Protein Engineering, Vol. 14, No. 9, 663-667,
September 2001
© 2001 Oxford University Press
Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region
1 Asahi Kasei Corporation, Shizuoka 410-2321, 2 Molecular Gene Technics, Genetic Resource Technology, Kyushu University, Fukuoka 812-8581 and 3 Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The thermostability enhancement of Flavobacterium meningosepticum glycerol kinase (FGK) by random mutagenesis in the subunit interface region was investigated. A single Escherichia coli transformant, which produced a more thermostable glycerol kinase than the parent enzyme, was obtained. The nucleotide sequence of the gene of the mutant enzyme (FGK2615) was determined, and the four amino acid replacements were identified as Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although the properties of FGK2615 were fundamentally similar to those of the parent enzyme, the thermostability and Km for ATP had changed. The thermostability of FGK2615 was apparently increased; the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation of FGK2615 was determined to be 72.1°C which was 3.1°C higher than that of the parent FGK. Four additional mutants each having a single amino acid replacement (Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys) were prepared and their thermostability and Km for substrates were evaluated. The effect of the substitution of Ser329 to Asp is discussed.
Keywords: Flavobacterium meningosepticum/glycerol kinase/random mutagenesis/thermostable mutant enzyme
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Glycerol kinase (GK, EC 2.7.1.30; ATP: glycerol 3-phosphotransferase) catalyzes the MgATP dependent phosphorylation of glycerol to yield glycero-3-phosphate. This enzyme plays a physiologically important role for the formation of glycero-3-phosphate in the biosynthesis of phospholipids. In addition, the enzyme is industrially important and useful for the clinical determination of the blood triglyceride level in combination with lipase, glycerol-3-phosphate oxidase and peroxidase (Fossati and Prencipe, 1982
). Up to now, GKs have been purified from several microorganisms (Bergmeyer et al., 1961; Hayashi and Lin, 1967; Comer et al., 1979; Charrier et al., 1997; Schweizer et al., 1997; Pasteris and Strasser de Saad, 1998) and a vertebrate (Wieland and Suyter, 1957), and subsequently characterized. The genes of GK from Escherichia coli (Pettigrew et al., 1988), Bacillus subtilis (Holmberg et al., 1990), Saccharomyces cerevisiae (Pavlik et al., 1993), Thermus flavus (Huang et al., 1998), Flavobacterium meningosepticum (Sakasegawa et al., 1998) and humans (Sargent et al., 1994) have been cloned into E.coli and the primary structures of their enzymes were determined. In particular, detailed studies of the relationship between the function and structure of E.coli GK have been carried out by X-ray crystallographic analysis of the regulatory complex of GK with the phosphocarrier protein IIIGlc (Hurley et al., 1993).
In a previous paper, we reported the purification of GK from F.meningosepticum (FGK) and characterized it (Sakasegawa et al., 1998). Although F.meningosepticum is a typical mesophile, the thermostability of FGK is higher than that of GK from a moderate thermophile, B.stearothermophilus (Comer et al., 1979) and comparable to that of GK from the extreme thermophile, T.flavus (Huang et al., 1998). The FGK has been cloned and abundantly produced from the recombinant E.coli cells. In addition, FGK is stable at 37°C for a long period of time. Such a high stability of FGK under various conditions is advantageous for easy preparation of the pure enzyme, for elucidation of the relationship between the structure and function, and for the diagnostic analysis of triglycerides and lipids in the serum.
Determination of the structural features contributing to the thermostability of many proteins has been approached both through comparison of the amino acid sequences and the crystal structures of homologous mesophilic and thermophilic proteins and through protein engineering. As a result, many structural features have been suggested and the stability of the thermophilic proteins can depend on the combination of these factors (for example, Matthews, 1993; Tanner et al., 1996; Auerbach et al., 1998; Tahirov et al., 1998; Britton et al., 1999; Hashimoto et al., 1999; Natesh et al., 1999; Vieille and Zeikus , 2001).
