Journal of Industrial Microbiology & Biotechnology (2001) 27, 234–240

D 2001 Nature Publishing Group 1367-5435/01 $17.00

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Purification and characterization of thermostable xylose(glucose)

isomerase from Bacillus thermoantarcticus
L Lama, B Nicolaus, V Calandrelli, I Romano, R Basile and A Gambacorta

Istituto per la Chimica di Molecole di Interesse Biologico, CNR, Via Campi Flegrei, 34, Pozzuoli 80078 ( Na ), Italy

Xylose isomerase produced by Bacillus thermoantarcticus was purified 73 - fold to homogeneity and its biochemical
properties were determined. It was a homotetramer with a native molecular mass of 200 kDa and a subunit
molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K m of 33 mM for xylose and also
accepted D - glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a
temperature of 8588 C. Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme
required divalent cations for its activity and thermal stability. Mn2 +, Co2 + or Mg2 + were of comparable efficiency for
xylose isomerase reaction, while Mg2 + was necessary for glucose isomerase reaction. Journal of Industrial Microbiology
& Biotechnology ( 2001 ) 27, 234 – 240.

Keywords: Bacillus; thermophile; xylose isomerase

Introduction tions of products and decreased viscosity of the substrate and

product stream. Therefore, thermostable xylose isomerases with
Various microorganisms are able to grow on hemicelluloses,
neutral or slightly acidic pH optima have potential industrial
which amount to 40% of the plant biomass [ 34 ], as a sole carbon
application.
source. Exoenzymes degrade the polymer to D - xylose, which is
Thermophilic microorganisms produce intrinsically thermo-
transported into the cell, isomerized to D - xylulose and then
stable enzymes, which have evolved and adapted to the extreme
phosphorylated to xylulose 5 - phosphate, which enters either the
environment of their natural habitats [ 2 ].
pentose phosphate pathway [ 10,15,18,20,21 ] or the phosphoke-
In the present work, D - xylose( glucose ) isomerase of the
tolase pathway [ 15 ]. Isomerization of D - xylose is carried out by
thermophilic bacterium Bacillus thermoantarcticus ( DSM 9572 )
D - xylose isomerase ( D - xylose keto - isomerase, EC 5.3.1.5 ). This
[ 33 ] was purified and the physicochemical and enzymatic proper-
enzyme has been used commercially because of its capacity to
ties were studied and compared between the two types of isomerase
produce a high - fructose corn - enriched syrup ( HFCS ) by
reactions.
converting D - glucose into fructose [ 3,34 ]. D - xylose isomerase
is also of industrial interest for the isomerization of D - xylose to
D - xylulose, which can be ultimately fermented to ethanol by
conventional yeasts [ 15 ]. Owing to the industrial significance of Materials and methods
the enzyme, xylose isomerases from various microorganisms
Strain and growth conditions
have been studied, and their catalytic and physicochemical B. thermoantarcticus ( DSM 9572 ) was isolated from Antarctic
properties have been reviewed [ 8,9 ]. Immobilization techniques geothermal soil near the crater of Mount Melbourne [ 33 ]. The
and an isomerization process with the enzyme have been strain was cultivated at 658C on a medium containing 0.6% yeast
described [ 17 ]. extract ( w / v ) and 0.3% ( w / v ) NaCl at pH 6.0 ( standard growth
Most commercially available xylose isomerases have been conditions ). Production of the enzyme was investigated on media
isolated from mesophilic microorganisms, including Streptomyces, containing 0.1% ( w / v ) yeast extract, 0.3% NaCl and 0.5% of a
Actinoplanes, Flavobacterium and Bacillus species [ 4,26 ]. These different carbon source: xylose, xylan, arabinose, galactose,
enzymes are generally thermostable, and are utilized in the fructose, yeast, maltose or glucose. Growth was followed by
immobilized form to enhance enzyme half - life [ 39 ]. These measuring the absorbance at 540 nm. Cells were harvested, in
enzymes require metal ions for their activity and stability, and the late exponential growth phase, by centrifugation at 9000g for
pH optima for enzyme activity are in the range 7.5 – 9.0. The 30 min.
reaction temperature used in the current industrial process for
sweetener production is limited to 608C because of by - product and
colour formation during reaction at high temperature and alkaline Enzyme assays
pH [ 7 ]. Reaction temperatures greater than 608C have the
advantage of faster reaction rates, higher equilibrium concentra- D -xylose isomerase: The formation of D - xylulose from
D - xylose was measured using the colorimetric assay of Dische and
Borenfreund [ 12 ] in which 20 l of the enzyme solution was added
to 20 l of a solution of 0.2 M D - xylose and 0.4 mM MnSO4 in 20
Correspondence: Dr L Lama, Istituto per la Chimica di Molecole di Interesse
mM Tris – HCl, pH 7.0, and incubated for 10 min at 808C. One unit
Biologico, CNR, Via Campi Flegrei, 34, Pozzuoli 80078 ( Na ), Italy ( U ) of enzyme activity was equal to the formation of 1 mol of
Received 18 March 2001; accepted 3 July 2001 D - xylulose per minutes at 808C.
 Xylose( glucose ) isomerase from Bacillus thermoantarcticus
L Lama et al

