The present invention describes the cloning and expression of a hyperthermostable xylose isomerase gene in an industrial host and the heat facilitated purification of the enzyme. The hyperthermostable enzyme has unique features sought by the fructose syrup industry.

FIELD OF THE INVENTION 
The present invention relates to enzymes. More particularly it relates to 
enzymes known as xylose isomerases or glucose isomerases. 
BACKGROUND OF THE INVENTION 
Xylose isomerase (EC 5.3.1.5) is an enzyme that catalyzes the reversible 
isomerization of D-xylose into D-xylose. It is also called glucose 
isomerase because of its ability to isomerase D-glucose into D-fructose, 
and it is therefore widely used in industry for the production of 
high-fructose corn syrup (HFCS). 
The isomerization of glucose reaches an equilibrium which is shifted 
towards fructose at high temperatures. Presently, the industrial 
isomerization process is performed at temperatures of about 58.degree. to 
60.degree. C., using moderately thermostable xylose isomerases, to produce 
40-42% fructose syrup. An additional step of chromatography allows the 
production of 55% fructose rich HFCS, which has a higher sweetening power 
than sucrose. 
Performing the isomerization at 90.degree. or 95.degree. C. would make 
possible the production of syrups containing 55% fructose, without the 
last chromatography step, but the half-life of the enzymes used today at 
those higher temperatures does not permit increasing the temperature of 
the isomerization reaction. Another advantage to performing the 
isomerization at high temperatures would be that it would decrease the 
risks of microbial contamination of the reactor. 
Previous attempts have been made to obtain more thermostable xylose 
isomerases, either by site-directed mutagenesis of moderately thermostable 
xylose isomerases, or by screening highly thermophilic organisms for 
xylose isomerase activity. However, none of those attempts have resulted 
in commercially useful hyperthermostable xylose isomerases. 
It obviously would be advantageous to have a hyperthermostable xylose 
isomerase and an efficient method of producing that enzyme in quantity. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to disclose a novel 
hyperthermostable xylose isomerase. 
It is a further object to disclose a method of producing the enzyme 
employing DNA encoding for the enzyme, plasmids containing the DNA, and 
bacteria into which the plasmids have been inserted and which produce the 
enzyme. 
It is a still further object to disclose a method of making fructose using 
the novel hyperthermostable xylose isomerase. 
The present invention relates to a hyperthermostable xylose isomerase, 
which is obtained from the microorganism Thermotoga neapolitana (DSM No. 
5068) and mutants thereof. The enzyme has the amino acid sequence SEQ ID 
NO:1 which is: 
__________________________________________________________________________ 
1 MAEFFPEIPK 
VQFEGKESTN 
PLAFKFYDPE 
EIIDGKPLKD 
HLKFSVAFWH 
51 
TFVNEGRDPF 
GDPTADRPWN 
RYTDPMDKAF 
ARVDALFEFC 
EKLNIEYFCF 
101 
HDRDIAPEGK 
TLRETNKILD 
KVVERIKERM 
KDSNVKLLWG 
TANLFSHPRY 
151 
MHGAATTCSA 
DVFAYAAAQV 
KKALEITKEL 
GGEGYVFWGG 
REGYETLLNT 
201 
DLGFELENLA 
RFLRMAVDYA 
KRIGFTGQFL 
IEPKPKEPTK 
HQYDFDVATA 
251 
YAFLKSHGLD 
EYFKFNIEAN 
HATLAGHTFQ 
HELRMARILG 
KLGSIDANQG 
301 
DLLLGWDTDQ 
FPTNVYDTTL 
AMYEVIKAGG 
FTKGGLNFDA 
KVRRASYKVE 
351 
DLFIGHIAGM 
DTFALGFKVA 
YKLVKDGVLD 
KFIEEKYRSF 
REGIGRDIVE 
401 
GKVDFEKLEE 
YIIDKETIEL 
PSGKQEYLES 
LINSYIVKTI 
LELR. 
__________________________________________________________________________ 
Thermotoga is a highly thermophilic organism, originating from geothermally 
heated sea floors, and deeply branched in eubacteria. A hyperthermostable 
xylose isomerase has been purified from the species T. maritima and has 
been shown to have an optimal activity at 105.degree.-110.degree. C. 
The T. neapolitana which produces the enzyme of the present invention has 
an optimum growth temperature of about 77.degree. C. and its growth rate 
is higher than 50% of the maximum at temperatures from 64.degree. to 
87.degree. C. (1). Its optimum growth pH is 7.5, and its growth rate is 
higher than 50% of the maximum at pHs from 6.0 to 9.0. 
The xylose isomerase that is produced by T. neapolitana is optimally active 
at 97.degree. C. and it retains 40% of its activity after incubation at 
90.degree. C. for 2 hours in the absence of substrate. It retains 60% of 
its activity after incubation at 80.degree. C. for 2 hours in the absence 
of substrate. It retains 55% of its activity after incubation at 
70.degree. C. for 4 hours in the absence of substrate and 90% of its 
activity after incubation at 50.degree. C. for 4 hours in the absence of 
substrate. 
