzyme might be a candidate for trehalose production.

The effects of metal ions and reagents on recombinant TreS activity
Recombinant TreS activity could be influenced by several ions and reagents at different levels. The enzyme was strongly inhibited by Hg 2+ , Zn 2+ and Cu 2+ for more than 40% and by SDS for about 90%. Other ions and chemical reagent, such as Mg 2+ , Fe 2+ , Na + , NH 4 + and EDTA, had no obvious effect, whereas Ca 2+ and Mn 2+ increased the activity slightly (Table 2). Besides, TreS was inactivated by addition of β-mercaptoethanol (β-ME), which supports the hypothesis of a structural dependence on disulfidebonds.

Conclusion
In summary, some TreS producing strains had been isolated from natural environment based on conserved domains and degenerate PCR. The treS gene from Enterobacter hormaechei was cloned by TAIL-PCR and expressed successfully. The characterization of the recombinant TreS suggested its potential application for trehalose production from maltose. Thus, the general applicability of this strategy for isolation of TreS producing strains and cloning of novel treS genes from natural sources was demonstrated.

Bacterial strains, media and culture conditions
Bacterial strains obtained from a Tibetan Plateau soil specimen were grown and enriched in nutrient broth medium (pepton1% w/v, beef extract 0.3% w/v, NaCl 0.5% w/v) at 30°C for 2 days. Arthrobacter aurescens, serving as a TreS positive control, was obtained from China General Microbiological Culture Collection Center (CGMCC1.1892).

Gene sequence analysis and strain screenings
All bacterial TreS sequences published in the NCBI Database were collected and analyzed by the multiple sequence alignment program CLUSTAL W2 http:// www.ebi.ac.uk/Tools/clustalw2. As shown in Figure 1, four degenerate primers (DF1, 2 and DR1, 2) were synthesized based on the conserved domains (see additional file 1 for the primer sequences).
DNA preparation of bacterial strains isolated from the soil specimen was performed by alkaline lysis [24]. Degenerate PCR was carried out according to the parameters optimized with the genome DNA template of Arthrobacter aurescens. PCR products were separated on a 1% agarose gel, recovered by using a Gel Extraction Kit (Tiangen, Beijing, China), and sub-cloned into the pMD18-T vector (TaKaRa, DaLian, China) for sequence analysis (SunBio, Beijing, China). The sequenced fragments were analyzed by BLAST http://www.ncbi.nlm.nih.gov/BLAST. All isolated positive strains were subsequently identified by their 16SrRNA sequence. and synthesized according to the core region amplified in degenerate PCR. The reaction parameters and the AD primers for TAIL-PCR were referred to previous reports [25,26]. The full length of treS was assembled from 3', 5' fragments and the core region. The deduced amino acid sequence was analyzed by DNAMAN 1.0.

Cloning of the treS gene of Enterobacter hormaechei and construction of the expression vector
The TreSF and TreSR primers (see additional file 1) were synthesized to introduce Nco I and Not I sites into the 3' and 5' ends of treS ORF, respectively. The PCR product was digested by Nco I and Not I, and inserted into the same digested pET30a(+) vector (Novagen, Cat No. 69909-3, Darmstadt, Germany) to generate His-tagged pET30a(+)-treS. The recombinant plasmid was confirmed by DNA sequencing and transformed to the E. coli BL21 (DE3) plysS.

Protein expression and purification
The E. coli BL21 (DE3) plysS transformed with pET30a(+)-treS was cultured in LB medium containing 50 μg/ml Kan and 34 μg/ml Cam in a shaker at 220 rpm and 37°C until an OD 600 of 0.6 was reached. These cells were induced with a final concentration of 0.5 mM IPTG and grown at 25°C for an addition of 4 h. Cell extracts were analyzed by 12.5% (w/w) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Gels with expressed protein were analyzed by a Molecular Imager ® Gel DocTM XR system 170-8170 (Bio-Rad, Hercules CA, USA) using the Quantity one-4.6.3 1-D analysis software.
For purification, cells were harvested by centrifugation and resuspended in lysis buffer (50 mM KH 2 PO 4 -K 2 HPO 4 , 500 mM NaCl, pH 7.9) followed by sonification and centrifugation at 12,000 × g for 20 min at 4°C to remove insoluble cell debris. The 6-His tagged protein in supernatant fraction was purified by using a Ni-NTA affin-IC assay of reaction products Figure 3 IC assay of reaction products. Reaction mixtures containing 400 μl purified TreS solution and 100 μl maltose (500 mM) substrate in 50 mM potassium phosphate buffer (pH 6) were incubated at 37°C for 2 h. The reaction mixtures were subsequently analyzed by IC, as described in the "Methods" section. Peak 1, trehalose; peak 2, glucose; peak 3, buffer reagent; peak 4, maltose.
Effect of pH and temperature on TreS activity Figure 4 Effect of pH and temperature on TreS activity. (A) Effect of pH on TreS activity. The enzyme activity of TreS at various pHs was assayed at 37°C in 50 mM potassium phosphate buffer (pH 2-10.5) for 30 min, using 100 mM maltose as a substrate; (B) Effect of temperature on TreS activity. The enzyme activity of TreS at various temperatures was assayed in 50 mM potassium phosphate buffer (pH 7) for 30 min, using 100 mM maltose as a substrate. ity chromatography column (NEB, Beijing, China). Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard [27].