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With the advent of directed evolution techniques, proteinengineering has received a fresh impetus. Engineering proteinsfor thermostability is a particularly exciting and challengingfield, as it is crucial for broadening the industrial use ofrecombinant proteins. In addition to directed evolution, avariety of partially successful rational concepts for engineeringthermostability have been developed in the past. Recentresults suggest that amino acid sequence comparisons ofmesophilic proteins alone can be used efficiently to engineerthermostable proteins. The potential benefits of the underlying,semirational ‘consensus concept’ are compared with those ofrational design and directed evolution approaches.

AddressesF Hoffmann-La Roche Ltd., Vitamins and Fine Chemicals Division,Department VFB, Building 203, CH-4070 Basel, Switzerland*e-mail: martin.lehmann@roche.com†e-mail: markus.wyss@roche.com

Current Opinion in Biotechnology 2001, 12:371–375

0958-1669/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

IntroductionBecause natural enzymes are adapted to their particularfunction in a living cell, in most instances they are poorlysuited for industrial applications that often encounterextremes of pH, temperature and/or salinity. Both for broadening the industrial applicability of enzymesand for furthering our understanding of protein structure/function relationships, protein engineering iscurrently a very active area of research. Interest in thissubject area has further increased following the emer-gence of high-throughput directed evolution techniques,which can be seen as test tube models of natural evolu-tion. This strong interest in protein engineering isreflected in two recent publications dedicated entirely tothis topic [1,2]. The present review focuses on engineer-ing proteins for thermostability and compares theadvantages and benefits of classical rational design prin-ciples, directed evolution, and a ‘consensus approach’that extracts valuable information from sequence comparisons of homologous (mesophilic) proteins alone.

Rational design principlesThe stability of a protein is determined by a multitude ofboth local and long-range interactions. In order to achievepronounced thermostabilisation, several substitutionseach with a relatively small effect usually need to be com-bined in a multiple mutant. This endeavour is facilitatedby the fact that in many cases the thermostabilisationeffects of individual mutations are independent and nearlyadditive (e.g., [3–8]).

One strategy for identifying thermostabilising mutationsinvolves the comparison of more stable proteins with lessstable ones, with the goal of identifying amino acidsequence patterns that correlate with thermostability [9].In a systematic study, Perl et al. [10••] compared the coldshock proteins from the thermophile Bacillus caldolyticusand the mesophile Bacillus subtilis, which differ in only 12out of 67 residues but display a considerable difference instability (15.8 kJ/mol at 70°C). Site-directed mutagenesisof all 12 residues in the Bacillus caldolyticus enzymerevealed that the difference in thermostability can be fullyaccounted for by only two amino acid substitutions(Glu3Arg, Glu66Leu) on the surface of the molecule. Inthis illustrative example, with small proteins that exhibithigh homology and a pronounced difference in thermosta-bility, it is noted that less than 20% of the amino acidsubstitutions actually contribute to the difference in stabil-ity. This observation highlights the problem of identifyingthe relevant thermostabilising mutations in larger and lesshomologous sets of proteins.

In other studies published last year, higher thermostabilitywas suggested to correlate with more proline and lessasparagine and glutamate residues [11], more arginines andtyrosines but less cysteine and serine residues, and withincreased numbers of salt bridges and sidechain–sidechainhydrogen bonds [12]. In addition, higher thermostabilitycorrelated with a larger fraction of residues in α helices andwith more arginine and less proline, cysteine and histidineresidues in those α helices [12]. The information accumu-lated to date indicates that nature relies on no singlestrategy for stabilisation (see [13]), and as a result manypublications in this area arrive at different and even con-flicting conclusions. The availability of more completegenome sequences may cause the reliability of the correla-tions to improve, but sequence statistics of this kind, whentaken alone, are still unlikely to provide useful informationfor the accurate prediction of thermostabilising mutations.

Thermostabilising mutations either increase the thermody-namic stability of a protein (i.e., they increase the free-energydifference between the unfolded and the folded state) orthey decrease the rate of unfolding by increasing the free-energy difference between the folded state and the transitionstate of unfolding (see [14]). To achieve either of these, various rational concepts have been proposed: to decreasethe entropy of the unfolded state by introducing additionaldisulfide bridges or by X→Pro mutations; to increase α-helixpropensity by Gly→Ala substitutions or by stabilisation of α-helix macrodipoles; to improve electrostatic interactionsbetween charged surface residues by introducing additionalsalt bridges or even salt-bridge networks, or by predicting

Engineering proteins for thermostability: the use of sequencealignments versus rational design and directed evolutionMartin Lehmann* and Markus Wyss†

thermostabilising mutations on the basis of calculations ofelectrostatic potentials. Again, because the predictive powerof these rational concepts is rather limited [15], the supposedthermostabilising mutations need to be tested individuallyby site-directed mutagenesis. This reduces speed andthroughput and thereby limits the sequence space amenableto testing.

