Báo cáo Y học: Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis Gene cloning and protein characterization doc

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis Gene cloning and protein characterization Anna LoÈnn 1 ,Ma  rk Ga  rdonyi 1 , Willem van Zyl 2 ,BaÈ rbel Hahn-HaÈ gerdal 1 and Ricardo Cordero Otero 2, * 1 Department of Applied Microbiology, Lund University, Sweden; 2 Department of Microbiology, University of Stellenbosch, Matieland, South Africa Random PCR mutagenesis was applied to the Thermus thermophilus x ylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild- type and mutated xylA genes w ere cloned and expressed in Escherichia coli HB101 using the v ector pGEM Ò-T Easy, and their physicochemical and catalytic properties were determined. T he optimum pH for xylose isomeriza- tion activity for the mut ants was  7.0, which is similar to the wild-type e nzy me. Compared w ith the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k cat )for D -xylose compared with the wild-type enzyme at 60 °C, but they did not s how any increase in catalytic eciency ( k cat /K m ). For D -glucose, both the k cat and the k cat /K m values for the mutants were increased compared w ith the wild-type enzyme. Furth ermore, the mutant enzymes exhibited up to 255 times higher inhibi- tion constants (K i ) for xylitol than the wild-type, indicat- ing t hat they are less in hibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of thewild-typeenzyme.Theresultsarediscussedintermsof increased molecular ¯exibility of the mutant enzymes at low temperatures. Keywords: xylose isomerase; cold adaptation; random mutagenesis; Saccharomyces cerevisiae; xylose fermentation. The use of ethanol from renewable raw materials is an attractive alternative for meeting increasing g lobal demand for liquid fuels because its combustion does not contribute to the greenhouse effect. For the industrial production of ethanol from pretreated and hydrolysed lignocellulose, the yeast Saccharomyces cerevisiae is the prime choice (reviewed i n [1]). Between 10 and 40% of lignocellulosic raw materials consists of pentoses [2], where xylose is the predominant portion. However, S. cerevisiae can not metabolize x ylose, only D -xylulose, an isomerization product of D -xylose. Xylose reductase (EC 1 .1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) from the xylose-fer- mentin g yeast Pichia stipiti s, have been introduced in to S. cerevisae to allow xylose fermentation to ethanol [3±5]. Fermentations r esulted in l ow ethanol yields and consi d- erable xylitol by-product f ormation. Xylose isomeras e ( XI) (EC 5 .3.1.5) i s u sed in the production of high-fructose corn syrup, where it catalyses the conversion of D -glucose to D -fructose [6]. The physiological function of the enzyme in v ivo is, however, the isomerization of the pentose D -xylose to D -xylulose. XI genes ( xylA) from several bacteria have been introduced into S. cerevisiae, including xylA from Escherichia coli [7,8], Actinoplanes missouriensis [9], Bacillus subtilis [9], Lactobacillus pentosus [10] and Clostridium thermosulfurogenes [11]. However, none of these attempts generated an active XI. The only xylA gene succe ssfully expressed in S . cerevi- siae was cloned from T. thermophilus [12]. This thermo- philic XI, with a temperature optimum at 85 °C, has a low a ctivity at 30 °C [12] which is the optimal growth temperature for S. cerevisiae. It would therefore be desirable to generate mutants of XI with improved kinetic properties at low temperatures. Random chemical muta- genesis has been used recently to obtain variants of the T. thermophilus 3-isopropylmalate-dehydrogenase [13], Sulfolobus solfataricus indolglycerol phosphate synthase [14] and t he mesophilic protease subtilisin BPN¢ [15±17], with increased activity at low temperatures. Error-prone PCR followed by DNA shuf¯ing resulted in the arti®cial evolution of cold-adapted mutants of a b-glycosidase from Pyrococcus furiosus [18] and a subtilisin-like protease from Bacillus sphaericus [19]. Here, we