Hyperthermostabilization of Bacillus licheniformis {alpha}-amylase and modulation of its stability over a 50°C temperature range

Nathalie Declerck1,2, Mischa Machius3, Philippe Joyet1, Georg Wiegand4, Robert Huber4 and Claude Gaillardin1

1 Génétique Moléculaire et Cellulaire, CNRS–URA1925, INRA–UMR216, F-78850 Thiverval-Grignon, France, 3 University of Texas Southwestern Medical Center, Dallas, TX 75390, USA and 4 Max-Planck-Institut für Biochemie,D-85152 Planegg-Martinsried, Germany

2 To whom correspondence should be addressed. Present address: Centre de Biochimie Structurale, CNRS-5048, INSERM-554, 29 rue de Navacelles, F-34090 Montpellier, France. E-mail: nathalie{at}cbs.cnrs.fr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacillus licheniformis {alpha}-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S–N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23°C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25°C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50°C through a few point mutations.

Keywords: bacterial {alpha}-amylase/enzyme engineering/mutagenesis/protein modelling/thermostability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The {alpha}-amylase from B.licheniformis (BLA) is an established model system for addressing questions having both fundamental and technological implications regarding protein thermostability. Although it is produced by a tempered soil bacteria, this amylolytic enzyme is highly thermostable and is therefore widely used in biotechnological processes, in particular for starch liquefaction at temperatures of up to 110°C (Vihinen and Mantsala, 1989Go). Over the last decade, a wealth of mutational, biochemical and structural data have been acquired in both academic and industrial laboratories investigating BLA thermal properties [for reviews see (Heslot, 1996Go; Nielsen and Borchert, 2000Go; Declerck et al., 2002Go)]. In our laboratory, we have constructed over 500 BLA variants bearing single or multiple amino acid substitutions (Declerck et al., 1990Go, 1995Go, 2000Go; Joyet et al., 1992Go). This mutational analysis, combined with structural studies (Declerck et al., 1995Go, 1997Go; Machius et al., 1995Go, 1998Go, 2003Go), allowed the probing of the contributions of various molecular features to BLA thermostability. In particular, a key role could be attributed to an unusual calcium/sodium binding site where amino acid substitutions drastically decreased the overall stability of the protein. In addition to thermosensitive mutations, we also identified several amino acid changes that render the enzyme even more thermostable than the wild-type.

In this paper, we report the cumulative effect of some of the most stabilizing and destabilizing mutations that we and others have identified in BLA. Comparison of the hyperthermostable and thermosensitive variants shows that the thermostability of BLA can be modulated over a temperature range of 50°C through a few point mutations. In addition, we show that the increased resistance of the enzyme towards high temperature is not necessarily accompanied by a decrease in its catalytic activity. The mutational effects were interpreted based on several crystal structures, including that of a hyperthermostable BLA variant that we recently determined (Machius et al., 2003Go).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Amylase genes, plasmids and strains

The sequence of the wild-type B.licheniformis {alpha}-amylase gene (bla) cloned in our laboratory (Joyet et al., 1984Go) is identical with that reported by Gray et al.(1986)Go. Construction of the mutations by site-directed mutagenesis has been described previously (Declerck et al., 1995Go, 2000Go). Multiple mutant bla genes were constructed by classical DNA recombination techniques using restriction sites originally present in bla or by using mutant genes as templates for additional rounds of site-directed mutagenesis. Wild-type and mutant bla genes were transferred into pINA9C9 (Declerck et al., 1995Go) to be expressed in Escherichia coli strain XacI (Normanly et al., 1986Go).

{alpha}-Amylase production and assay

Wild-type and mutant BLAs were produced in E.coli and extracted from the cell periplasm by osmotic shock as described previously (Declerck et al., 2000Go). Production of the mutant amylases was similar to the wild-type levels except for the variants carrying the H156Y and/or N172R mutations, which were recovered in significantly smaller amounts (10–30% of wild-type). Purified preparations of some amylase variants were kindly provided by T.V.Borchert (Novozymes). {alpha}-Amylase activity was measured in microtitration plates at room temperature (Declerck et al., 2000Go) using 1% soluble starch (Merck) or the synthetic chromogenic substrate (4',6-ethylidene-p-nitrophenyl-{alpha},D-maltoheptaoside) from a Sigma Diagnostic Amylase kit (Sigma) in 50 mM MOPS buffer, pH 7, 2 mM CaCl2, 50 mM NaCl.