Directed evolutionThe term ‘directed evolution’ encompasses a series ofexperimental techniques that reproduce, on an acceleratedtimescale in the test tube, the evolution of natural diversi-ty and environmental adaptation. This is achieved throughmutation and recombination and by giving the process a‘direction’ towards the optimisation of one or more proper-ties of interest. Either a selective pressure is applied, or ineach round of mutagenesis and/or recombination thelibrary of variants obtained is screened for the desired trait.

One frequently applied strategy comprises repeatedrounds of random mutagenesis, starting with a given par-ent gene of interest. After each round, the best mutant(s)is (are) selected and used as parent sequence(s) in the following round of random mutagenesis. Typically, ratherlow mutation frequencies are employed to suppress accu-mulation of neutral or even deleterious mutations [13,14].In a recent example of this type, two cycles of randommutagenesis and screening yielded two mutants of phos-pholipase A1 with six and seven amino acid substitutionseach; the mutants displayed increases in their temperatureof half-inactivation of 7 and 11°C, respectively [16].

Since the introduction of DNA shuffling in 1994 [17,18],most directed evolution experiments encompass one ormore cycles of recombination between a set of homologoussequences. These experiments may use a set of improvedvariants of a given enzyme, obtained by random mutagene-sis, or a set of naturally occurring, homologous genes isolatedfrom wild-type organisms. DNA shuffling was shown to bevery powerful in recombining favourable mutations and ineliminating deleterious or neutral mutations.

Combinations of random mutagenesis and/or DNA (family)shuffling have been used successfully to engineer proteinthermostability [19–21,22•,23–27]. In these examples,increases in ‘thermostability’ of 14–20°C were attained with7–19 amino acid substitutions (relative to the parent protein), which were accumulated over up to eight genera-tions of directed evolution. Two important conclusions can be drawn from these results. Firstly, the increases in thermostability obtained by directed evolution have, as yet,been no more impressive than the best examples of rationaldesign (e.g., [28,29]), although they may have required lesstime and/or less effort. Secondly, despite improved equip-ment that allows higher throughput and increasedautomation, the sequence space amenable to testing is stillrather limited. Key prerequisites for larger steps and longerwalks in sequence space include the efficient elimination

of neutral and deleterious mutations, higher frequencies ofrecombination between homologous genes, recombinationevents in stretches of low amino acid sequence identity(e.g., [30••]), and powerful selection tools for improvedtraits. Although longer walks in sequence space might notactually be required for increases in thermostability of20–30°C, they may be desirable for more dramatic increasesin stability and will definitely be advantageous for optimis-ing other enzyme properties, such as reaction mechanism orsubstrate specificity.

The consensus conceptA third, semirational approach for engineering thermosta-bility is based on the hypothesis that at a given position inan amino acid sequence alignment of homologous pro-teins, the respective consensus amino acid contributesmore than average to the stability of the protein than thenonconsensus amino acids. Consequently, substitution ofnonconsensus by consensus amino acids may be a feasibleapproach for improving the thermostability of a protein.

Although Pantoliano et al. [3] were the first to apply the‘consensus concept’, showing that the consensus-typemutation Met50Phe increased the unfolding temperature(Tm) of subtilisin BPN′ by 1.8°C, it was five years later thatSteipe et al. [31] offered a possible theoretical explanation,based on statistical thermodynamics, for the feasibility ofthis approach. Steipe et al. [31] predicted ten individual,stabilising mutations for the immunoglobulin variable VLdomain, of which six were indeed stabilising. Furtherexperiments on the immunoglobulin VL and VH domainsas well as on a catalytic single-chain Fv fragment con-firmed these results and showed that the combination of aset of stabilising mutations in a multiple variant providedadditive thermostabilising effects [5,32–34].

In addition to applying the consensus concept to sets ofhomologous amino acid sequences, Steipe and coworkersalso tried to apply the concept to structural motifs. Thehypothesis that the most frequently occurring residuesin specific positions of β-turn motifs increase the foldingstability of a protein was experimentally confirmed foran immunoglobulin VL domain [32]. In contrast, whenthe concept was applied to surface-exposed loops andturns, which were assumed to be stabilised primarily by local interactions, only less stable variants wereobserved, suggesting that this particular application ofthe consensus concept does not allow reliable predictionof thermostabilising mutations.

