Article pubs.acs.org/JAFC
Creation of Metal-Independent Hyperthermophilic L-Arabinose Isomerase by Homologous Recombination Young-Ho Hong,†,∥ Dong-Woo Lee,§,∥ Yu-Ryang Pyun,# and Sung Haeng Lee*,⊗ †
CJ Foods R & D, CJ Cheiljedang Corporation, Seoul 100-749, Korea Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea # Department of Biotechnology, Yonsei University, Seoul 120-749, Korea ⊗ Department of Cellular and Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-759, Korea §
S Supporting Information *
ABSTRACT: Hyperthermophilic L-arabinose isomerases (AIs) are useful in the commercial production of D-tagatose as a lowcalorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn2+-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAIbased hybrids containing the N-terminus (residues 1−89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn2+, indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of D-tagatose. KEYWORDS: L-arabinose isomerase, metal, thermostability, activity, chimera
■
and AI24 revealed that, although their primary structures were very different (73% similarity but have different temperature dependence. In addition, we included a variety of N- and C-terminally truncated mutants of thermophilic GSAI as model enzymes. Using these chimeric and truncated AIs we sought to address the following questions: (i) What is the difference between mesophilic and hyperthermophilic AIs in terms of metal requirements? (ii) What are the roles of metal ions in catalysis and thermostability? (iii) Do these roles overlap functionally and structurally, or are they separate? (iv)
Which regions are responsible for the differences in metal dependence of hyperthermophilic AIs compared to mesophilic AIs? With these aims in mind, we characterize the chimeric AIs and discuss here how the metal-mediated conformational changes and folding stability are related to the catalysis and structural thermostability of hyperthermophilic AIs compared to their mesophilic counterparts.
■
MATERIALS AND METHODS
Bacterial Strains and Culture Conditions. E. coli DH5α was used as host for the construction of hybrids and expression vectors, and E. coli BL21 (DE3) was used for expression. The plasmid pGEMT Easy vector (Promega, Madison, WI) was used as a cloning and sequencing vector, and pET-22b (+) (Novagen, San Diego, CA) was used for expression. To express the wild-type and mutant enzymes, E. coli BL21 cells transformed with appropriate expression constructs were grown at 37 °C in 1 L of Luria−Bertani (LB) medium containing 100 μg of ampicillin per milliliter, induced in midexponential phase (A600 = 0.6) with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), grown for an additional 5 h, and harvested by centrifugation (10000g at 4 °C for 20 min). Bacterial pellets were stored at −70 °C. Construction of Chimeric and Truncated AIs. To construct chimeric AIs, the expression vectors pET-BHAI, pET-GSAI, and pETTMAI containing B. halodurans, G. stearothermophilus, and T. maritima araA genes, respectively, were used as templates for polymerase chain reaction (PCR) as described previously.6,22 The hybridized AI genes were generated by recombination of two gene fragments from different AI coding sequences by PCR.38,39 For example, as shown in Figure 1, hybrid I, composed of 89 N-terminal amino acid residues of BHAI and 407 C-terminal residues of TMAI, was constructed as follows: two 12940
dx.doi.org/10.1021/jf203897a | J. Agric.Food Chem. 2011, 59, 12939−12947
Journal of Agricultural and Food Chemistry
Article
purified further using a Superdex 200 HR 12/60 gel filtration column pre-equilibrated with 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The purified enzymes were dialyzed against 20 mM Tris-HCl (pH 7.5) and stored at 4 °C. Protein concentrations were determined by the bicinchoninic acid method with bovine serum albumin as a standard. 