235
D -glucose isomerase: The test for D - glucose
isomerase Tris – HCl, pH 7.0. The column was washed with 10 ml of the same
activity was the same as that for D - xylose isomerase, with the buffer and the enzyme was eluted with a linear gradient 0 – 0.6 M
exception that D - xylose and MnSO4 were replaced by 0.4 M NaCl ( 30 ml ) at a flow rate of 0.5 ml / min.
D - glucose, 10 mM MgSO4 and 1 mM CoCl2 in 20 mM Tris – HCl,
pH 7.0. Electrophoresis and M r determination
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Protein content ( SDS - PAGE ) was performed as described by Laemmli [ 19 ].
Protein content was determined by the method of Bradford [ 5 ] by Native PAGE was performed without SDS, and Tris – HCl buffer
using the Bio - Rad protein assay with bovine albumin as a standard. ( pH 8.6 ) was used during polyacrylamide gel preparation.
Staining of protein bands was done with Coomassie brilliant
blue R - 250 ( Bio - Rad ). M r values of purified xylose isomerase
HPAE - PAD
were determined by gel filtration on a Sephacryl S - 200 column
The products of reaction were identified and quantitated by HPAE -
( 2.549 cm ); Blue Dextran ( M r 2,000,000 ) and Pharmacia Gel
PAD Dionex, equipped with Carbo - Pac PA - 1 column, using
Filtration Calibration Kit ( high and low molecular weight ) were
100 mM NaOH as solvent system [ 31 ].
used as M r standards.
Subunit M r values were estimated by SDS - PAGE using the
Enzyme purification Pharmacia high - and low - molecular - weight electrophoretic stand-
ards.
Preparation of the crude extract: Wet cells ( 5 g ) were lysed The isoelectric point of the enzyme was determined by using the
by freezing and thawing, suspended in 20 mM Tris – HCl, pH 7.0 Mini Protean II ( Bio - Rad ) system with IEF Ready Gel ( range pH
( 25 ml ) and, after ultrasonic treatment ( Heat Systems Instrument ) 3 – 10 ). Protein bands were made visible by Coomassie brillant
for 4 min, treated with 0.2 mg of DNase and 6 mg of lysozyme at Blue G - 250 staining.
378C for 30 min. The resulting suspension was centrifuged at
34,000g for 30 min. The dialysed ( 20 mM Tris – HCl, pH 7.0, Metal ion effects on enzyme activity and stability
containing 0.4 mM MgCl2 and 1 mM CoCl2 ) supernatant was taken To prepare a metal - ion - free enzyme solution, xylose isomerase
as crude extract. purified from B. thermoantarcticus was dialyzed at 48C for 24 h
against 20 mM Tris – HCl, pH 7.0, containing 10 mM EDTA,
Heat treatment: The crude extract of B. thermoantarcticus was followed by dialysis against buffer without EDTA ( two changes ).
heated for 15 min at 808C, in the presence of 10 mM MgSO4 and Enzyme activity was determined in the presence of various
1 mM CoCl2, and cooled to 48C. The soluble fraction was concentrations of MgCl2, MnCl2 and CoCl2 using the standard
recovered after centrifugation at 15,000g for 20 min. assay.
Thermostability was determined by measuring residual activity
Ion exchange chromatography: The above enzyme prepa- under optimum assay conditions after preincubation of the enzyme
ration was loaded on a column ( 2.540 cm ) of Q - Sepharose Fast at different temperatures in the presence of various metal ions.
Flow pre - equilibrated with 20 mM Tris – HCl, pH 7.0, containing
0.5 mM CoCl2 and 5 mM MgSO4. The column was washed with
Effect of temperatures and borate on isomerization
100 ml of the same buffer at flow rate of 2.5 ml / min and eluted with
a 5 - bed - vol linear NaCl salt gradient ( 0 – 0.6 M ) in the same
ratio
The isomerization reaction was carried out at different temperatures
buffer. The active fractions were pooled and concentrated by
at pH 7.0. This reaction was also conducted at 708C in the presence
ultrafiltration ( YM 30 Amicon ).
of different quantities of sodium tetraborate ( 0 – 0.5 M ). All
reaction products were quantified by HPAE - PAD.
Gel filtration: Concentrated fractions were applied to a column of
Sephacryl S - 200 ( 1.580 cm ). Equilibration and elution were
carried out with 20 mM Tris – HCl, pH 7.0, containing 0.5 mM
CoCl2 and 5 mM MgSO4 at flow rate of 0.4 ml / min. The active
fractions were pooled and concentrated as above. Table 1 Induction of synthesis of D - xylose ( XI ) and glucose ( GI )
isomerase from B. thermoantarcticus
Hydrophobic interaction: A saturated ammonium sulphate
solution was added to the enzyme solution to give a final Carbon source ( 0.5% w / v ) Specific activity ( U / mg )
concentration of 1.3 M. This solution was applied to a 1.516
XI GI
cm Phenyl - Sepharose CL - 4B column, previously equilibrated
with 20 mM Tris – HCl, pH 7.0, containing 1.3 M ( NH4 )2SO4. The
Xylose 0.4 0.18
column was washed with 2 bed vol of the same buffer, and eluted Xylan 0.136 0.009
with a 10 - bed - vol linear gradient of 1.3 – 0 M ( NH4 )2SO4 at a flow Arabinose 0.0085 0
rate of 0.5 ml / min. Fractions containing enzyme activity were Galactose 0.048 0
pooled, concentrated as above and dialysed overnight against Fructose 0.041 0
Yeast 0.018 0.007
20 mM Tris – HCl, pH 7.0. Maltose 0.007 0.003
Glucose 0 0.005
Fast protein liquid chromatography: The enzyme was
further purified by FPLC on Mono Q HR / 5 / 5 column in 20 mM Values are the means of those from three independent assays.
 Xylose( glucose ) isomerase from Bacillus thermoantarcticus
L Lama et al