A set of systematic studies of the consensus concept hasalso been provided by Fersht’s group. In one study [35],p53 homologues from 23 species were aligned, and 20 nonconsensus residues of human p53 were mutated individually to the respective consensus residue. Thechanges in stability ranged from +1.27 to –1.49 kcal/mol,and the theoretical sum of the stability changes of the 20individual mutations was no more than –0.48 kcal/mol.

372 Protein technologies and commercial enzymes

Four stabilising mutations were combined in a quadruplemutant (Met133Leu, Val203Ala, Asn239Tyr, Asn268Asp),which was stabilised by 2.65 kcal/mol and displayed anincrease in Tm of 5.6°C.

In two other studies [36•,37], the consensus concept wasapplied to GroEL minichaperones (i.e., fragments ofGroEL capable of facilitating protein folding). A sequencealignment of 130 sequences of homologous chaperonin 60proteins revealed that 31 (out of approximately 150)amino acids of the Escherichia coli minichaperoneGroEL(193–345) occur with a frequency of <35% at theirrespective positions in the alignment. Each of theseresidues was replaced individually with the most fre-quently occurring residue(s), yielding a total of 34 singlemutants which displayed differences in stability relativeto wild type ranging from +1.55 to –1.78 kcal/mol.Summing up the stability effects of all the individualmutations yields a theoretical overall difference in stabili-ty of –0.30 kcal/mol, which is unlikely to be significantlydifferent from zero. Among the 34 amino acid substitu-tions analysed, 13 were stabilising (38% success rate), fivewere neutral, and 16 were destabilising. However, whenconsidering for substitution only those residues that occurwith a frequency of <20% (rather than <35%) at therespective position in the alignment, 13 out of 18 muta-tions were stabilising (72% success rate), two were neutral,and only three were destabilising. Two multiple variantseach combining a set of six stabilising mutations were sta-bilised by 6.99 and 6.15 kcal/mol, and displayed Tmincreases of 18.6 and 14.2°C relative to wild-typeGroEL(193–345).

No attempt has been made by either Steipe’s or Fersht’sgroup to apply the consensus concept over the entiresequence of a protein. In fact, on the basis of the data ofNikolova et al. [35] and Wang et al. [36•] (see above), onemight be inclined to assume that such an attempt will fail,because stabilising and destabilising mutations may coun-terbalance each other. Very much to the contrary are theresults of Lehmann et al. [38••] describing the design of aconsensus phytase. In this study an appropriate computerprogram was used to calculate an entire consensussequence from 13 homologous amino acid sequences ofwild-type phytases from mesophilic fungi. A syntheticgene was constructed from the consensus sequence, andrecombinant expression of this gene gave rise to a consen-sus phytase (consensus phytase-1) that was 15–26°C morethermostable than all of its parents [38••]. Subsequently,incorporation of additional wild-type sequences in thealignment yielded consensus phytase-10, which displayed32 amino acid differences relative to consensus phytase-1and a further 7.4°C increase in Tm (M Lehmann et al.,unpublished data). Both consensus phytases differ in atleast 80 amino acids from any of their parents. This clearlyshows that the consensus concept allows multiple aminoacid exchanges to be combined in a single step in order toprovide a significantly improved variant of the enzyme.

Remarkably, the overall correlation often observedbetween increases in thermostability and decreases incatalytic turnover rate at ambient temperature does notapply to the consensus phytases; instead, the opposite istrue (M Lehmann et al., unpublished data).

In summary, the consensus approach allows much largersteps in sequence space than most directed evolution tech-niques reported to date. In addition, at present, increases inthermostability of up to 30°C seem possible in a single shot.

ConclusionsIn the case of engineering proteins for thermostability,researchers are in the enviable situation of being able tochoose between three different, apparently equally successful, strategies: rational design, directed evolution,and the construction of (semirational) synthetic consensusgenes. Rather than playing off one approach against theothers, future efforts should focus on how to best combinethese alternative approaches in order to access a larger frac-tion of the total sequence space and to approach the globaloptimum for a given trait in a consistent and direct way. Afirst successful study of this type has been reported byCherry et al. [39•]. In their endeavour to improve the stability of a haem peroxidase for laundry applications, fourmutations were rationally designed: one to increase theenzyme’s thermostability and three to increase resistanceto oxidative damage. The combination of these mutationswith favourable amino acid exchanges identified in direct-ed evolution experiments yielded a final mutant with 174times the thermal stability and 100 times the oxidative stability of the wild-type haem peroxidase.