report on random PCR mutagenesis to create cold-adapted T. thermophilus XI. The character- ization of the physicochemical and c atalytic properties of three c old-adapted XIs that exhibited up to 9 times higher k cat for xylose than the wild-type enzyme at 60 °C are described. Correspondence to B. Hahn-Ha È gerdal, Department of Applied Microbiology, Lund University, PO Box 124, SE-221 00 Lund, Sweden. Fax: + 46 46 2224203, Tel.: + 46 46 2228428, E-mail: Barbel.Hahn-Hagerdal@tmb.lth.se Abbreviations: XI, xylose isomerase. *Present address: Institute for Wine Biotechnology, University of Stellenbosch, Private Bag XI, Matieland 7602, South Africa. (Received 28 May 2001, revised 23 October 2001, accepted 25 October 2001) Eur. J. Biochem. 269, 157±163 (2002) Ó FEBS 2002 MATERIALS AND METHODS Chemicals All chemicals were obtained from commercial suppliers and used as described by the manufacturer. D (+)-xylose was obtained f rom S igma (Steinheim, Germany) and sorbitol dehydrogenase from Boehringer Mannheim (Mannheim, Germany). Strains and plasmids Escherichia coli HB101(F-hsdS20ara-1 recA13 proA12 lacY1 galK2 rspL20 mtl-1xyl-5) [20] was used for cloning of the mutated XIs using pGEMÒ-T Easy vector (Promega, Madison, WI, USA). PCR mutagenesis Random mutagenesis of the XI gene (xylA) was performed under conditions described previously [21] using the PCR primers 5¢-TGATC AATGTACGAGCCCAAACC-3¢ and 5¢-TGATCACCCCCGCACC-3¢, which directly ¯ank the xylA gene. Both primers contained the restriction endonuc- lease site for BclI (underlined). The PCR contained: 1 ´ PCR buffer (BIOTAQä), 0.2 m M dATP, 0.2 m M dGTP, 1 m M dCTP, 1 m M dTTP, 1.5 m M MgCl 2 ,0.5m M MnCl 2 ,0.15l M of both primers, 0.02 n M template DNA and 5 U Taq DNA polymerase (BIOTAQä)inatotal volume of 100 lL. PCR was performed in a Thermal Cycler (PerkinElmer 2400) for nine cycles: 30 s at 94 °C, 30 s at 50 °Cand45sat68°C. The PCR products were then puri®ed using H igh Pureä PCR Product (Boehringer Mannheim). DNA sequencing Analysis of the mutated sequences was carried out using ABI PRISMÒ Big Dyeä Terminator cycle sequencing ready reaction kits with an ABI PRISMä 377 DNA sequencer (PE/Applied Biosystems). Both t he coding and the noncoding strands were sequenced to ensure the reliable identi®cation of all mutations. Growth conditions and preparation of cell extract from E. coli E. coli HB101 h arbouring the plasmids pGEM Ò -T Easy containing the wild-type and the mutated XI genes w ere grown at 37 °C in 50 mL Luria±Bertani medium [22] containing 100 lgámL )1 ampicillin. The cells were har- vested by centrifugation in the stationary phase of growth and wash ed once w ith i ce-cold d istilled w ater. W ashed cells were resuspended in 100 m M triethanolamine, pH 7.0, 65 kUámL )1 lysozyme, 0.25 mgámL )1 DNAse and 1 m M phenylmethanesulfonyl¯uoride in dimethylsulf- oxide. The s olutions were kep t at room temperature for 1 h and then on ice for 2 h before storing in a freezer at )20 °C. Cell extracts were thaw ed on ice, cell d ebris was removed by centrif ugation (15 000 g for 1 5 min at 4 °C) and the supernatant was used as the crude enzyme preparation. Protein determination Protein concentration was determined using the Pierce protein reagent with bovine serum albumin as standard [23]. Page SDS/PAGE was performed as previously described [24]. Immunochemical determination of XI Rabbit antiserum against XI from Streptomyces rubiginosus was prepared by Antibody AB (So È dra Sandby, Sweden) and immunoblotting was performed as described previously [25]. Brie¯y, 2 lg of cell-free e xtract together with 2±50 ng of puri®ed XI from S. rubiginosus were resolved by SDS/ PAGE and were then electrophoretically transferred onto a poly(vinylidene di¯uoride) membrane (Bio-Rad, Hercules, CA, USA). The blotted proteins were identi®ed immuno- chemically by sequential addition of anti-XI serum followed by goat anti-(rabbit IgG) Ig conjugated with alkaline phosphatase (Bio-Rad, Hercules, CA, USA). The secondary antibody was detected with a S torm 860Ò (Pharmacia