40 Purity and size were analyzed by sodium dodecyl sulfate (SDS)−12.5% polyacrylamide gel electrophoresis (PAGE) and visualized with Coomassie Blue R-250.41 Native−PAGE was performed as described previously.22 Enzyme Assay. AI activity was assayed by measuring the increase in L-ribulose. Unless otherwise noted, the standard reaction mixture contained 50 mM 4-morpholinepropanesulfonic acid (MOPS) buffer (pH 7.5 at room temperature), 0.2 mL of enzyme preparation at a suitable dilution, 0.1 M L-arabinose, and distilled water to a final volume of 1.25 mL. Reactions were incubated at appropriate temperatures for 20 min and stopped by cooling on ice. L-Ribulose was quantified by the cysteine−sulfuric acid−carbazole method, 42 and the absorbance was measured at 560 nm. One unit of isomerase activity is defined as the amount of enzyme that produces 1 μmol of product per minute under the assay conditions. Physicochemical Characterization. Metal ions were removed from purified AIs (as isolated) by treatment with 10 mM EDTA at 25 °C for 4 h followed by dialysis against 10 mM Tris-HCl buffer (pH 7.5) overnight with several changes of buffer. The effect of metal ions on AI activity was determined by adding 1 mM Mn2+ to the dialyzed enzymes (apo protein), and AI activity was assayed as described above. The divalent metal content of the as-isolated and EDTA-treated samples was determined by high-resolution inductively coupled plasma (ICP)−mass spectrometry (Elan 6100, Perkin-Elmer) on a PlasmaQuad 3 instrument at Korea Basic Science Institute. The effect of divalent metal ions on the temperature dependence of AI was measured under the standard assay conditions except that the reaction temperatures ranged from 30 to 90 °C. Kinetics. The kinetic parameters of wild-type and mutant AIs were determined in the same reaction mixtures as described above, except that AIs were assayed over 1 min to obtain the initial reaction rates. The concentrations of L-arabinose and Mn2+ ranged from 0 to 40 mM and from 0 to 1 mM, respectively. Kinetic data were obtained by fitting data with a Michaelis−Menten equation using the Origin program (version 8.0). Circular Dichroism (CD). CD measurements were made with a Jasco J-810 spectropolarimeter with a Peltier temperature-controlled cuvette holder. Wild-type and mutant enzymes (0.3 mg/mL) were preincubated at 25 and 65 °C in 10 mM Tris-HCl buffer (pH 7.5) containing 100 mM L-arabinose with and without Mn2+ until no further CD signal was observed. The CD spectra of enzyme samples in a cuvette with a 0.1 cm path length were recorded in the far-UV region (190−240 nm). Scans were collected five times at 0.1 nm intervals with a 1 nm bandwidth. Each spectrum was corrected by subtracting the spectrum of the solution containing the buffer used, with and without Mn2+. Unfolding was monitored by CD ellipticity at 222 nm from 25 to 105 °C at a heating rate of 1 °C/min. Protein concentration was 0.3 mg/mL in 10 mM Tris-HCl (pH 7.5). The ratio of denatured to total protein in the transition range, fd = (εN − ε)/(εN − εU), was calculated from the CD signal ε relative to the signals of the native and unfolded baseline values of εN and εU, respectively. Melting temperature (Tm) is defined as the midpoint of AI unfolding.
overlapping gene fragments were amplified separately by PCR using two pairs of primers, BHAI-F (5′-CATATGATGTTACAAACGAAGCCA-3′), introducing a NdeI site (underlined), and internal TMAI-R (5′- ATGGAGAGTCCCCGTATCCACATCTTTGCA-3′), and internal BHAI-F (5′-ACGGGGACTCTCCATAAA-3′) and TMAI-R (5′-AAGCTTTCATCTTTTCA-AAAGCCCCCAG-3′), introducing a HindIII site (underlined). PCR was performed as described previously,6,22 and PCR products of the appropriate sizes were purified on 1% (w/v) agarose gel using a QIAEX II gel extraction kit (Qiagen GmbH, Hilden, Germany). Next, the amplified DNA fragments served as templates for amplifications to generate the complete hybrid gene by PCR. The second PCR was performed with 20 ng of each overlapping gene fragment from the previous amplifications, 200 μM dNTP mix, and 2.5 U of Turbo Pfu DNA polymerase in a total volume of 50 μL without primers. After initial denaturation for 4 min at 94 °C, six cycles (denaturation at 94 °C for 30 s, annealing at 37 °C, extension at 72 °C for 2 min) were run. After that, 10 pmol each of a pair of primers (BHAI-F and TMAI-R) was added to the second PCR mixtures, and then 25 cycles of the third PCR (denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 1 min), followed by a final extension step of 5 min at 72 °C, were performed to generate