Applied and Environmental Microbiology, July 2004, p . 4318-4325, Vol . 70, No . 7

The XIs can be classified into two groups, based on their size, amino acid sequence similarity, and divalent cation preference . Group I includes XIs from genera such as Streptomyces (15, 35, 38, 39), Actinoplanes (21, 29, 36), Thermus (9), and Arthrobacter (27, 37) . The average length of their polypeptide chains is 380 to 390 amino acids, and they have about 60% amino acid sequence identity, with the active-site residues being highly conserved . The enzymes from Klebsiella (14), Escherichia (22, 34), Lactobacillus (3, 4, 24, 40), Lactococcus (13), Clostridium (25), Bacillus (28), Staphylococcus (33), and Thermoanaerobacter (23) are classified in group II . They are 440 to 460 amino acids long and show more than 50% amino acid sequence identity among the group members . Although they share only 20 to 30% amino acid sequence identity with group I XIs, the active-site residues in group I and group II enzymes are highly conserved (Table 1) . The structures of monomeric, homodimeric, and homotetrameric XIs from a number of microbial sources including Streptomyces (5-7), Actinoplanes (1, 21, 29, 36), Arthrobacter (37), Bacillus (28), and Thermus (9) were solved by X-ray crystallography . A structural feature of monomeric XIs is that they consist of an (/ß)₈ barrel having an active site and a C-terminal loop region . There are two ways of forming homodimers (15, 17, 27) . One is the barrel-to-barrel facing way, in which the C-terminal loop of one subunit extends to the barrel domain of the other subunit, forming an active dimer . The active site of XI is completed when a conserved amino acid residue, Phe, is provided from the other subunit (37) . A dimer can also be formed when two subunits are parallel to and leaning against each other, forming an inactive dimer . In the inactive dimer conformation, the active sites of two subunits face in the opposite direction, with the result that they cannot share the Phe residue, while their C-terminal loops are forming the active dimers in the manner described above . The quaternary structure of a tetrameric enzyme is a dimer of an active dimer and an inactive dimer, and it can be dissociated into the dimers by mild treatment with a denaturant such as urea .

 
In the lactic acid bacteria, the ability to metabolize xylose is a function of their original habitats . Most of the xylose-metabolizing lactic acid bacterial genera such as Leuconostoc and Lactobacillus were isolated from fruits, vegetables, and fermented vegetables, in which D-xylose is the primary constituent of xylan and, therefore, is the primary carbon source for the microorganisms . On the other hand, only a few strains of Lactococcus lactis subsp . lactis, which has been used for dairy fermentation, have been reported as xylose-utilizing bacteria (18) . Some L . lactis subsp . lactis and Lactococcus lactis subsp . cremoris strains show only a trace of XI (13) . In previous studies, genotypic and phenotypic investigations into D-xylose metabolism were conducted with several L . lactis strains . L . lactis subsp . lactis strains CM56, MS39, 210, FB61, and 61 possessed a xylA gene but could not utilize D-xylose as a sole carbon source . Also L . lactis subsp . cremoris strains 160, BO32, and MS44 had a xylA gene of a larger than usual size due to an insertion, and they were all Xyl^– . Two strains, L . lactis subsp . lactis 210 and L . lactis subsp . lactis IO-1, were selected for further study .

L . lactis subsp . lactis 210, which is a commercial starter culture, does not utilize D-xylose but does produce an inactive XI, while L . lactis subsp . lactis IO-1, isolated from a kitchen sink, can grow on D-xylose as a sole carbon source . It produces an active XI whose expression is regulated by D-xylose (19) . In this study, the amino acid sequence differences between the XIs from strains IO-1 (XylA⁺) and 210 (XylA^–) were examined, and mutants of these enzymes were constructed to characterize the effects of the different residues on enzymatic activity and solubility .

Media and culture conditions.
E . coli was grown in Luria broth (Sigma, St . Louis, Mo.) plus chloramphenicol or ampicillin at 37°C with shaking . For induction, E . coli transformants were grown to an optical density at 600 nm of 0.5 to 0.6, and then isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 100 µM . The culture was incubated for another 3 h, and then cells were harvested . Lactococci were grown at 30°C without agitation . M17 medium (Difco, Detroit, Mich.) supplemented with 0.5% glucose was used to culture lactococcal strains . A mixture of 0.4% D-xylose and 0.1% D-glucose was used for induction of XI overexpression in lactococcal strains .