In this study, to obtain more thermostable FGK and further information about the enhancement of thermostability, the enzyme was investigated based on the tertiary structure of E.coli GK (Hurley et al., 1993) by the random mutagenesis that focused on the subunit interface region.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Materials
Glycerol-3-phosphate oxidase was purchased from Asahi Kasei Corporation (Tokyo, Japan). ATP, ampicillin and the peroxidase were obtained from Sigma Chemical (St Louis, MO), and the brain heart infusion (BHI) was from Difco Laboratories, (Detroit, Mich.). Plasmid pUC118, restriction endonucleases, DNA modifying enzymes and all other genetic engineering kits were purchased from Takara (Kyoto, Japan), while N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS) was from Dojindo (Kumamoto, Japan). DEAE-Sepharose Fast Flow, Phenyl-Sepharose Fast Flow, Q-Sepharose Fast Flow and Sephadex G-25 were obtained from Pharmacia, Sweden. All other chemicals were analytical grade products from Nacalai Tesque (Kyoto, Japan).
Determination of enzymatic activity
The enzyme assay was carried out as previously described (Sakasegawa et al., 1998). The standard reaction mixture contained 50 mM Tris–HCl buffer (pH 8.0), 1 mM glycerol, 4 mM ATP, 4 mM MgCl2, 10 units of glycerol-3-phosphate oxidase, 4.5 units of peroxidase, 1.5 mM 4-aminoantipyrine, 1.5 mM DAOS and 20 µl of enzyme in a final volume of 1.02 ml. The reaction mixture was incubated at 37°C in a cuvette with a 1.0-cm light path and the increase in absorbance at 600 nm [
M (molar absorption coefficient) = 17.5/mM/cm] was spectrophotometrically measured. One unit (U) of enzyme was defined as the amount of enzyme catalyzing the formation of 1 µmol of glycerol-3-phosphate per minute at 37°C. Specific activity was expressed as U/mg protein. Protein was measured with a Bio-Rad protein assay kit using bovine serum albumin as the standard protein.
The apparent kinetic parameters, Km and kcat, for glycerol and ATP were estimated from the Lineweaver–Burk plots. The concentrations of ATP and glycerol used for the kinetic parameters determination were in the ranges of 0–2.0 and 0–0.05 mM, respectively.
Random mutagenesis
Random mutagenesis of the FGK was carried out using a Mutan-K kit according to the method of Kunkel (Kunkel, 1985). A 2.4 kb EcoRI–SphI fragment containing the FGK gene was excised from an expression plasmid of the wild-type FGK that was previously constructed (Sakasegawa et al., 1998). After filling up of the EcoRI site by use of a DNA Blunting kit, the fragment was subcloned into the SmaI–SphI site downstream of the lac promoter of pUC118. This recombinant plasmid was incorporated into E.coli CJ236 and the cell was transformed to yield the uracil-containing single-strand DNA. Mix primers for mutagenesis (5'-CAT GAC GCG GCA NNN GTA NNN NNN CTT GCG NNN NNN GTT AAG GAT AAT-3'; N indicates a mixture of A, G, C and T) were synthesized using an Applied Biosystems Model 394 DNA synthesizer and were annealed to the single-strand DNAs. The second DNAs were synthesized using the T4 DNA polymerase. After ligation with E.coli ligase, the E.coli BMH71-18mutS was transformed with the double-stranded heteroduplex DNA. The introduced base substitution was confirmed by DNA sequencing.
Isolation of the mutant exhibiting higher thermostable GK
Transformants were spread onto 3.7% BHI agar plates containing 50 mg/l ampicillin and 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). After the plates were incubated at 30°C overnight, each colony was picked up and grown at 30°C in 3.7% BHI liquid medium containing 50 mg/l ampicillin and 1 mM IPTG. The cells were harvested, suspended in 20 mM potassium phosphate buffer (pH 7.0) and disrupted by sonication. The supernatant obtained by centrifugation (8000 g, 20 min) was heated at 73.5°C for 30 min. The heat stability was evaluated by comparison with the remaining activities after heat treatment.
Site-directed mutagenesis
Four additional mutants were obtained according to the method described by Kunkel et al. (Kunkel et al., 1987). Sequences of the primers used for the mutagenesis were 5'-CAT GAC GCG GCA GAC GTC AGT ACT CTT GCG GGC AGC GTT AAG GAT AAT-3', 5'-CAT GAC GCG GCA GAA GTC GAC ACT CTT GCG GGC AGC GTT AAG GAT AAT-3', 5'-CAT GAC GCG GCA GAA GTA AGT GCA CTT GCG GGC AGC GTT AAG GAT AAT-3' and 5'-CAT GAC GCG GCA GAA GTA AGT ACT CTT GCG GGC AAG GTT AAG GAT AAT-3' for Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys, respectively.