the full-length hybrid gene. The amplified PCR products were purified as described above, ligated into pGEM-T Easy Vector, and transformed into competent E. coli DH5α cells. Transformants containing the pGEM-T Easy Vector harboring the appropriate hybrid gene were selected on LB medium− ampicillin plates containing 0.01% 5-bromo-4-chloro-3-indolyl-β- Dgalactopyranoside (X-Gal). Plasmid DNA was isolated from the transformants and verified by sequencing. The plasmid DNA isolated was digested with NdeI and HindIII, purified, and ligated into the NdeI and HindIII sites of the pET-22b (+) vector to yield pET-hybrid I. This resulting product was transformed into E. coli BL21 (DE3). All hybrids were constructed as described above, and the primers used for the construction of hybrids are listed in Supplementary Table 1 of the Supporting Information. To construct truncated AIs, mutants were created by PCR using pET-GSAI as a template. The N-terminally truncated forms of GSAI were obtainted using the following forward primers, which introduced restriction sites (BamHI and NdeI). The Nterminal deletion mutants were created by deletion of the C-terminal sequence and introduction of a Met sequence for start codon using specific primers (Supplementary Table 1, Supporting Information). The C-terminally truncated mutants of GSAI were created by addition of five premature stop codons using reverse primers that have a HindIII site (Supplementary Table 1 of the Supporting Information). The PCR and molecular techniques for truncated mutants were carried out as described previously.6,22,38 AI Purification. The purification procedures for wild-type AIs were described previously.6,22 To purify various chimeric and truncated mutant AIs, cell pellets were resuspended in 50 mL of 20 mM Tris-HCl buffer (pH 8.0) and disrupted by sonication, and the lysates were centrifuged at 14000g for 20 min to remove cell debris. For thermostable wild-type and mutant AIs, the supernatants were heated at 70 °C for 20 min, and the denatured E. coli proteins were removed by centrifugation (20000g for 20 min). The resulting supernatants were filtered through 0.2 μm filters and loaded onto Hiprep 16/10 Q-XL columns (20 mL) equilibrated with 20 mM TrisHCl buffer (pH 8.0). The columns were washed with the same buffer, and a linear gradient of NaCl (from 0 to 1 M) was applied at 5 mL/ min. Next, ammonium sulfate ((NH4)2SO4) was added to the active pooled fractions to a final concentration of 1.5 M in 20 mM Tris-HCl buffer (pH 8.0), and the samples were applied to Phenyl-Sepharose columns (20 mL) equilibrated with 1.5 M (NH4)2SO4 in 20 mM TrisHCl buffer (pH 8.0). The absorbed proteins were eluted with a descending gradient (from 1.5 to 0 M (NH4)2SO4) at 0.5 mL/min. The pooled fractions containing AI activity were dialyzed overnight against 20 mM Tris-HCl buffer (pH 8.0) and applied to a Mono Q HR 5/5 column equilibrated with the same buffer at 0.5 mL/min. Elution was conducted with a linear gradient of NaCl (from 0 to 0.5 M). Active fractions were pooled and concentrated with an ultrafiltration device (10 kDa cutoff), and the concentrates were
■
RESULTS Generation of Functional AI Chimeras. According to the crystal structure of the ECAI monomer,24 AIs comprise three domains: the N-terminal domain (1−176 amino acids), central domain (177−327), and C-terminal domain (328− 500). Intriguingly, the crystal structure of ECAI suggests that the N-terminal domain of ECAI is involved in the region contacting other subunits.24,43 In addition, alignment of the amino acid sequences of various AIs revealed that the N12941
dx.doi.org/10.1021/jf203897a | J. Agric.Food Chem. 2011, 59, 12939−12947
Journal of Agricultural and Food Chemistry
Article
