Characterization of an L-arabinose isomerase from Bacillus coagulans NL01 and its application for D-tagatose production
© The Author(s). 2016
Received: 22 January 2016
Accepted: 21 June 2016
Published: 30 June 2016
L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed.
The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn2+ at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM-1 min-1 and 1.0 mM-1 min-1 respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L-1 and 250 g L-1 D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h).
In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn2+. Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.
D-tagatose is a natural rare ketohexose that possesses 92 % of the sweetness, but only 38 % of the calories of sucrose . Since it attained GRAS (Generally Recognized As Safe) status under U.S. Food and Drug Administration (FDA) regulations, D-tagatose has become a promising functional sweetener on the food market. Until now, it has been used in the productions of confectionery, soft drinks and health foods for improving the flavors and reducing the calories. It also shows positive attributes in treatment of type II diabetes and hyperglycemia. Currently, one mature method for D-tagatose production is the direct isomerization of D-galactose into D-tagatose with metal hydroxides as the chemical catalysts under basic conditions . This process was applied into commercial food grade D-tagatose production by Arla Food Company between 2002 and 2006 . Nevertheless, it has been gradually dismissed because of the drastic reaction conditions and high cost of the subsequent purification steps. Another method is the enzymatic process that mainly depends on L-arabinose isomerase (AI, EC 220.127.116.11). AIs can isomerize D-galactose to D-tagatose in one step at a milder environment. The method has some significant advantages over the chemical process, such as a lower alkali dosage and less unexpected by-products .
Several reports indicated that high reaction temperature is a favorable condition for D-tagatose formation [8, 9]. Numerous AIs from thermophile strains have been reported since the year 2000 including Thermotoga maritima, Thermotoga neapolitan, Anoxybacillus flavithermus, Bacillus stearothermophilus US100 [8–11]. Their optimal temperatures were within 80 to 95 °C. High operation temperature causes browning reaction and formation of by-products in catalysis, which is a big obstacle for isolation of the target product . Therefore, many researchers changed their interests to AIs from mesophilic Bacillus strains such as Bacillus licheniformis, Bacillus subtilis, Bacillus halodurans and Bacillus coagulans [12–15]. These enzymes could adapt to moderate temperatures from 50 °C to 60 °C. They possessed inherently high k cat/K m values for the natural substrate L-arabinose. Therefore, their applications mainly focused on the synthesis of L-ribulose. Only B. halodurans AI was reported to show a k cat/K m of 0.4 mM-1 min-1 toward D-galactose. Relevant data about the properties of Bacillus AIs in D-tagatose production are insufficient and needed to be complemented.
In this study, an araA gene from the B. coagulans NL01 was cloned and expressed in Escherichia coli BL21 (DE3). The biochemical properties of purified B. coagulans AI (BCAI) were comprehensively studied. Whole cells of E. coli expressing BCAI were used to produce D-tagatose under high D-galactose concentrations in order to test its actual bioconversion capacity.
Results and discussion
Over-expression and purification of BCAI
Purification of BCAI expressed in recombinant E. coli
Specific activity(U mg-1)
HisTrap HP 5 mL column
Effects of temperature and pH on activity of BCAI
To investigate the effect of pH, enzyme assays were carried out at a series of pH from 2.2 to 9.0. The relative activity of BCAI reached the maximal value at pH 7.5 (Fig. 3b) and decreased by less than 10 % at pH 8.0 to 9.0. By contrast, the activity was weaker at acidic conditions. It decreased severely when the pH dropped to 5.0 as most of moderate alkaline AIs did previously, possibly because some side chain groups close to its substrate binding sites were difficult to ionize under this condition .
It has been experimentally proved that the optimum pH (pHopt) of AIs is affected by some crucial residues with polar groups, for example the E268 residue of B. halodurans AI (BHAI, pHopt =8.0) and the equivalent D269 of L. fermentum AI (LFAI, pHopt =6.5) [13, 17]. Modifications of the two residues to lysine (K) resulted that the pHopt of BHAI and LFAI decreased to 7.0 and 5.0, respectively [19, 20]. Protein sequence alignment showed D268 in BCAI was the counterpart of E268 of BHAI and D269 of LFAI (Fig. 2a). It could be presumed that if the D268 residue was changed to lysine, the pHopt of BCAI would probably decrease to a lower value.