Thermostability tests

The thermostability of the wild-type and mutant BLAs was tested by measuring residual amylase activities after incubation at high temperature followed by a cooling period on ice. Thermostability is thus referred to here as the behaviour of the different enzymes towards irreversible thermal inactivation, which was previously shown for BLA to follow first-order kinetics (Tomazic and Klibanov, 1988Go; Declerck et al., 1997Go). Low pH and calcium concentrations were used for the incubation conditions in order to reduce the time and temperature required for denaturation and thereby facilitate the test procedure. At least two independent tests were performed with periplasmic extracts 20-fold diluted in 50 mM sodium acetate, pH 5.6, 0.1 mM CaCl2. For kinetic studies, the samples were incubated for various times in a water-bath at 85 or 95°C, cooled on ice for 15 min and assayed for amylase activity using the synthetic substrate before and after incubation. The half-lives (t1/2) of the enzymes were then calculated from the slope of the inactivation curves obtained by linear regression of log(residual activity) versus incubation time. Thermostability profiles were obtained by incubating the samples for 10 min at various temperatures in a water-bath or an oil-bath and by plotting the residual amylase activity versus incubation temperature. The T50 values corresponding to the temperature at which 50% of the enzyme activity is irreversibly lost were estimated from the thermostability profiles.

Protein modelling

Protein modelling was performed based on the crystal structure of the calcium-free form of wild-type BLA [(Machius et al., 1995Go) PDB access code (#) 1BPL], the 190F–264S–265Y mutant BLA [(Machius et al., 1998Go) PDB# 1BLI], the 133V–190F–209V–264S–265Y hyperthermostable mutant BLA [(Machius et al., 2003Go) PDB# 1OB0], and also on the crystal structure of chimeric Bacillus amyloliquefaciens {alpha}-amylase (BAA)/BLA in its native form (PDB# 1E3X) and in complex with a decasaccharide (PDB# 1E3Z) (Brzozowski et al., 2000Go). Graphic representation, in silico mutagenesis, assessment of local conformational changes and minimization were performed using TURBO-FRODO (http://afmb.cnrs-mrs.fr/TURBO_FRODO). Modelling of the calcium-bound wild-type BLA structure was performed using 1BLI as a starting model and replacing Phe190, Ser264 and Tyr265 by Asn or Gln residues as observed in 1BPL and/or 1E3X. Slight modifications of the backbone and side chain conformations of the loop comprising residues 264 to 267 were performed manually in order to better match the wild-type structure. Modelling of the 8-fold hyperthermostable mutant BLA was performed using 1OB0 as a starting model and replacing Val133, His156, Asn172 and Ala181 by Ile, Tyr, Arg and Thr, respectively. No adjustment of surrounding residues was necessary to accommodate the substituting side chains. The oligosaccharide was then modelled in the substrate binding cleft of both structures as observed in the 1E3Z complex structure. Most backbone and side chain atoms forming the binding site in 1E3Z are superimposable with their counterpart in the BLA structures with a root mean square deviation (r.m.s.d.) of <0.5 Å.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Combination of stabilizing mutations

We have previously reported the construction of hyperthermostable BLA variants carrying stabilizing mutations at positions 133 and 209 (Declerck et al., 1990Go; Joyet et al., 1992Go). Introduction of all possible amino acid side chains in replacement of His133 and Ala209 enabled us to select Ile and Val, respectively, as the best substituting residues at these sites (Declerck et al., 1995Go). More recently we have identified new stabilizing mutations by replacing the amide side chain of some Asn/Gln residues (Declerck et al., 2000Go). It had been shown that BLA inactivation at high temperature was accompanied by deamidation processes. It was therefore proposed that the replacement of some labile Asn or Gln side chains may lead to thermostabilization (Tomazic and Klibanov, 1988Go). There was as yet no information on which Asn/Gln residues would be the most prone to deamidation inflicting deleterious damage on the enzyme molecule. Mutational analyses had suggested that BLA thermostability determinants were mostly located in a protein region spanning the variable B domain and the subsequent portion of the central A domain (Declerck et al., 1997Go). We therefore independently mutated seven Asn/Gln residues in this region and replaced them with various amino acids. Beneficial amino acid replacements were observed at positions 172 and 190, the Asn190Phe (N190F) mutation being by far the most thermostabilizing single mutation we ever obtained in BLA (Declerck et al., 2000Go). We also replaced the contiguous Gln264 and Asn265 simultaneously by Ser and Tyr, respectively, the residues found in the homologous {alpha}-amylase from Bacillus stearothermophilus (Gray et al., 1986Go). The characterization of this Q264S–N265Y tandem mutation has not previously been reported.

