Abstract: A NADH-dependent mannitol dehydrogenase gene (mtlD) was cloned from Pseudomonas fluorescens, subcloned into an expression vector (pDEST110) and entered into different strains of E. coli to compare their protein expression and the enzyme specific activity. Purifications were accomplished by Ni2+-NTA affinity chromatography. Using this approach, the efficiency of purification process significantly increased (up to 90%) so that the purified enzyme gave a sharp single band (55 kDa) in SDS-PAGE. The results showed that among the strains, BL21 (DE3) plysS exhibited the maximum expression level of MDH(mannitol dehydrogenase) (11 mg L-1). Results from activity assay with fructose as substrate also showed that in this strain the specific activity of 63 U mg-1 protein monitored for the enzyme, the record not reported before. Resazurin staining also indicated that the enzyme reduced fructose, whereas oxidized other substrates including mannitol, sorbitol and arabitol under optimal assay condition. From HPLC analysis it was showed for the first time that the enzyme could convert substrate isomaltulose to the specific products, GPM and GPS. Interestingly, because of the high specificity of the enzyme for substrate, the method can be used as an alternative approach to substitute non-specific conventional method of isomalt production.
INTRODUCTION
Mannitol is an acyclic hexitol and the most abundant sugar alcohol found in nature (Horer et al., 2001). It is produced in different organisms such as bacteria, fungi and higher plants (Jennings et al., 1998) where it is used as a source of carbon, a storage compound for energy and a reducing equivalent (Voegele et al., 2005). It also functions as a compatible solute during osmoregulation to respond to oxidative stress (Stoop and Pharr, 1994; Conde et al., 2007). D-mannitol is widely utilized in food and pharmaceutical industries as an excellent bulking agent (Soetaert et al., 1999). This product has been preferred by consumers because of its safety for diabetics and its tooth friendly. Mannitol sweetness is about half of sucrose and is not metabolized by human body and therefore considered as a low-calorie sweetener (Ronda et al., 2005).
Methodologically, the mass production system of mannitol involves in reducing fructose using Ni2+ ion as catalyst in presence of hydrogen gas. Due to the low selectivity of the catalyst, the procedure finally results in equal amounts of sorbitol and mannitol which must be separated through additional stages (Brünker et al., 1997). An alternative method is to use microorganisms and their enzymes to produce mannitol without producing any additional sorbitol. This is based on synthesizing mannitol by NAD(P)H-dependent mannitol dehydro- genase (MDH)s belong to dehydrogenase reductases family. Based on sequence analysis data, protein size and coenzyme-binding motifs, the family divided into three groups including as Short-Chain Dehydrogenase Reductase (SDRs), Medium-Chain Dehydrogenase Reductase (MDRs) and Long-Chain Dehydrogenase Reductase (LDRs). The SDRs bear a typical coenzyme-binding site, GXXXGXG and an active site YXXXK (Nidetzky et al., 1996). The MDR enzymes are zinc dependent and have the N-terminal coenzyme binding motif, GXGXXG (Otte and Lengeler, 2001). Despite an overall sequence identity as low as 10%, the LDR enzymes have a conserved KXXXXNXXG motif (Lee et al., 2003). MDHs from bacteria are considered as members of the LDR family with 66 recognized members (Lee et al., 2003).
Isomalt is also a natural sugar substitute, a sugar alcohol which is primarily used for its sugar-like physical properties. It has only a small impact on blood sugar levels, does not promote tooth decay and has one half the calories of sugar. It also has an effect like dietary fiber in the gut and produces butyric acid and propionic acid that act as antioxidants. It is an odorless, white and crystalline substance containing about 5% water of crystalline. Isomalt has minimal cooling effect lower than many other sugar alcohols. It is unusual as it is a natural sugar alcohol that is produced from beets. Isomalt (6-O-α-D-glucopyranosid-D-fructose) is manufactured in a two-stage process in which sugar (Saccharose) is first transformed into isomaltulose, a reducing disaccharide that is then hydrogenated using a Raney metal catalytic converter. The final product -isomalt- is an equimolar composition of 6-O-α-D-glucopyranosid-D-sorbitol (1,6-GPS) and 1-O-α-D-glucopyranosid-mannitol-dihydate (1,1-GPM-dihydrate). The molecular changes that occur during these steps make isomalt to have more chemical and enzymatical stabilities than sucrose. Isomalt stability is the reason for many of its health benefits and the large variety of improved products (Ndindayino et al., 2002).