terminal (1−101 amino acids based on the numbering of ECAI), central (208−274), and C-terminal (430−497) regions of all the AIs were more variable than the remaining regions 6,24 (see Supplementary Figure 1 and Supplementary Table 2 in the Supporting Information). Therefore, to investigate whether the N- and C-terminal regions are involved in metal-mediated conformational changes and catalysis of hyperthermophilic AIs, we generated four chimeric AIs that had either their N-terminal or C-terminal regions replaced by the corresponding regions of their mesophilic counterparts (Figure 1A,B). Although we did not employ any rational computational strategy to minimize the possibility of disruption of parental residue−residue contacts in the recombinants, we used highly conserved regions selected by primary and secondary structural analyses as hybridization sites (Figure 1A,B). The four chimeric AIs expressed in E. coli BL21 (DE3) were purified in a catalytically active, soluble form as shown in Figure 1C. As expected, the molecular masses of the purified chimeric AIs were estimated to be 57 kDa as judged by SDS−PAGE (Figure 1C). Effect of Mn2+ on Chimeric AI Activity. Table 1 presents measurements of the isomerization activities of EDTA-treated
region of BHAI did not alter the metal dependence of a hyperthermophilic AI. In addition, although hybrid III (BH116/GS381), which contains the C-terminal residues of GSAI, had relatively low activity, it is clear that metal ions significantly enhanced the activity of its apo enzyme. Effect of Temperature on the Catalytic Activity of Chimeric AIs. Our previous thermodynamic analysis demonstrated that the apo and holo forms of hyperthermophilic AIs have different melting temperatures (Tm), indicating that metal ions contribute to the structural stability of AIs.44 Therefore, the temperature dependence of the activity profiles of the chimeras was compared with that of the wild-type AIs in the presence and absence of Mn2+. To obtain the apo enzymes (60 °C) in hybrid I but not in hybrid III (BH116/GS381), both originating from TMAI, displayed different metal dependencies and permitted us to draw two important conclusions: (1) the C-terminal region (∼70 residues) plays a key role in the metalmediated conformational changes but not in the structural stability of the enzymes; and (2) this region could be a key target for engineering hyperthermophilic AIs that no longer have metal requirements but retain activity at elevated temperatures. This is an important requirement for the biological production of D-tagatose in the food industry.6 This conclusion was supported by our observations of hybrids II (TM424/BH72) and IV (GS426/BH73) because hybrid IV (GS426/BH73) also retained activity at high temperatures (>55 °C) but no longer displayed metal-mediated conformational changes, whereas hybrid III (BH116/GS381), with the 116 N-terminal residues of BHAI, retained a well-defined thermophilic requirement for metal ions, like GSAI. Because the metal dependencees of their structural stability were quite different, it seems that the N-terminus plays a role in determining structural stability. Indeed, in contrast to hexameric E. coli AI, hyperthermostable AIs including mesophilic BHAI have tetrameric structures as their native forms,22 and when the N-terminal region was truncated by the deletion of six residues, in contrast to GSAI-CΔ20, which retains its native structure, their tetrameric structures disassembled to yield dimeric populations, as shown by GSAI-NΔ4 (Figure 4). This result strongly suggests that the N-terminus of thermostable AIs contributes to the oligomeric structure. These chimeras may provide guidelines for predicting the key residues responsible for specific functions. Because homologous AIs from hyperthermophilic and mesophilic organisms have nearly identical sequences and overall structures,6,24,46 it is difficult to identify the important structural differences between them. Nonetheless, the properties of the chimeras and
Figure 4. SDS−PAGE (left) and native−PAGE (right) analyses of GSAI and truncated mutant AIs. Purified recombinant proteins were analyzed by SDS−12.5% PAGE and native−10% PAGE as described previously.22 Construction of the truncated AIs is described under Materials and Methods.
clearly showed that GSAI-CΔ20 retained the native tetrameric structure of the wild-type enzyme, whereas GSAI-NΔ4 existed exclusively as dimers, suggesting that the N-terminal region of GSAI plays an important role in the stabilization of the tetrameric native structure of AI. Thus, these data strongly support that the N-terminal region of hyperthermophilic AIs contributes to the structural stability of the tetrameric enzymes.