The Q264S–N265Y mutation was recombined with the His133Ile (H133I), Ala209Val (A209V) and N190F stabilizing mutations. Figure 1AGo shows the inactivation curves obtained by measuring the residual amylase activity of the BLA variants after different times of incubation at 85°C. The 264S–265Y variant is significantly more thermostable than the wild-type enzyme: under the conditions used for incubation (pH 5.6, 0.1 mM CaCl2), the half-life (t1/2) of BLA is raised from 5 min for the wild-type to 11 min for the variant (Table IGo). This is, however, a limited gain of stability compared with that induced by the N190F mutation alone or in combination with the H133I–A209V double mutation: the t1/2 values are 31, 51 and 257 min for the 190F, 133I–209V and 133I–190F–209V variants, respectively. Nevertheless, the stability of these variants can be further improved by the Q264S–N265Y mutation: addition of this mutation doubles the half-life of the 190F variant whereas the hyperthermostable variant combining the five mutations remains fully active after 2 h of incubation at 85°C (Figure 1AGo). When incubated at 95°C, the t1/2 value for the 133I–190F–209V–264S–265Y variant is 25 min, whereas that of the 190F variant is only 4 min and the wild-type is deactivated immediately (Figure 1BGo). These results are in good agreement with unfolding kinetics and pseudo-melting curves performed with purified preparations of some BLA variants (Machius et al., 2003Go).



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Fig. 1. Time course of irreversible thermal inactivation at 85°C (A) or 95°C (B), pH 5.6, 0.1 mM CaCl2, of wild-type BLA (•) and its variants bearing stabilizing mutations: 264S–265Y ({triangleup}); 190F ({blacksquare}); 133I–209V ({square});133I–264S–265Y ({blacklozenge}); 133I–190F–209V ({blacktriangleup});133I–190F–209V–264S–265Y ({lozenge});133I–156Y–172R–181T–190F–209V–264S–265Y ({blacktriangledown}).

 

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Table I. Effect of stabilizing or destabilizing substitutions on BLA thermal behaviour
 
Amylolytic activity of hyperthermostable variants

BLA variants bearing the Q264S–N265Y mutation were purified and compared with the wild-type enzyme for their activity on starch or a chromogenic maltoheptaosidic substrate (Figure 2Go). Experiments were carried out under conditions where wild-type BLA is fully stable (25°C, 2 mM CaCl2, pH 7) in order to measure the impact of the mutations on the catalytic function alone. Surprisingly, a significant increase in amylolytic activity was brought about by the Q264S–N265Y mutation: the activity of the double mutant BLA was about 30–40% higher than that of the wild-type. However, the beneficial effect of this mutation was masked when introducing the other, more stabilizing mutations at positions 133, 190 and 209, even though these mutations individually were not previously found to alter significantly the enzyme’s catalytic function (Declerck et al., 1997Go, 2000Go). Introduction of the N190F mutation is the most deleterious, resulting in a triple mutant that is only slightly more active than wild-type BLA and further addition of stabilizing mutations at positions 133 and 209 entirely abolishes the beneficial effect of the Q264S–N265Y mutation. As a result, the 5-fold mutant BLA exhibits amylolytic activity similar to wild-type. More detailed enzymatic studies are required to understand the catalytic behaviour of the mutant amylases.



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Fig. 2. Relative activity of purified wild-type and mutant BLAs on starch (white bars) or synthetic maltoheptosidic substrate (black bars) and incubated at room temperature (25°C) in 50 mM MOPS buffer, pH 7, 2 mM CaCl2, 50 mM NaCl.