236

Figure 1 ( a ) Production of D - xylose isomerase from B. thermoantarcticus. Cell density of culture ( open symbols ); enzyme activity ( closed
symbols ). ( b ) Glucose isomerase from B. thermoantarcticus. Cell density of culture ( open symbols ); enzyme activity ( closed symbols ). ( c ) Effect of
temperature on D - xylose ( XI ) and D - glucose ( GI ) isomerase activity. ( d ) Effect of pH on purified D - xylose ( XI ) and D - glucose ( GI ) isomerase
activity at 808C. Enzyme activities were assayed under standard conditions in 50 mM glycine buffer, pH 3.0, 4.5, 7.5, 8.5, 9.0, 10.0; 50 mM sodium
acetate buffer, pH 4.0, 5.0, 5.5; 50 mM phosphate buffer, pH 6.0, 6.5; Tris – HCl buffer, pH 7.0, 7.5, 8.0, 8.5. ( e,f ) Effect of borate on equilibrium
displacement of isomerization. Isomerization was carried out at 708C with 1 M D - xylose ( e ) or D - glucose ( f ) and sodium tetraborate ( 0 – 0.5 M ).
 Xylose( glucose ) isomerase from Bacillus thermoantarcticus
L Lama et al

Table 2 Purification of D - xylose / glucose isomerase from B. thermoantarcticus 237

Purification step Total protein ( mg ) Total activity ( U ) Specific activity ( U / mg ) Yield ( % ) Purification ( fold )

GI XI GI XI GI XI GI XI GI

Crude extract 135 49.6 12 0.37 0.09 100 100 1 1

Heat treatment ( 808C15 min ) 49.5 51.3 12.7 1.0 0.26 103 106 2.7 2.9
Q - Sepharose FF 12.8 47.2 11.7 3.7 0.91 95 97.5 10 10.1
Sephacryl S - 200 8.5 47 11.6 5.5 1.36 94.8 96.7 14.9 15.1
Phenyl Sepharose CL - 4B 2.3 29.5 7.4 12.8 3.22 59.5 61.7 34.6 35.8
Mono Q / FPLC 0.4 10.8 2.8 27 7 21.8 23.3 73 78

Results and discussion Physicochemical properties

The enzymic activity of D - xylose isomerase was measured at
Strain and induction of the D - xylose isomerase activity various temperatures utilizing D - xylose and D - glucose as sub-
The antartic thermophilic microorganism B. thermoantarcticus strates. For both substrates, the optimum temperature was 908C
classified as a novel species of Bacillus [ 33 ] on the basis on the ( Figure 1c ) and Arrhenius plots yielded straight lines up to a
phenotypic, taxonomic and genetic studies possesses an endocel- temperature of 858C and declined thereafter.
lular D - xylose( glucose ) isomerase that can be induced by growth The optimum pH with both substrates was around 7.0 and
under certain conditions. showed 80% of its maximum activity at pH 6.0 ( Figure 1d ).
In many organisms, D - xylose isomerase was induced by its Stability of the xylose isomerase as a function of pH was tested by
substrate D - xylose [ 24,30,41 ]. Induction of the enzyme by a range preincubation of the enzyme at 258C at different pH values for 2 h.
of compounds was tested in B. thermoantarcticus. The relative After incubation, the activities of xylose isomerase and glucose
activity obtained is shown in Table 1. D - xylose was the most potent isomerase were measured at pH 7.0 — both activities were stable in
inducer of both D - xylose isomerase and D - glucose isomerase. the broad pH range 5.5 – 10.0 — and readily denatured at pH values
The cost of enzyme production is an important factor in the lower than 5.0.
evaluation of its suitability for industrial application. Intensive
efforts have been made to optimise the fermentation parameters for
the production of xylose isomerase with a view to developing an
economically feasible technology; one aspect of this is the
optimization of the fermentation medium. Xylose is very expensive
and hence impractical for use on a commercial scale.
B. thermoantarcticus grew on xylan, a cheaper medium whose
presence in the growth medium enhanced the expression of xylose
isomerase about eightfold with respect to standard growth medium.
The organism possesses extracellular xylanase and - xylosidase
[ 21 ] that are able to degrade xylan in xylose. Maximum activity of
the xylose isomerase was expressed in a late exponential growth
phase ( Figure 1a and b ).

