- Research article
- Open Access
A trehalase from Zunongwangia sp.: characterization and improving catalytic efficiency by directed evolution
© Cheng et al. 2016
- Received: 9 July 2015
- Accepted: 15 January 2016
- Published: 29 January 2016
Trehalases have potential applications in several fields, including food additives, insecticide development, and transgenic plant. In the present study, we focused on a trehalase from the marine bacterium Zunongwangia sp., which hydrolyzes trehalose to glucose.
A novel gene, treZ (1590 bp) encoding an α, α-trehalase of 529 amino acids was cloned from Zunongwangia sp., and TreZ was found to have an optimal activity at 50 °C and pH 6. The activity of TreZ was increased by the presence of NaCl, showing the highest activity (136 %) at 1 M NaCl. A variant C4 with improved catalytic activity was obtained by error-prone PCR and followed by a 96-well plate high-throughput screening. The variant C4 with two altered sites (Y227H, and R442G) displayed a 3.3 fold increase in catalytic efficiency (k cat/K m, 1143.40 mmol−1 s−1) compared with the wild type enzyme (265.91 mmol−1 s−1). In order to explore the contribution of the mutations found in variant C4 to the increased catalytic activity, two mutants Y227H and R442G were constructed by site-directed mutagenesis. The results showed that the catalytic efficiencies of Y227H and R442G were 416.78 mmol−1 s−1 and 740.97 mmol−1 s−1, respectively, indicating that both mutations contributed to the increased catalytic efficiency of variant C4. The structure modeling and substrate docking revealed that the substitution Y227H enlarged the shape of the binding pocket, to improve the binding of the substrate and the release of the products; while the substitution R442G reduced the size of the side chain and decreased the steric hindrance, which contributed to channel the substrate into the active cavity easier and promote the release of the product.
In this study, a novel trehalase was cloned, purified, characterized, and engineered. A variant C4 with dramatically improved catalytic activity was obtained by directed evolution, and the mutation sites Y227H and R442G were found to play a significant role in the catalytic efficiency. The overall results provide useful information about the structure and function of trehalase.
- Marine bacterium
- Error-prone PCR
- Site-directed mutagenesis
- Catalytic efficiency
Trehalose is a non-reducing disaccharide in which the two glucose units are linked in α-α-1, 1-glycosidic linkage. It is widely distributed in nature and mainly isolated from bacteria, fungi, insects, invertebrates, and plants . In living cells trehalose acts as a carbon source , a signal molecule [3, 4], an essential component of cell wall [5, 6]. It also protects the membranes under stress conditions [7, 8].
In the present work, Zunongwangia sp., a marine bacterium, which survives under high hydrostatic pressure and a low temperatures environment was explored for trehalase production . In Zunongwangia sp. trehalose is not only a source of energy but also associated with high salt-tolerance and cold adaptation . α, α-trehalases (EC 184.108.40.206) are the enzymes that specifically hydrolyze trehalose to glucose.
Based on the amino acid sequence of known and hypothetical proteins, α, α-trehalases are classified into three glycoside hydrolase (GH) families 15, 37, and 65 (http://www.cazy.org/) [10, 11]. These families belong to the clan GH-L or GH-G, but share the same classic (α/α)6 barrel fold, and an inverted reaction mechanism . A previous study showed that over-expression of plant trehalase in Arabidopsis decreased the trehalose levels to recover from drought stress . In another study, trehalose accumulation was reported to have toxic effect in Cuscuta reflexa , suggesting that the transfer of trehalase gene into plants will contribute to protect the plants under such conditions. Trehalase deficiency is a metabolic disorder in which human body is not able to convert trehalose into glucose . Individuals suffering from this deficiency experience vomit, abdominal discomfort and diarrhea after consumption of trehalose rich food . These reports indicated that trehalase is both a therapeutic enzyme and potential food additive.
In 2007, the three-dimensional structure of periplasmic trehalase (Tre37) from E. coli was explored . Silva et al. first provided the evidence (site-directed mutagenesis) that trehalase from Spodoptera frugiperda, a member of GH family 37, has an aspartate residue (D322) and a glutamate residue (E520) as the general acid and base catalysts, respectively . Error-prone-PCR and Site-directed mutagenesis are commonly used to generate a large mutant library with modified sequences and to examine the role of specific residues in enzyme activity [19, 20].
To date, to the best of our knowledge, only Silva et al.  have reported the mutants of trehalase from Spodoptera frugiperda and determined the active site. The objectives of present study were to characterize a novel Trehalase (TreZ) from Zunongwangia sp. and to improve its activity by directed evolution.