■
DISCUSSION Catalytically functional chimeric AIs were generated by recombining fragments of parental genes encoding different AIs (BHAI, GSAI, and TMAI) that share >73% amino acid similarity (Supplementary Figure 1, Supporting Information).6,22 These chimeric enzymes were classified as mesophilic and hyperthermophilic on the basis of several criteria, including apparent Topt, metal requirement, conformational changes, apparent Tm, and kinetic parameters Km and Vmax. These data demonstrated that the hyperthermophilic chimeras, such as TMAI and GSAI, required divalent metal ions for folding at elevated temperatures, indicating that metal-mediated conformational changes of apo enzymes are prerequisite for catalytic activity and/or substrate binding. The results strongly suggest that metal-mediated conformational changes are essential for the proper folding of hyperthermophilic AIs, but not of their mesophilic counterparts. The metal dependence of the conformational changes did not show a close correlation to the thermostability of the hyperthermophilic AIs. Many studies6,12,22,25,45 indicate that 12945
dx.doi.org/10.1021/jf203897a | J. Agric.Food Chem. 2011, 59, 12939−12947
Journal of Agricultural and Food Chemistry
Article
(10) Cheetham, P. S. J.; Wootton, A. N. Bioconversion of D-galactose into D-tagatose. Enzyme Microb. Technol. 1992, 15, 105−108. (11) Cheng, L.; Mu, W.; Zhang, T.; Jiang, B. An L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization. Appl. Microbiol. Biotechnol. 2010, 86, 1089−1097. (12) Lee, S. J.; Lee, D. W.; Choe, E. A.; Hong, Y. H.; Kim, S. B.; Kim, B. C.; Pyun, Y. R. Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum. Appl. Environ. Microbiol. 2005, 71, 7888−7896. (13) Tewari, Y. B.; Steckler, D. K.; Goldberg, R. N. Thermodynamics of isomerization reactions involving sugar phosphates. J. Biol. Chem. 1988, 263, 3664−3669. (14) Vieille, C.; Hess, J. M.; Kelly, R. M.; Zeikus, J. G. xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl. Environ. Microbiol. 1995, 61, 1867−1875. (15) Farber, G. K.; Glasfeld, A.; Tiraby, G.; Ringe, D.; Petsko, G. A. Crystallographic studies of the mechanism of xylose isomerase. Biochemistry 1989, 28, 7289−7297. (16) Korndorfer, I. P.; Fessner, W. D.; Matthews, B. W. The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution. J. Mol. Biol. 2000, 300, 917−933. (17) Leang, K.; Takada, G.; Ishimura, A.; Okita, M.; Izumori, K. Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli. Appl. Environ. Microbiol. 2004, 70, 3298−3304. (18) Seemann, J. E.; Schulz, G. E. Structure and mechanism of Lfucose isomerase from Escherichia coli. J. Mol. Biol. 1997, 273, 256− 268. (19) Boulter, J. R.; Gielow, W. O. Properties of D-arabinose isomerase purified from two strains of Escherichia coli. J. Bacteriol. 1973, 113, 687−696. (20) Cho, E. A.; Lee, D. W.; Cha, Y. H.; Lee, S. J.; Jung, H. C.; Pan, J. G.; Pyun, Y. R. Characterization of a novel D-lyxose isomerase from Cohnella laevoribosii RI-39 sp. nov. J. Bacteriol. 2007, 189, 1655−1663. (21) van Staalduinen, L. M.; Park, C. S.; Yeom, S. J.; Adams-Cioaba, M. A.; Oh, D. K.; Jia, Z. Structure-based annotation of a novel sugar isomerase from the pathogenic E. coli O157:H7. J. Mol. Biol. 2010, 401, 866−881. (22) Lee, D. W.; Choe, E. A.; Kim, S. B.; Eom, S. H.; Hong, Y. H.; Lee, S. J.; Lee, H. S.; Lee, D. Y.; Pyun, Y. R. Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus. Arch. Biochem. Biophys. 2005, 434, 333−343. (23) Allen, K. N.; Lavie, A.; Glasfeld, A.; Tanada, T. N.; Gerrity, D. P.; Carlson, S. C.; Farber, G. K.; Petsko, G. A.; Ringe, D. Role of the divalent metal ion in sugar binding, ring opening, and isomerization by D-xylose isomerase: replacement of a catalytic metal by an amino acid. Biochemistry 1994, 33, 1488−1494. (24) Manjasetty, B. A.; Chance, M. R. Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production. J. Mol. Biol. 2006, 360, 297−309. (25) Patrick, J. W.; Lee, N. Purification and properties of an Larabinose isomerase from Escherichia coli. J. Biol. Chem. 1968, 243, 4312−4318. (26) Chouayekh, H.; Bejar, W.; Rhimi, M.; Jelleli, K.; Mseddi, M.; Bejar, S. Characterization of an L-arabinose isomerase from the Lactobacillus plantarum NC8 strain showing pronounced stability at acidic pH. FEMS Microbiol. Lett. 2007, 277, 260−267. (27) Rhimi, M.; Ilhammami, R.; Bajic, G.; Boudebbouze, S.; Maguin, E.; Haser, R.; Aghajari, N. The acid tolerant L-arabinose isomerase from the food grade Lactobacillus sakei 23K is an attractive D-tagatose producer. Bioresour. Technol. 2010, 101, 9171−9177. (28) Takata, G.; Poonperm, W.; Rao, D.; Souda, A.; Nishizaki, T.; Morimoto, K.; Izumori, K. Cloning, expression, and transcription
truncated mutants, together with detailed analysis of a multiplesequence alignment, has pointed to the idea that the role of metals in stabilizing AIs is separate from their effect on catalytic activity. This is supported by the finding that the irreversible unfolding of the chimeric enzymes is altered depending on the N-terminal region (89 residues) (see Tm values in Table 2), but not the intrinsic catalytic activities. Although the 3D structure of hyperthermophilic AI is not yet established, the fact that homologous mesophilic and hyperthermophilic AIs have different metal requirements for catalysis and stability strongly suggests that mimicking nature by using a region-specific evolution may be a feasible way to engineer hyperthermophilic AIs that have no metal requirement for industrial demands.
■
ASSOCIATED CONTENT
S Supporting Information *
Additional figure and tables. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
■
REFERENCES
Corresponding Author *Phone: +82-62-230-6381. Fax: +82-62-233-6337. E-mail:
[email protected]. Author Contributions ∥ These authors contributed equally to this work. Funding This work was supported in part by research funds from Chosun University (2008) and Grants 2009-0064846 and 2010-0020772 (the Happy Tech. program) from the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology to S.H.L. and the KRIBB Research Initiative Program, Daejeon, Korea, to D.W.L. (1) Oh, D. K. Tagatose: properties, applications, and biotechnological processes. Appl. Microbiol. Biotechnol. 2007, 76, 1−8. (2) Livesey, G.; Brown, J. C. D-Tagatose is a bulk sweetener with zero energy determined in rats. J. Nutr. 1996, 126, 1601−1609. (3) Espinosa, I.; Fogelfeld, L. Tagatose: from a sweetener to a new diabetic medication? Expert Opin. Invest. Drugs 2010, 19, 285−294. (4) Barili, P. L.; Berti, G.; Catelani, G.; D’Andrea, F.; Miarelli, L. New syntheses of D-tagatose and of 1,5-anhydro-D-tagatose from D-galactose derivatives. Carbohydr. Res. 1995, 274, 197−208. (5) Eeadle, J. R.; Saunders, J. P.; Wajda, T. J. Process for manufacturing tagatose. U.S. Patent 5078796, 1992. (6) Lee, D. W.; Jang, H. J.; Choe, E. A.; Kim, B. C.; Lee, S. J.; Kim, S. B.; Hong, Y. H.; Pyun, Y. R. Characterization of a thermostable Larabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima. Appl. Environ. Microbiol. 2004, 70, 1397−1404. (7) Hong, Y. H.; Lee, D. W.; Lee, S. J.; Choe, E. A.; Kim, S. B.; Lee, Y. H.; Cheigh, C. I.; Pyun, Y. R. Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing Larabinose isomerase from Thermotoga neapolitana. Biotechnol. Lett. 2007, 29, 569−574. (8) Jorgensen, F.; Hansen, O. C.; Stougaard, P. Enzymatic conversion of D -galactose to D -tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii. Appl. Microbiol. Biotechnol. 2004, 64, 816−822. (9) Lim, B. C.; Kim, H. J.; Oh, D. K. Tagatose production with pH control in a stirred tank reactor containing immobilized L-arabinose rom Thermotoga neapolitana. Appl. Biochem. Biotechnol. 2008, 149, 245−253. 12946