The xylose isomerase retains 100% of its activity at pHs of from 6.8 to 7.3 
for at least 30 minutes. It has an optimum pH for activity of 7.1, but 
also shows: 
More than 70% activity between pHs 5.8 and 7.7 
More than 80% activity between pHs 6.1 and 7.6 
More than 90% activity between pHs 6.6 and 7.4 
The xylose isomerase of the present invention has the following additional 
physiochemical properties: 
(1) Reactivity: It isomerizes xylose to produce D-xylulose and D-glucose to 
produce D-fructose. 
(2) Activity: It has a specific activity of greater than 21 U/mg of 
purified enzyme. 
(3) The enzyme is composed of identical subunits of about 50,000 daltons, 
as measured by SDS polyacrylamide gel electrophoresis. 
The preferred method of the present invention for producing the enzyme, 
comprises, isolating and purifying chromosomal DNA from T. neapolitana 
strain 5068, partially digesting the chromosomal DNA with a restriction 
enzyme, ligating the DNA into a plasmid vector, transforming the E. coli 
with the ligation mixture, growing the E. coli and isolating the enzyme 
from the E. coli. The enzyme can then be purified by heating it to about 
90.degree. C. for about 150 minutes. Most E. coli proteins are denatured 
during this treatment and the last E. coli proteins can be eliminated by 
chromatographic steps to obtain the purified xylose isomerase. 
The novel isolated hyperthermostable xylose isomerase gene has the 
nucleotide sequence of SEQ ID NO:2. The nucleotide sequence SEQ ID NO:2 
is: 
__________________________________________________________________________ 
1 
GTCGACGCAA 
AGGTCGTGAC 
GGGTGGAAAC 
ATAAACGTTC 
AGCTGGGAAC 
51 
TGTGTCCTCG 
GCTGCTGTTG 
AAGGAACATA 
CGTTATCGAA 
GTTGGACAAT 
101 
TCTCTGGAAC 
GGTCACATCC 
GAGCTTGATG 
TCAAGATCCG 
CCGTTGTCCT 
151 
CAGCACCCCT 
TCCGTACACC 
CTGTCATCCT 
TCACAACGGG 
GATGAAGGGA 
201 
TCCGTTTCCC 
ACAGCGAAAG 
ATCCCCTGGT 
GGAACGGTGT 
CTATGTGTGT 
251 
CACTATCCAC 
AATGTTTTGC 
TTCTGTCCCT 
GCCGGGAATG 
ATTGCAAGCA 
301 
GATTCGACCT 
CCAAATTCCG 
TTCTGGTCTT 
TTGTGTCATG 
ACGCTCAACA 
351 
GTGTATCCCA 
TCTTTTTGAG 
AAGTTCCTCC 
AGCCAGTCGG 
CCTTCTCTTT 
401 
CTCTCCAGGT 
CCACCGAAGA 
CTGGATTCAC 
CGAATTGATC 
GATATGAACC 
451 
TTTTCAGCGA 
ATCTACCATT 
TCGTCTTTCA 
ATTCTTCTAT 
CTTTCTTGTT 
501 
ATCTCCATCT 
GAAACACCTC 
CCAAGTACAA 
GTATATCTCT 
CCAAAAAAAT 
551 
ATTTGAAATG 
ACCCCAGGGA 
ATTTTATATA 
ATTGATTGAT 
AGAAAAAATT 
601 
TAGGGAGGTG 
TTCACATGGC 
TGAATTCTTT 
CCAGAAATCC 
CGAAAGTGCA 
651 
GTTCGAAGGC 
AAAGAAAGCA 
CAAATCCACT 
TGCGTTCAAG 
TTCTACGATC 
701 
CAGAAGAGAT 
CATCGACGGC 
AAACCCCTCA 
AGGACCATCT 
GAAGTTCTCC 
751 
GTTGCCTTCT 
GGCACACCTT 
CGTGAACGAG 
GGAAGGGATC 
CCTTCGGAGA 
801 
CCCAACGGCC 
GATCGTCCCT 
GGAACAGGTA 
CACCGATCCC 
ATGGACAAGG 
851 
CTTTTGCAAG 
GGTGGACGCC 
CTTTTTGAAT 
TCTGCGAAAA 
ACTCAACATC 
901 
GAGTACTTCT 
GCTTCCACGA 
CAGAGACATC 
GCTCCCGAGG 
GAAAAACGCT 
951 
GAGGGAGACA 
AACAAAATTT 
TGGACAAAGT 
AGTGGAGAGA 
ATCAAAGAGA 
1001 
GAATGAAAGA 
CAGCAACGTG 
AAGCTCCTCT 
GGGGTACTGC 
AAACCTCTTT 
1051 
TCCCACCCAA 
GGTACATGCA 
TGGTGCAGCG 
ACAACCTGCA 
GTGCTGATGT 
1101 
TTTTGCGTAC 
GCGGCCGCCC 
AGGTGAAAAA 
AGCCCTTGAG 
ATCACCAAAG 
1151 
AACTTGGAGG 
AGAAGGGTAC 
GTCTTCTGGG 
GTGGAAGAGA 
AGGATACGAA 