Effects of metallic ions on activity and thermostability of BCAI
From the two perspectives of enzyme activity and thermostability, it seemed that external Mn2+ was not essential for the raw purified BCAI. Since a low amount of metallic ions can increase the purity and safety of products, BCAI will show its unique value in food-grade D-tagatose production.
Kinetic parameters of BCAI and molecular docking studies
Kinetic parameters of AIs
V max (U mg-1)
K m (mM)
k cat/K m (min-1 mM-1)
V max (U mg-1)
K m (mM)
k cat/K m (min-1 mM-1)
B. coagulans NL01
P. pentosaceus PC-5
Although molecular docking studies had been implemented on BLAI before, the result was not very representative because BLAI did not possess any D-galactose activity in experiments. Most AIs such as BCAI were able to catalyze L-arabinose and D-galactose simultaneously. As shown in Fig. 6c, two hydrogen bonds (2.15 Å, 2.34 Å) existed between the C2 hydroxyl group of L-arabinose and the oxygens of E306. One hydrogen bond (1.96 Å) was found between the C1 hydroxyl group and the oxygen of E331. According to the presumed catalytic mechanism of E. coli AI and B. licheniformis AI [26, 27], L-arabinose was firstly transformed to an enediol intermediate and then L-ribulose was formed. During the first step, protons were transferred through C1 and C2 of the substrate with the assistances of E306 and E331. The docking result here confirmed that E306 and E331 of BCAI played important roles of targeting the C2 and C1 hydroxyl groups of L-arabinose. The hydrogen bond interactions between L-arabinose and the residues were sufficiently strong. By contrast, the interactions for D-galactose were weaker. Among the two hydrogen bonds (2.35 Å, 1.94 Å) found in Fig. 6d, only the bond between the C3 hydroxyl group of D-galactose and E306 could promote the reaction according to the putative mechanism exposed above. E331 residue did not orientate the C1 hydroxyl group of D-galactose correctly, which would cause difficulties on proton transfer and slowed formation of enediol intermediate. This could be an explanation for why the formation of D-tagatose was always slower than that of L-ribulose in most AI catalyses.
Meanwhile, the C-DOCKER energies for the docking poses of L-arabinose and D-galactose were -9.39 kcal/mol and -7.07 kcal/mol respectively. A lower value indicates a more favorable binding, thus further confirming that D-galactose is poorer fit in the active site pocket of BCAI than L-arabinose.
Conversion of D-galactose to D-tagatose by using whole cells of recombinant E. coli
Since BCAI could isomerize D-galactose to D-tagatose, the feasibility of D-tagatose production was further studied. It was complicated to use purified enzyme as biocatalyst in industry. Instead, whole cells of recombinant E. coli was constructed and selected as a suitable biocatalyst for D-tagatose production.
Based on the above experiments, the time course of tagatose production at 150 g L-1 and 250 g L-1 galactose were performed under the optimal conditions. After 32 h biotransformation, the concentrations of D-tagatose were 48.1 g L-1 and 55.5 g L-1 respectively (Fig. 7d). The conversion rates were 32.1 and 22.2 % respectively. During the next 16 h, the conversion rate for 250 g L-1 D-galactose rose up a little to 27.2 %. The achieved conversion rates were attractive for industrial D-tagatose production. Although immobilized AIs from G. stearothermophilus, T. mathranii and T. neapolitana [28–30] had been used to produce D-tagatose before, the process in this study was easier to operate and due to the enzyme purification and immobilization steps were eliminated. It was a one-pot bioconversion process and introductions of high cell density cultivation and continuous reactors could hopefully improve its feasibility in the future.
In this study, an AI from B. coagulans NL01 was comprehensively studied. It showed a broad adaptability to moderate high temperatures. Its original dependency on added metallic ions such as Mn2+ was considerably low. Besides, molecular modelling of BCAI trimer combined with docking studies was used to understand its substrate specificity more deeply. Finally, a simple bioconversion system was established using whole cells of recombinant E. coli harboring BCAI as the biocatalyst. Attractive D-galactose conversion rates and D-tagatose productions were obtained.