In this study, we cloned a mannitol dehydrogenase gene from Pseudomonas fluorescens DSM 50106, subcloned it into an expression vector, expressed the deduced protein in different strains of E. coli and purified the enzyme by affinity chromatography to test the best MDH expression system among the strains.
As the other part of this research, we studied the in vitro effect of the purified enzyme on a new substrate, isomaltulose for the first time and showed that the enzyme effectively could convert the substrate to isomalt, a favorable sugar substitute.
MATERIALS AND METHODS
Microorganisms and E. coli strains: The strains of E. coli used in this study were: BL21(DE3)PlysS, JM109, Origami (DE3) and M15 (Table 1). The strains were grown in LB liquid medium or on agar plates with appropriate antibiotics. Pseudomonas fluorescens DSM 50106 was grown at 30 °C.
Molecular cloning: Total genomic DNA from Pseudomonas fluorescens DSM 50106 was extracted according to Sambrook method (Sambrook and Russell, 2001). One pair of primer (Forward: 5`-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GAA ACT GAA TAA GCA GAA -3` and Reverse: 5`-GGG GAC CAC TTT GTA CAA AGC TGG GTC TTA AAC CGG TTT CTT CAG GTG -3`) was designed and synthesized by Metabion company (Germany) according to conserved regions of previously reported bacterial LDRs. PCR performed using the following protocol: DNA was denatured at 95 °C for 3 min followed by 10 cycles of amplification (95 °C for 30 sec, 56 °C for 30 sec, 68 °C for 2 min), then by 30 cycles (95 °C for 30 sec, 58.5 °C for 30 sec, 68 °C for 2 min) and finally by 4 min at 72 °C. GATEWAY ® system was used to create the expression vector, mtlD-pDEST110 (Fig. 1) according to manufacture`s instruction (Invitrogen, USA). The resulting plasmid was entered into E. coli strains by electroporation (Bio-Rad, Gene Pulser XcellTM, USA). The resulting plasmid mtlD-pDEST encoded an MDH protein by c-terminal His6 tag.
| Table 1: | Bacterial strains used in this study |
| Fig. 1: | The genomic map of expression plasmid pDEST110-mtlD. The gene was located under the control of T7 promoter. The deduced protein contained His-tags at C-terminal |
Expression of MDH: Ten milliliter of an overnight culture of each transformed E. coli strains was transferred into 1 L of LB broth supplemented with appropriate antibiotics.
The cultures were shacked in 250 rpm at 37 °C until the OD600 reached to about 0.5. Expression of the recombinant mtlD gene was induced by addition of isopropyl-1-thio-β-D-galactoside (IPTG) to a final concentration of 1 mM. Incubation was continued for a period of 4 h, after which cells were harvested by centrifugation at 4000 rpm for 15 min at 4 °C (Eppendorf 5010 R, Hamburg, Germany) followed by washing twice with buffer A (5mM K3PO4, 300 mM NaCl, 10% Glycerol, pH 7.3). The pellet resuspended in 10 mL of the additional buffer A. Bacterial disruption performed by adding 2 mg mL-1 lysozyme (Fluka) and the cell debris was removed by centrifugation at 14000 rpm for 1 h in 4 °C.
Purification of recombinant protein: All steps were performed at 4 °C. Purification of mannitol 2-dehydrogenase protein from supernatant was performed by Fast Protein Liquid Chromatograph (FPLC) system (Sykam, Germany). The system consisted of S 2100 solvent delivery system, S 5111 injektor valve bracket, S 3210 UV/visible detector and Ni2+-NTA affinity chromatography column (Amersham Hi-Trap 1 mL). The column was first equilibrated with buffer A and then the whole extract was loaded on the column. The elution was performed by a linear gradient condition from a mixture of 100% buffer A to 100% buffer B (buffer A +500 mM imidazol) within 39 min. Flow rate was 1 mL min-1 and absorbance at 280 nm were measured. The fraction loaded on acrilamide gel (12%) as previously described by Lammlie. The MDH-containing fractions were pooled and dialyzed against 50 mM assay buffer (K2HPO4 pH 7.3). After dialysis, the protein solution was concentrated to 1 mL with an ultra-centrifugation filter (Amicon) and used as purified enzyme during the following experiments. Protein concentration was measured by the method of Bradford with bovine serum albumin (Sigma) as standard and stored in 10% glycerol (v/v) at -80 °C.
