Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei
© Yue et al; licensee BioMed Central Ltd. 2009
Received: 20 January 2009
Accepted: 12 June 2009
Published: 12 June 2009
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.
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%.
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.
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 . In the animal kingdom it is abundant and e.g. insects use it as a source for glucose to provide sufficient energy during flight . 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 .
At present, many trehalose synthesizing enzymes systems have been reported in microorganisms . There are three pathways widely distributed among them: (1) Phosphate-based enzyme complex systems: Trehalose is synthesized through the transfer of the glucosyl moiety from UDP-glucose to glucose-1-phosphate, forming the intermediate trehalose-6-phosphate which was then hydrolyzed by phosphatase to yield free trehalose [7, 8]. (2) A two-step enzyme system with maltooligosyl trehalose synthase (TreY) and maltooligosyl trehalose trehalohydrolase (TreZ) were found for trehalose biosynthesis in Arthrobacter sp. , Brevibacterium helvolum  and Bradyrhizobium . (3) TreS synthesizing trehalose from maltose in one step was found only in bacteria . We are most interested in TreS as a simple biocatalyst for trehalose production. Up to now, a few treS genes from different bacterial species have been reported to be cloned, expressed and characterized, including Pseudomonas stutzeri CJ38 , Pimelobacter sp. R48 , Mycobacterium smegmatis , Arthrobacter aurescens , Propionibacterium freudenreichii , Corynebacterium glutamicum ATCC13032  and some thermophilic strains [19–21].
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 . 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
Strains identified by 16SrRNA and protein sequence identities
Amplified size (bp/aa)
Blast result of core regions
Genbank accession No.
DF2 & DR2
DF2 & DR2
DF2 & DR2
DF1 & DR1
DF1 & DR2
DF1 & DR2
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, encoding 541 amino acids with a predicted molecular mass (Mr) of 61.8 kDa. As analyzed by NCBI BLAST, the full-length ORF of the Enterobacter hormaechei treS gene showed the highest similarity with several encoding sequences of TreS from Klebsiella pneumoniae 342 (CP000964, 815/1161, 70%), Pseudomonas stutzeri A1501 (NC000304, 386/569, 67%) and Pseudomonas aeruginosa PA7 (CP000744, 730/1119, 65%).
All of the conserved regions mentioned above were found in Enterobacter hormaechei TreS (Figure 1). Besides the GH16 domain, other active sites of glycosidase (H...D...E...HD) were also observed in most bacterial TreS (Figure 1), as reported by Chen . It is also reported that a glycosidase of Thermobifida fusca had TreS activity . Therefore, it was suggested that TreS might employ a hydrolysis mechanism.
Expression and purification of recombinant TreS
67 mg of recombinant TreS were purified from the supernatant of cell lysate containing 589 mg of crude protein. Soluble recombinant protein was effectively purified at a yield of 11.4% (w/w) of the total soluble protein. The final preparation showed a single band with an Mr of approximately 65 kDa (Figure 2).
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 , but close to that of the recombinant TreS of Mycobacterium smegmatis .
The effect of pH and temperature on recombinant TreS activity
The effects of metal ions and reagents on recombinant TreS activity
Effects of metal ions and chemicals on the activity of recombinant TreS
Relative activity (%)
Relative activity (%)
52 ± 4.7
57 ± 2.2
90 ± 2.4
98 ± 1.3
101 ± 4.3
103 ± 7.9
10 ± 1.1
59 ± 1.3
98 ± 5.8
92 ± 5.9
111 ± 10.7
Kinetics of recombinant TreS activity
The K m values for recombinant E. hormaechei TreS were found to be 25 mM for maltose and 75 mM for trehalose. V max values of 1.4 mM/min/mg protein for maltose and 3.2 mM/min/mg protein for trehalose were calculated, respectively. With regard to these results, the recombinant TreS had a higher affinity to maltose and a favorite reaction direction toward the synthesis of trehalose. Interestingly, all reported TreS enzymes share the feature of a reversible conversion at different degrees.
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).
E. coli Top10 was used for construction of recombinant plasmids. E. coli BL21 (DE3) plysS (Novagen, Germany) was used as expression host and grown in LB medium containing 50 μg/ml kanamycin (Kan) and 34 μg/ml chloramphenicol (Cam) after transformation.
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 . 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.
Cloning of the treS gene of Enterobacter hormaechei and construction of the expression vector
TAIL-PCR was performed to amplify the 3' and 5' fragments of Enterobacter hormaechei treS. The PU and PD primers (see additional file 1) for TAIL-PCR were designed 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.
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 OD600 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 KH2PO4-K2HPO4, 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 affinity chromatography column (NEB, Beijing, China). Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard .
Activity assay of recombinant TreS
The catalytic reaction was performed in a mixture containing the TreS solution and 100 mM maltose in 50 mM phosphate buffer (pH 6) at 37°C for 2 h. Samples were taken at different time intervals during the reaction and boiled for 10 min to stop the reaction. The reaction mixtures were separated by thin layer chromatography on G-60 TLC silica plates (Merck, Darmstadt, Germany) using a solvent system of 1-butanol/pyridine/water (4:5:1, v/v). Products released by TreS were quantified by IC using a Dionex2500 system equipped with a CarboPacPA™ 20 column. One unit (U) of TreS was defined as the amount of enzyme required to produce 1 μmol trehalose per min under the specified conditions . The conversion rate was calculated by the ratio of the trehalose product to the amount of maltose substrate.
Properties of recombinant TreS
The optimum pH of TreS was assayed by incubating the purified enzyme with 100 mM maltose substrate in 50 mM potassium phosphate buffer at pH 2 to 10.5 and 37°C for 30 min, respectively. The optimum temperature for TreS activity was determined at 10 to 60°C using the same buffer at pH 7 for 30 min, respectively. To determine the effect of metal ions and different chemical reagents on TreS, its activity was also assayed in the presence of these ions or compounds at 1 mM, respectively.
Determination of kinetic parameters
The Michaelis-Menten (K m ) and maximum activity (V max ) constant for recombinant TreS were determined with different concentrations of substrates ranging from 1 to 200 mM, and the data were plotted according to the Lineweaver and Burk method.
Nucleotide sequence accession number
The nucleotide sequence of Enterobacter hormaechei treS has been submitted to GenBank under accession no. FJ215664.
We are grateful to Dr. Volker Jäger for his insightful comments and language improvement on the manuscript. This research was supported by a key project of the Chinese international science and technology cooperation program (Grant No.2005DFA31070) and the National 863 Program.
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