Introduction
Oxidoreductases are a class of enzymes that transfer electrons from natural or artificial donors to acceptors. Glucose dehydrogenase (GDH) is a type of oxidoreductase that uses NAD(P)+ as its cofactor. GDH can catalyze the oxidation of β-ᴅ-glucose to ᴅ-glucono-δ-lactone when a cofactor is available [12].
GDH has been used in biochip and biosensor applications for the enzymatic determination of ᴅ-glucose in samples. This enzyme is also used to supply NAD(P)H for the enzymatic production of useful chiral compounds using reductases [7,10,22]. For example, some yeast reductases converted ethyl-4-chloro-3-oxobutanoate (ECOB) into (R)- or (S)-ethyl-4-chloro-3-hydroxybutanoate (ECHB) using NADPH. Both (R)- and (S)-ECHB are chiral building blocks used as precursors for many pharmaceutical compounds [8,28].
Compared with other commonly used NAD(P)H regeneration systems, such as formate dehydrogenase from Candida boidinii [24] and phosphite dehydrogenase from Pseudomonas stutzeri WM88 [26], GDH derived from Bacillus species exhibits a higher specific activity and dual cofactor specificity [2,13,16,17]. Until now, Bacillus GDHs have been isolated from B. subtilis, B. megaterium, B. amyloliquefaciens, and B. thuringiensis and used in bioconversion reactions [6,8,20].
The crystal structure of the GDH (GDH-BM) from B. megaterium IWG3 was determined at 1.7Å resolution, which showed that the enzyme consisted of four identical subunits, similar to those of other short-chain reductases/dehydrogenases [27]. The GDH-BM tetramer dissociates into inactive monomers at pH 9.0, which is attributed mainly to the weakness of the Q-axis interface. Rational design and site-directed mutagenesis studies have been performed to develop mutant enzymes with the narrowest substrate specificity and high catalytic activity and thermostability [18,25].
A polyhistidine (His)-tag is an amino acid motif that consists of at least six histidine residues located at the N- or C-terminus of a protein [9,14]. His-tags are useful in affinity purification, subcellular localization, ELISA, western blotting, and immobilization. His-tagging can sometimes affect the activity of the expressed enzyme [19]. Although His-tag engineering has been extensively implemented for protein purification, the effect of His-tagging on different enzyme terminal regions has not been studied well.
In this study, the glucose 1-dehydrogenase B gene (gdhB) from B. thuringiensis subsp. kurstaki was cloned and expressed in E. coli BL21 (DE3). We added His-tags on the N- or C-terminus during the cloning step. The activities and biochemical properties of the GDH-BTs were studied. GDH-BTs are used as coupling enzymes in yeast reductase-mediated chiral synthesis reactions.
Materials and Methods
Bacterial Strains and Plasmid
B. thuringiensis subsp. kurstaki (KCCM 11429) was purchased from the Korean Culture Center of Microorganisms (KCCM) and used as the source of gdh gene (GenBank ID CP010005, AJK39257). E. coli XL-1 Blue and BL21 were used as the hosts for cloning and expression of the gdh gene. Plasmids pGEM-T, pET-22, and pET- 28 were used as the vectors for the cloning and expression of the gdh gene.
Cloning of the GDH Gene
For PCR cloning of the B. thuringiensis gdhB gene, three primers were designed: primer F, 5’-CATATG TAT AGT GAT TTA GCA-3’; primer R1, 5’-GGATCC TTA CCC ACG CCC AGC TTG AAA-3’; and primer R2, 5’-GGATCC GA CCC ACG CCC AGC TTG AAA-3’. Primers F and R1 were used to clone gdhWT and gdhN-His. Primers F and R2 were used to clone gdhC-His. PCRs were conducted using B. thuringiensis chromosomal DNA under the following conditions: pre-denaturation (94℃ for 10 min); 35 cycles of denaturation (95℃ for 30 sec), annealing (37℃ for 30 sec), and elongation (74℃ for 90 sec); and post-elongation (72℃ for 10 min).