Expression and purification of the mutants
The mutated FGKs were expressed in E.coli DH5
. All purification procedures were done at room temperature, and 50 mM Tris–HCl buffer (pH 8.0) was used as the standard buffer throughout the purification. The recombinant strain producing each of the mutated FGKs was cultured in a 3.7% BHI liquid medium with 50 mg/l ampicillin and 1 mM IPTG at 37°C for 17 h. The cells were suspended in the buffer and disrupted by sonication. The cell debris was removed by centrifugation (8000 g, 10 min), followed by heat treatment at 65°C for 30 min. After centrifugation (8000 g, 15 min), the supernatant solution was put on a DEAE-Sepharose Fast Flow column equilibrated with the buffer. The enzyme was eluted with a linear gradient of 0–0.5 M KCl. The fractions with activity were collected, solid (NH4)2SO4 was added (20%), and the solution was put on a Phenyl-Sepharose Fast Flow column equilibrated with the buffer containing 20% (NH4)2SO4. The enzyme was eluted with a linear gradient of the concentration from 20 to 0% (NH4)2SO4. The desalted enzyme solution was then put on a Q-Sepharose Fast Flow column equilibrated with the buffer. The bound enzyme was eluted with a linear gradient of 0–0.5 M KCl. The active fractions were collected, concentrated and desalted by passage through a Sephadex G-25 column.
Heat inactivation
The enzyme solution (25 µg/ml in 50 mM Tris–HCl buffer pH 8.0) was incubated at various temperatures for certain periods of time, followed by rapid cooling. The remaining activity after the treatment was assayed. The kd [first-order rate constant (s–1)] of heat inactivation was calculated as described elsewhere (Suzuki et al., 1980).
Structural model construction of FGK and FGK2615
The FGK sequence has shown a 60% homology with that of E.coli GK (Sakasegawa et al., 1998). The crystal structure of the complex of E.coli GK with the unphosphorylated form of IIIGlc (an allosteric inhibitor of the phosphotransferase system) has already been determined by Hurley et al. (Hurley et al., 1993). The E.coli GK coordinates (1glb) were obtained from the Brookhaven Protein Data Bank as a template. The model was constructed using the InsightII/Homology software package available from Molecular Simulation Inc., San Diego, CA. Side-chain substitutions were automatically done by the InsightII program. After the substitutions, structure relaxation was performed with the steepest descent method under the conditions restricting the relative position of C. The optimization was done using the conjugate gradient method until the maximum derivative was less than 0.01 kcal/Å. All geometry optimization operations were performed using the consistent valence force field in the program Discover available from Molecular Simulation Inc.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Random mutagenesis in the subunit interface region and isolation of higher thermostable mutant FGK
The region from Lys320 to Val335 was chosen as the target for the random mutagenesis to increase the thermal stability. In this region, we chose especially the residues at the positions of Glu327, Ser329, Thr330, Gly333 and Ser334 for the random mutagenesis to introduce the further increase of hydrophobicity in this region and to elevate thermostability of the enzyme (Figure 1). Thermal stability of the enzyme in the crude extract obtained from each of 2000 clones was examined. As a result, we found one colony producing a higher thermostable GK than the FGK. The remaining activity of the crude mutant enzyme (FGK2615) was 74% after heat treatment at 73.5°C for 30 min, but that of FGK was 52% under the same conditions. The thermostability of the other transformants was similar to or lower than that of the FGK. A recombinant plasmid was extracted from these clone cells and the DNA sequence of the insert fragments was determined. The changes in eight bases (5'-CAT GAC GCG GCA GAT GTA GAT GCA CTT GCG GGC AAG GTT AAG GAT AAT-3'; the italic letters indicate the positions of nucleotide mutations) were found in the gene which resulted in the replacement of four amino acid residues in the sequence of FGK2615 (Figure 1).
|
Purification of the mutant enzyme and its characterization
FGK2615 was purified approximately 200-fold with a high yield of 75% to show homogeneity on SDS–PAGE. The thermostability of FGK2615 was compared with that of the parent enzyme (Figure 2). The FGK2615 clearly exhibited a higher thermostability than the parent enzyme. The optimum pH, specific activity, temperature depending on the activity and the effect of pH on the stability for the mutant enzyme were similar to those of the parent enzyme. The Km values of FGK2615 for glycerol and ATP were determined to be 3.6 and 521 µM, respectively. This shows that the Km for the ATP of FGK2615 is approximately twice that of FGK (249 µM) but a substantial change in Km for glycerol was not observed between FGK (3.5 µM) and FGK2615. No significant alternations in the kcat values for the ATP of FGK and FGK2615 were observed (Table I).