1201 
ACACTCCTCA 
ACACGGACCT 
TGGATTCGAA 
CTTGAAAACC 
TCGCCCGCTT 
1251 
CCTCAGAATG 
GCTGTGGATT 
ATGCAAAAAG 
GATCGGTTTC 
ACCGGACAGT 
1301 
TCCTCATCGA 
ACCAAAACCG 
AAAGAACCCA 
CCAAACACCA 
GTACGACTTC 
1351 
GACGTTGCAA 
CCGCCTATGC 
CTTCCTGAAG 
AGCCACGGTC 
TCGATGAATA 
1401 
CTTCAAATTC 
AACATCGAGG 
CAAACCACGC 
CACACTCGCC 
GGTCACACCT 
1451 
TCCAGCACGA 
ACTGAGAATG 
GCAAGGATCC 
TTGGAAAACT 
CGGAAGCATC 
1501 
GATGCAAACC 
AGGGAGACCT 
TCTTCTTGGA 
TGGGACACCG 
ATCAGTTCCC 
1551 
AACAAACGTC 
TACGATACAA 
CCCTTGCAAT 
GTACGAAGTG 
ATAAAAGCGG 
1601 
GAGGCTTCAC 
AAAAGGTGGG 
CTCAACTTCG 
ATGCGAAGGT 
GAGGAGGGCT 
1651 
TCTTACAAAG 
TGGAGGACCT 
CTTCATAGGG 
CACATAGCGG 
GAATGGACAC 
1701 
CTTTGCACTC 
GGTTTCAAGG 
TGGCATACAA 
ACTCGTGAAG 
GATGGTGTTC 
1751 
TGGACAAATT 
CATCGAAGAA 
AAGTACAGAA 
GTTTCAGGGA 
GGGCATTGGA 
1801 
AGGGACATCG 
TCGAAGGTAA 
AGTGGATTTT 
GAAAAACTTG 
AAGAGTATAT 
1851 
AATAGACAAA 
GAAACGATAG 
AACTTCCATC 
TGGAAAGCAA 
GAATACCTGG 
1901 
AAAGCCTCAT 
CAACAGTTAC 
ATAGTGAAGA 
CCATTCTGGA 
ACTGAGGTGA 
1951 
AACAGAGTGT 
GAAGTTCTTG 
AATCTTCGAA 
GATTACTTCT 
TCTGGCACTG 
2001 
ATTGCGGCTG 
GAATCTCAGT 
GATCATAGTC 
GTATCCAACC 
GGGAAAACAG 
2051 
GGTGAAATTT 
CCAGAAGGAG 
AGATTGTGAT 
AACTGACGGA 
GAAAGATCTC 
2101 
TGAAACTTCG 
TGTCGAGATA 
GCGAACACTC 
CTTTTTTTCG 
TTCGATCGGT 
2151 
CTGATGTACA 
GAAAGAGCAT 
CCCGGATGAC 
TTCGGGATGC 
TCTTTGTTTT 
2201 
TGAAGAAGAT 
ACAAGAAGCG 
GCTTCTGGAT 
GAAGAACACC 
TACGTTCCCC 
2251 
TCGAAATCGC 
CTTCATAGAC 
AGAAACGGCA 
TCGTATTTTC 
CATTCAGGAG 
2301 
ATGGAGCCAT 
GCGAAAAAGA 
ACCCTGCAAG 
GTTTACTACG 
CACCAAAGCC 
2351 
GTTCAGATAC 
GCTCTTGAAG 
TGAAAAGAGG 
TTTTTTCGAA 
AGGCATGGAT 
2401 
TTGGAGTGGG 
AAGCCGTGTC 
CTGATAGAAA 
AGTAGCGGTA 
CTTTCAAACA 
2451 
AAAACGTATG 
GAATCTTCAT 
CTTCTTTGCC 
TCGTACATTC 
TCGAGTCAGC 
2501 
CATCTTCAGA 
AGTTCTTCTA 
GA. 
__________________________________________________________________________ 
The novel recombinant plasmid comprises a compatible vector containing the 
DNA sequence of SEQ ID NO:2. A compatible vector is one into which the 
gene can be inserted and which can be introduced into a suitable host for 
production of the enzyme. 
The preferred method of preparing D-fructose comprises enzymatically 
treating D-glucose with the hyperthermostable xylose isomerase of the 
present invention at a temperature of about 80.degree. C. to about 
100.degree. C. at a pH of about 5.8 to 7.7 to obtain a syrup containing up 
to about 55% D-fructose. 
The achievement of the above and other objects and advantages of the 
present invention will be apparent to those skilled in the art from the 
description of the drawings, the preferred embodiment and the experimental 
work.

DESCRIPTION OF PREFERRED EMBODIMENT 
The hyperthermostable enzyme from Thermotoga neapolitana strain 5068 (DSM 
No. 5068.) is more stable at high temperatures (90.degree. C.) than the 
enzymes from conventional or moderate thermophilic organisms. During the 
production of high-fructose corn syrup, the use of a highly thermostable 
xylose isomerase in the glucose isomerization process allows the reaction 
to proceed at higher temperatures, so that syrups with a higher fructose 
content can be obtained due to chemical equilibrium. 