 
Further increase in thermostability

We have investigated the ability of three other stabilizing mutations to increase further the thermostability of the above-mentioned hyperthermostable variants. We had previously found that the Asn172Arg (N172R) mutation induced a 2-fold increase in BLA half-life at 80°C (Declerck et al., 2000Go), but its effect in combination with other stabilizing mutations had never been tested. The His156Tyr (H156Y) and Ala181Thr (A181T) mutations were identified at Novozymes (Denmark) and were already known to improve the performance of BLA in industrial processes when combined with other stabilizing mutations that we identified (Bisgard-Frantzen et al., 2000Go).

Introduction of these three mutations together significantly increased the thermostability of the already highly thermostable mutant BLA: the t1/2 value measured at 95°C was raised from 25 min for the 5-fold mutant to over 100 min for the variant combining all the thermostabilizing mutations (Figure 1BGo). However, this 8-fold mutant enzyme is not the most thermostable BLA variant that we engineered. Comparison of the stability profiles of the BLA variants incubated at different temperatures (Figure 3Go) shows that the 7-fold mutant lacking the N172R mutation exhibits the highest gain in stability: the T50 value (the temperature at which the amylase activity is reduced by 50% for a 10-min incubation) for the 133I–156Y–181T–190F–209V–264S–265Y variant is 106°C, whereas that of the 8-fold mutant is only 102°C (Table IGo). Under the same conditions, the T50 value for the 172R single mutant is around 85°C, i.e. 2°C higher than that of the wild-type enzyme. The N172R mutation is thus slightly beneficial when introduced as a single mutation but detrimental to BLA thermostability when combined with the other stabilizing mutations. By contrast, the Q264S–N265Y double mutation that enhanced the T50 value to the same extent as the N172R mutation always improved the enzyme performance when combined with the other stabilizing mutations (Table IGo).



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Fig. 3. Temperature resistance profiles of wild-type BLA (•, dotted line) and its variants: 264S–265Y ({triangleup}); 172R ({blacksquare}); 133I–209V ({square});133I–190F–209V ({blacktriangleup});133I–190F–209V–264S–265Y ({lozenge});133I–156Y–172R–181T–190F–209V–264S–265Y ({blacktriangledown}); H133I–H156Y–A181T–N190F–A209V–Q264S–N265Y ({circ}); 237D (x); 204K (+); 204K–D237D ({star}V).

 
Combination of destabilizing mutations

Two of the most destabilizing mutations that we identified in BLA have been combined: the Asp204Lys (D204K) mutation and the Lys237Asp (K237D) mutation. These mutations were originally designed to probe the existence of a salt bridge between the two residues in the wild-type structure, as predicted from earlier modelling studies (Declerck et al., 1995Go). Disruption of the salt bridge was expected to destabilize the enzyme carrying any of the single mutations, whereas we expected to restore, at least partly, BLA stability by constructing the double mutant in which a reciprocal salt bridge could be formed between the two mutated residues.

The effects of the mutations on the enzyme stability are shown in Figure 3Go. Both single mutations induced a drastic reduction of the enzyme half-inactivation temperature: compared with wild-type; the T50 value was lowered by as much as 20°C for the 204K variant and 15°C for the 237D variant. Contrary to what we expected, the double mutant supposed to carry the reciprocal salt bridge was in fact even more thermolabile than the single mutants: the T50 value of the 204K–237D variant was reduced by another 5 and 10°C compared with the single mutants and by 25°C compared with the wild-type enzyme.

Modelling of wild-type and multiple mutant BLA structures

In order to gain insight into the molecular impact of the multiple mutations on BLA stability and activity, we modelled the liganded structure of the native enzyme and of the 8-fold mutant combining all the stabilizing mutations that we introduced (Figure 4Go). Several BLA structures have been determined by crystallography, including that of a calcium-free form of the wild-type (Machius et al., 1995Go; Hwang et al., 1997Go) and of the calcium-liganded 190F–264S–265Y mutant (Machius et al., 1998Go). Very recently we have solved the crystal structure of the 133V–190F–209A–264S–265Y hyperthermostable mutant, also in the presence of calcium (Machius et al., 2003Go). In addition, we used the crystal structure of an active chimeric {alpha}-amylase consisting of residues 1–300 from B.amyloliquefaciens {alpha}-amylase (BAA) and residues 301–483 from BLA (Brzozowski et al., 2000Go). The liganded structure of the BAA/BLA chimera, solved in the presence of calcium and the inhibitor acarbose, exhibits a Ca–Na–Ca metal triad identical with that seen in BLA (Machius et al., 1998Go) and a decasaccharide transglycosylation product bound in the active site cleft. Based on these structures, we modelled the structure of wild-type BLA and of the hyperthermostable BLA variant carrying the H133I, H156Y, N172R, A181T, N190F, A209V, Q264S and N265Y mutations, in complex with calcium and the oligosaccharide seen in the BAA/BLA chimera (see Materials and methods for details). Modelling of the oligosaccharide in the BLA binding cleft was facilitated by the fact that, although most of the liganding residues in the chimera are from BAA, they are strictly conserved in BLA and can be perfectly superimposed with their counterparts in the BLA structures.