Amersham, Uppsala, Sweden) using a chemi¯uorescent substrate ECF (Pharmacia Amersham). Data analysis was performed using IMAGE QUANT Ò software (Pharmacia Amershamm), giving a quantitative measurement of the amount of XI in the cell-free extracts. These data were used with the maximum velocity ( V max )tocalculatek cat . Enzyme assays A two-step XI standard assay (0.5 mL) was modi®ed from [26]. A substrate concentration of 700 m MD -xylose was used at 60 °C in 200 m M triethanolamine at pH 7.0 in the presence of 10 m M MnCl 2 and crude enzyme preparations. Glucose isomerase activity was assayed under the same reaction conditions as those used in the XI assay, except that glucose instead of xylose was used in the reaction mixture. The reactions were stopped by adding 150 lL 50% trichloroacetic acid, and then 2 M Na 2 CO 3 was added to neutralize the solutions. The isomerization products, xylu- lose or fructose, were reduced at pH 7.0 (37 °C) with 0.04 U sorbitol dehydrogenase (SDH) or 0.5 U SDH, respectively, and 0.15 m M NADH using a COBAS MIRA plus (Roche, Mannheim, Germany). The rate of disappearance of NADH was followed at 340 nm and the amount of D -xylulose and D -fructose determined from calibration curves. One unit o f isomerase activity was de®ned as the amount of crude enzyme r equired to produce 1 lmol of product per minute under the assay conditions employed. The speci®c activity (Uámin )1 ámg )1 ) was determined from the a ctivity and the protein co ncentration of the crude enzyme preparations. Kinetic parameters The kinetic parameters, V max (lmolámin )1 ámg )1 )and Michaelis constant (K m ,m M ), were determined from Michaelis±Menten plots of speci®c activities at various substrate concentrations. Typically, duplicate measure- ments at 6±10 concentrations of substrate s panning the value of K m were used to determine the value of K m .The 158 A. Lo È nn et al. (Eur. J. Biochem. 269) Ó FEBS 2002 concentration of XI in the c ell-free extracts was determined immunochemically using a molecular mass of 44 000 kDa [27], to allow calculation of the catalytic rate constant (k cat ) from the relationship k cat  V max /[E 0 ], where [E 0 ]  tot al enzyme concentration [28]. The K i (m M ) for xylitol was d etermined by i ncubating crude enzyme pre paration s in different xylose concentra- tions (20±600 m M ) at different ®xed xylitol concentrations. By plotting the speci®c activities for each xylitol concentra- tion against the xylose concentrations, K i was determined using the equation K m ¢  K m á(1 + i/K i )[29],wherei is the xylitol concentration (m M )andK m ¢ the a pparent K m value at a certain concentration of xylitol. PH pro®le The effect of pH on the a ctivity of t he wild-typ e a nd mutated enzymes was investigated in the pH range 5±10 in 700 m M xylose, 10 m M MnCl 2 and a buffer prepared b y mixing acetate, Pipes, Hepes and glycine, to a ®nal concentration o f 5 0 m M each [30]. The pH was adjusted at 60 °C with NaOH. Above pH 7.0 corrections were made for the chemical isome rization of D -xylose. Temperature pro®le The temperature pro®les for the wild-type XI and mutated XIs were m easured at temperatures between 30 and 95 °C. Above 60 °C corrections were made for the chemical isomerization of D -xylose. Preparation of metal-free XI and metal ion effects on enzyme activity Metal-free enzymes were prepared as previously described [26]. N o isomerase activity was observed in the absence of Mn 2+ ,Mg 2+ or Co 2+ . The effect of metal ions on XI activity was determined by adding 10 m M ®nal concentra- tion of either CoCl 2 ,MnCl 2 or MgCl 2 to the metal-free enzyme preparations in the assay mixture. Enzyme stability The temperature stability of the wild-type XI and mutated XIs was investigated by incubating metal-free crude enzyme preparations in 200 m M triethanolamine, pH 7.0 with 10 m M MnCl 2 in airtight tubes at 70 °C. At different times, 100-lL samples were withdrawn and stored on ice until the residual activity was determined. RESULTS Isolation of XI mutants with increased activity at low