MDH activity assay: The activity of MDH was determined spectrophotometrically by monitoring the change in A340 upon oxidation or reduction of NAD(H) at 25 °C. The reaction mixture (1 mL) consisted of 2 mM NADH, 100 mM fructose and 20 μL enzyme solution in 50 mM potassium phosphate buffer, pH 7.3. The reaction was started by adding fructose and the mixture allowed standing for 30 sec. Reverse (oxidation) activity of the enzyme also assayed via determination of increasing absorption in A340. The activity was finally expressed as unit per milligram of protein. Effect of the enzyme on different polyol sugars were also showed spectrophotometrically on the conditions explained earlier.
Activity staining: MDH activity was monitored during MDH purification using Resazurin staining described by Perrot et al. (2003) with some modifications. Briefly, The staining mixtures consisted of 100 mM fructose, 50 mM K2HPO4, 15% agarose, 100 μL purified enzyme and 2 mM NADH were incubated at 25 °C for 30 min.
HPLC and TLC: HPLC (High Performance Liquid Chromatography) and TLC (Thin Layer Chromatography) were used to monitor the enzyme activity, control assays in the absence of enzyme or sugar were performed in parallel. Sugar quantification was carried out by injecting the samples (20 μL) to HPLC (Sykam, Germany) equipped with a CROMRESIN Ca++, IEX, 8 μm column (300x, 8 mm). Elution was performed by a mobile phase of 100% double distilled water (HPLC-grade) within 30 min at 70 °C at a flow rate of 0.5 mL min-1. Effluents was monitored by using a refractive index detector (Sykam, S 3580, Germany). Integrations were carried out with the HPLC software package (Chromstar 6.3).
Silica-gel Thin Layer Chromatography (TLC) performed to determine the presence of sugar polyols. Four microliter of each sample spotted on a thin layer chromatography alufoil plate (Silica Gel 60 F254, Merck) and developed with ethylacetate/acetic acid/ methanol/water (60:15:15:10) as the mobile phase. A solution of 0.5% KMnO4 in 1 M NaOH was sprayed on TLC sheet to stain the sugars. The sheet was heated at 95 °C for 3 min before visualization. Each standard sample (fructose and mannitol) was prepared from Sigma, dissolved in ddH2O up to 1 M (final concentration) and loaded (2 μ) on TLC.
RESULTS AND DISCUSSION
Molecular cloning: Five clones were choosen according to growth on the ampicilin agar plate and used for gene sequencing. All the sequencing results were the same. This similarity attributes to the use of AccuPrime™ Pfx DNA polymerase with a high specificity and fidelity causing no point mutation. This sequence had 100% similarity with the results of Brünker et al. (1997) (Fig. 2).
Overexpression of MDH in different strains of E. coli: The results from cloning and sequencing showed that we successfully obtained a MDH gene from P. fluorescens. This fragment had a high identity to another gene has been isolated by Bruncker et al. (1997). In order to express the enzyme in different strains of E. coli, the resulting construct was subcloned in a destination vector, pDEST110 to create mtlD-pDEST110. Different strains of E. coli transformed with the plasmid mtlD-pDEST110 to select the best strain in terms of MDH expression. The results showed that the treatment of bacteria for 4 h with IPTG at 37 °C obtained the highest expression level of the enzyme during the experiments and that among the strains, BL21 (DE3) plysS exhibited the maximum expression level (11 mg L-1). Therefore, this genotype was confirmed to be the best strain for expression of MDH in both laboratory and industrial applications.
| Fig. 2: | Sequencing of mtlD gene cloned in this study: small and capital letters show the sequence of cloned gene and primer, respectively |
Purification of recombinant MDH: Affinity chromatography with Ni2+-NTA resins has been used for the purification of a variety of proteins (Whitford, 2007). This method is one of the most important techniques for high purification of proteins, especially those of analytical and pharmaceutical interest. Principally, proteins with a stretch of histidin residues at N, C or both terminals have a high affinity to Ni2+ matrix. The method was applied previously to purify the MDH protein with six histidin tag at the C-terminal (Brünker et al., 1997). Methodologically, MDH was bound to Ni2+ ions and then eluted with buffer B. The protein contamination was eluted by loading resin with buffer A containing imidazol. It was showed that approximately pure mannitol dehydrogenase was eluted using 335 mM imidazol (Fig. 3). Practically, we did not use any precipitation step in order to protect the minor deals of MDH protein. We also loaded supernatant on the column twice and then kept the column on ice for 30 min. This approach significantly increased the efficiency of purification process (up to 90%). As it is showed in Fig. 3, the purified enzyme gave a single band (55 kDa) in SDS-PAGE.