The preferred method of producing the enzyme broadly comprises expressing 
an active and thermostable enzyme in a mesophilic organism which allows 
the addition of a heat treatment step, which improves dramatically the 
purification procedure. 
In an especially preferred practice of the method of the invention 
chromosomal DNA from T. neapolitana strain 5068 is purified by the method 
of Goldberg and Ohman (2). The chromosomal DNA is partially digested with 
the restriction enzyme Sau3AI. The 3-7 kb fragments are isolated from a 
sucrose gradient (10-40%), and ligated into plasmid vector pUC18, which is 
cut by BamHI and dephosphorylated with calf intestine alkaline phosphatase 
to form a hybrid plasmid. E. Coli SURE strain is transformed with the 
ligation mixture, and recombinants which contain the xylose isomerase gene 
are selected by colony hybridization using a PCR-generated homologous 
probe (3). Lastly, positive clones are tested for xylose isomerase 
activity at 90.degree. C. 
A further understanding of the invention can be obtained from the following 
description of the experimental work we performed. 
EXPERIMENTAL 
MATERIALS AND METHODS 
Bacterial strains and plasmids. 
Thermotoga neapolitana strain 5068 was used as a source of chromosomal DNA 
to construct the library. Escherichia coli Sure strain (e14.sup.- (mcrA), 
.DELTA.(mcrCB-hsdSMR-mrr)171, sbcC, recB, recJ, umuC::Tn5 (kan.sup.r), 
uvrC, supE44, lac, gyrA96, relA1, thi-1, endA1, [F' proAB, lacI.sup.q 
Z.DELTA.M15, Tn10, (tet.sup.r)]) (Stratagene, La Jolla, Calif.) was used 
for the cloning experiments, and E. coli xyl.sup.- mutant HB101 (F.sup.-, 
hsdS20, ara-1, recA13, proA12, lacY1, galK2, rpsL20, mtl-1, xyl-5) (4) was 
used for the purification of the recombinant xylose isomerase. Plasmids 
pUc18 (Pharmacia, Piscataway, N.J.) and pBluescriptIIKS+ (pBSIIKS+) and 
(Stratagene, La Jolla, Calif.) were used as cloning vectors. Plasmids 
constructed in this study are shown in FIG. 1. 
Media and growth conditions. 
T. neapolitana cultures were grown in modified ASW (5) at 80.degree. C. E. 
coli cultures were grown in Luria broth (10 g tryptone, 5 g yeast extract, 
5 g NaCl per liter), except for the purification of the recombinant xylose 
isomerase, where Terrific Broth (TB) was used (6). The antibiotics 
ampicillin and kanamycin (kan) were used at 100 .mu.g/ml and 25 .mu.g/ml 
respectively. 
DNA preparation and genomic library construction. 
Chromosomal DNA from T. neapolitana strain 5068 was purified by the method 
of Goldberg and Ohman (2). The chromosomal DNA was partially digested with 
the restriction enzyme Sau3AI. The 3-7 kb fragments were isolated from a 
sucrose gradient (10-40%), and ligated into plasmid vector pUC18, which 
was cut by BamHI and dephosphorylated with calf intestine alkaline 
phosphatase. E. Coli (SURE strain) was transformed with the ligation 
mixture by electroporation (3). 
Manipulation of DNA. 
Plasmid DNA purification, restriction analysis, PCR reaction, and colony 
and DNA hybridization were performed by conventional techniques (6) (3). 
The following oligonucleotides (obtained from the Michigan State 
University Macromolecular Facility) were used for PCR reactions: 5'-CCA 
AGC TTN ACN CAY CCN GTN TTY AAR GA3' (A, encodes the peptide FTHPVFKD), 
where the AAGCTT sequence creates a HINDIII site; 5'-GAR CCN AAR CCN AAY 
GAR CCG CGG-3' (B, encodes the peptide EPKPNEP), where the CCGCGG sequence 
creates a SacII site; 5'-GGT CTA GAR AAY TAY GTN TTY TGG GGN GG-3' (C, 
encodes the peptide ENYVFWGG), where the TCTAGA sequence creates an XbaI 
site. DNA was recovered from agarose gels with the Geneclean II kit (BIO 
101, La Jolla, Calif.). Plasmid pTNE2 was stabilized by cloning the 
kan.sup.r cartridge from pUC-4K (Pharmacia, Piscataway, N.J.) into the 
pTNE2 unique SalI site, giving rise to plasmid pTNE2::kan. 
Nucleotide sequence determination. 
Sequential deletions of pTNE2 were created by the exonuclease III digestion 
procedure of Henikoff (7). Sequences were determined, on both strands, by 
the dideoxy chain termination technique (8), using the Sequenase Version 
2.0 kit (U.S. Biochemical Corp., Cleveland, Ohio). The sequencing data 
were analyzed using the Sequence Analysis Software Package of the Genetics 
Computer Group, version 5 (University of Wisconsin) (9). Hydrophobic 
cluster analysis (HCA) of the amino acid sequences was performed as 
described by Gaboriaud et al. (10) and Lemesle-Varloot et al. (11) with 
the HCA plot program, version 2, from Doriane (Le Chesnay, France). 
Enzyme purification. 