Reagents and chemicals.
All restriction enzymes were purchased from New England Biolabs (Beverly, Mass.) . T4 DNA ligase was from Gibco BRL Life Technologies (Grand Island, N.Y.), and AmpliTaq was from Perkin-Elmer (Foster City, Calif.) . Chemicals were obtained from Sigma Chemical Co .

PCR.
The primers used in this study are listed in Table 2 . The primers (NFX and BRX) used to amplify the wild-type and mutated xylA genes were designed on the basis of the nucleotide sequence of xylA from Lactobacillus brevis (3) . They included BamHI (BRX) or NcoI (NFX) restriction sites that are compatible with the multiple cloning site of pET-19b . The primers were synthesized at the Cornell University BioResource Center (Ithaca, N.Y.) . The 100-µl PCR mixture contained 1x PCR buffer (10 mM Tris-HCl [pH 8.8], 50 mM KCl, 1% Triton X-100), 4 µl of IO-1 or 210 crude cell lysate, 50 pmol of each primer, 5 nmol of deoxynucleoside triphosphate, 75 nmol of MgCl₂, and 4 U of AmpliTaq DNA polymerase . The PCR mixture was cycled in a Perkin-Elmer GeneAmp PCR System 2400 thermocycler with a program of one cycle of 4 min at 94°C; 30 cycles of 1 min at 94°C, 1 min at 55°C, and 1.5 min at 72°C; and a final cycle of 10 min at 72°C .

 
Cloning of wild-type and mutated xylA genes.
The 100-µl PCR products were purified using a Qiagen purification kit (Qiagen Inc., Chatsworth, Calif.) and resuspended in 50 µl of sterile water . About 20 µg of the purified PCR products was digested by the restriction enzymes BamHI and NcoI, in a final volume of 50 µl along with 800 ng of pET-19b at 37°C . The reaction mixture was purified, lyophilized, and resuspended in 11 µl of sterile water . Ligation mixture contained the digested 100 ng of PCR product and 10 ng of pET-19b, 1 U of T4 DNA ligase (Gibco, Gaithersburg, Md.), and 3 µl of 5x ligase buffer (250 mM Tris-HCl [pH 7.6], 50 mM MgCl₂, 5 mM dithiothreitol, 25% [wt/vol] polyethylene glycol 8000) and was incubated at 16°C for 20 h . The ligation mixture was transformed into competent E . coli BL21(DE3)pLysS, which was prepared by the calcium chloride method (30) .

All the clones were sequenced at the Cornell University BioResource Center using an ABI 377 automated DNA sequencer . Lasergene software (DNAStar, Inc., Madison, Wis.) was used to analyze the sequences .

Site-directed mutagenesis.
A two-step PCR method using megaprimers was used to conduct site-directed mutagenesis (31) . Briefly, oligonucleotides containing the mutated sequence(s) were synthesized and used in the first-round PCR with the primer BRX . The first-round PCR product, a partial xylA fragment containing the mutated sequence(s), was purified and used as a megaprimer in the second-round PCR with the forward primer NFX, generating a complete xylA gene containing the desired mutation . The conditions for the second PCR were modified by extending the annealing time from 1 to 4 min and by lowering the primer concentration from 50 to 10 pmol .

Enzyme purification.
The purification of the recombinant XIs overproduced in the E . coli strain was conducted according to the protocol of Yamanaka and Takahara (40) with modifications . The induced cells were harvested, washed, resuspended in a 50 mM triethanolamine (TEA) buffer (pH 7), and lysed by sonication (Heat Systems-Ultrasonics Inc., Farmingdale, N.Y.) . The lysed cells were centrifuged at 14,300 x g for 20 min . MnCl₂ was added to the supernatant to a final concentration of 70 mM, and the supernatant was held at 55°C for 10 min . The precipitate was removed by centrifugation at 12,000 x g for 20 min . The enzyme was precipitated with 60% saturation of ammonium sulfate . The precipitate was resuspended in 50 mM TEA buffer (pH 7) and dialyzed overnight against the same buffer containing 1 mM MnCl₂ . The dialyzed solution was applied to a Q Sepharose (Pharmacia, Uppsala, Sweden) fast flow ionic column . The fractions eluted between 0.2 M and 0.3 M NaCl were pooled and then applied to a Sephadex G-200 (1.5- by 90-cm) column (Pharmacia) . The active fractions eluted with 50 mM TEA were pooled and applied to the ionic column and gel filtration again for higher purification . The active fractions after the second round of the ionic column and gel filtration were pooled, dialyzed, and concentrated using an Ultrafree-4 centrifugal filter and tube (Millipore Corporation, Bedford, Mass.) .