|
|
Heat inactivation
FGK and FGK2615 followed the first-order kinetics of heat inactivation at pH 8.0 and gave linear Arrhenius plots of the kd values (Figure 3). The T50 values (the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation) were calculated to be 69.0 and 72.1°C, respectively (Table I
).
|
Construction and characterization of additional mutants
Four additional mutants were constructed by site-directed mutagenesis, i.e. E327D (Glu327 to Asp), S329D (Ser329 to Asp), T330A (Thr330 to Ala) and S334K (Ser334 to Lys). Of the four mutants, the three mutants of E327D, T330A and S334K exhibited a similar thermostability to the parent enzyme (Figure 3). On the other hand, the S329D showed a substantially increased thermostability; the T50 (72.6°C) for the S329D was compatible with that (72.1°C) for the FGK2615 (Table I). In addition, the S329D showed a Km (586 µM) value for ATP which was also similar to that (521 µM) of the FGK2615.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study, we succeeded in elevating the thermostability of FGK by random mutagenesis in the subunit interface region. The obtained mutant FGK (FGK2615) followed first-order kinetics during heat inactivation at pH 8.0 and gave a linear Arrhenius plot of the kd values (Figure 3
). The T50 value calculated for FGK2615 (72.1°C) was higher than that of the parent enzyme (69.0°C, Table I). The higher value of T50 for FGK2615 suggests that the mutant enzyme may have a more stabilized structure than the parent enzyme. It is considered that the stabilized structure of the mutant enzyme results in the alteration of the thermostability and catalytic properties of the parent enzyme. We discuss below the possible molecular mechanisms concerning the higher thermostability of the mutant enzyme.
In recent years, based on three-dimensional structures, many molecular and structural characteristics of the highly thermostable enzyme have been analyzed by pairwise comparisons with homologous proteins which have different thermal stabilities (for example, Tanner et al., 1996; Auerbach et al., 1998; Sanz-Aparicio et al. 1998; Tahirov et al., 1998; Britton et al., 1999; Hashimoto et al., 1999; Natesh et al., 1999). As a result, several mechanisms and factors have been found to be responsible for the higher thermostability, i.e. hydrophobic interactions, ion-pairs, hydrogen bonds, helix capping, solvent accessible surface areas, etc. (Matthews, 1993; Vieille and Zeikus, 2001). Among them, the large contribution of hydrophobic interactions in the subunit interface to the stability has been demonstrated with some thermophilic enzymes (Biro et al., 1990; Kirino et al., 1994; Erduran and Kocabiyik, 1998). Escherichia coli GK is known to exist in the equilibrium state between the functional dimer and tetramer subunit structures for the physiological concentration (de Riel and Paulus, 1978). In the GK structure, three types of intersubunit interactions are observed (Hurley et al., 1993). Although the entire hydrophobic profile (Kyte and Doolittle, 1982) of FGK is very similar to that of the E.coli GK, the FGK exhibits higher hydrophobicity only in part of the subunit–subunit interface region than that of E.coli GK; the subunit–subunit interface region is Lys320 to Ala332 for FGK, and the region with higher hydrophobicity in FGK compared to E.coli GK is Asp324 to Val335 (Figures 1 and 4). For this point, the FGK is clearly different from the E.coli GK (Sakasegawa et al., 1998). We postulated that this region may be responsible for the higher thermostability of the FGK and then carried out the random mutagenesis in the region of FGK as the strategy for increasing the thermostability. As a result, one clone, which produces a mutant enzyme with a higher thermostability than the parent FGK, was obtained and four amino acid substitutions, Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys, were identified in the mutant enzyme (Figure 1). Unlike our prediction, the replacement in the mutant enzyme did not result in a hydrophobicity increase in the interface region. We tried the construction of additional enzyme mutants in order to determine which substitution among the four amino acids was exclusively responsible for the increased thermostability. The result clearly showed that only the mutation of Ser329 to Asp and not the other three mutations is responsible for the increased thermostability of FGK2615 and S329D (Figure 3). This indicates that the change in the side chain of Ser329 to Asp mainly produces the change of its nature.