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Fig. 4. (A) Stereo view of the modelled structure of BLA in complex with metal ions and a decasaccharide moiety (shown in blue) in the substrate binding site. Domain B (residues 101 to 205) is coloured green. The mutation sites are shown as large red spheres and calcium and sodium ions as small orange and yellow spheres, respectively. (B) Close-up stereo view highlighting side chains at and around the H133I, N172R, N190F and Q264S–N265Y mutation sites in the model of wild-type BLA (black side chains) or the 8-fold hyperthermostable mutant (red side chains). (C) Close-up view as in (B) after 180° rotation, highlighting side chains at and around the H156Y, A181T, A209V, D204K and K237D mutation sites.

 
Figure 4Go shows the location of the mutation sites in the model of the liganded BLA structure. As reported previously (Joyet et al., 1992Go; Declerck et al., 2000Go), all the mutations that affect BLA thermostability in one way or another are clustered in domain B (the long excursion containing residues 101–205 protruding from the central {alpha}8 barrel forming domain A) and its interface with domain A. This region contains the substrate binding cleft as well as the metal triad. None of the mutated side chains is in direct contact with another mutated residue or with the metal ions, except for Asp204 and Lys237, which form a salt bridge and are part of the metal liganding cage. Amino acid substitutions at these sites are extremely destabilizing and may alter the overall structure of the enzyme. Modelling of the mutant structure was therefore not attempted. By contrast, all the stabilizing mutations are located at the surface of the molecule and can be accommodated without perturbing surrounding residues (Figure 4B and CGo). Indeed, comparison of wild-type and mutants in experimentally determined structures reveals no or only very small conformational changes.

Detailed analysis and structural interpretation of the mutational effect on BLA stability at positions 133, 190, 209 and 264–265 have already been performed. Based on earlier protein modelling and the recently determined crystal structure of the 5-fold mutant, it has been proposed that the enhancement of stability resulted mainly from the stabilization of a ß-sheet (position 133), increased hydrophobic packing of surface indentations (positions 133 and 209), aromatic–aromatic interactions (positions 190 and 265) and the removal of possibly deamidating residues (positions 190, 264 and 265) (Declerck et al., 1995Go, 1997Go; Machius et al., 1998Go, 2003Go). In addition, our present model of the 8-fold mutant suggests possible explanations of the stabilizing effect at position 156, 172 and 181. All three residues are located in domain B and their side chains are exposed to the solvent. At position 172, substitution of an arginine for the asparaginyl side chain, in addition to removing a potential deamidation site, may lead to the formation of a stabilizing salt bridge with Asp164. At position 181, the Ala to Thr substitution may be beneficial not only because it replaces an apolar residue at the surface by a more thermodynamically favourable residue, but also because the threonine side chain could make a stabilizing interaction with Asp204 and thereby extend the interaction network that stabilizes the metal binding site. At position 156, the stabilizing effect of the His to Tyr substitution, replacing a charged residue at the surface by a more hydrophobic residue, appears rather intriguing. However, as seen in Figure 4CGo, the side chain of residue 156 belongs to an extensive cluster of aromatic side chains involving residues at the domain surface, its interior, domain A and the metal binding site. The tyrosine side chain may improve stacking and reinforce the network of aromatic–aromatic interactions that is probably crucial for maintaining the proper folding of this region.