For comparison purposes Thermoanaerobacterium thermosulfurigenes 4B 
recombinant xylose isomerase was purified from E. coli HB101 carrying 
plasmid pCMG11-3, as described by Lee et al. (12), except that: i) 50 mM 
MOPS (4-morpholinepropanesulfonic acid) (pH7.0) containing 5 mM MgSO.sub.4 
plus 0.5 mM CoCl.sub.2 was used as buffer; ii) a (NH.sub.4).sub.2 SO.sub.4 
fractionation step was added after the heat treatment (13); iii) ion 
exchange chromatography was performed on Q-Sepharose Fast Flow, and 
proteins were eluted with a linear NaCl gradient (0.0-0.3M). 
T. neapolitana recombinant xylose isomerase was purified from E. coli HB101 
carrying plasmid pTNE2::kan as described above, except that: i) the cells 
were grown in TB supplemented with kan; ii) the cell extract was heat 
treated at 90.degree. C. for 2 h 30 min in an oil shaking bath. 
Protein concentration was determined routinely by the method of Bradford 
(14), using the Bio-Rad protein reagent. Bovine serum albumin was used as 
the standard. Protein concentration of pure enzyme preparations was 
estimated at 280 nm, on denaturated enzyme. The extinction coefficient 
(e=52950) was calculated from the sequence, using the Sequence Analysis 
Software Package of the Genetic Computer Group program. Enzyme fractions 
were analyzed by SDS-12% polyacrylamide gel electrophoresis, and 
visualized by Coomassie blue staining. 
Enzyme assays. 
Cell extracts prepared by sonication and purified preparations were used as 
enzyme sources. To determine the effect of temperature on glucose 
isomerase specific activity, glucose isomerase activity was measured by 
incubating the enzyme (0.02-0.1 mg/ml) in 100 mM MOPS (pH 7.0 at room 
temperature), 1 mM CoCl.sub.2 and 0.8M glucose for 20 min. According to 
.DELTA.pKa/.DELTA.t of MOPS buffer which is -0.011 (US Biochemical, 
Cleveland, Ohio, catalog 1993, p. 290), the pH was 6.3 at 90.degree. C. 
The reaction was stopped by cooling the tubes in ice. The fructose was 
assayed by the cysteine-carbazole/sulfuric acid method (15). One unit of 
isomerase activity is defined as the amount of enzyme that produced 1 
.mu.mole of product/min under the assay conditions. 
Thermostability studies. 
The enzyme was incubated at different temperatures in the presence of 100 
mM MOPS (pH 7.0), 1 mM CoCl.sub.2 at room temperature for different 
periods of time. Thermoinactivation was stopped by cooling the tubes in a 
water bath equilibrated at room temperature. The residual glucose 
isomerase activity was measured in the conditions described above. 
pH studies. 
The effect of pH on glucose isomerase activity was measured using the 
standard protocol described above for enzyme assays, except that the MOPS 
buffer was substituted by acetate 100 mM (pH 4.0-5.7), PIPES 
(piperazine-N, N'-bis-[2-ethanesulfonic acid]) 100 mM (pH 6.0-7.5), or 
EPPS (N-[2-hydroxyethyl]piperazine-N'-[3-propanesulfonic acid]) 100 mM (pH 
7.5-8.7). All pHs were adjusted at room temperature, and the 
.DELTA.pKa/.DELTA.t for acetate, PIPES, and EPPS (0.000, -0.0085, and 
-0.011 respectively) (16, USB catalog 1993, p. 290) were taken into 
account for the results (FIG. 6). The effect of pH on enzyme stability was 
measured by incubating the enzyme (0.5-1.0 mg/ml) at 90.degree. C. for 30 
min in acetate 100 mM (pH 4.0-5.7), PIPES 100 mM (pH 6.0-7.5), or EPPS 100 
mM (pH 7.5-8.7), in the presence of 0.5 mM CoCl.sub.2. The inactivation 
was stopped by cooling the tubes in a water bath equilibrated at room 
temperature. The residual glucose isomerase activity was measured at pH 
7.0, using the standard protocol. The enzyme was diluted ten-fold in the 
reaction mixture. 
Cloning of the xylose isomerase gene. 
The first attempts to clone T. neapolitana by complementation of a 
xyl.sup.- strain of E. coli were unsuccessful. This result was expected, 
since no xylose isomerase activity could be detected with the xylose 
isomerase purified from T. maritima, at temperatures compatible with E. 
coli growth (5). On the other hand, no hybridization signal could be 
detected in T. neapolitana genomic DNA with the Thermoanaerobacterium 
thermohydrosulfuricum 4B xylA gene as a probe. 
We therefore tried to amplify a DNA fragment, internal to the T. 
neapolitana xylA gene, and use it as a probe to screen a plasmid library. 