| Fig. 3: | SDS-PAGE of MDH after affinity chromatography. Extracted Protein fractions of transformed E. coli Bl21(DE3)plysS derived by chromatography, loaded on a 12% SDS-PAGE. M: Molecular weight standard was obtained from Fermentase company. Lane 1, 2: crude extract, Lane 3: crude extract after loading on Ni2+-NTA column, Lanes 5-8: Pure eluted mannitol dehydrogenase ≈ 55 kDa |
Assay activity: The activity of the purified enzyme was determined from A340 using a spectrophotometer. With Fructose as a substrate, the specific activity of 63 U mg-1 protein was monitored for the enzyme in strain BL21(DE3) plysS. According to this data, this value was higher than any records reported previously (Brünker et al., 1997). Comparing to the values yielded in other strains, this high enzymatic activity was significantly related to the higher expression of the protein in strain BL21 (DE3) plysS.
| Fig. 4: | HPLC chromatograms; A: chromatogram of MDH mixture reaction in the presence of isomaltulose and NADH. Appeared peaks (GPM and GPS) are two necessary fractions of sugar substitute isomalt. B: chromatogram of isomalt standard (standard retention times: isomaltulose 12.1 min, 1,1 GPM 15.1 min, 1,6 GPS 17.4 min) |
Therefore, it is concluded again that this strain has the most potential for producing of the enzyme among the strains we used and as a result it can be used in large scale protein purification. Resazurin staining was performed to confirm the reductive or oxidative state of the reaction. When the reaction was reductive, the reaction mixture turned to white, whereas in the case of oxidative reactions there was no change in color of mixture. The staining finally showed that the enzyme was active in reducing fructose whereas, oxidized other substrates including mannitol, sorbitol and arabitol under optimal assay conditions.
Substrate and product analysis by TLC and HPLC: HPLC analysis showed a significant decrease in fructose concentration in the reaction mixture containing NADH, accompanied by the presence of mannitol detected on TLC (Fig. 5).
To test the possible activity of MDH on isomaltulose, HPLC performed to analysis the presence of the specific products, GPM and GPS. In the reaction mixtures with NADH, substrate or enzyme, two new peaks with the retention characteristics of GPM and GPS was formed in the chromatograms (Fig. 4). This means that MDH accepts its substrate. This was the first time that in vitro activity of mannitol 2-dehydrogenase on isomaltulose was demonstrated. Due to the high specificity of MDH in this approach, the technique can be finally used as a new and alternative method for large scale isomalt production instead of non-specific production systems. It was previously claimed that mannitol 2-dehydrogenase from Pseudomonas fluorescens DSM 1056 can accept sugars with 5-6 carbons as substrate (Kavanagh et al., 2002, 2003). In present study, we showed that the enzyme can also act as substrate like isomaltulose with 12-carbon backbone. We suggest that it attributes to the thermodynamic stability of the ligand. In general, sugars in cyclic form in solvent are more stable than linear form. Stability of glucose in cyclic form to linear form is 90:10, whereas stability of cyclic fructose to linear fructose is 70:30. By adding a cyclic form to the linear form, stability of the cyclic form will be increased. By these descriptions it will be appeared that why mannitol 2-dehydrogenase can affect on isomaltulose. Demonstration of MDH using PDB (Protein Data Bank) site and Weblab Viewer Pro. software showed that MDH in presence of isomaltulose and NADH as substrates had a binding site with enough space for isomaltulose (Fig. 6) not for trehalose which is a trisaccharide.
| Fig. 5: | Enzyme activity assay figured by Thin Layer Chromatography (TLC), Lane 1: standard mannitol, Lane 2: sample of the enzyme assay with fructose as a substrate, Lane 3: standard of fructose |
| Fig. 6: | The MDH structure with NADH molecule on the left as a cofactor and isomaltulose on the right as a substrate illustrated using Weblab Viewer Pro. MDH has enough space for isomaltulose in substrate binding site |
ACKNOWLEDGMENTS
We are grateful to ABRICI`s researchers for their helpful aid during this study. We also express our sincere thanks to M. Shariati of Isfahan University supporting parts of assays.