The purified PCR products were ligated into a pGEM-T vector and transformed into competent E. coli XL-1 blue cells. The resulting recombinant gdh-T vectors were isolated and digested with NdeI and BamHI for a second cloning into pET vectors. gdhWT and gdhC-His were ligated into pET-22 vectors, and gdhN-His into the pET-28 vector. All three vectors were transformed into competent E. coli XL-1 blue cells. The resulting recombinant gdh-pET vectors (Fig. 1) were isolated and used for the GDH-BT production experiment.
Fig. 1.Construction of expression vectors for B. thuringiensis GDH. (A) The gdhWT and gdhC-His genes were inserted into the pET22 plasmid. (B) The gdhN-His gene was inserted into the pET28 plasmid. (C) GDH-BTWT has 261 amino acid residues. GDH-BTN-His and GDH-BTC-His have His-tags on their N-terminal and C-terminal parts, respectively.
Expression of Recombinant GDH-BT in E. coli BL21 (DE3)
Expression of gdhWT, gdhN-His, and gdhC-His was conducted by transforming gdh-pET vectors into E. coli BL21 (DE3) competent cells. E. coli transformants were cultured at 37℃ in Luria-Bertani medium containing 1 mM ampicillin until the OD600 increased to 0.5. After isopropyl-β-ᴅ-thiogalactopyranoside (1 mM) was added, the cultures were incubated at 20℃ with agitation (230 rpm) for 18 h. The E. coli cells were harvested and washed with potassium phosphate buffer (10 mM, pH 7.5) and lysed by the ultrasonic disruption, followed by centrifugation at 12,000 rpm for 10 min at 4℃. Supernatants were taken and protein concentrations were determined by the Bradford method. To check the expression of GDH-BT enzymes, SDS-PAGE was performed.
GDH Enzyme Activity Assays
Quantification of GDH activity was performed by adding 10 μl of enzyme solution into the 0.99 ml assay mixture including 100 mM Tris-HCl buffer (pH 7.5), 10 mM glucose, and 0.2 mM NADP+. Assays were initiated by measuring the increasing OD340 rate due to the formation of NADPH for 10 min. This reaction was performed at 30℃. One unit of GDH enzyme was defined as the amount of enzyme that catalyzes the formation of 1 μmol NADPH per minute under assay conditions. Specific activity was expressed as unit per milligram of protein.
Characterization of the GDH-BT Enzyme
The optimal temperature of the GDH-BT enzyme was studied at various temperatures between 30℃ and 70℃ under standard conditions. To determine the thermostability, the enzyme solution was pre-incubated in Tris-HCl buffer (100 mM, pH 7.5) at 30-70℃ for 30 min and the residual activity measured.
To investigate the optimal pH of the GDH-BT enzyme, five buffer systems were used: 50 mM sodium citrate (pH 4.0-6.0), 50 mM potassium phosphate (pH 6.0-8.0), 50 mM Tris/HCl (pH 7.0-9.0), 50 mM Gly/NaOH (pH 9.0-11.0), and KCl/NaOH (pH 12.0-13.0). The pH stability was determined by pre-incubating the enzyme in pH 2.0-13.0 using 50 mM KCl/HCl (pH 2.0), 50 mM Gly/HCl (pH 3.0), and the above buffers at 30℃ for 30 min and measuring the residual activity.
Substrate specificity was studied using eight different substrates, glucose, fructose, mannose, galactose, xylose, sucrose, lactose, and maltose, under the standard condition.
For kinetic studies, the initial reaction rates were measured for two cofactors (NADP+ and NAD+) and one substrate (glucose). Various concentrations of NADP+ (0.02-1 mM) and NAD+ (0.05-1 mM) were used with a fixed glucose concentration (10 mM). Then, various concentrations of glucose (0.5-50 mM) were used with a fixed concentration of NADP+ (0.2 mM). The kinetic parameters were evaluated by the Lineweaver-Burk plot method.