We expect that the consensus concept will prove to be, andwill thus be generally recognised as, an additional valuableinstrument in our tool box for designing more ther-mostable enzymes for all types of industrial applications.Construction of a single consensus protein has the poten-tial to provide a similar increase in thermostability as manymonths or years of rational design or as directed evolutioncampaigns involving multiple generations and analysis ofthousands of mutants.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Dalboge H, Borchert TV (Guest Eds): Protein engineering ofenzymes. Biochim Biophys Acta 2000, 1543:203-455.

2. Arnold FH (Guest Ed): Evolutionary protein design. Adv ProteinChem 2001, 55:1-438.

3. Pantoliano MW, Whitlow M, Wood JF, Dodd SW, Hardman KD,Rollence ML, Bryan PN: Large increases in general stability forsubtilisin BPN′′ through incremental changes in the free energy ofunfolding. Biochemistry 1989, 28:7205-7213.

4. Serrano L, Day AG, Fersht AR: Step-wise mutation of barnase tobinase. A procedure for engineering increased stability of proteinsand an experimental analysis of the evolution of protein stability.J Mol Biol 1993, 233:305-312.

Engineering proteins for thermostability Lehmann and Wyss 373

5. Ohage E, Steipe B: Intrabody construction and expression. I. Thecritical role of VL domain stability. J Mol Biol 1999, 291:1119-1128.

6. von der Osten C, Branner S, Hastrup S, Hedegaard L, Rasmussen MD,Bisgard-Frantzen H, Carlsen S, Mikkelsen JM: Protein engineering ofsubtilisins to improve stability in detergent formulations. JBiotechnol 1993, 28:55-68.

7. Akasako A, Haruki M, Oobatake M, Kanaya S: High resistance ofEscherichia coli ribonuclease HI variant with quintuplethermostabilizing mutations to thermal denaturation, aciddenaturation, and proteolytic degradation. Biochemistry 1995,34:8115-8122.

8. Shih P, Kirsch JF: Design and structural analysis of an engineeredthermostable chicken lysozyme. Protein Sci 1995, 4:1063-2072.

9. Haney PJ, Badger JH, Buldak GL, Reich CI, Woese CR, Olsen GJ:Thermal adaptation analyzed by comparison of protein sequencesfrom mesophilic and extremely thermophilic Methanococcusspecies. Proc Natl Acad Sci USA 1999, 96:3578-3583.

10. Perl D, Müller U, Heinemann U, Schmid FX: Two exposed amino acid•• residues confer thermostability on a cold shock protein. Nat Struct

Biol 2000, 7:380-383.The cold shock proteins from the thermophile Bacillus caldolyticus and themesophile Bacillus subtilis differ in only 12 out of 67 residues. Systematicsite-directed mutagenesis revealed that only two of these 12 residuesaccount for the considerable difference in stability between the two proteins.For some of the other 10 residues, the B. caldolyticus protein was even sta-bilised when the respective amino acid from the less thermostable B. sub-tilis protein was introduced. These results question the usefulness ofsequence comparisons between thermophilic and mesophilic or psy-chrophilic proteins for accurately predicting (thermo-) stabilising mutations.

11. Sriprapundh D, Vieille C, Zeikus JG: Molecular determinants ofxylose isomerase thermal stability and activity: analysis ofthermozymes by site-directed mutagenesis. Protein Eng 2000,13:259-265.

12. Kumar S, Tsai CJ, Nussinov R: Factors enhancing proteinthermostability. Protein Eng 2000, 13:179-191.

13. Wintrode PL, Arnold FH: Temperature adaptation of enzymes:lessons from laboratory evolution. Adv Protein Chem 2001,55:161-225.

14. Steipe B: Evolutionary approaches to protein engineering.Curr Top Microbiol Immunol 1999, 243:55-86.

15. Spector S, Wang A, Carp SA, Robblee J, Hendsch ZS, Fairman R,Tidor B, Raleigh DP: Rational modification of protein stability bythe mutation of charged surface residues. Biochemistry 2000,39:872-879.

16. Song JK, Rhee JS: Simultaneous enhancement of thermostabilityand catalytic activity of phospholipase A1 by evolutionarymolecular engineering. Appl Environ Microbiol 2000, 66:890-894.