To design primers for PCR, the amino acid sequences of the xylose 
isomerases from Actinoplanes missouriensis (17), Ampullariella (18), 
Streptomyces rubuginosus (19), S. violaceoniger (20), Bacillus subtilis 
(21), Lactobacillus brevis (22), L. pentosus (23), Thermoanaerobacterium 
thermosulfurigenes 4B, Staphylococcus xylosus (24), and E. coli (25) have 
been aligned (not shown) to identify highly conserved regions. Two main 
families of highly conserved enzymes were identified, the first included 
the Streptomyces, Ampullariella and A. missouriensis and the second 
included Bacillus, the Lactobacilli, and T. thermosulfurigenes. Even if 
they were more related to family II xylose isomerases, E. coli and S. 
xylosus enzymes still stood apart, and were not taken into account in the 
following. Only the sequence EPKPN/KEP (positions 232 to 238 in T. 
thermosulfurigenes enzyme) was conserved among the proteins of the 2 
families. The sequence ENYVFWG (positions 183 to 189 in T. 
thermosulfurigenes enzyme) was conserved only in family II, whereas the 
sequence FTHPVFKD (positions 94 to 100 in A. missouriensis enzyme) was 
conserved only in family I. Since we did not know to which family the T. 
neapolitana enzyme would belong, we designed two sets of primers to 
correspond to the 2 families. With primers A plus B we expected a 294 bp 
PCR product, and with primers C plus B we expected a 180 bp PCR product. 
With T. neapolitana genomic DNA as the template, only the PCR reaction 
using primers C plus B gave rise to a single band of the expected size in 
a reproducible way. This PCR product has been cloned into the XbaI-SacII 
sites of pBSIIKS+ and sequenced. It encodes a 54 residues polypeptide, 80% 
identical to the part of T. thermosulfurigenes xylose isomerase chosen for 
the amplification. This PCR fragment was used as a probe to screen a T. 
neapolitana genomic library. Among 15,000 clones that were screened by 
colony hybridization, only 6 hybridized strongly with the probe, and were 
completely isolated. Restriction analysis of these clones revealed that 
they all overlapped. All of them showed xylose isomerase activity. 
One of them, clone pTNE1 (FIG. 1), was chosen for further studies. 
Additional hybridizations with restriction digests of pTNE1 showed that a 
750 bp EcoRI-BamHI fragment carried the homology with the PCR probe. 
Several sub-clones were constructed, and tested for xylose isomerase 
activity (FIG. 1). Compared to pTNE1, the same level of activity was 
detected with both pTNE2 and 3. Since the insert was in opposite 
orientation in pTNE2 and pTNE3, when referring to the lacZ promoter, the 
xylose isomerase gene carried by the insert is probably expressed from its 
own promoter. No activity could be detected with R3 and R9. 
Nucleotide sequence of the xylose isomerase gene. 
The 2.4 kb SalI-XbaI insert of pTNE2 has been sequenced. One open reading 
frame (ORF) of 1332 nt, encodes a 444 AA polypeptide (calculated molecular 
weight: 50,892), 70% identical to the 4B xylose isomerase. This ORF is 
preceded by a Shine-Dalgarno sequence, GGAGGT, which exactly matches the 
T. maritima 16S rRNA sequence 3'-CCUCCA-5' (26). A potential promoter, 
TTGAA (-35) TATAAT (-10), corresponding to the consensus defined for T. 
maritima (27) is present 63 bp upstream of the ATG start codon. An 
inverted repeat located 213 pb downstream of xylA might be involved in 
termination of transcription. No homology was found upstream and 
downstream of xylA with E. coli xylB. The genetic organization of the 
xylose metabolism genes therefore seems different from what has been 
described in other microorganisms, where xylA and xylB are usually 
co-transcribed. The overall G+C content of xylA is 47% (Thermotogales have 
a genomic G+C content of 40 to 46%). The G+C content in codon position 3 
is 59% (it is 60% for T.maritima omp.alpha.); this content is 15% higher 
than what is expected in an organism with 46% genomic G+C. 
Comparison of the protein sequences. 
The T. neapolitana xylose isomerase clearly belongs to the xylose isomerase 
family of type II, including the enzymes from E. coli, B. subtilis, C. 
thermohydrosulfuricum, and T. thermosulfurigenes strain 4B. Enzymes of 
type I are shorter by 40 to 50 residues at the N-terminal. The catalytic 
triad (His101, Asp104, Asp339), as well as almost all the other residues 
involved either in substrate or metal binding are conserved among the two 
protein families. 
Biochemical characterization of T. neapolitana xylose isomerase. 
Since plasmid pTNE2 was unstable in E. coli HB101, reliable amounts of T. 
neapolitana xylose isomerase were purified from HB 101(pTNE2::kan) 
cultures, grown in the presence of kanamycin. Heat treatment of the cell 
extracts for 2 h 30 min at 90.degree. C. was a highly efficient step. The 
main contaminating protein was further removed by ammonium sulfate 
fractionation, and a last step of ion exchange chromatography allowed to 
purify the protein to homogeneity (not shown). The purified xylose 
isomerase is a tetramer composed of identical subunits of 50,000 Da. 
The cloned xylose isomerase is optimally active at temperatures as high as 
94.degree.-100.degree. C. 
By the exercise of the method of the present invention a highly 
thermostable and thermophilic xylose isomerase has been cloned from the 
hyperthermophile T. neapolitana 5068. This enzyme clearly belongs to the 
xylose isomerase family II. Although 70% identical to the 
Thermoanaerobacterium thermosulfurigenes 4B enzyme and, according to the 
HCA comparison, showing highly conserved secondary structures, the 
Thermotoga xylose isomerase is unexpectedly optimally active at 
temperatures 15.degree. C. higher than the 4B enzyme (95.degree. C. versus 
80.degree. C.). 