Purification of D - xylose / glucose isomerase

The enzyme activity was stable at room temperature for several
hours; therefore, all steps of purification were performed at that
temperature. Also, the enzyme activity was stable to freezing and
thawing with no loss of enzyme activity.
The effects of various techniques on the specific activity, fold
purification and yield are shown in Table 2. The last purification
step was significant for both activities; in fact, purification was
about twofold higher than after the previous step. D - glucose
isomerase activity co - purified exactly with D - xylose isomerase
activity. The purified enzyme was homogeneous by the detection of
a single protein band on SDS - PAGE and native PAGE. An
approximate molecular mass of 200 kDa was estimated from the gel
filtration step. SDS - PAGE analysis showed a single band with a Figure 2 SDS - PAGE of purified D - xylose ( glucose ) isomerase from
molecular mass of about 47 kDa ( Figure 2, lane 2 ), suggesting that B. themoantarcticus. Lanes 1 and 3 contain M r marker proteins: myosin
the enzyme was a homomeric tetramer, which is the most common ( 212,000 ), 2 - macroglobulin ( 170,000 ), - galactosidase ( 116,000 ),
phosphorylase b ( 97,000 ), transferrin ( 76,000 ), serum albumin
oligomer form for xylose isomerases [ 22 ]. ( 66,200 ), glutamic dehydrogenase ( 53,000 ), ovoalbumin ( 45,000 ),
Isoelectric focusing of the purified D - xylose isomerase gave a carbonic anhydrase ( 31,000 ). Lane 2 contains purified D - xylose
single protein band corresponding to an isoelectric point of 4.8. ( glucose ) isomerase ( 5 g ). Lane 4 contains crude extract.
 Xylose( glucose ) isomerase from Bacillus thermoantarcticus
L Lama et al

238 Table 3 The effect of metal ions on thermal stability of EDTA - treated xylose ( glucose ) isomerase from B. thermoantarcticus

Preincubation Thermal stability ( % residual ) with

of enzyme
No metal added Co2 + ( 1 mM ) Mg2 + ( 5 mM ) Mg22 ++ ( 5 mM ) Mn2 + ( 5 mM ) Mn22 ++ ( 5 mM )
Co ( 1 mM ) Co ( 1 mM )

XI GI XI GI XI GI XI GI XI GI XI GI

708C24 h 38 15 100 141 102 111 104 130 103 0 104 28

808C1 h 35 15 88 84 98 124 104 106 100 0 101 53
908C30 min 0 0 50 44 13 20 74 102 70 0 80 39

D - xylose( glucose ) isomerase from B. thermoantarcticus was substrate. This reflected the presumed physiological function of the
remarkably stable and resistant to high temperatures. Therefore, this enzyme, which acted in the organism to produce xylulose, which
enzyme, stable at acidic pH values, may increase the efficiency of was subsequently metabolized via either the pentose phosphate
the process and may reduce the possibilities of by - product pathway or phosphoketolase pathway.
formation [ 16 ]. Xylose isomerases typically require the presence of divalent
In addition, the combination of saccharification and isomer- metal cations such as Mn2 + , Mg2 + or Co2 + as essential