Protein analysis.
A 1-ml aliquot of E . coli recombinant cells was centrifuged at 14,000 x g for 5 min and resuspended in 100 µl of a 50 mM TEA buffer (pH 7) containing 10 µl of lysozyme (50 mg/ml) . The suspension was incubated at 37°C for 1 h and centrifuged, and then the supernatant was saved for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The total protein concentration of the supernatant was measured using a Bio-Rad kit (Bio-Rad Laboratories, Richmond, Calif.) . About 5 µg of total protein was used for the SDS-PAGE and separated by electrophoresis (100 V for 90 min) on a 12% polyacrylamide gel .

For Western blotting, lactococcal strains IO-1 and 210 were harvested, washed, lysed, and used for SDS-PAGE . After separation, the proteins were transferred to a nitrocellulose membrane in a transfer buffer containing 20% (vol/vol) methanol, 3.03 g of Tris/liter, and 14.4 g of glycine/liter at 0.04 A overnight . The immunoblotting was performed in four steps with a washing step between each one . The membrane was washed in TBST (Tris-buffered saline [TBS] buffer plus 1% Tween 20) three times with agitation for 5 min each time and washed once in TBS (pH 7.5) (29.22 g of NaCl and 2.41 g of Tris per liter) for 5 min . Then, the membrane was blocked with 0.4% bovine serum albumin in TBS buffer at room temperature for an hour and washed . The blocked membrane was treated with 1:500-diluted primary antibody (rabbit anti-E . coli XI polyclonal antibody) in TBS for an hour at room temperature, washed, and incubated with 1:1,000-diluted goat anti-rabbit immunoglobulin G (alkaline phosphatase conjugate) in TBS buffer containing 0.4% bovine serum albumin . The membrane was washed and incubated in 10 ml of Sigma Fast 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium solution (0.15 mg of 5-bromo-4-chloro-3-indolylphosphate/ml, 0.3 mg of nitroblue tetrazolium/ml, 100 mM Tris buffer [pH 9.5], 5 mM MgCl₂) for 5 min, and the reaction was stopped by rinsing the membrane with water .

Enzyme assays.
XI activity was qualitatively measured using the cysteine-carbazole assay (12) . Whole-cell lysate was used, and the amount of xylulose produced was calculated with a standard curve which was made using different concentrations of xylulose . The K_m and k_cat values were determined by a coupled enzymatic assay with D-sorbitol dehydrogenase (Boehringer Mannheim, Indianapolis, Ind.) (20) . The reaction mixture contained 0.2 to 0.3 U of XI (1 U was defined as the amount of enzyme that converts 1 µM D-xylose to D-xylulose/min), 1 U of D-sorbitol dehydrogenase (1 U was defined as the amount of enzyme that oxidizes 1 µM NADH/min), 2 to 30 mM D-xylose, and 0.15 mM NADH (Boeringer Mannheim) . The oxidation rate of NADH was measured at 340 nm with a DU Series 600 spectrophotometer (Beckman Instruments, Fullerton, Calif.) . Kinetic parameters, K_m (millimolar) and k_cat (per second), were calculated from a 1/v versus 1/S plot .

 
Wild-type XIs of 210 and IO-1.
Cell crude extracts of lactococcal strains IO-1 and 210 induced by D-xylose were prepared, and the cell debris and supernatant were separated by centrifugation and then analyzed by SDS-PAGE . Western blotting showed that the XI enzymes were expressed in both strains (Fig . 1) . Western blotting, however, showed two differences between IO-1 and 210 . First, the total amount of 210 XI in the extract was less than that of IO-1 . Second, immunoreactive material was found in the cell debris but not in the supernatant from L . lactis subsp . lactis 210, suggesting that the enzyme is insoluble (Fig . 1, lanes 1 and 2) . Their activities were measured using the cysteine carbazole method . 210 XI displayed no activity compared to that of the blank, while IO-1 XI was active .