Strains, plasmids and reagents
B. coagulans NL01 was stored in our lab and preserved at -80 °C . E. coli BL21 (DE3) was used as the expression host. Plasmid pEASY-Blunt (TransGen Biotech, China) and pETDuet-1 (Novagen) were used for gene cloning and gene expression respectively. FastPfu DNA polymerase was purchased from TransGen Biotech (China). HisTrap HP 5 mL column was from GE Health Life Science (USA). D-galactose, D-tagatose and L-arabinose were acquired from TCI (Japan) and L-ribulose was acquired from Carbosynth (United Kingdom).
Construction of recombinant E. coli
The B. coagulans L01 strain was cultured in LB medium for 12 h. Its genomic DNA was extracted by using TIANamp Bacteria DNA Kit (TIANGEN, Beijing) and then was used as the template DNA of PCR amplication. The primers used for cloning the araA gene were 5′-CGCGGATCCGATGTTGAAAATAAAAGA-3′ (forward primer) and 5′-CCGGAATTCTGTTAAAGAAGTGCATC-3′ (reverse primer). The underlined sequences represent restriction sites BamH I and EcoR I respectively. The PCR product was ligated with pEASY-Blunt cloning vector. The resulting recombinant plasmid was sequenced by BGI Tech. (Shanghai, China). Then, both the recombinant cloning plasmid and the expression vector pETDuet-1 were digested with BamH I and EcoR I, and the araA gene was cloned into the multiple cloning sites of pETDuet-1 to generate the recombinant expression plasmid, pETDuet-araA. Finally, the plasmid was transformed into E. coli BL21 (DE3) for expression.
Overexpression of the araA gene and enzyme purification
The E. coli BL21 (DE3) harboring pETDuet-araA gene was grown in LB medium with shaking at 37 °C until OD600 reaches 0.6-0.8. IPTG was added into the medium with a final concentration of 0.5 mM for the recombinant protein expression. After incubation at 25 °C and 200 rpm for 8 h, cells were harvested by centrifugation and resuspended in phosphate buffer solution (PBS, 50 mM, pH 7.4). Cell disruption was carried out by sonication and the obtained solution was centrifugated at 10,000 × g for 15 min at 4 °C to remove insoluble cell debris. The supernatant was used as crude cell extract. For the following purification, the crude extract was heated at 60 °C in order to remove host proteins. Then it was filtered by a 0.22-μm filtering membrane and loaded on a HisTrap HP 5 mL column and equilibrated with binding buffer (20 mM sodium phosphate, 500 mM sodium chloride, and 20 mM imidazole, pH 7.4). The target protein (BCAI) was eluted with 60 % binding buffer and 40 % elution buffer (20 mM sodium phosphate, 500 mM sodium chloride, 500 mM imidazole, pH 7.4). Purity of the protein was assessed by 11.25 % SDS-PAGE. Estimation of molecular mass of multimeric protein was carried out by using 4–16 % Native-PAGE. Gels were visualized by Coomassie Blue R250 staining. The expected band size of BCAI monomer and hexamer was predicted by Compute pI/Mw tool (http://web.expasy.org/compute_pi/). Purified BCAI was stored at 4 °C for biochemical property studies.
Enzyme assay and protein determination
The activity of BCAI was measured by determining the amount of formed keto sugar (L-ribulose or D-tagatose). Under standard conditions, 1 mL reaction mixture contained 100 mM L-arabinose or D-galactose, 50 mM Tris-HCl buffer (pH 7.5), 1 mM MnCl2 and 100 μL of enzyme solution at a suitable concentration. The reaction mixture was incubated at 60 °C for 20 min. Samples were cooled on ice for stopping the reaction. The amount of L-ribulose or D-tagatose was determined by cysteine-carbazole-sulfuric-acid method and the absorbance at 560 nm . One unit of AI acitivity was defined as the amount of enzyme producing 1 μmol keto sugar per min under the conditions above. The protein concentration was determined by the Bradford (Sigma) method using bovine serum albumin for calibration.
Effects of temperature, pH and metallic ion on purified BCAI
The effect of temperature on activity of purified BCAI was determined by testing the activities at temperatures from 40 °C to 90 °C at pH 7.5. The effect of pH was determined by testing the activities at pH 2 to 9 and the optimal temperature obtained above. Two buffer systems, disodium hydrogen phosphate-citric acid (50 mM, pH 2.2 to 7.0) and Tris-HCl (50 mM, pH 7.0 to 9.0) were used to get desired pH ranges.