temperatures One-step mutagenesis was used to screen for mutant XIs with improved activity at low temperatures. The mutated XI fragments were cloned i nto the vector E. coli pGEMÒ-T Easy and transformed into the E. coli HB101 (xyl-5)strain to generate a mutant library. Transformants were replica plated on McConkey agar plates, complemented with 1% xylose and c ultivated at 3 7 °C overnight. A fter a further 2 days o f incubation at 30 °C, the p H indicator in t he medium allowed detection and quanti®cation of red acid- producing colonies. Three candidate mutants, termed M-1021, M-1024 a nd M-1026 were identi®ed. Colonies of these three were a d eeper red on the McConkey/xylose medium than were wild-type xylA colonies (suggesting higher XI activity). DNA s equencing revealed t hat the mutants exhibited approximately 80% transitions (T to C) and 20% transversions (A to C or T). XI from T. thermophilus is a homotetrameric enzyme with a 387-residue subunit. Each monomer c omprises two domains: the larger N-terminal domain (domain I, residues 1±321), which f olds into a (b/a) 8 barrel, and t he smaller C-terminal domain ( domain II, residues 322±387), which consists of loops and helices (Fig. 1) [31]. Domain II extends from domain I and makes extensive contacts with a neighbouring subunit. M-1021 contained two mutations in domain II; E372G and V 379A. M-1024 possessed two mutations, one in domain I (F163L) and one in domain II (E372G). M-1026 carries one mutation in domain II that is shared by M-1024 and M-1021; E372G. The locations of the amino-acid substitutions in the original tertiary structure of XI are shown i n Fig. 1. Neither the substrate-binding sites (H53, D56 and K 182) nor th e metal-binding sites (E180, E216, H219, D244, D254, D256 and D286) were affected by the mutations in the mutant enzymes. Properties of the mutant enzymes Temperature pro®les. XI from T. thermophilus has a temperature optimum around 95 °C [30]. To investigate whether the mutations caused any change in the tempera- ture optimum the temperature pro®les were investigated from 30 to 95 °C (Fig. 2). The temperature optimum for M-1024 and M-1026 was around 5 °C higher t han t he optimum for the wild-type (90 °C). For M-1021 the temperature optimum was somewhat lower,  75 °C. At 30 °C the speci®c activity was higher for the mutants than for the wild-type XI. Due to the overall low activity of the enzymes at this temperature, the physicochemical and kinetic characterization of the wild-type and mutant enzymes was carried out at 60 °C. PH pro®les. XI from T. thermophilus shows a pH optimum around 7.0 [30]. To examine w hether the mutations altered the pH dependence for xylose isomerization, the activity of Fig. 1. Structure of one subunit of T. thermophilus XI. The amino acids 372, 379 and 163 are identi®ed to show the position of the mutations. Ó FEBS 2002 Mutant xylose isomerases (Eur. J. Biochem. 269) 159 each m utant enzyme was measured as a function of pH (Fig. 3). The activity of each enzyme relative t o t he maximum activity was plotted as a percentage against pH. The pH dependence of the enzyme activity was examined at a substrate concentration well above K m , where the velocity of the r eaction is p roportional to k cat . The pH activity pro®les of the mutants were broader, and extended into the alkaline region, compared with the wild-type XI. The wild- type showed no XI activity at pH 9 and 10. For M-1024 and M-1026 the speci®c activit y at pH 9 and 10, was 66 and 45%, and 62 and 31%, of the maximum, respectively. The pH optima for the mutant XIs were not signi®cantly different from that of the wild-type, i.e. around 7.0. Eect of metal ions. XIs r equire two metal ions to be bound to the active site of each monomer in order to exhibit enzyme activity [32]. However, XIs from different organisms require different metals for optimal activity [33], a nd XI from T. thermophilus requires e ither M g 2+ or Mn 2+ for 100% activity [30]. Metal ions are not only essential for the catalytic mechanism, but they also co ntribute to the