Gel Permeation Chromatography (GPC)
Before measuring the molecular size of the three GDH-BT enzymes, they were partially purified using two sequential steps: ammonium sulfate (AS) precipitation and PD-10 column chromatography (GE Healthcare Bio-Science AB, USA). First, the proteins that precipitated between 0% and 30% AS saturation were removed, while proteins that precipitated between 30% and 70% AS saturation were collected and dissolved in Tris-HCl buffer (100 mM, pH 7.5).
The enzyme solution (2.5 ml) was loaded onto a PD-10 desalting column (bed volume, 8.3 ml) and eluted with 3.5 ml of Tris-HCl buffer (100 mM, pH 7.5). The active GDH-BT fractions were collected and utilized in GPC.
The column used in GPC analysis was a Superose 12 10/300 GL column (GE Healthcare Bio-Science AB) and the buffer used was Tris-HCl (100 mM, pH 7.5), with a flow rate of 0.5 ml/min. The injection volume was 0.5 ml and the running time was 64 min. Detection of protein samples was carried out using a UV detector at 280 nm wavelength. The standard for this analysis was Gel Filtration Standard Protein (Bio-Rad Laboratories, USA) that comprised thyroglobulin (MW 670,000 Da), γ-globulin (MW 158,000 Da), ovalbumin from chicken (MW 44,000 Da), horse myoglobulin (MW 17,000), and vitamin B12 (MW 1,350 Da). The partition coefficient (Kav) was calculated by Kav = (Ve-Vo)/(Vt-Vo), where Ve, Vo, and Vt are the elution volume, void volume, and total bed volume, respectively.
Homology Modeling of GDH-BT
The amino acid sequence of GDH-BT (GenBank ID, AJK39257) was submitted to the SWISS-MODEL homology modeling server [1,3] and modeling was performed in the automatic mode. The homology model was constructed based on the crystal structure (PDB ID, 3AY6) of GDH-BM (GenBank ID, P40288), which had an amino acid identity of 88.51% to GDH-BT.
Enzymatic Coupling Reaction Using GDH-BT and Reductase Enzymes
To check the cofactor regeneration efficiency, the conversion rate of the enzymatic reduction of ECOB was evaluated as described in previous studies [8,28]. For the coupling reactions, 30 mM ECOB, 45 mM ᴅ-glucose, 1 mM NADP+, 40 U of yeast reductase, and 80 U of GDH-BTWT (or GDH-BTN-His) were mixed in 10 ml of Tris-HCl buffer (100 mM, pH 7.5). The mixture was put into a titration vessel with thermostat jacket (Metrohm, Switzerland) and incubated at 25 ℃ with stirring at 400 rpm (728 Magnetic stirrer; Metrohm). The pH of the reaction mixture was monitored with a pH meter (Mettler Toledo, USA) and maintained at 7.0 to 7.5 during the reaction period by adding 1 M NaOH. ECHB was extracted with ethyl acetate and dried via evaporation. After the acetylation and washing steps, the organic phase was subsequently analyzed using a gas chromatography system equipped with a Chirasil-dex column (Varian, USA). The column temperature was increased from 70℃ to 180℃ at a rate of 5°C/min and maintained for 2 min at 180°C. The totals of the product quantities in the reaction mixtures were calculated by comparing the retention times and peak areas of the standard (R)- and (S)-alcohol peaks.
Results and Discussion
Cloning and Expression of the GDH Gene (gdh)
The glucose 1-dehydrogenase B (gdhB) gene from B. thuringiensis subsp. kurstaki is reported to have 786 nucleotides. In this research, the gene was amplified using two different pairs of primers as described in the Material and Methods section. Two PCR products were then used to facilitate the cloning of gdhB into two cloning vectors (Figs. 1A and 1B). The pET-22 vector was used to express both GDH-BTWT and GDH-BTC-His, whereas the pET-28 vector was used to express GDH-BTN-His. GDH-BTWT has 261 amino acid residues and the calculated molecular mass is 28,048 Da (Fig. 1C). GDH-BTN-His was designed to have an extra 20 amino acids at its N-terminus and its molecular mass is 30,212 Da, whereas GDH-BTC-His was designed to have an extra 22 amino acids at its C-terminus and its molecular mass is 30,457 Da.