dx.doi.org/10.1021/jf203897a | J. Agric.Food Chem. 2011, 59, 12939−12947
Journal of Agricultural and Food Chemistry
Article
analysis of L-arabinose isomerase gene from Mycobacterium smegmatis SMDU. Biosci., Biotechnol., Biochem. 2007, 71, 2876−2885. (29) Prabhu, P.; Tiwari, M. K.; Jeya, M.; Gunasekaran, P.; Kim, I. W.; Lee, J. K. Cloning and characterization of a novel L-arabinose isomerase from Bacillus licheniformis. Appl. Microbiol. Biotechnol. 2008, 81, 283−290. (30) Banerjee, S.; Anderson, F.; Farber, G. K. The evolution of sugar isomerases. Protein Eng. 1995, 8, 1189−1195. (31) Kim, B. C.; Lee, Y. H.; Lee, H. S.; Lee, D. W.; Choe, E. A.; Pyun, Y. R. Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: bioconversion of D-galactose to Dtagatose using the enzyme. FEMS Microbiol. Lett. 2002, 212, 121−126. (32) Cheng, L.; Mu, W.; Jiang, B. Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation. J. Sci. Food Agric. 2010, 90, 1327−1333. (33) Rhimi, M.; Bejar, S. Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive Larabinose isomerase from the Bacillus stearothermophilus US100 strain. Biochim. Biophys. Acta 2006, 1760, 191−199. (34) Lehmann, M.; Wyss, M. Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution. Curr. Opin. Biotechnol. 2001, 12, 371−375. (35) Drummond, D. A.; Silberg, J. J.; Meyer, M. M.; Wilke, C. O.; Arnold, F. H. On the conservative nature of intragenic recombination. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5380−5385. (36) Thulasiram, H. V.; Erickson, H. K.; Poulter, C. D. Chimeras of two isoprenoid synthases catalyze all four coupling reactions in isoprenoid biosynthesis. Science 2007, 316, 73−76. (37) Carbone, M. N.; Arnold, F. H. Engineering by homologous recombination: exploring sequence and function within a conserved fold. Curr. Opin. Struct. Biol. 2007, 17, 454−459. (38) Yon, J.; Fried, M. Precise gene fusion by PCR. Nucleic Acids Res. 1989, 17, 4895. (39) Horton, R. M.; Hunt, H. D.; Ho, S. N.; Pullen, J. K.; Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 1989, 77, 61−68. (40) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76−85. (41) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680− 685. (42) Dische, Z.; Borenfreund, E. A new spectrophotometric method for the detection and determination of keto sugars and trioses. J. Biol. Chem. 1951, 192, 583−587. (43) Zhu, W.; Chance, M. R.; Manjasetty, B. A. Crystal structure of Mn2+-bound Escherichia coli L-arabinose isomerase (ECAI) and implications in protein catalytic mechanism and thermostability. J. Young Invest. 2007, 17 (www.jyi.org). (44) Lee, D. W.; Hong, Y. H.; Choe, E. A.; Lee, S. J.; Kim, S. B.; Lee, H. S.; Oh, J. W.; Shin, H. H.; Pyun, Y. R. A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic L -arabinose isomerases: the effects of divalent metal ions on protein stability at elevated temperatures. FEBS Lett. 2005, 579, 1261−1266. (45) Yamanaka, K. L-Arabinose isomerase from Lactobacillus gayonii. Methods Enzymol. 1975, 41, 458−461. (46) Jaenicke, R.; Bohm, G. The stability of proteins in extreme environments. Curr. Opin. Struct. Biol. 1998, 8, 738−748. (47) Greenfield, N. J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2006, 1, 2876−2890.
12947
dx.doi.org/10.1021/jf203897a | J. Agric.Food Chem. 2011, 59, 12939−12947