stabilization of the native structure, which is especially important for thermophilic enzymes. The effect of different bivalent metal ions (Mn 2+ ,Mg 2+ and Co 2+ ) on the EDTA-treated enzymes was investigated (Table 1). The wild-type and mutated XIs were most effectively activated by Mn 2+ and, to a smaller degree, by Mg 2+ and Co 2+ . The wild-type showed 88 and 74% of the maximum activity with Co 2+ and Mg 2+ , respectively. The mutants, on the other hand, were less activated by Co 2+ and Mg 2+ . Kinetic properties of D -xylose and D -glucose isomeriza- tion. The kinetics of D -xylose and D -glucose isomerization were determined from crude enzyme preparations at 60 °C, pH 7.0, and at metal-ion satu ration (Mn 2+ ) (Table 2 ). The K m values for D -xylose were up to 26 times higher for the mutants, and the catalytic rate constants (k cat )wereupto nine times higher than for the wild-type enzyme. The catalytic ef®ciency (k cat /K m )for D -xylose for M- 1026 was 6% higher than that of the wild-type, while for the other mutants it was lower. As for the wild-type X I, the mutants had a lower K mand higher k cat for D -xylose than for D -glucose. The K m for glucose for M-1021 and M-1024 was lower, by as much as three times, than for the wild-type enzyme. For M-1026, on the other hand, the K m was higher than that of the wild-type enzyme. T he k cat and the k cat /K m values for D -glucose were up to ®ve and seven times higher, respectively, for all the mutants, than for the wild-type XI. Inhibition by xylitol. The extended a cyclic forms of the substrates xylose and glucose have binding closely resem- bling that observed for the acyclic polyol inhibitor xylitol [34,35]. Competitive i nhibition is thus expected and has previously been reported [36,37]. K i for xylitol for the three mutant enzymes was between seven ( M-1021) and 255 (M-1024) times higher, than for the wild-type enzyme (Table 2), indicating that the mutant enzymes are not inhibited by xylitol to the same extent as t he wild-type enzyme. Thermal stability. To determine whether the m utations producing a change in the temperature dependence of XI activity also affected the thermal stability of the mutated enzymes, the r esidual a ctivities w ere m easured afte r heat treatment at 7 0 °C for various lengths of t ime (Fig. 4). Investigations of the m etal-free enzyme preparations in buffer at saturated metal concentration (Mn 2+ ) showed that the wild-type XI and the mutated XIs retained almost Fig. 3. The relative activity at dierent values of pH for the mutated XIs and the wild-type XI: (e) wild-type; (d) M-1021; (,) M-1024; and (j) M-1026. The scale of relative activity (%) indicates the percentage of experimental value at various pH relative to the maximum value of each enzyme. Table 1. Eect of various bivalent cations (10 m M ) o n the activity of EDTA-treated enzymes. The % relative activity is shown compared to the speci®c activity with 10 m M MnCl 2 at 60 °C which was set to 100% for each enzyme. Enzyme Co 2+ Mg 2+ Wild type 87.9 73.8 M-1021 7.4 23.2 M-1024 33.6 22.7 M-1026 34.6 38.9 Temperature ( o C) 20 30 40 50 60 70 80 90 100 Relative activity (% of maximum) 0 20 40 60 80 100 120 Fig. 2. The relative activity at dierent temperatures for the mutated XIs and the wild-type XI: (e) wild-type; (d) M-1021; (,) M-1024; and (j) M-1026. The scale of relative activity (%) indicates the percentage of experimental values at various temperatures relative to the maxi- mum value of each enzyme. 160 A. Lo È nn et al. (Eur. J. Biochem. 