The amino acid sequence of GDH-BTWT was compared with other Bacillus GDHs. It had identities of 98% and 97% with the GDH from B. thuringiensis M15 (DQ515911) and B. thuringiensis konkukian 97-27 (AAT63738), respectively. It also had identities of 88%, 82%, and 81% with B. megaterium IWG3 (P40288), B. subtilis (ABR21558), and B. amyloliquefaciens (KOS50696) GDHs, respectively.
Three recombinant plasmids were transformed into E. coli BL21 (DE3) cells and IPTG induction was then performed. SDS-PAGE analysis showed that all GDH-BTWT, GDH-BTN-His, and GDH-BTC-His were produced mainly as soluble forms (Fig. 2). The sizes of the GDH-BTs increase slightly in the order of GDH-BTWT, GDH-BTN-His, and GDH-BTC-His as expected. Specific activities of E. coli cell-free extracts were measured to be 6.6 U/mg (GDH-BTWT), 5.5 U/mg (GDH-BTN-His), and 0.020 U/mg (GDH-BTC-His). The specific activities of GDH-BTWT and GDH-BTN-His were considerably higher than that of GDH-BTC-His. This result suggests that the intact C-terminal carboxyl group of GDH-BT is important for enzyme activity. Previous literature documents that C-terminal His-tagging on YedY from the sulfite oxidase family induced an asymmetric structure containing five monomers with disordered C-terminals. Therefore, it destabilized the terminal residues and impaired enzyme folding and activity [23].
Fig. 2.SDS-PAGE of GDH-BT enzymes expressed in E. coli cells. (A) E. coli proteins with expressed GDH-BTWT. (B) E. coli proteins with expressed GDH-BTN-His. (C) E. coli proteins with expressed GDH-BTC-His. M, protein size markers; Ppt, insoluble proteins; Sup, soluble proteins; arrows, GDH-BT enzymes.
Effects of Temperature and pH on Enzyme Activity and Stability The GDH activity of the wild type and His-tagged enzymes was measured at 30-70℃. The optimum temperature of GDH-BTWT was 55℃ and its activity was comparably high in the range of 45-65℃ (Fig. 3A). GDH-BTN-His and GDH-BTC-His had optimum temperatures of 65℃ and 60℃, respectively, and showed a substantial change in activity depending on assay temperatures.
Fig. 3.Effects of temperature and pH on GDH activity and stability. (A) GDH activities were measured at different temperatures. (B) Residual activities were measured at 30℃ after pre-treatment at different temperatures for 30 min. (C) GDH activities were measured at different pHs. (D) Residual activities were measured at pH 7.5 after pre-treatment at different pHs for 30 min. ○, GDH-BTWT; ●, GDH-BTN-His; □, GDH-BTC-His.
The thermostability of the wild-type and His-tagged enzymes was tested by measuring residual activities after incubation at 30-75℃ for 30 min (Fig. 3B). GDH-BTWT maintained high activity (>85%) up to 65℃, whereas GDH-BTN-His and GDH-BTC-His maintained high activities only up to 45℃, and then their activities decreased dramatically above 45℃. This result suggests that GDH-BT is a thermostable enzyme. That is, it was more resistant against heat inactivation compared with other Bacillus GDHs. GDH from Bacillus sp. G3 was stable up to 40℃, whereas GDH from B. thuringiensis M15 was stable up to 45℃ in 60 min incubation experiments [4,6]. Our experiments also demonstrate that the intact N- and C-termini are required for GDH-BT to maintain stability at high temperatures. Previous studies concerning the laccase enzyme showed that the His-tagged enzyme had thermal instability compared with the wild-type enzyme [11].