Representative of the plasmids and viral vectors which can be used in the 
method of the present invention are the following: pUC plasmids and 
derivatives like pTZ18-19, pBluescript, with high plasmid copy number per 
cell (200-300); pBR322 and derivatives, with lower copy number (20-50); 
plasmids with low copy number (5-10) like pMMB67EH (Furste et al., 1986) 
(28); and plasmids able to replicate in B. subtilis or other food-safe 
strains. 
In general any plasmid can be used in which the gene can be inserted; which 
is stable when transformed into a bacteria and which will cause the 
bacteria to express the enzyme in a recoverable form. 
Representative of the bacteria which can be employed in addition to the 
HB101 strain of E. coli are the following: other E. coli strains (no 
background glucose isomerase activity originating from E. coli can be 
detected after the heat treatment included in our protocol); B. subtilis 
strains, and other food-safe bacterial strains. 
It will be apparent to those skilled in the art that a number of changes 
and modifications can be made without departing from the spirit and scope 
of the invention. Therefore, it is intended that the invention only be 
limited by the claims. 
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48:119-131. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2522 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GTCGACGCAAAGGTCGTGACGGGTGGAAACATAAACGTTCAGCTGGGAACTGTGTCCTCG60 
GCTGCTGTTGAAGGAACATACGTTATCGAAGTTGGACAATTCTCTGGAACGGTCACATCC120 
GAGCTTGATGTCAAGATCCGCCGTTGTCCTCAGCACCCCTTCCGTACACCCTGTCATCCT180 
TCACAACGGGGATGAAGGGATCCGTTTCCCACAGCGAAAGATCCCCTGGTGGAACGGTGT240 
CTATGTGTGTCACTATCCACAATGTTTTGCTTCTGTCCCTGCCGGGAATGATTGCAAGCA300 
GATTCGACCTCCAAATTCCGTTCTGGTCTTTTGTGTCATGACGCTCAACAGTGTATCCCA360 
TCTTTTTGAGAAGTTCCTCCAGCCAGTCGGCCTTCTCTTTCTCTCCAGGTCCACCGAAGA420 
CTGGATTCACCGAATTGATCGATATGAACCTTTTCAGCGAATCTACCATTTCGTCTTTCA480 
ATTCTTCTATCTTTCTTGTTATCTCCATCTGAAACACCTCCCAAGTACAAGTATATCTCT540 
CCAAAAAAATATTTGAAATGACCCCAGGGAATTTTATATAATTGATTGATAGAAAAAATT600 
TAGGGAGGTGTTCACATGGCTGAATTCTTTCCAGAAATCCCGAAAGTGCAGTTCGAAGGC660 
AAAGAAAGCACAAATCCACTTGCGTTCAAGTTCTACGATCCAGAAGAGATCATCGACGGC720 
AAACCCCTCAAGGACCATCTGAAGTTCTCCGTTGCCTTCTGGCACACCTTCGTGAACGAG780 
GGAAGGGATCCCTTCGGAGACCCAACGGCCGATCGTCCCTGGAACAGGTACACCGATCCC840 
ATGGACAAGGCTTTTGCAAGGGTGGACGCCCTTTTTGAATTCTGCGAAAAACTCAACATC900 
GAGTACTTCTGCTTCCACGACAGAGACATCGCTCCCGAGGGAAAAACGCTGAGGGAGACA960 
AACAAAATTTTGGACAAAGTAGTGGAGAGAATCAAAGAGAGAATGAAAGACAGCAACGTG1020 
AAGCTCCTCTGGGGTACTGCAAACCTCTTTTCCCACCCAAGGTACATGCATGGTGCAGCG1080 
ACAACCTGCAGTGCTGATGTTTTTGCGTACGCGGCCGCCCAGGTGAAAAAAGCCCTTGAG1140 
ATCACCAAAGAACTTGGAGGAGAAGGGTACGTCTTCTGGGGTGGAAGAGAAGGATACGAA1200 
ACACTCCTCAACACGGACCTTGGATTCGAACTTGAAAACCTCGCCCGCTTCCTCAGAATG1260 
GCTGTGGATTATGCAAAAAGGATCGGTTTCACCGGACAGTTCCTCATCGAACCAAAACCG1320 
AAAGAACCCACCAAACACCAGTACGACTTCGACGTTGCAACCGCCTATGCCTTCCTGAAG1380 
AGCCACGGTCTCGATGAATACTTCAAATTCAACATCGAGGCAAACCACGCCACACTCGCC1440 
GGTCACACCTTCCAGCACGAACTGAGAATGGCAAGGATCCTTGGAAAACTCGGAAGCATC1500 