Characteristics of treZ gene, TreZ and mutants
The substrate specificity and kinetic parameters of TreZ and mutants
The substrate specificity of TreZ
Specific activity (U/mg)
Steady-state kinetic parameters for wild-type TreZ and mutant
K m (mmol l−1)
k cat (s−1)
k cat /K m (mmol−1 s−1)
0.99 ± 0.04
263.25 ± 0.91
265.91 ± 1.21
0.3793 ± 0.03
433.69 ± 0.82
1143.40 ± 1.67
0.7194 ± 0.05
299.83 ± 1.39
416.78 ± 1.57
0.4317 ± 0.03
319.88 ± 0.95
740.97 ± 1.64
Effects of metal ions and chemical reagents on TreZ
Effect of metal ions and chemical reagents on TreZa
Concentration/Relative activity (%)
106.64 ± 1.58c
127.44 ± 6.69
94.71 ± 1.58
158.92 ± 0.69
125.97 ± 1.07
112.76 ± 8.14
92.46 ± 2.38
100.85 ± 1.02
116.15 ± 5.36
105.27 ± 5.69
134.43 ± 4.20
126.31 ± 7.24
65.67 ± 0.12
7.17 ± 0.46
4.91 ± 2.15
94.55 ± 3.60
42.23 ± 0.08
38.46 ± 3.30
47.47 ± 4.58
3.91 ± 2.47
19.96 ± 2.27
18.86 ± 0.50
14.3 ± 3.44
105.25 ± 2.67
135.54 ± 3.59
111.94 ± 0.63
70.48 ± 5.36
78.75 ± 9.23
44.45 ± 5.26
155.65 ± 4.78
76.71 ± 6.00
55.75 ± 8.77
139.02 ± 0.82
9.09 ± 2.02
Substrate docking analysis of TreZ and mutants
In the present study, TreZ from Zunongwangia sp. was isolated and expressed in E. coli BL21 (DE3). The optimum temperature for TreZ is 50 °C, which is higher than that of many previously reported trehalases, such as those in Apis mellifera L 7 °C , Rhodotorula rubra 30 °C , Saccharomyces cerevisiae 40 °C , and Rhizopus microsporus var. rhizopodiformis 45 °C . However, a trehalase from a thermophilic bacterium Rhodothermus marinus has a higher optimal temperature of 88 °C than TreZ .
Another remarkable characteristic of TreZ is its extreme salt-tolerance (Fig. 3). The enzyme was found to be active over a wide NaCl concentration range (0–5 M exhibiting the maximum activity at 1 M NaCl (136 %) and retaining more than 100 % of its original activity at 0.5–3.5 M NaCl. This behavior is similar to that of a xylanase and two amylases from Zunongwangia sp., which showed the optimum activity at 3, 1.5 and 2 M NaCl, respectively [27–29]. TreZ retains activity with or without NaCl and thus differs from other halophilic enzymes which require salt to remain active and stable, indicating that TreZ is a salt-tolerant enzyme. To date, several reports have been published about the trehalases, whose activity was enhanced by Na+ [30, 31]. Furthermore, TreZ is relatively stable after 24 h incubation in high salinity conditions (Fig. 3). A similar salt tolerance behavior was also observed in xylanase and amylases from Zunongwangia sp. [27–29]. Additionally, TreZ activity was also enhanced by K+, Ni+, Mg2+, Ca2+, and Ba2+ and strongly inhibited by Fe3+, Cu2+, Zn2+ and ADP (10 mM).
In this study, we improved the catalytic efficiency of TreZ (1143.4 mmol−1 s−1) by directed evolution. When ompared to previously reported trehalases in terms of the catalytic efficiency of mutant C4 is higher than the trehalases (262–730 mmol−1 s−1) from Metarhizium strains  and Tre37 (485 mmol−1 s−1) from E.coli , but lower than the trehalase (1273 mmol−1 s−1) from Apis mellifera L  and trehalase (2400 mmol−1 s−1) from Spodoptera frugiperda .
Despite many studies trehalases, only a few further engineered trehalases have been reported. Silva et al. reported that three Arg residues (R169, R222 and R287) are essential for trehalase activity from Spodoptera frugiperda . In this study, a mutant C4 (Y227H and R442G) dramatically improved catalytic activity by directed evolution. To understand the relationship between single mutant site and catalytic efficiency, the single site mutants were constructed separately, respectively. The results showed that Y227H and R442G played an important role on the catalytic efficiency of trehalase.