In our model of BLA in complex with the oligosaccharide, residues 264 and 265 are very close to the last visible glucose moiety bound at the +3 subsite of the enzyme. This observation and the fact that mutations at these positions modify the amylolytic function of BLA imply that residues 264 and/or 265 are involved in substrate binding. Our modelling suggests that both substituting residues may improve substrate binding: the hydroxyl group of the serine side chain introduced at position 264 may provide an additional hydrogen bond at the +3 subsite, whereas the tyrosine side chain at position 265 may provide a stacking platform and/or hydrogen bond for the binding of a glucose moiety at the +4 subsite. Alternatively, the increased activity of the mutant enzyme may be due to the disruption of a hydrogen bond between Gln264 in domain A and Glu189 in domain B, which is contacting the substrate. In the BAA/BLA chimeric structure, as in the modelled wild-type structure of BLA, this bond spans over the entrance of the binding cleft and its disruption may therefore loosen the active site region and thereby increase the enzyme’s activity.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The design of thermostable enzymes is among the most spectacular achievements of protein engineering. Since the pioneering work of Perry and Wetzel (Perry and Wetzel, 1984Go), the advent of site-directed mutagenesis methodology has yielded an increasing number of protein thermostabilization through rational design or empirical means [for examples and reviews see (Matsumura et al., 1986Go; Das et al., 1989Go; Pantoliano et al., 1989Go; Matthews, 1995Go; Heslot, 1996Go; Allen et al., 1998Go; Nielsen and Borchert, 2000Go; Shibuya et al., 2000Go; Sakasegawa et al., 2001Go)]. The engineering work that we and others have accomplished on BLA demonstrates that even in the case of an enzyme that is naturally highly thermostable there is room for substantial improvement. However, some of the general rules that have been successfully used for the thermostabilization of mesophilic proteins do not seem to apply to thermophilic proteins. For instance, the engineering of disulfide bridges should not be considered to achieve hyperthermostabilization since damaging oxidation of cysteinyl residues may occur at high temperature (Perry and Wetzel, 1987Go). Our work on BLA also seems to contradict two general concepts of protein stability, namely (i) amino acid substitutions at the protein surface usually have little impact on protein stability and (ii) hydrophobic residues at solvent-exposed sites are not favourable. In the hyperthermostable variants that we constructed, all the mutation sites are located on the surface and, in five cases, the stability increase results from the incorporation of more hydrophobic residues. As discussed elsewhere in detail (Machius et al., 2003Go), all the mutated residues are involved in forming intricate, cooperative interaction networks, in particular around the metal triad. At such sites, numerous and complex forces are at work and determine stability, therefore the mutational effects are difficult to predict and even apparently thermodynamically unfavourable mutations should be explored.

Additivity of the mutational effects is the general rule observed when combining stabilizing or destabilizing mutations (Matsumura et al., 1986Go; Gregoret and Sauer, 1993Go; Zhang et al., 1995Go). This property is taken as an indication that the amino acid changes inflict only local structural perturbations and that they introduce (un)favourable interactions that (de)stabilize independently the protein structures against thermal denaturation. In our BLA variants, the incremental stabilization observed when adding more beneficial substitutions is in good agreement with the fact that all the mutation sites are far apart from each other and no large conformational changes are induced. Although each of the individual mutations led to a rather limited gain in stability, the half-life at 85°C of the 5-fold mutant BLA that we first constructed was at least 100 times longer than that of the wild-type enzyme. We could then further increase the thermostability of this variant by adding two other stabilizing substitutions (H156Y and A181T). However, the enhancement of stability brought about by these mutations was not always strictly additive. In the case of the H156Y mutation, it was higher than expected, suggesting possible synergistic positive effects of this mutation when combined with the other stabilizing mutations. Conversely, the N172R mutation, which alone increases the T50 of the wild-type by 2°C, reduced the T50 of the 7-fold mutant by 4°C, indicative of a synergistic negative effect taking place in the multiple mutant. Examination of the BLA mutant structure did not offer a structural interpretation for this phenomenon. Modelling of an arginine side chain at position 172 suggests that it may form a stabilizing salt bridge with the nearby Asp164, but the destabilizing effects observed in the multiple mutant are difficult to rationalize. The closest mutated residue is Phe190, yet the distance between the C{alpha} atoms of the two residues is more than 17 Å, so that direct interactions between side chain atoms are most unlikely.

