Protein Engineering, Vol. 13, No. 4, 259-265,
April 2000
© 2000 Oxford University Press
Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis
1 Department of Food Science and Human Nutrition and 2 Department of Biochemistry, Michigan State University, East Lansing, MI 48824 and 3 Michigan Biotechnology Institute, Lansing, MI 48909, USA
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Abstract |
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Xylose isomerases (XIs) from Thermoanaerobacterium thermosulfurigenes (TTXI) and Thermotoga neapolitana (TNXI) are 70.4% identical in their amino acid sequences and have a nearly superimposable crystal structure. Nonetheless, TNXI is much more thermostable than TTXI. Except for a few additional prolines and fewer Asn and Gln residues in TNXI, no other obvious differences in the enzyme structures can explain the differences in their stabilities. TNXI has two additional prolines in the Phe59 loop (Pro58 and Pro62). Mutations Gln58Pro, Ala62Pro and Gln58Pro/Ala62Pro in TTXI and their reverse counterpart mutations in TNXI were constructed by site-directed mutagenesis. Surprisingly, only the Gln58Pro mutation stabilized TTXI. The Ala62Pro and Gln58Pro/Ala62Pro mutations both dramatically destabilized TTXI. Analysis of the three-dimensional (3D) structures of TTXI and its Ala62Pro mutant derivative showed a close van der Waal's contact between Pro62-C
and atom Lys61-Cß (2.92 Å) thus destabilizing TTXI. All the reverse counterpart mutations destabilized TNXI thus confirming that these two prolines play important roles in TNXI's thermostability. TTXI's active site has been previously engineered to improve its catalytic efficiency toward glucose and increase its thermostability. The same mutations were introduced into TNXI, and similar trends were observed, but to different extents. Val185Thr mutation in TNXI is the most efficient mutant derivative with a 3.1-fold increase in its catalytic efficiency toward glucose. With a maximal activity at 97°C of 45.4 U/mg on glucose, this TNXI mutant derivative is the most active type II XI ever reported. This `true' glucose isomerase engineered from a native xylose isomerase has now comparable kinetic properties on glucose and xylose.
Keywords: site-directed mutagenesis/thermal stability/thermozyme/xylose isomerase
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Introduction |
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Xylose isomerase (D-xylose ketol isomerase; EC 5.3.1.5) (XI) is an intracellular enzyme found in a number of bacteria that utilize xylose as carbon substrate for growth (Chen, 1980
). XI converts D-xylose to D-xylulose in vivo and also catalyzes the conversion of D-glucose to D-fructose in vitro (Takasaki et al., 1969). This latter activity is used in industry for the production of high fructose corn syrup (HFCS), and xylose (i.e. glucose) isomerase is one of the largest volume commercial enzymes used today (Lee and Zeikus, 1991). Thermostable XIs with neutral or slightly acidic pH optima have a potential for industrial applications with the advantages of faster reaction rates, higher fructose concentrations at equilibrium, decreased viscosity of substrate and product streams and less problems of by-products formation (Lee and Zeikus, 1991).
Two XI groups have been identified; type I enzymes are shorter than type II enzymes by about 50 amino acids at their N-terminus (Vangrysperre et al., 1990). Xylose isomerases are significantly stabilized and activated in the presence of divalent cations, especially by Mg2+, Co2+ and Mn2+. Two type II XIs have been studied extensively in our laboratory: one from a thermophile, Thermoanaerobacterium thermosulfurigenes (TTXI) and the other from a hyperthermophile, Thermotoga neapolitana (TNXI). The genes encoding these enzymes (xylA genes) were cloned, sequenced and expressed in Escherichia coli (Lee et al., 1990a,b; Meng et al., 1991; Vieille et al., 1995). TNXI has a higher turnover number, a lower Km for glucose and is more thermostable than any other known type II xylose isomerases (Vieille et al., 1995). Although TNXI and TTXI are highly similar (70.4% identity), TNXI is significantly more thermostable than TTXI with an optimum temperature of 95°C (85°C for TTXI) and melting temperature in the presence of 0.5 mM Co2+ of 126°C (82°C for TTXI) (Vieille et al., 1995). No obvious differences in the enzyme structures can explain the differences in their stabilities (Gallay et al., manuscript in preparation) except for a few additional prolines and fewer Asn and Gln residues in TNXI (as seen from its alignment with TTXI).