 
The IO-1 XI was purified, and the kinetics of the purified enzyme were measured . The native 210 XI could not be purified, because the protein always precipitated with the cell debris . The optimal temperature for IO-1 XI activity was around 65°C, and activity decreased dramatically above 74°C (Fig . 2) . The K_m and k_cat of purified IO-1 XI were 2.25 mM and 184/s, respectively (Table 4) .

 
The wild-type XIs that were cloned into pET-19b, designated pET-IO-xylA and pET-210-xylA, were expressed in E . coli . 210 XI expressed in E . coli precipitated with cell debris and was inactive, similar to the enzyme found in the native host strain, while IO-1 XI was soluble and active (Table 3; Fig . 3C, lane 3) . 210 XI was never found in a soluble form despite modification of induction conditions such as reducing the agitation speed, shortening the induction time, lowering the temperature, and decreasing the IPTG concentration .

 
XI mutants of 210 and IO-1.
Among the six amino acids that differ between the 210 and IO-1 XIs, five residues (R202, Y218, V275, T388, and E407) of 210 XI were individually changed to the corresponding residue from IO-1 XI by site-directed mutagenesis, and the effects on activity and solubility of 210 XI were examined . Ala247 of 210 XI was not changed because Ala is the conserved residue in group II XIs . None of the mutants gained any detectable activity except the 210 XI V275A mutant . This mutant showed about 24% relative activity compared to the wild-type IO-1 XI activity (Table 3) . Three different 210 XI double mutants, R202M/Y218D, R202M/V275A, and Y218D/V275A, were constructed . The R202M/V275A and Y218D/V275A mutants showed levels of activities similar to that of the V275A single mutant, 26 and 24%, respectively . The R202M/Y218D mutant did not gain any activity (Table 3) . A triple 210 XI mutant, R202M/Y218D/V275A, was constructed . It showed about 60% relative activity and was soluble (Table 3; Fig . 3B, lane 7) . The K_m of the mutant was 3.46 mM, and its k_cat was 142/s (Table 4) .

Several IO-1 XI single mutants including S247A, S388T, and K407E were constructed . The S388T mutation in IO-1 XI resulted in a complete loss of its activity (Table 3), and the mutant protein was insoluble (Fig . 3C, lane 5) . To confirm the absence of any unintended secondary mutations, the S388T mutation was mutated back to T388S, which resulted in restoration of full activity to IO-1 XI . Residues 247 and 407 are the only differences between the S388T IO-1 mutant and the 210 XI R202M/Y218D/V275A mutant (Table 3) . These differences therefore define the activity and solubility of the 210 XI R202M/Y218D/V275A mutant and IO-1 XI S388T mutant . While the former is an active, soluble protein, the latter is inactive and insoluble . To examine the effect of the two residues, a second mutation (either S247A or K407E) was added to the IO-1 XI S388T mutant . Two double mutants of IO-1 XI, S247A/S388T and S388T/K407E, showed 27 and 50% relative activity, respectively (Table 3) . Also, the IO-1 XI S388T/K407E mutant clearly produced a soluble protein (Fig . 3C, lane 8) . The K_m and k_cat of the IO-1 XI S388T/K407E mutant were 4.4 mM and 124/s, respectively (Table 4) . On the other hand, the single and double mutations S247A and K407E had no significant effect on the activity and solubility of IO-1 XI (Table 3; Fig . 3C, lane 9) .

The 210 XI always precipitated with cell debris when it was expressed in the native lactococcal strain or in E . coli (Fig . 3A, lane 3) . All of the inactive 210 mutant XIs and the inactive IO-1 XI S388T mutant expressed in E . coli also precipitated with cell debris (Fig . 3) . Expression in E . coli under alternative induction conditions (lower IPTG or lower temperature) did not yield soluble protein (data not shown) . It is therefore difficult to separate the lack of activity of this protein from its insolubility .