|
The increased thermostability may be explained by the stabilization of tertiary structure rather than the formation of a subunit–subunit interaction for the FGK2615 (S329D). As one way to understand the tertiary structure stabilization, the improvement of the electrostatic interaction in the vicinity of the substituted amino acid by the addition of a negative charge has been considered. Several examples have been reported that the ion-pair may play a key role in the maintenance of the high enzyme stability (Auerbach et al., 1998
; Sanz-Aparicio et al., 1998; Tahirov et al., 1998; Hashimoto et al., 1999). However, based on the three-dimensional structure of the FGK2615 (S329D) predicted from the sequence homology and the three-dimensional structure of E.coli GK, the formation of a new ion-pair by the substitution of Ser329 for Asp in FGK2615 (S329D) was not observed (Figure 5).
|
As an additional mechanism to increase the thermostability for FGK2615 (S329D), we estimated the formation of a new hydrogen bond. The contribution of hydrogen bonds to the stabilization of protein structures has been reported (Marqusee and Sauer, 1994
; Peterson et al., 1999). A comparison analysis on the structure of different D-glyceraldehyde-3-phosphate dehydrogenases from psychrophile, thermophiles and hyperthermophile has revealed that the increase in the number of hydrogen bonds formed between the charged side chain and neutral partners is well correlated to the increase in the enzyme thermostability (Tanner et al., 1996). From the analysis of the structural model of FGK, we have found that the substitution of Ser329 for Asp in FGK may give rise to the formation of a new charged-neutral hydrogen bond between the side chains of Asp329 and Ser414 (Figure 5). The distance between the carboxylate oxygen of Asp329 and the hydrogen atom of Ser414 is shorter than 3.0 Å and the donor–hydrogen–acceptor angle is below 20° (linear hydrogen bond has an angle of 0° in this convention), which makes it possible to form the charged-neutral hydrogen bond. On the other hand, the distances are too large to form a hydrogen bond between the side chain of Ser329 and those of Ser414 of FGK and Asp329 and those of Asn415 of FGK2615 (S329D); distances were approximately 5.0 and 4.5 Å, respectively. This supports the fact that the new formation of the charged-neutral hydrogen bond between Asp329 and Ser414 contributes to the stabilization of the tertiary structure and increased thermostability.
|
Notes |
|---|
4 To whom correspondence should be addressed. E-mail: sakasegawa.sb{at}om.asahi-kasei.co.jp
|
Acknowledgments |
|---|
We thank Ms N.Take and Y.Yasuda for their excellent technical assistance.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Auerbach,G., Ostendorp,R., Prade,L., Korndorfer,I., Dams,T., Huber,R. and Jaenicke,R. (1998) Structure, 6, 769–781.[Medline]
Bergmeyer,H.U., Holz,G., Kauder,E.M., Mollering,H. and Wieland,O. (1961) Biochem. Z., 333, 471–480.
Biro,J., Fabry,S., Dietmaier,W., Bogedain,C. and Hensel,R. (1990) FEBS Lett., 275, 130–134.[ISI][Medline]
Britton,K.L. et al. (1999) J. Mol. Biol., 293, 1121–1132.[ISI][Medline]
Charrier,V., Buckley,E., Parsonage,D., Galinier,A., Darbon,E., Jaquinod,M., Forest,E., Deutscher,J. and Claiborne,A. (1997) J. Biol. Chem., 272, 14166–14174.
Comer,M.J., Bruton,C.J. and Atkinson T. (1979) J. Appl. Biochem., 1, 259–270.
de Riel,J.K. and Paulus,H. (1978) Biochemistry, 17, 5134–5140.[Medline]
Erduran,I. and Kocabiyik,S. (1998) Biochem. Biophys. Res. Commun., 249, 566–571.[ISI][Medline]
Fossati,P. and Prencipe,L. (1982) Clin. Chem., 28, 2077–2080.
Hashimoto,H., Inoue,T., Nishioka,M., Fujiwara,S., Takagi,M., Imanaka,T. and Kai,Y. (1999) J. Mol. Biol., 292.707–716.[ISI][Medline]
Hayashi,S. and Lin,E.C.C. (1967) J. Biol. Chem., 242, 1030–1035.