Proteins can be stabilized by decreasing their entropy of unfolding (Matthews et al., 1987). Prolines, with their pyrrolidine ring, can only adopt a few configurations. They restrict the configurations allowed for the preceding residue, and they can decrease the entropy of a protein's unfolded state. Substituting Pro for another carefully chosen residue can thus increase protein stability, provided that the newly introduced proline does not create volume interferences, and does not destroy stabilizing non-covalent interactions. The effect of prolines on protein stabilization has been studied by site-directed mutagenesis. The two T4 lysozyme mutants Ala82Pro (Matthews et al., 1987) and Ile3Pro (Dixon et al., 1992) illustrate the importance of carefully selecting the mutation location. With mutation Ala82Pro, the two conditions listed above were addressed, and the mutation stabilized the protein mainly by decreasing its entropy of unfolding. In mutant Ile3Pro, the substitution eliminated a hydrogen bond and the hydrophobic interactions created by Ile. The degree of enthalpic destabilization was greater than the entropic stabilization and the mutation was destabilizing. In a study by Allen et al. (1998) of Aspergillus awamori glucoamylase, three proline mutants—Ser30Pro, Asp345Pro and Glu408Pro—were constructed. The Ser30Pro mutation stabilized the protein because the mutation site allowed a residue conformation compatible with a proline, and because residue 29 (a valine) could adopt one of the conformations allowed for the residue preceding a proline (Matthews et al., 1987). The Glu408Pro mutation was destabilizing because the mutation site did not allow any conformation required for proline, whereas the Asp345Pro mutation did not affect stability due to the fact that the substitution destroyed the 
-helix dipole in that region. Prolines 58 and 62 in TNXI are present in a large loop in which 14 hydrophilic residues surround Phe59. The corresponding TTXI residues are Gln58 and Ala62. The Phe59 loop participates in building the neighboring subunit's active site (Farber et al., 1989; Whitlow et al., 1991). In the present report we test the hypothesis that substituting Gln58 and Ala62 with prolines will stabilize TTXI, and that the reverse mutations will destabilize TNXI.
In a previous study, genetic engineering was used to improve TTXI activity on glucose (not the natural substrate): the enzyme's substrate-binding pocket was enlarged and the water-accessible surface area in the active site was altered (Meng et al., 1991, 1993). Substituting Trp138 with Phe, a smaller residue, decreased TTXI's Km for glucose and increased its catalytic efficiency (kcat/Km) on glucose 2.6-fold. This mutant enzyme was less active than the wild-type TTXI on xylose. Interestingly, this mutation doubled TTXI's half-life at 85°C (Meng et al., 1993). Substituting Val185 with Thr, a polar residue, improved TTXI's catalytic efficiency on glucose. This improvement was thought to result from the creation of an additional hydrogen bond to glucose's C6-OH group. The double mutation Trp138Phe/Val185Thr vastly improved TTXI's catalytic efficiency on glucose (5.7-fold). In this study, we will introduce these mutations in TNXI to determine if TNXI activity and stability can be further enhanced by genetic engineering. All mutations will be characterized by their kinetic parameters, optimum temperatures, half-lives at 85 or 95°C, and temperatures of 50% precipitation in order to compare their thermal stability and activity.
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Materials and methods |
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Bacterial strains and chemicals
Escherichia coli strain BL21 (DE3) (Novagen, Madison, WI) was used to overexpress the recombinant T.thermosulfurigenes and T.neapolitana xylA genes cloned in the pET23a and pET22b+ vectors, respectively (Novagen). Media and growth conditions were the same as described by Vieille et al. (1995). Medium components and all other chemicals were reagent grade.