The crystallographic structures of a few group II XIs have been submitted to the Protein Data Base (PDB) (PDB accession numbers 1A0C, 1A0D, and 1A0E) . The active-site residues and residues mediating subunit interactions in the group I XIs are conserved in the group II XIs (2, 14, 25, 32) . The active-site residues of the Streptomyces rubiginosus XI (38) are conserved through the 20 group I and II XIs including the 210 and IO-1 XIs (Table 1) . In addition, the residues (D24, R140, L200, A201, and A224) having a role in the subunit interactions are also conserved or similar, except that the 210 XI has V275 instead of A224 (Table 1) . The three-dimensional structure of a monomeric 210 XI was predicted by SwissProt homology modeling (http://www.expasy.org/spdbv/text/modeling) with the XIs of Thermoanaerobacter and Thermotoga as templates (Fig . 4) . Superimposition of the -backbones of the predicted 210 XI and those of Thermoanaerobacter and Thermotoga XIs showed no significant deviation between them (data not shown) . Also the six amino acids that are different between 210 and IO-1 do not appear to be directly involved in substrate or metal binding . However, these residues could affect the subunit binding . The alignment suggests that the structure of 210 and IO-1 XI is similar to those of other group I and II XIs and that the influence of the six amino acid differences on activity is not a result of a catalytic defect but more likely a significant structural perturbation .

 
Residue A275 of IO-1 XI is highly conserved in group I and II XIs, and the corresponding residue in group I XIs (for example, A224 of the Streptomyces) is located in a turn structure and is involved in subunit interaction that leads to an active tetramer (5) . The 210 XI has, however, Val instead of Ala at the corresponding residue, and Val is least likely to promote a turn in the protein structure (10) . The V275 might destabilize the turn structure or the subunit interactions or both, inhibiting the formation of an active tetramer (Table 3) .

Neither the single nor the double mutation of residues 202 and 218 of 210 XI gave rise to an active, soluble enzyme . When the two mutations were combined with the V275A mutation, however, there was a significant increase in the activity and solubility of 210 XI (Table 3; Fig . 3B, lane 7) . The prevalent amino acid at position 202 in the group II XIs is Met, while an Arg is found in 210 XI . The predominant amino acid at position 218 is Asp or Asn in the group I and II XIs, while the 210 XI has Tyr . The R group of Tyr is bulky and hydrophobic compared to Asp or Asn and cannot participate in electrostatic interactions . Although these two residues do not directly contribute to the activity and solubility of the group I and II XIs, all three residues (202, 218, and 275) appear to be important for enzymatic activity and solubility and changes to them rendered the 210 XI soluble and active .

Amino acid residues 247 and 407 distinguish the 210 XI R202M/Y218D/V275A mutant from the IO-1 XI S388T mutant . When A247 and E407 are present in the 210 XI R202M/Y218D/V275A mutant, the enzyme is soluble and active, while S247 and K407 resulted in an inactive and insoluble protein in the IO-1 XI S388T mutant (Fig . 3B, lane 7, and 3C, lane 5) . The S247A/K407E mutation suppressed the defective S388T mutation in IO-1, restoring the activity and solubility . To determine which of these two residues is responsible for the suppression, the S247A and K407E mutations were added to the IO-1 S388T mutant, separately . The IO-1 XI S388T/K407E mutant showed significantly increased activity and solubility, while the IO-1 XI S247A/S388T mutant showed only slightly increased activity (Table 3; Fig . 3C, lanes 7 and 8) . Therefore, the K407E mutation suppressed the S388T mutation on the IO-1 XI . Amino acid residues 388 and 407 are located in the C-terminal loop that plays an important role in the homologous oligomerization of the subunits, leading to an active tetramer (29) . This suggests that the S388T mutation on the IO-1 XI probably causes some critical structural defect which results in an insoluble and inactive protein . K407E did not affect the activity or the solubility of wild-type IO-1 XI (Table 3; Fig . 3C, lane 6) . Probably the K407E mutation compensates for the predicted volumetric distortion in the structure as a result of the S388T mutation .

In conclusion, the 210 XI seems to have lost its activity and solubility due in part to the cumulative effect of the three mutations R202/Y218/V275 . The S388 residue of the IO-1 XI appears to play a critical role in maintaining enzymatic activity and solubility . There may exist an interaction between the 388 and 407 residues, and the K407E mutation suppresses the S388T mutation .

Present address: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853 .