ization is an ideal development in the progress of HFCS production, cofactors for their catalytic activity [ 1,6,25 ]. The effect of
and it is likely to be put into operation once an acid - stable enzyme various metal ions on the activity of EDTA - treated enzymes
was discovered. from B. thermoantarcticus is illustrated in Table 4. Treatment of
Because of the high thermostability and the slightly acidic pH purified enzyme with EDTA resulted in an almost complete loss
optimum for the enzyme, xylose isomerase from B. thermoantarc- of enzyme activity. However, the activity could be restored by
ticus could be a better candidate for the production of high - fructose the addition of metal ions. In particular, increasing amounts of
syrup and ethanol [ 23 ]. Mn2 + , Mg2 + or Co2 + ( each up to 10 mM ) were able to restore
The effect of various metals on thermal stability of EDTA - only 60 – 80% of the original xylose isomerase activity. For
treated xylose / glucose isomerase from B. thermoantarcticus is glucose isomerase, 10 mM Mg2 + was required to restore 80% of
shown in Table 3. The xylose isomerase requires Mg2 + , Mn2 + or the original activity, whereas Mn2 + did not enhance enzyme
Co2 + for high thermal stability; glucose isomerase needed Mg2 + or activity. As is common with other isomerases, 10 mM Mn2 + , in
Co2 + , while Mn2 + had no effect. The combination of Mn2 + and combination with 1 mM Co2 + , had the ability to restore full
Co2 + showed an inhibitory effect for glucose isomerase. xylose isomerase activity, while 10 mM Mg2 + plus 1 mM Co2 +
restored total glucose isomerase activity.
Catalytic properties The results of equilibrium conversion of D - xylose and
D - glucose at different temperatures are shown in Table 5.
The K m and V max values were obtained from Lineweaver – Burk
plots of specific activities at various substrate concentrations. For Temperature had a profound influence on the equilibrium
xylose isomerase, K m and V max were 33 mM and 57 U / mg, concentration of products.
respectively, while for glucose isomerase, they were 167 mM and Xylose isomerase from B. thermoantarcticus isomerized
6.3 U / mg. The enzyme preference for xylose as substrate over xylose to xylulose up to an 80:20 equilibrium ratio, while
glucose was illustrated by a lower K m and higher V max toward this glucose to fructose equilibrated up to 50:50, as also reported by
Chen [ 9 ].
It is well known that equilibrium of the enzyme - catalyzed
isomerization between aldose and ketose was shifted to the ketose
Table 4 The effect of various metal cations on the activity of EDTA -
side when borate was added to the reaction mixture [ 32 ].
treated xylose ( XI ) / glucose ( GI ) isomerase purified from B. thermo-
antarcticus Isomerization of D - xylose and D - glucose with various
amounts of sodium tetraborate was shown in Figure 1e and f.
Metal Isomerase activity ( % of maximum ) The shift in equilibrium in both cases was almost linearly