Site-directed mutagenesis and other DNA techniques
All DNA manipulations were performed using established protocols (Sambrook et al., 1989; Ausubel et al., 1993). Point mutations were introduced into the T.neapolitana and T.thermosulfurigenes xylA genes using the QuickChangeTM Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and M13 single-stranded site-directed mutagenesis (Ausubel et al., 1993). Mutagenic oligonucleotides (Table I) were synthesized by the Macromolecular Structure Facility (Department of Biochemistry, Michigan State University). Mutations were verified by DNA sequencing using the Thermosequenase Sequencing kit (US Biochemical, Cleveland, OH).
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Protein purification
Recombinant enzymes were purified using the procedure of Vieille et al. (1995) followed by two additional steps. Partially purified enzymes were applied to a Q-Sepharose column (2.5x15 cm) equilibrated with MOPS 50 mM (pH 7.0) containing 5 mM MgSO4 and 0.5 mM CoCl2 (buffer A) and enzymes were eluted using a 500 ml 0–250 mM NaCl gradient in buffer A. The pooled fractions from the Q-Sepharose column were concentrated in a stirred ultrafiltration cell (MW cut-off 30 kDa) (Amicon, Beverly, MA), dialyzed twice against buffer A, applied to a Polybuffer column (Pharmacia, Uppsala, Sweden) equilibrated with 25 mM histidine–HCl (pH 6.2) and eluted using a pH (6.0–4.0) gradient according to the manufacturer's instructions. The active fractions were pooled, concentrated in a stirred ultrafiltration cell (Amicon), and dialyzed twice against buffer A. Concentrated, homogenous enzymes were dispensed and stored frozen at –70°C.
Xylose isomerase assays
Xylose isomerase activity was routinely assayed with glucose as the substrate. The enzyme (0.06 mg/ml) was incubated in 50 mM MOPS (pH 7.0 at room temperature) containing 1 mM CoCl2 and 1 M glucose at 60°C for 20 min. The reaction was stopped by cooling the tubes in ice. The amount of fructose produced was determined by the cysteine–carbazole–sulfuric acid method (Dische and Borenfreund, 1951). To determine the effect of temperature on XI activity, the assay mixtures were incubated at the temperatures of interest in a Perkin-Elmer Cetus GeneAmp PCR system 9600 (Perkin Elmer, Norwalk, CT) for 20 min. To determine the kinetic parameters, assays were performed in the presence of either 80–1400 mM glucose or 20–900 mM xylose. The amounts of fructose and xylulose produced were determined as above. Absorbances were measured at 537 and 560 nm for xylulose and fructose, respectively. One unit of isomerase activity is defined as the amount of enzyme that produced 1 µmol product per min under the assay conditions.
Thermostability assays
The time course of irreversible thermoinactivation was measured by incubating the enzyme (0.5–5 mg/ml) in 10 mM MOPS buffer (pH 7.0) containing 50 µM CoCl2 (buffer B) at 85°C (TTXI derivatives) or 95°C (TNXI derivatives) in a Perkin-Elmer Cetus GeneAmp PCR system 9600 for various amounts of time, and by determining the residual glucose isomerase activity at 65 (TTXI derivatives) or 80°C (TNXI derivatives). The first order rate constant, k, of irreversible thermoinactivation was obtained by linear regression in semi-log coordinates. Enzyme half-life was calculated from the equation: t1/2 = ln 2/k.
Heat-induced enzyme precipitation
Heat-induced enzyme precipitation was monitored from 25 to 100°C by light scattering (
= 580 nm) using protein solutions (0.2 mg/ml) in buffer B. Absorbance measurements were conducted in 0.3 ml quartz cuvettes (path length = 1.0 cm), using a Gilford Response spectrophotometer (Corning, Oberlin, OH) equipped with a Peltier cuvette heating system. The increasing thermal gradient was 1.0°C min–1. The temperature of 50% precipitation was the temperature at which the OD580 equals half of the difference between the baseline and the maximum ODs.
Analysis of TTXI and TNXI three-dimensional structures
Enzymes were visualized on an IRIS-4D25 computer (Silicon Graphics Computer System, Mountain View, CA) using the INSIGHT II graphic program (Biosym Technologies, San Diego, CA). Proteins Data Bank (PDB) files (#1A0C for TTXI and #1A0E for TNXI) were obtained from the Protein Data Bank website (www.rcsb.org/pdb).