Holmberg,C., Beijer,L., Rutberg,B. and Rutberg,L. (1990) J. Gen. Microbiol., 136, 2367–2375.[Medline]
Huang,H., Kabashima,T., Ito,K., Yin,C., Nishiya,Y., Kawamura,Y. and Yoshimoto,T. (1998) Biochim. Biophys Acta, 1382, 186–190.[Medline]
Hurley,J.H., Faber H.R., Worthylake,D., Meadow,N.D., Roseman,S., Pettigrew,D.W. and Remington,S.J. (1993) Science, 259, 673–677.[Abstract]
Kirino,H., Aoki,M., Aoshima,M., Hayashi,Y., Ohba,M., Yamagishi,A., Wakagi,T. and Oshima,T. (1994) Eur. J. Biochem., 220, 275–281.[ISI][Medline]
Koga,Y., Morikawa,M., Haruki,M., Nakamura,H., Imanaka,T. and Kanaya,S. (1998) Protein Eng., 11, 1219–1227.
Kunkel,T.A. (1985) Proc. Natl Acad. Sci. USA, 82, 488–492.
Kunkel,T.A., Roberts,J.D. and Zakour R.A. (1987) Methods Enzymol., 154, 367–382[ISI][Medline]
Kyte,J. and Doolittle,R.F. (1982) J. Mol. Biol., 157, 105–132.[ISI][Medline]
Marqusee,S. and Sauer,R.T. (1994) Protein Sci., 3, 2217–2225.[Abstract]
Matthews,B.W. (1993) Annu. Rev. Biochem., 62, 139–160.[ISI][Medline]
Natesh,R., Bhanumoorthy,P., Vithayathil,P.J., Sekar,K., Ramakumar,S. and Viswamitra,M.A. (1999) J. Mol. Biol., 288, 999–1012.[ISI][Medline]
Pasteris,S.E. and Strasser de Saad,A.M. (1998) Lett. Appl. Microbiol., 27, 93–97.
Pavlik,P., Simon,M., Schuster,T. and Ruis,H. (1993) Curr. Genet., 24, 21–25.[ISI][Medline]
Peterson,R.W., Nicholson,E.M., Thapar,R., Klevit,R.E. and Scholtz,J.M. (1999) J. Mol. Biol., 286, 1609–1619.[ISI][Medline]
Pettigrew,D.W., Ma,D.P., Conrad,C.A. and Johnson,J.R. (1988) J. Biol. Chem., 263, 135–139.
Sakasegawa,S., Yoshioka,I., Koga,S., Takahashi,M., Matsumoto,K., Misaki,H. and Ohshima,T. (1998) Biosci. Biotechnol. Biochem., 62, 2388–2395.[Medline]
Sanz-Aparicio,J., Hermoso,J.A., Martinez-Ripoll,M., Gonzalez,B., Lopez-Camacho,C. and Polaina,J. (1998) Proteins, 33, 567–576.[ISI][Medline]
Sargent,C.A., Young,C., Marsh,S., Ferguson-Smith,M.A. and Affara,N.A. (1994) Hum. Mol. Genet., 3, 1317–1324.
Schweizer,H.P., Jump,R. and Po,C. (1997) Microbiology, 143, 1287–1297.[Abstract]
Suzuki,Y., Nakamura,N., Kishigami,T. and Abe,S. (1980) J. Biochem., 87, 745–751.
Tahirov,T.H., Oki,H., Tsukihara,T., Ogasahara,K., Yutani,K., Ogata,K., Izu,Y., Tsunasawa,S. and Kato,I. (1998) J. Mol. Biol., 284, 101–124.[ISI][Medline]
Tanner,J.J., Hecht,R.M. and Krause,K.L. (1996) Biochemistry, 35, 2597–2609.[Medline]
Vieille,C. and Zeikus,G.J. (2001) Microbiol. Mol. Biol. Rev., 65, 1–43.
Wieland,O. and Suyter,M. (1957) Biochem. Z., 329, 320–331.[ISI][Medline]
Received January 15, 2001; revised June 20, 2001; accepted June 23, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Declerck, M. Machius, P. Joyet, G. Wiegand, R. Huber, and C. Gaillardin Hyperthermostabilization of Bacillus licheniformis{alpha}-amylase and modulation of its stability over a 50{degrees}C temperature range Protein Eng. Des. Sel., April 1, 2003; 16(4): 287 - 293. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||