GATGCAAACCAGGGAGACCTTCTTCTTGGATGGGACACCGATCAGTTCCCAACAAACGTC1560 
TACGATACAACCCTTGCAATGTACGAAGTGATAAAAGCGGGAGGCTTCACAAAAGGTGGG1620 
CTCAACTTCGATGCGAAGGTGAGGAGGGCTTCTTACAAAGTGGAGGACCTCTTCATAGGG1680 
CACATAGCGGGAATGGACACCTTTGCACTCGGTTTCAAGGTGGCATACAAACTCGTGAAG1740 
GATGGTGTTCTGGACAAATTCATCGAAGAAAAGTACAGAAGTTTCAGGGAGGGCATTGGA1800 
AGGGACATCGTCGAAGGTAAAGTGGATTTTGAAAAACTTGAAGAGTATATAATAGACAAA1860 
GAAACGATAGAACTTCCATCTGGAAAGCAAGAATACCTGGAAAGCCTCATCAACAGTTAC1920 
ATAGTGAAGACCATTCTGGAACTGAGGTGAAACAGAGTGTGAAGTTCTTGAATCTTCGAA1980 
GATTACTTCTTCTGGCACTGATTGCGGCTGGAATCTCAGTGATCATAGTCGTATCCAACC2040 
GGGAAAACAGGGTGAAATTTCCAGAAGGAGAGATTGTGATAACTGACGGAGAAAGATCTC2100 
TGAAACTTCGTGTCGAGATAGCGAACACTCCTTTTTTTCGTTCGATCGGTCTGATGTACA2160 
GAAAGAGCATCCCGGATGACTTCGGGATGCTCTTTGTTTTTGAAGAAGATACAAGAAGCG2220 
GCTTCTGGATGAAGAACACCTACGTTCCCCTCGAAATCGCCTTCATAGACAGAAACGGCA2280 
TCGTATTTTCCATTCAGGAGATGGAGCCATGCGAAAAAGAACCCTGCAAGGTTTACTACG2340 
CACCAAAGCCGTTCAGATACGCTCTTGAAGTGAAAAGAGGTTTTTTCGAAAGGCATGGAT2400 
TTGGAGTGGGAAGCCGTGTCCTGATAGAAAAGTAGCGGTACTTTCAAACAAAAACGTATG2460 
GAATCTTCATCTTCTTTGCCTCGTACATTCTCGAGTCAGCCATCTTCAGAAGTTCTTCTA2520 
GA2522 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 444 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetAlaGluPhePheProGluIleProLysValGlnPheGluGlyLys 
151015 
GluSerThrAsnProLeuAlaPheLysPheTyrAspProGluGluIle 
202530 
IleAspGlyLysProLeuLysAspHisLeuLysPheSerValAlaPhe 
354045 
TrpHisThrPheValAsnGluGlyArgAspProPheGlyAspProThr 
505560 
AlaAspArgProTrpAsnArgTyrThrAspProMetAspLysAlaPhe 
65707580 
AlaArgValAspAlaLeuPheGluPheCysGluLysLeuAsnIleGlu 
859095 
TyrPheCysPheHisAspArgAspIleAlaProGluGlyLysThrLeu 
100105110 
ArgGluThrAsnLysIleLeuAspLysValValGluArgIleLysGlu 
115120125 
ArgMetLysAspSerAsnValLysLeuLeuTrpGlyThrAlaAsnLeu 
130135140 
PheSerHisProArgTyrMetHisGlyAlaAlaThrThrCysSerAla 
145150155160 
AspValPheAlaTyrAlaAlaAlaGlnValLysLysAlaLeuGluIle 
165170175 
ThrLysGluLeuGlyGlyGluGlyTyrValPheTrpGlyGlyArgGlu 
180185190 
GlyTyrGluThrLeuLeuAsnThrAspLeuGlyPheGluLeuGluAsn 
195200205 
LeuAlaArgPheLeuArgMetAlaValAspTyrAlaLysArgIleGly 
210215220 
PheThrGlyGlnPheLeuIleGluProLysProLysGluProThrLys 
225230235240 
HisGlnTyrAspPheAspValAlaThrAlaTyrAlaPheLeuLysSer 
245250255 
HisGlyLeuAspGluTyrPheLysPheAsnIleGluAlaAsnHisAla 
260265270 
ThrLeuAlaGlyHisThrPheGlnHisGluLeuArgMetAlaArgIle 
275280285 
LeuGlyLysLeuGlySerIleAspAlaAsnGlnGlyAspLeuLeuLeu 
290295300 
GlyTrpAspThrAspGlnPheProThrAsnValTyrAspThrThrLeu 
305310315320 
AlaMetTyrGluValIleLysAlaGlyGlyPheThrLysGlyGlyLeu 
325330335 
AsnPheAspAlaLysValArgArgAlaSerTyrLysValGluAspLeu 
340345350 
PheIleGlyHisIleAlaGlyMetAspThrPheAlaLeuGlyPheLys 
355360365 
ValAlaTyrLysLeuValLysAspGlyValLeuAspLysPheIleGlu 
370375380 
GluLysTyrArgSerPheArgGluGlyIleGlyArgAspIleValGlu 
385390395400 
GlyLysValAspPheGluLysLeuGluGluTyrIleIleAspLysGlu 
405410415 
ThrIleGluLeuProSerGlyLysGlnGluTyrLeuGluSerLeuIle 
420425430 
AsnSerTyrIleValLysThrIleLeuGluLeuArg 
435440 
__________________________________________________________________________