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Results |
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Proline substitutions in the Phe59 loop of TTXI and TNXI
TNXI Pro58 and Pro62 are present in a large loop in which 14 hydrophilic residues surround Phe59 (Figure 1). According to crystallographic data, Phe59 (Phe26 in Actinoplanes XI) participates in the architecture of the neighboring subunit's active site (Farber et al., 1989; Whitlow et al., 1991). The corresponding residues in TTXI, Gln58 and Ala62, have backbone dihedral angles [(–66.57, –14.49) and (–57.91, 138.87), respectively] (Gallay et al., manuscript in preparation) allowed for prolines (Nicholson et al., 1988). With backbone dihedral angles of (–139.73, –175.10) and (–93.32, 176.20), respectively, TTXI residues Asp57 and Lys61 are in extended conformations, the most common conformation for residues preceding prolines (Nicholson et al., 1988). This structural information suggests that mutations Gln58Pro and Ala62Pro would not create unfavorable backbone conformations and that they could stabilize TTXI.
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Mutation Gln58Pro did not affect the optimum temperature for TTXI activity (i.e. 85°C), whereas mutation Ala62Pro and double mutation Gln58Pro/Ala62Pro decreased it by 7 and 12°C, respectively (Figure 2A
). Mutation Gln58Pro stabilized TTXI at 85°C: the enzyme's half-life was extended from 69 to 99 min (a 43% increase). With half-lives of 6.2 and 21 min at 85°C, respectively, Ala62Pro and Gln58Pro/Ala62Pro mutant TTXIs were significantly less thermostable than the wild-type enzyme (Figure 3A). These surprising results indicate that Pro in position 62 destabilizes TTXI. Similar results were obtained in precipitation experiments. Mutation Gln58Pro increased TTXI's temperature of 50% precipitation by 6°C, whereas mutations Ala62Pro and Gln58Pro/Ala62Pro decreased it by approximately 4 and 3°C, respectively (Table II).
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, TTXI; 
, Gln58Pro; 
, Ala62Pro; 
, Gln58Pro/Ala62Pro. (B) TNXI and its mutant derivatives:
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The reverse counterpart mutations in TNXI (Pro58Gln and Pro62Ala, and double mutation Pro58Gln/Pro62Ala) decreased the optimum temperature for TNXI activity (Figure 2B
). Not surprisingly, thermoinactivation curves revealed that mutation Pro58Gln decreased TNXI's half-life from 69.3 to 49.5 min (29% decrease). Both mutations Pro62Ala and Pro58Gln/Pro62Ala decreased TNXI's half-life to 11.6 min (83% decrease) which confirmed that these mutations were all destabilizing as we expected (Figure 3B). Again, these results were confirmed by precipitation experiments. The temperature of 50% precipitation of both Pro62Ala and Pro58Gln were 95.7°C, 1.1°C lower than that of wild-type TNXI. Double mutant Pro62Ala/Pro58Gln precipitated at an even lower temperature than each single mutant enzyme, suggesting that these destabilizing effects are additional.
The kinetic features of the wild-type and mutant xylose isomerases with glucose and xylose as substrates were determined at 65°C for the TTXI series and at 80°C for the TNXI series (Table III). With the exceptions of mutations Ala62Pro and Gln58Pro/Ala62Pro in TTXI, the mutations in TTXI and TNXI Phe59 loops did not significantly alter the enzymes' catalytic properties. The Ala62Pro mutation increased both TTXI's Vmax and Km on glucose, leaving its catalytic efficiency on glucose almost unchanged. This mutation had a much stronger effect on TTXI activity on xylose. It increased its affinity for xylose and its catalytic efficiency approximately 2.5-fold. Mutation Gln58Pro/Ala62Pro had a more pronounced effect on TTXI activity on glucose. A twofold increase in its Km for glucose decreased its catalytic efficiency almost threefold. These results indicate that mutations Ala62Pro and Gln58Pro/Ala62Pro altered TTXI's catalytic features and at the same time destabilized the enzyme.