The GDH activities of the wild-type and His-tagged enzymes were measured between pHs 4.0 and 13.0 (Fig. 3C). The optimum pH of both GDH-BTWT and GDH-BTN-His was 9.0. GDHN-His showed a high activity in a broad pH range of 8.0-10.0. GDH-BTC-His had quite different properties compared with GDH-BTWT and GDH-BTN-His, as it had high enzymatic activity in the range of 10.0-11.0. The wild-type and His-tagged enzymes exhibited high stability in a broad range of pHs (Fig. 3D). GDH-BTWT and GDH-BTN-His were stable from pH 3.0 to 11.0, whereas GDH-BTC-His was stable from pH 2.0 to 12.0. This result revealed that these GDH-BTs exhibited high stability against a wide range of pHs compared with the other Bacillus GDHs, such as GDH from Bacillus sp. G3 and B. thuringiensis M15, which were stable up to pH 9.0 and 8.0, respectively [6].
Substrate Specificity and Kinetic Parameters
Regarding the substrate specificity, GDH-BTWT and GDH-BTN-His had high activity toward glucose as a substrate, but showed no or little activity toward most other sugars (Table 1). This narrow substrate specificity corresponds well to the previous report that B. thuringiensis M15 GDH had very narrow substrate specificity [4,5]. This is different from other bacterial GDH enzymes that possess broad substrate specificity. For example, the GDH enzyme from Pseudomonas fluorescens possesses broad substrate specificity for glucose, xylose, mannose, galactose, and maltose. Thus, GDH-BT is unique among many GDHs by its narrow substrate specificity [5].
Table 1.N/D, not detected.
On the other hand, GDH-BTC-His showed enzymatic activity toward glucose and lactose, with the relative enzymatic activity of 100% and 88.33%, respectively. This result suggests that the C-terminal His-tag affected the enzyme’s active site and changed the substrate-binding site. Previous studies also reported that His-tagging, either on the N- or C-terminus, alters enzyme properties, such as substrate specificity, although the detailed mechanism of the alteration has not been revealed [15,21]. Moreover, C-terminal His-tagged thioesterase I shows lower catalytic activity toward palmitoyl-CoA and p-nitrophenyl dodecanoate compared with the wild type. Nevertheless, it showed higher catalytic activity toward p-nitrophenyl acetate [15].
The Vmax and KM values of GDH-BTWT and GDH-BTN-His toward glucose were quite different from those of GDH-BTC-His, as expected (Table 2). The KM values of GDH-BTWT and GDH-BTN-His were much lower than that of GDH-BTC-His. The Vmax values of GDH-BTWT and GDH-BTN-His were much higher than that of GDH-BTC-His. The kinetic results toward NADP+ were very similar to the results obtained with glucose. In the case of NAD+, GDH-BTWT and GDH-BTN-His had higher Vmax values than GDH-BTC-His; however, all enzymes showed similar KM values.
Table 2.Kinetic parameters of GDH enzymes toward substrates.
These kinetic studies demonstrated that glucose and NADP+ bound more securely to GDH-BTWT and GDH-BTN-His than GDH-BTC-His and that turnover rates of GDH-BTWT and GDH-BTN-His were higher than that of GDH-BTC-His. In addition, the kinetic results showed that both NADP+ and NAD+ could be used as cofactors.
Gel Permeation Chromatography
The GPC experiment with GDH-BTWT, GDH-BTN-His, and GDH-BTC-His showed that their partition coefficients (Kav) were 0.311, 0.373, and 0.373, respectively (Fig. 4). From the standard curve, their molecular masses were calculated to be 56.4, 30.9, and 30.9 kDa, respectively. As the molecular mass of the GDH-BT monomer was calculated based on an amino acid sequence of 28,048 Da, GDH-BTWT seems to be a dimer, and GDH-BTN-His and GDH-BTC-His are monomers. This correlated with a previous report that the native form of B. thuringiensis M15 GDH is a dimer [4,5]. The X-ray crystal structure of B. megaterium GDH consists of four identical subunits [18,27]. In this respect, the quaternary structure of GDH-BT (dimer) is different from that of GDH-BM (a tetramer).