1. Allen, K . N., A . Lavie, A . Glasfeld, T . N . Tanada, D . P . Gerrity, S . C . Carlson, G . K . Farber, G . A . Petsko, and D . Ringe. 1994 . 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 33:1488-1494.
2. Batt, C . A., A . C . Jamieson, and M . A . Vandeyar. 1990 . Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase . Proc . Natl . Acad . Sci . USA 87:618-622.
3. Bor, Y.-C. 1993 . Cloning and characterization of xylose operon from Lactobacillus brevis . Ph.D . dissertation . Cornell University, Ithaca, N.Y.
4. Bor, Y.-C., C . Moraes, S . Lee, W . L . Crosby, A . J . Sinskey, and C . A . Batt. 1992 . Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase . Gene 114:127-131.
5. Carrell, H . L., B . H . Rubin, T . J . Hurley, and J . P . Glusker. 1984 . X-ray crystal structure of D-xylose isomerase at 4 Å resolution . J . Biol . Chem . 259:3230-3236.
6. Carrell, H . L., H . Hoier, and J . P . Glusker. 1994 . Modes of binding substrates and their analogues to the enzyme D-xylose isomerase . Acta Crystallogr . D 50:113-123.
7. Carrell, H . L., J . P . Glusker, V . Burger, F . Manfre, D . Tritsch, and J.-F . Biellmann. 1989 . X-ray analysis of D-xylose isomerase at 1.9 Å: native enzyme in complex with substrate and with a mechanism-designed inactivator . Proc . Natl . Acad . Sci . USA 86:4440-4444.
8. Chaillou, S., Y.-C . Bor, C . A . Batt, P . W . Postma, and P . H . Pouwels. 1998 . Molecular cloning and functional expression in Lactobacillus plantarum 80 of xylT encoding the D-xylose-H⁺ symporter of Lactobacillus brevis . Appl . Environ . Microbiol . 64:4720-4728.
9. Chang, C., B . C . Park, D.-S . Lee, and S . W . Suh. 1999 . Crystal structures of thermostable xylose isomerases from Thermus caldophilus and Thermus thermophilus: possible structural determinants of thermostability . J . Mol . Biol . 288:623-634.
10. Creighton, T . E. 1993 . Proteins, 2nd ed . W . H . Freeman and Company, New York, N.Y.
11. Davis, E . O., and P . J . F . Henderson. 1987 . The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli K12 . J . Biol . Chem . 262:13928-13932.
12. Dische, Z., and E . Borenfreund. 1951 . A new spectrophotometric method for the detection and determination of keto sugars and trioses . J . Biol . Chem . 192:583-587.
13. Erlandson, K . A. 1999 . Strain-specific detection and characterization of the xyl and xyn loci in Lactococcus lactis . Ph.D . dissertation . Cornell University, Ithaca, N.Y.
14. Feldmann, S . D., H . Sahm, and G . A . Sprenger. 1992 . Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12 . Mol . Gen . Genet . 234:201-210.
15. Gailwad, S., H . Rao, and V . Deshpande. 1993 . Structure-function relationship of glucose/xylose isomerase from Streptomyces: evidence for the occurrence of inactive dimer . Enzyme Microb . Technol . 15:155-157.
16. Heath, E . C., J . Hurwitz, B . L . Horecker, and A . Ginsburg. 1958 . Pentose fermentation by Lactobacillus plantarum . I . The cleavage of xylulose-5-phosphate by phosphoketolase . J . Biol . Chem . 231:1008-1029.
17. Hess, J . M., V . Tchernajenko, C . Vielle, J . G . Zeikus, and R . M . Kelly. 1998 . Thermotoga neapolitana homotetrameric xylose isomerase is expressed as a catalytically active and thermostable dimer in Escherichia coli . Appl . Environ . Microbiol . 64:2357-2360.
18. Ishizaki, A., and T . Ohta. 1989 . Batch culture kinetics of L-lactate fermentation employing Streptococcus sp . IO-1 . J . Ferment . Bioeng . 67:46-51.
19. Kahl, W . E. 1997 . Variation in D-xylose metabolism among Lactococcus lactis strains . M.S . thesis . Cornell University, Ithaca, N.Y.
20. Kersters-Hilderson, H., M . Callens, W . Vangrysperre, and C . K . De Bruyne. 1986 . Kinetic characterization of D-xylose isomerases by enzymatic assays using D-sorbitol dehydrogenase . Enzyme Microb . Technol . 9:145-148.