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Site-directed mutagenesis in TNXI active site
The Val185Thr mutant and the double mutant Trp138Phe/Val185Thr TNXI derivatives had the same optimum temperature (97°C) as wild-type TNXI (Figure 4A). Interestingly, mutation Val185Thr almost doubled TNXI specific activity on glucose at 97°C (from 26.0 to 45.6 U/mg). Although the Trp138Phe mutation decreased TNXI optimum temperature to 87°C, the mutant enzyme still retained almost 80% of its activity at 97°C. The stability of these mutant enzymes was studied at 95°C (Figure 4B). With a half-life of 69.3 min, the Val185Thr mutation did not affect TNXI stability at 95°C. The Trp138Phe mutant (half-life of 87 min) and the Trp138Phe/Val185Thr double mutant (half-life of 99 min) enzymes were 25 and 43% more stable, respectively, than wild-type TNXI.
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When kinetic parameters were concerned, these three TNXI mutant enzymes showed the same trends as their equivalents in TTXI (Table III
). They all showed improved catalysis on glucose and poorer catalysis on xylose. Most of the catalytic efficiency increases were due to lower Km's for glucose. Major differences existed though, between TTXI and TNXI derivatives. Whereas W138F is the best mutation in terms of increasing TTXI catalytic efficiency on glucose, its effect on TNXI catalytic efficiency on glucose is only marginal. On the other hand, mutation Val185Thr is better at increasing TNXI catalytic efficiency on glucose, than at increasing TTXI's. These differences are surprising, knowing that TTXI and TNXI active site structures are almost completely superimposable. |
Discussion |
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The high degree of similarity between TTXI and TNXI and their significantly different thermostabilities and thermophilicities make comparative studies of these enzymes attractive for understanding the key molecular features responsible for their stability and activity differences (Vieille et al., 1995
). Multiple factors have been identified (Vieille and Zeikus, 1996) that can be responsible for a protein's high thermostability. They include packing efficiency, hydrophobic interactions, loop stabilization, reduction of entropy of unfolding, electrostatic interactions, etc. In our case, no obvious differences in TTXI and TNXI structures could explain their different thermostabilities except for a few additional prolines and fewer Asn and Gln residues in TNXI compared with TTXI.
Two additional prolines are found in a large `Phe59 loop' region in TNXI (Figure 1). These prolines are substituted with Gln and Ala in TTXI. A protein can be stabilized by engineering proline into selected sites thereby decreasing the protein's conformational entropy of unfolding (Allen et al., 1998). In order for a proline substitution to stabilize a protein, at least four criteria have to be considered: (i) the mutation site should allow one of the conformations allowed for proline; (ii) the preceding residue should be able to adopt one of the conformations allowed for the residue preceding a proline; (iii) proline should not create volume interferences and (iv) proline should not destroy stabilizing non-covalent interactions (Nicholson et al., 1988). TTXI Gln58 and Ala62 have backbone dihedral angles allowed for prolines, and Asp57 and Lys61 have dihedral angles allowed for residues preceding prolines. Despite these first two conformational requirements being satisfied, only mutation Gln58Pro stabilized TTXI. As seen in Figure 5A, the conformation of Gln58's side chain is very close to that of the proline pyrrolidine ring. No volume interference is created by the Gln58Pro mutation. In the wild-type TTXI structure, Gln58 is not involved in any potentially stabilizing, non-covalent interactions (not shown). No enthalpic destabilization is expected with mutation Gln58Pro, and the stabilization it provides to TTXI probably entirely results from a reduction of the entropy of unfolding. On the other hand, the Ala62Pro mutation had a destabilizing effect on TTXI. In TTXI, Ala62's side chain points toward a large cavity, and it is not in close vicinity of any other residues. So the Ala62Pro mutation does not eliminate any stabilizing non-covalent interactions. Detailed analysis of the Ala62Pro mutation modeled into the TTXI structure (Figure 6) suggests that Pro62's pyrrolidine ring (C atom) is in close contact (within 2.92 Å) with Lys61's side chain (Cß atom). Carbon atoms have van der Waal's radii of 1.70–1.78 Å in protein. Optimal van der Waal's interactions between two carbon atoms would take place at approximately 3.4–3.5 Å. The unfavorable van der Waals contact between Pro62-C and Lys61-Cß probably leads to local conformational changes. Not only are these changes destabilizing, they also affect the active site structure and the enzyme's interaction with the substrate. The overall destabilizing nature of mutation Ala62Pro indicates that the conformational destabilization of the native enzyme more than cancels the benefits of a potential decrease in unfolding entropy.