The structure modeling analysis and substrate docking (Fig. 4a, b, c and d) of Y227H revealed a subtle modification of the shape of the binding pocket. According to Morley et al. , the replacement like Y227H far away from the active site may remodel the site arrangement and lead to fine alterations in the protein backbone and side chain, which altered the secondary structure of protein, and also produced a subtle change in the shape of the binding pocket, finally leading to dramatic changes in the catalytic activity of enzyme. The modeled TreZ and mutant structures (Fig. 4a) and the docking analysis of the substrate (Fig. 4e, f) showed that the residue 442 is in the vicinity of the entrance to the active site. In R442G, the replacement of arginine by glycine reduced the size of the side chain of residue 442. Due to the reduction of the side-chain functional groups, the nearest distance between residue 442 and residue 509 become longer, which decreased the steric hindrance, contributed to channel the substrate into the central binding pocket easier and promoted the release of product, finally leading to the improvement of the catalytic efficiency of trehalase.
In this study, a novel salt-tolerant trehalase from Zunongwangia sp. was cloned, purified, characterized, and engineered. Moreover, we found that the mutation sites Y227H and R442G make synergic contributions to the catalytic activity of mutant C4 and explored that the single mutant site affects the catalytic activity. These results provided useful some insight into the relationship between structure and function of the trehalase.
Bacterial strains, plasmids and medium
D-trehalose and D-glucose were purchased from Sigma-Aldrich (St. Louis, MO, USA); Restriction endonucleases from Takara (Japan); T4 DNA ligase and pfu DNA polymerase from Transgene (Beijing, China); and DNA purification Kits from Axygen (USA). The PreScission protease and the GST-binding Resin were procured from GE Healthcare (USA) and Merck (Germany), respectively. GST-Bind Purification kit was purchased from Novagen (Germany).
Zunongwangia sp. was isolated from seawater. E. coli strains DH5α and BL21 (DE3) were used as gene cloning and protein expression hosts, respectively. The plasmid vector for cloning and expression was pGEX-6P-1 (GE Healthcare, USA). Zunongwangia sp. was grown in Luria-Bertani (LB) medium containing 20 g l−1 NaCl at 26 °C. The strains of E. coli were grown on LB medium or LB agar plates at 37 °C with Ampicillin (100 μg ml−1).
Cloning of treZ gene
The genomic DNA of Zunongwangia sp. was purified and used as a template for amplification of TreZ gene (treZ). The primers were designed on the basis of putative TreZ gene from Zunongwangia profunda SM-A87 (GenBank CP001650) which was sequenced by Qin et al. . The pair of primers (TreZ-F, TreZ-R) was listed in Additional file 1: Table S1. The amplification was performed by following the PCR(s) program: (i) 94 °C for 4 min, (ii) 30 cycles of 94 °C for 30 s, 49 °C for 30 s, and 72 °C for 96 s, and (iii) 72 °C for 10 min. The amplified products were purified and digested with EcoR I/Xho I and ligated into the same restriction site of pGEX-6p-1 to generate recombinant plasmid pGEX-6p-treZ. The pGEX-6P-treZ was transformed into E. coli BL21 (DE3) for protein expression and purification. The nucleotide and protein sequences were analyzed using the BLAST tool in the NCBI website. Multiple sequence alignment was performed using the DNAMAN software package.
Construction of mutant library
Error-prone PCR was used to construct the randomly mutant treZ gene library. The PCR mixture (100 μl) was composed of Taq buffer containing 5 mM MgCl2, 20 ng template pGEX-6p-treZ, 1 mM dNTPs, 0.2 mM dTTP and dCTP, 0.2 mM MnCl2, 2.5 units of Taq DNA polymerase, and 0.4 μM primers (TreZ-F and TreZ-R). The PCR reaction was carried out at an initial temperature of 94 °C for 4 min, followed by 30 cycles of (94 °C for 30 s, 46 °C for 30 s, and 72 °C for 96 s) and a final elongation at 72 °C for 10 min. The amplified PCR products were digested by EcoR I and Xho I and cloned into plasmid pGEX-6p-1, which were transformed into E. coli DH5α to obtain the mutant library.
Screening of library
Transformants of the TreZ mutant library were spread on LB plates containing 100 μg ml−1 ampicillin and incubated for 14 h at 37 °C. The colonies were picked up with sterile toothpick(s) and resuspended separately in a 96-deep-well plate containing 0.6 mL liquid LB medium and 100 μg ml−1 ampicillin. After incubation for 20 h at 37 °C, 180 μl of fresh LB medium (0.1 mM IPTG and T7 phage) was added to each well [34, 35], followed by incubation for 6 h at 28 °C under shaking at 180 r.p.m. Subsequently, each cell suspension from the 96-deep well (150 μl) was transferred to another 96-deep-well flat-bottom block with each well containing 250 μl of trehalose (20 mM). After incubation for 10 min at 50 °C, reaction was stopped by adding 200 μl of DNS. Finally, the mixture was boiled for 5 min, and absorbance was determined at A540 using a Multiskan Spectrum spectrophotometer (Thermo Scientific, Vantaa, Finland). The clones with a higher absorbance value than that of the wild-type enzyme were selected for further evaluation.