269) Ó FEBS 2002 full activity after 8 h of incubation. The mutants showed a drop in residual activity after 24 h, and after 56 h of incubation between 54 and 74% of their maximum activity remained. T he wild-type still had 95% residual activity after 56 h of incubation. Clearly, the mutated XIs were more sensitive to heat treatment at 70 °C than the wild-type XI. DISCUSSION The goal of the present study was to generate ef®cient cold- adapted XIs from T. thermophilus, with improved kinetic properties at low temperatures. Random PCR mutagenesis was performed in the gene encoding the enzyme (xylA)and a mutant library was constructed. When the resulting proteins were screened, we obtained three cold-adapted mutants: E372G/V379A (M-1021), E372G/F163L (M-1024) and E372G ( M-1026), with hig her k cat values than the wild-t ype XI for D -xylose at 60 °C. All mutations obtained were located on the enzyme surface, and not close to t he active site. Amino-acid substitution distant f rom the catalytic ce ntre or in the major substrate b inding site of enzymes c an lead to cold adaptation [38]. It has been proposed that variations in the enthalpy and entropy of conformational changes of impor- tance in binding and catalysis can be due to sequence changes outside the active sites. In the evolutionary adaptation of k cat and K m in response to acute temperature changes, these e ffects should play an i mportant role [39]. The effect of mutation in a single amino acid on the kinetic properties r eported h ere has been seen before. There are reports that almost all the p sychrophilic character of some cold-adapted enzymes is due to a single amino-acid substitution. A single difference in the sequence at a subunit contact site was the cause of differences in the temperature± K m relationship or stability between closely related ®sh LDH [40]. In addition, nearly all the improvement in the catalytic ef®ciency of a mutated Vibrio marinus triosephos- phate is omerase was due to replacement of a completely conserved Ser in the phosphate binding helix by Ala in the psychrophilic enzyme [41]. There are, however, no structural features that can be correlated exclusively to cold adapta- tion. Structural explanations for cold adaptation can not be generalized. There is no single structural characteristic that accounts for the simultaneously appearing low stability and increased catalytic ef®ciency, proposed to be a consequence of high molecular ¯exibility. T he origin o f the increased enzyme activity and red uced stability lies i n a partic ular region of the molecule rather than, for example, a general reduction in intramolecular interactions. A clear correlation seems to exist between cold adaptation and a reduction in the number of interactions between structural domains or subunits [42]. There is a close relationship between molecular ¯exibility and function. Thermophilic enzymes are rigid and require elevated temperatures in order to gain suf®cient molecular ¯exibility for activity. Their molecular structure must thus be balanc ed between the requirements f or stability and dynamics. We propose that the sequence changes underly- ing t he adaptation of T. thermophilus XI mutants to temperatures lower than their optimal temperature, allow a higher degree of ¯exibility in a reas that move during catalysis. Higher ¯exibility in these areas should increase k cat by reducing the energetic cost of a conformational change from the apoenzyme to the holoenzyme. By increasing k cat and K m , the catalytic ef®ciency of most cold-adapted enzymes increases, compared with t he warm-adapted ones. k cat increases because of the ability of cold-adapted enzymes to reduce the free energy of activation compared with warm- adapted homologues. The increased K m istheresultofa more ¯exible conformation [39]. Kinetic analysis d emon- strated that the increase in the relative activity in the mutated XIs for xylose at low temperatures was indeed caused by an increase in k cat and not by a decrease in the K m value. This suggests that the mutant enzymes did not acquire higher af®nity for the substrate than the wild-type enzyme at lower temperatures. The k cat /K m values for xylose for the mutated X Is only improved for M-1026. This was due to the large increase in the K m values for xylose. The Table 2. K inetic properties of wild-type XI and mutated XIs. Xylose Glucose Xylitol K i (m M ) K m (m M ) k cat (s )1 ) k cat /K m (s )1 ám M )1 ) K m (m M ) k cat (s )1 ) k cat /K m (s )1 ám M )1 ) Wild-type 3.44  0.4 46.6 13.6 146.8  12.3 16.3 0.11 4.6 M-1021 25.1  4.0 257.5 10.3 52.0  4.9 39.9 0.77 33.2 M-1024 89.4  8.4 381.6 4.3 130.8  17.1 66.9 0.51 1174 M-1026 28.7  3.3 412.4 14.4 171.8  10.0 88.7 0.47 68.7 Fig. 4. Thermal stability of wild-type XI and mutated XIs. Metal-free enzyme preparations were incubated at 70 °C in 200 m M triethanol- amine, pH 7.0, 10 m M MnCl 2 , and residual activities of aliquots were recorded as a function of time using xylose as a substrate: (e) wild- type; (d) M-1021; (,) M-1024; (j) M-1026. Ó FEBS 2002 Mutant xylose isomerases (Eur. J. Biochem. 