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Another key molecular structure–function feature studied in this paper are residues in the active sites. Meng et al. (1991) have shown that mutation Trp138Phe significantly increased TTXI catalytic efficiency on glucose (2.7-fold increase). This increase was suggested to result from a better accommodation of glucose in the substrate-binding site because Trp was substituted by a smaller residue, Phe. This substitution was also shown to double TTXI's half-life at 85°C (Meng et al., 1993
). The higher thermostability may be explained as the consequence of a reduction of the water-accessible hydrophobic surface area. Another mutation that has been shown to increase TTXI's catalytic efficiency on glucose was Val185Thr. This increase was attributed to additional hydrogen bonding of Thr to glucose's C6-OH. When these mutations were introduced into TNXI, we observed the same trends in terms of catalytic features. All the mutant enzymes, including the double mutant Trp138Phe/Val185Thr, had a higher catalytic efficiency for glucose than wild-type TNXI. These increases mainly resulted from a much lower Km for glucose than that of TNXI. On the other hand, all these mutant TNXIs showed a lower catalytic efficiency for xylose like they also did in TTXI. Trp138Phe was the mutation that had the most significant effect on TTXI's catalytic efficiency on glucose. Here, the TNXI Val185Thr mutant derivative had the highest catalytic efficiency on glucose. This result came as a surprise since the active sites of the two XIs were almost completely superimposable. As seen in Figure 7, Trp138 and Val185 are almost completely superposed in TTXI and TNXI. The conformations of the neighboring residues are also extremely conserved. The most significant difference between TTXI and TNXI active sites is the position of metal II (i.e. the catalytic metal, a Co2+ in both enzymes): the two cations are 1.23 Å apart. This shift of the catalytic metal might affect the enzyme–substrate binding properties and/or the catalytic metal's reactivity during catalysis, thus explaining why there was a difference in mutation effects on TTXI and TNXI catalytic activities on glucose. All TNXI mutant derivatives were as stable as (Val185Thr) or more stable than wild-type TNXI (Trp138Phe and Trp138Phe/Val185Thr). These mutations did not increase TNXI stability to the same extent as they did in TTXI (Meng et al., 1993). Mutation Trp138Phe might have the same stabilizing potential in TNXI as in TTXI, but another TNXI molecular feature probably becomes limiting for stability before the full stabilization potentially provided by the mutation can be reached. These findings prove that it is possible to further stabilize hyperthermophilic proteins. To our best knowledge, with a maximal activity at 97°C of 45.4 U/mg on glucose, the TNXI Val185Thr mutant derivative is the most active type II xylose isomerase ever reported.
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In conclusion, our data show that genetic engineering approaches can be utilized to identify amino acid residues responsible for extreme thermal stability and high catalytic activity of xylose (glucose) isomerase thermozymes. Both prolines 58 and 62 appear to stabilize TNXI's Phe59 loop; whereas TTXI's thermal stability can be enhanced by the Gln58Pro mutation. TNXI's thermal stability was further enhanced by the Trp138Phe substitution. The Val185Thr mutation significantly enhanced TTXI's and TNXI's Vmax and kcat/Km for glucose isomerization to fructose. This significant catalytic enhancement of glucose isomerase activity was made possible in large part by the template enzyme's naturally evolved function as xylose isomerase. Genetic engineering altered the active site only to better accommodate glucose as the substrate.
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Notes |
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4 To whom correspondence should be addressed at: Michigan Biotechnology Institute, Collins Road, Lansing, MI 48909, USAEmail: zeikus{at}mbi.org
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Acknowledgments |
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This research was supported by grant 95-29047 from the National Science Foundation and by the MSU Crop and Food Bioprocessing Research Center. We gratefully acknowledge Dr Kaillathe Padmanabhan for his generous help and helpful discussions on the 3D structure of XIs.
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Received August 19, 1999; revised January 27, 2000; accepted January 27, 2000.
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