The D204K and K237D mutations are among the most disruptive single amino acid replacements we observed in BLA. We had predicted the destabilizing effect of these mutations from earlier modelling studies that had suggested the existence of a salt bridge between Asp204 and Lys237 (Declerck et al., 1995Go). Later, the determination of the calcium-bound BLA crystal structure showed that indeed the side chain of Asp204 is salt bridging Lys237, but it is also liganding the non-conserved calcium ion of the metal triad (Machius et al., 1998Go). The metal triad is unique to BLA and its homologous bacterial amylases (Brzozowski et al., 2000Go; Suvd et al., 2001Go). The involvement of Asp204 and Lys237 in calcium binding was therefore not predicted in our model based on the structure of Taka-amylase from Aspergillus oryzae (Matsuura et al., 1984Go). The highly deleterious effect induced by the mutations at these sites is due to the disruption of the electrostatic interaction network entrapping the metal ions rather than the sole disruption of the salt bridge between the two residues. The D204K/K237D double mutation that was supposed to restore this salt bridge failed to restore the thermostability, because it could not recreate an efficient binding network around the metal ions.

On the one hand, we were able to combine up to seven amino acid substitutions that together increased the temperature of half-inactivation of BLA by 23°C. On the other hand, it is possible to lower the T50 value by about 25°C by combining two destabilizing mutations. We could therefore modulate the thermostability of this bacterial amylase over a range of temperature of nearly 50°C through only nine point mutations. There are other examples that illustrate the feasibility of drastically elevating or reducing the thermostability of an enzyme through a few amino acid changes (Matsumura et al., 1986Go, 1989Go; Das et al., 1989Go; Allen et al., 1998Go; Mikami et al., 1999Go). By contrast, adjustments of the thermostability of natural proteins seems to occur through the accumulation of numerous mutations with limited individual impact, as suggested by comparing homologous proteins from psychrophilic, mesophilic, thermophilic and hyperthermophilic organisms (Jaenicke and Bohm, 1998Go; Cambillau and Claverie, 2000Go; D’Amico et al., 2001Go; Vieille and Zeikus, 2001Go; Bertoldo and Antranikian, 2002Go). Concomitantly, natural enzymes tend to adjust their conformational flexibility in order to reach optimum catalytic efficiency in the temperature range at which they are supposed to operate [although this might not be the case for BLA (Fitter et al., 2001Go)]. Thermophilic proteins are usually more rigid and psychrophilic proteins more flexible than their mesophilic counterparts (Danson et al., 1996Go; Lonhienne et al., 2001Go). Consequently, heat-adapted enzymes are often poor catalysts at moderate temperatures. By contrast, enzyme thermostabilization achieved by protein engineering through point mutations does not necessarily lead to a reduction in the catalytic efficiency. None of the individual stabilizing mutations that we introduced in BLA was found to reduce the enzyme’s activity significantly and the Q264S–N265Y mutation even improved the amylolytic function. The benefit of this tandem mutation is lost in the 5-fold hyperthermostable mutant, suggesting that, as observed in naturally engineered proteins, the accumulation of stabilizing mutations tends to rigidify the entire molecule. Nevertheless, this should neither alter the amylolytic properties of the variants at high temperature nor hamper their use in industrial processes such as starch liquefaction. Indeed, the stabilizing mutations turned out to improve the performances of BLA at high temperatures, low calcium concentration and acidic pH (Shaw et al., 1999Go; Bisgard-Frantzen et al., 2000Go), offering great opportunities for optimizing biotechnological applications (Nielsen and Borchert, 2000Go).

Conclusions

The engineering work performed on BLA over the last decade provides a good example of the extent to which an enzyme can be remodelled in order to improve its natural performance and fulfil industrial requirements. Contrary to expectations, the thermal resistance of this highly thermostable {alpha}-amylase is far from being maximized in the wild-type enzyme and there seem to exist many ways to increase the thermostability even further. A fair set of stabilizing substitutions have already been found in BLA and many more may be identified in the future. Given the success achieved so far, it may even be possible to increase BLA thermostability beyond the most thermostable enzymes found in hyperthermophiles.


    Acknowledgments
 
This paper is dedicated to the memory of Professor Henry Heslot, who promoted the genetic studies on B.licheniformis {alpha}-amylase in our laboratory and very early envisioned protein engineering as a powerful tool for improving the performance of industrial enzymes. Our work on BLA would never have been accomplished without his support and enthusiasm.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received January 21, 2002; revised January 30, 2003; accepted February 5, 2003.





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