Fig. 4.Gel permeation chromatography of GDH-BT enzymes. Partition coefficients (Kav) were calculated as (Ve-Vo)/(Vt-Vo). Open circles, standard proteins, and closed circles, GDH-BT enzymes.
This GPC result suggests that intact N- and C-termini are required for the GDH-BT enzyme to form a dimer structure.
Homology Model of GDH-BT
A homology model of GDH-BT was constructed to explain the relationship between the protein structure and catalytic activity. The protein sequence alignment of GDH-BT and GDH-BM (1WG3) showed 88.51% identity. Therefore, the homology model of GDH-BT was constructed through the SWISS-MODEL homology modeling server using the GDH-BM structure (PDB ID 3AY6) as the template. However, the X-ray crystal structure of GDH-BM was a tetramer form (Fig. 5A), whereas GDH-BT was a dimer form. Therefore, we could not build a homology model of the GDH-BT dimer form based on the GDH-BM structure. Instead, we built a homology model of the GDH-BT single subunit (Fig. 5B). It contained the central parallel β-sheet with connecting α-helices, which is a typical Rossmann superfamily structure, and both N- and C-terminals protrude out from the monomer surface.
Fig. 5.X-ray crystal structure of GDH-BM and homology model of GDH-BT. (A) The X-ray crystal structure of GDH-BM tetramer (PDB ID, 3AY6) is shown. Each subunit is shown in green, cyan, dark-salmon, and yellow-orange. (B) Homology model of the GDH-BT single subunit, constructed based on GDH-BM by SWISS MODELER. α-Helixes are shown in cyan, the β-sheet is in red, and loops are in magenta. (C) GDH-BT dimer model. Each subunit is shown in green and cyan.
According to the crystal structure of GDH-BM (Fig. 5A), N-terminals of each subunit locate at interfaces between two subunits, and C-terminals of each subunit insert into the neighboring subunit and interact directly with glucose [18]. Based on the GDH-BM structure, the GDH-BT monomer model (Fig. 5B), and our experimental results (Fig. 4 and Table 2), we suggested the GDH-BT dimer model to be as shown in Fig. 5C. In this model, N-/C-terminals locate at the interface between two subunits, and N-/C-terminal extra tagging might interfere with dimer formation.
Coupling Reaction Using GDH-BT and Reductase
The GDH-BT/reductase coupling reaction (Fig. 6A) was performed with a time course and analyzed using a GC system equipped with a chiral column. When the coupling reaction was performed by GDH-BTWT with an initial substrate concentration of 30 mM, approximately 90% of the ECOB added was converted within 30 min (Fig. 6B), implying that the NADPH was recycled at least 27 times by GDH-BTWT. When the same reaction was performed by GDH-BTN-His, the conversion yield was almost the same as GDH-BTWT (Fig. 6C). In both cases, (R)-ECHB was produced with an enantiomeric excess value of 98%, exhibiting the efficient catalytic capability of the GDH-BT/reductase system.
Fig. 6.Coupling reaction of GDH-BT and reductase for NADPH recycling. (A) The GDH-BT enzyme produced NADPH, which is used as the cofactor in the reductase-mediated reduction reaction. (B) The conversion yield using GDH-BTWT and reductase was measured with a time course. (C) The conversion yield using GDH-BTN-His and reductase was measured with a time course.
In summary, gdhB from B. thuringiensis subsp. kurstaki was cloned and GDH-BTWT, GDH-BTN-His, and GDH-BTC-His were expressed in E. coli BL21 (DE3) cells. Native GDH-BT WT was thermostable and in a dimer form, whereas GDH-BTN-His and GDH-BTC-His were less thermostable and in monomeric forms. Enzyme kinetic study suggests that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BTWT and GDH-BTN-His regenerated NADPH as a cofactor in a reductase-mediated chiral synthesis reaction, suggesting that they can be used as catalysts in fine-chemical and pharmaceutical industries.
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