To investigate the effect of metal ions on BCAI activity, purified BCAI was dialyzed against 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA at 25 °C for 3 h. Then, the buffer was changed to 50 mM Tris-HCl (pH 7.5) for another dialysis of 36 h. Metallic ions were added into reaction mixture containing EDTA-treated BCAI at a final concentration of 0.5 mM (or 0.5 to 4 mM when studying effect of Mn2+ concentration). Enzyme assay was carried out at the standard condition without adding other metallic ions.
To investigate the thermostability, the raw purified BCAI was divided into two groups. One is incubated without Mn2+ and the other is incubated in presence of 0.5 mM Mn2+. The incubations are at 60 to 90 °C and pH 7.5 for 120 min. The enzyme activity was measured under the standard condition without addition of Mn2+.
Determination of kinetic parameters
Kinetic parameters were determined using a 50 mM Tris-HCl buffer (pH 7.5), and 12.5 to 700 mM substrate (L-arabinose or D-galactose) and incubation for 20 min at 60 °C. The reaction was stopped by cooling on ice and the amount of L-ribulose or D-tagatose was determined by cysteine-carbazole-sulfuric-acid method.
Molecular modelling and docking studies
The template structures for comparative modelling were searched from RCSB PDB database (http://www.rcsb.org/). The structure of BCAI trimer was constructed with the MODELLER program and validated by the Profiles-3D tool in Discovery Studio 4.0 package (DS 4.0, BIOVIA, San Diego, CA). Then the protein structure typed with CHARMm  force field and the substrate structures typed with MMFF94 force field  were subjected to energy minimizations using Conjugate Gradient Descent algorithm. Then, the substrate molecules were docked into the binding pocket of BCAI by using CDOCKER module . The docking poses with the lowest interaction energy were selected for the analysis of orientation and binding interaction.
Optimization of D-tagatose transformation conditions using recombinant E. coli cells containing BCAI
10 mL reaction mixtures were prepared in a 50 mL centrifuge tube containing 50 mM Tris-HCl buffer (pH 7.5). The optimal cell concentration was determined by adding 0.6 to 6 g L-1 recombinant E. coli cells to the reaction mixtures and carrying out an incubation at 60 °C for 15 h. The optimal reaction temperature was determined by incubating the reaction mixtures at 40 °C to 80 °C with the optimal concentration of E. coli cells. Then, a gradient of D-galactose concentrations (50 to 250 g L-1) were set for investigating their effect on the conversion rate. The time courses were tested with the selected D-galactose concentrations at the optimal conditions. Samples were taken periodically and analyzed by a high-performance liquid chromatography (HPLC) system (Agilent 1200 series, USA).
The amount of D-galactose and D-tagatose was measured by a HPLC equipped with a Waters Sugar-pak1 column (6.5 × 300 mm) and a refractive index detector (SHIMADZU). Deionized water was used as mobile phase at a flow rate of 0.4 mL min-1 and a column temperature of 80 °C .
AI, L-arabinose isomerase; BCAI, Bacillus coagulans NL01 AI; DCW, Dry Cell Weight; EDTA, Ethylenediaminetetraacetic acid; HPLC, High-performance liquid chromatography; IPTG, Isopropyl β-D-thiogalactopyranoside; PCR, Polymerase Chain Reaction
We thank for Dr. Bingfang He from Nanjing Tech University for offering Discovery Studio Package 4.0 software.
The research project was financially supported by the National Natural Science Foundation of China (51561145015, 31300487), the Natural Science Foundation of Jiangsu Province of China (BK20130970) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Availability of data and materials
The dataset of sequences supporting the conclusions of this article is available in the GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Accession number of B. coagulans NL01 araA gene: KX356659. Protein ID of B. coagulans NL01 L-arabinose isomerase: ANJ21429.
WM participated in the design of the experiments, data analysis and writing the manuscript. LW carried out the experiments of whole cell catalysis; ZY participate in the experiments of molecular biology. ZZ participated in data analysis and modifying the manuscript; JO coordinated the whole project and completed the manuscript. All authors read and proved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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