21. Lambeir, A.-M., M . Lauwereys, P . Stanssens, N . T . Mrabet, J . Snauwaert, H . van Tilbeurgh, G . Mattyssens, I . Lasters, M . De Maeyer, S . J . Wodak, J . Jenkins, M . Chiadmi, and J . Janin. 1992 . Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis . 2 . Site-directed mutagenesis of the xylose binding site . Biochemistry 31:5459-5466.
22. Lawlis, V . B., M . S . Dennis, E . Y . Chen, D . H . Smith, and D . J . Henner. 1984 . Cloning and sequencing of the xylose isomerase and xylulokinase genes of Escherichia coli . Appl . Environ . Microbiol . 47:15-21.
23. Liu, S.-Y., J . Wiegel, and F . C . Gherardini. 1996 . Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 289 . J . Bacteriol . 178:5938-5945.
24. Lokman, B . C., P . Van Santen, J . C . Verdoes, J . Kruse, R . J . Leer, M . Posno, and P . H . Pouwels. 1991 . Organization and characterization of the three genes involved in D-xylose catabolism in Lactobacillus pentosus . Mol . Gen . Genet . 230:161-169.
25. Meaden, P . G., J . Aduse-Opoku, J . Reizer, A . Reizer, Y . A . Lanceman, M . F . Martin, and W . J . Mitchell. 1994 . The xylose isomerase-encoding gene (xylA) of Clostridium thermosaccharolyticum: cloning, sequencing and phylogeny of XylA enzymes . Gene 141:97-101.
26. Mortlock, R . P. 1984 . The development of catabolic pathways for the uncommon aldopentose . Plenum Publishing Corporation, New York, N.Y.
27. Rangarajan, M., B . Asboth, and B . S . Hartley. 1992 . Stability of Arthrobacter D-xylose isomerase to denaturants and heat . Biochem . J . 285:889-898.
28. Rasmussen, H., T . La Cour, J . Nyborg, and M . Schulein. 1994 . Crystallization and preliminary investigation of xylose isomerase from Bacillus coagulans . Acta Crystallogr . D 50:231-233.
29. Rey, F., J . Jenkins, J . Janin, I . Lasters, P . Alard, M . Classens, G . Mattyssens, and S . Wodak. 1988 . Structural analysis of the 2.8 Å model of xylose isomerase from Actinoplanes missouriensis . Proteins Struct . Funct . Genet . 4:165-172.
30. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
31. Sarkar, G., and S . Sommer. 1990 . The "megaprimer" method of site-directed mutagenesis . BioTechniques 8:404-407.
32. Schmiedel, D., M . Kintrup, E . Kuster, and W . Hillen. 1997 . Regulation of expression, genetic organization and substrate specificity of xylose uptake in Bacillus megaterium . Mol . Microbiol . 23:1053-1062.
33. Sizemore, C., E . Buchnere, T . Rygus, C . Witke, F . Goetz, and W . Hillen. 1991 . Organization, promoter analysis and transcriptional regulation of the Staphylococcus xylosus xylose utilization operon . Mol . Gen . Genet . 227:377-384.
34. Song, S., and C . Park. 1997 . Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator . J . Bacteriol . 179:7025-7032.
35. Suekane, M., M . Tamura, and C . Tomimura. 1978 . Physicochemical and enzymatic properties of purified glucose isomerase from Streptomyces olivochromogenes and Bacillus stearothermophilus . Agric . Biol . Chem . 42:909-917.
36. Tilbeurgh, H., J . Jenkins, M . Chiadmi, J . Janin, S . J . Wodak, N . T . Mrabet, and A.-M . Lambeir. 1992 . Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis . 3 . Changing metal specificity and the pH profile by site-directed mutagenesis . Biochemistry 31:5467-5471.
37. Varsani, L., T . Cui, M . Rangarajan, B . S . Hartley, J . Goldberg, C . Collyver, and D . M . Blow. 1993 . Arthrobacter D-xylose isomerase: protein-engineered subunit interface . Biochem . J . 291:575-583.
38. Whitaker, R . D. 1995 . Kinetic and structural studies of the Streptomyces rubiginosus D-xylose isomerase . Ph.D . dissertation . Cornell University, Ithaca, N.Y.
39. Wong, H . C., Y . Ting, H . C . Lin, F . Reichert, K . Myambo, K . W . K . Watt, P . L . Toy, and R . J . Drummond. 1991 . Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus . J . Bacteriol . 173:6849-6858.
40. Yamanaka, K., and N . Takahara. 1977 . Purification and properties of D-xylose isomerase from Lactobacillus xylosus . Agric . Biol . Chem . 41:1909-1915.

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