XI GI

Table 5 Effect of temperature on equilibrium of isomerization of D - xylose

None 8 2
Mg2 + ( 1 mM ) 40 45 isomerase and D - glucose isomerase
Mg2 + ( 5 mM ) 70 65
Mg2 + ( 10 mM ) 80 82 Temperature ( 8C ) Conversion ( % )
Mn2 + ( 1 mM ) 55 0
Mn2 + ( 5 mM ) 59 0 X1 GI
Mn2 + ( 10 mM ) 62 0
Co2 + ( 1 mM ) 35 0
Co2 + ( 5 mM ) 48 0 60 17 15
Co2 + ( 10 mM ) 60 0 70 21 33
Mg2 + ( 10 mM ) Co2 + ( 1 mM ) 90 105 80 27 48
Mn2 + ( 10 mM ) Co2 + ( 1 mM ) 110 0
Values are the means of three independent assays.
 Xylose( glucose ) isomerase from Bacillus thermoantarcticus
L Lama et al

Table 6 Physico - chemical properties of xylose / glucose isomerases from thermophilic microorganisms 239

Microorganisms Molecular Optimum Optimum pH Thermostability

weight ( kDa ) temperature ( 8C )

B. thermoantarcticus 200 90 7.0 908C30 min

B. stearothermophilus 130 80 7.5 – 8.0 758C10 min
Thermot. maritima 200 105 – 110 6.5 – 7.5 1008C10 min
Thermot. neapolitana 200 95 7.1 half - life 958C24 min
Thermus thermophilus 200 95 7.0 858C8 h
Thermus aquaticus HB8 196 85 7.5 708C1 month
Thermoanaerobacterium saccharolyticum B6A - R1 200 80 7.0 – 7.5 858C1 h
Thermoanaerobacterium JW / SL - YS 489 200 80 6.8 808C1 h
Clostridium thermosulfurogenes 200 80 7.0 – 7.5 858C1 h
Streptomyces sp. ( PLC ) 183 80 7.0 538C10 days
Streptomyces sp. SK 185 95 6.0 808C5 h

affected by the quantity of tetraborate; the introduction of Acknowledgements

additional tetraborate did not increase the equilibrium concen-
This work was partially supported by PNRA. We thank E. Esposito
tration of products when more than 80% of substrates had been
and E. Pagnotta for technical assistance.
converted. This saturation phenomenon was also observed by
Takasaki [ 38 ].

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