The site-directed mutagenesis was carried out by one-step overlap PCR, using the plasmid pGEX-6p-treZ as a template and primers designed from pairs of complementary oligonucleotides containing desired mutants (Additional file 1: Table S1). The PCR program was set as follows: denaturation at 97 °C for 2 min, 20 cycles of 20 s at 95 °C, 30 s at 54 °C, 1 min 40 s at 72 °C, followed by a 7-min extension at 72 °C and 10-min preservation at 15 °C . After PCR reaction, the amplified products were mixed with Dpn I to digest the wild-type templates, followed by incubation for 12 h at 37 °C, and then transformed into E. coli DH5α cells. The selected transformants were sequenced, and transformed into E. coli BL21 (DE3) for enzyme expression and purification.
Protein expression and purification
The recombinant plasmid pGEX-6P-treZ and mutants were expressed in E. coli BL21 (DE3) cells. A single colony grown on an LB plate (containing 100 μg ml−1 ampicillin) was used to prepare the seed culture, 20 ml of which was transferred to 1 L of LB medium (containing 100 μg ml−1 ampicillin) and grown at 37 °C until the absorbance reached 0.6 at 600 nm. Then gene expression was induced by adding IPTG (0.1 mM) into the culture, and further incubated at 18 °C for 16 h. Cells were harvested by centrifugation and resuspended in cold phosphate-buffered saline (PBS, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.0) followed by cell disruption using a French pressure cell technique. The cell debris was spun down at 12,000 × g for 30 min at 4 °C, followed by purifying the glutathione-S-transferase (GST) tagged TreZ protein in the supernatant, and adding the 3C protease (PreScission, Pharmacia) to remove the GST tag using GST fusion protein purification kit according to manufacturer’s instruction. Finally, the purified protein was eluted in 1 ml of PBS (pH 7.0). All purification steps were carried out at 4 °C. The homogeneity of purification and the molecular mass of the enzyme were determined by 12 % SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the protein concentration was measured by the Bradford method  using bovine serum albumin (BSA) as standard.
Enzymatic assay and kinetic parameters assay
The activity of TreZ was determined by a modified method reported by Pereira et al. . The amount of reducing D-glucose was determined by the 3,5-dinitrosalicylic acid (DNS) method and GOD-POD method [39, 40]. One unit (U) of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of glucose in one minute under standard assay conditions. The specific activity was expressed as units per mg protein (U/mg).
The substrate specificity was tested by using the substrates of trehalose, sucrose, maltose, lactose and cellobiose. The enzyme assay was carried out under standard assay conditions.
The K m and k cat values of TreZ and the mutants were determined by measuring the enzyme activity using trehalose ranging from 0.2 to 4.0 mM L−1 under optimal reaction conditions. The data were estimated by the Lineweaver-Burk plot method using Graph-pad Prism software.
Effects of temperature, pH, metal ions, and chemical reagents on trehalase activity
The effect of temperature on enzyme activity was assayed at a temperature range from 20 to 75 °C. To determine the thermostability, the enzyme was incubated for 1 h at the indicated temperatures, and residual activities were measured under standard assay conditions. The effect of pH on the purified TreZ was evaluated under TreZ activity assay conditions at the optimal temperature in a pH range of 4.0–9.0, using two different buffers:0.2 M Na2HPO4/0.1 M citric acid buffer for pH 4.0–8.0, and 0.05 M Na2B4O7 · 10H2O/0.2 M NaOH buffer for pH 8.0–9.0.
The effect of salt concentration on enzyme activity was tested in a Tris–HCl buffer (50 mM; pH 7.0) containing different concentrations of NaCl (0–5 M) at 50 °C. The influence of NaCl on enzyme stability was studied by measuring the residual activity after pre-incubation of enzyme solution with 0–4 M NaCl at 20 °C for 24 h. Untreated enzyme was considered as control. The effects of different metal ions and chemical reagents on enzyme activity were evaluated under optimal conditions at 1, 5 and 10 mM concentration; the enzyme without any reagent treatment was taken as control.
The three dimensional (3D) structures of TreZ and mutants were predicted by SWISS-MODEL (http://swissmodel.expasy.org/)  and modeled based on the reported crystal structure of Trehalase Tre37 from E. coli (PDB code: 2WYN). Docking analyses of substrate with TreZ and mutants were performed by Molecular Operating Environment (MOE) 2009 (Chemical Computing Group Inc., Montreal, Canada).
This work was supported by grants from National Science Foundation of China (No. 31270162) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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