269) 161 speci®c activity, or turnover number, k cat , re¯ects the catalytic potential at saturated substrate concentrations. The quantity, k cat /K m , is the catalytic ef®ciency that re¯ects the overall conversion of substrate to product. It has been suggested that the catalytic ef®ciency, k cat /K m ,providesa better approximation o f catalytic activity at physiological substrate concentrations, w hich are usually below satura- tion [43]. In lignocellulosic hydrolysate the concentration of xylose can vary considerably. The concentration of xylose inside the cell, on the o ther hand, remains unknown, and i s probably dependent on the xylose transporters. In natural xylose fermenting yeasts, the ®rst xylose converting enzyme (XR) has a K m for xylose between 10 and 100 m M [44±46]. Recombinant S. cerevisiae expressing XR from P. stipitis has been shown to ferment xylose [3±5]. Therefore it is reasonable to assume that t he mu tated XIs with K m for xylose between 25 and 89 m M will be able to support a functional xylose metabolic pathway. For glucose, all mutated XIs had both h igher k cat and k cat /K m values. These results indicate that we obtained improved kinetic constants at 60 °Cfor D -glucose isomer- ization, but not to the same extent for D -xylose isomeriza- tion. Clearly, the mutated XIs were also thermally sensitive at 70 °C, indicating that these m utations might confer ther- molabile characteristics on the enzyme. It has been reported previously that the thermostability of proteins can be altered by single amino-acid substitution [47,48], but it is not yet clear which these a mino acids are [49]. I t has also be en suggested that higher catalytic ef®ciency in naturally occurring cold-adapted enzymes is associated with lower thermal stability, due to the higher molecular ¯exibility at lower temperatures [ 43,50,51]. The low stability at high temperatures is therefore regarded as a necessary conse- quence of cold adaptation. The reduced thermal stability of the mutated XIs i s not a problem for xylose fermentation because fermentation occurs at moderate (30±40 °C) tem- peratures and the yeast is continuously producing the enzyme during the fermentation process. However, the higher k cat at moderate temperatures is essential for obtaining xylose fermentation rates compatible with indus- trial processes [12]. All mutants showed a dramatic increase in K i for xylitol, which is an inhibitor of XI. This may be a very important trait in the fermentation of xylose to ethanol, as S. cerevisiae produces xylitol from xylose via unspeci®c aldose reductases [52,53]. 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Tra È ,K.,CoderoOtero,R.R.,vanZyl,W.H.&Hahn-Ha È gerdal, B. (2000) Genetically engineered yeast and mutants thereof for the ecient fermentation o f the lignocellu lose hydrolys ates. Interna- tional patent application, WO 99/54477. Ó FEBS 2002 Mutant xylose isomerases (Eur. J. Biochem. 269) 163 . Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis Gene cloning and protein characterization Anna LoÈnn 1 ,Ma  rk Ga  rdonyi 1 , Willem van Zyl 2 ,BaÈ. of thewild-typeenzyme.Theresultsarediscussedintermsof increased molecular ¯exibility of the mutant enzymes at low temperatures. Keywords: xylose isomerase; cold adaptation; random mutagenesis; Saccharomyces cerevisiae; xylose fermentation. The. A Ê Streptomyces rubiginosus structures with xylitol and D -xylose. Proteins 9, 153±173. 36. Yamanaka, K. (1969) Inhibition of D -xylose isomerase by penti- tols and D -lyxose. Arch. Biochem. Biophys.