17. Stemmer WPC: Rapid evolution of a protein in vitro by DNAshuffling. Nature 1994, 370:389-391.

18. Stemmer WPC: DNA shuffling by random fragmentation andreassembly: in vitro recombination for molecular evolution. ProcNatl Acad Sci USA 1994, 91:10747-10751.

19. Giver L, Gershenson A, Freskgard PO, Arnold FH: Directed evolutionof a thermostable esterase. Proc Natl Acad Sci USA 1998,95:12809-12813.

20. Hoseki J, Yano T, Koyama Y, Kuramitsu S, Kagamiyama H: Directedevolution of thermostable kanamycin-resistance gene: aconvenient selection marker for Thermus thermophilus. J Biochem1999, 126:951-956.

21. Kikuchi M, Ohnishi K, Harayama S: Novel family shuffling methodsfor the in vitro evolution of enzymes. Gene 1999, 236:159-167.

22. Spiller B, Gershenson A, Arnold FH, Stevens RC: A structural view• of evolutionary divergence. Proc Natl Acad Sci USA 1999,

96:12305-12310.The three-dimensional structure of the parent wild-type p-nitrobenzylesterase was compared with that of a thermostabilised mutant that wasobtained by eight generations of directed evolution; the mutant displayed13 amino acid substitutions and a 17°C increase in Tm relative to the wildtype. Remarkably, the mutations exerted their influence on the esterasestructure over large distances, in a manner that would be difficult (or insome cases even impossible at present) to predict. The loops with thelargest structural changes were not generally the sites of mutations. These

results therefore question the reliability of rational approaches that oftenneglect long-range effects.

23. Zhao H, Arnold FH: Directed evolution converts subtilisin E into afunctional equivalent of thermitase. Protein Eng 1999, 12:47-53.

24. Gershenson A, Schauerte JA, Giver L, Arnold FH: Tryptophanphosphorescence study of enzyme flexibility and unfolding inlaboratory-evolved thermostable esterases. Biochemistry 2000,39:4658-4665.

25. Kim GJ, Cheon YH, Kim HS: Directed evolution of a novel N-carbamylase/D-hydantoinase fusion enzyme for functionalexpression with enhanced stability. Biotechnol Bioeng 2000,68:211-217.

26. Miyazaki K, Wintrode PL, Grayling RA, Rubingh DN, Arnold FH:Directed evolution study of temperature adaptation in apsychrophilic enzyme. J Mol Biol 2000, 297:1015-1026.

27. Arnold FH: Combinatorial and computational challenges forbiocatalyst design. Nature 2001, 409:253-257.

28. van den Burg B, Vriend G, Veltman OR, Venema G, Eijsink VGH:Engineering an enzyme to resist boiling. Proc Natl Acad Sci USA1998, 95:2056-2060.

29. Malakauskas SM, Mayo SL: Design, structure and stability of ahyperthermophilic protein variant. Nat Struct Biol 1998, 5:470-475.

30. Coco WM, Levinson WE, Crist MJ, Hektor HJ, Darzins A, Pienkos PT,•• Squires CH, Monticello DJ: DNA shuffling method for generating

highly recombined genes and evolved enzymes. Nat Biotechnol2001, 19:354-359.

The authors describe a novel strategy for the molecular evolution of proteins,termed random chimeragenesis on transient templates (RACHITT). Thestrategy relies on the ordering, trimming and joining of randomly cleavedparental DNA fragments annealed to a transient, full-length, single-strandedpolynucleotide scaffold. This method may lead to a significant improvementof recombination frequency and a more comprehensive exploitation ofsequence space than previously reported DNA shuffling strategies.Chimeric libraries averaging 14 cross-overs per gene were obtained, com-pared with the one to four cross-overs obtained with previous techniques. Inaddition, four cross-overs per gene occurred in regions of 10 or fewer basesof sequence identity. No unshuffled parental clones or duplicated siblingchimeras were detected, and only a few inactive clones were identified.

31. Steipe B, Schiller B, Plückthun A, Steinbacher S: Sequencestatistics reliably predict stabilizing mutations in a proteindomain. J Mol Biol 1994, 240:188-192.

32. Ohage EC, Graml W, Walter MM, Steinbacher S, Steipe B: ββ-Turnpropensities as paradigms for the analysis of structural motifs toengineer protein stability. Protein Sci 1997, 6:233-241.

33. Ohage EC, Wirtz P, Barnikow J, Steipe B: Intrabody constructionand expression. II. A synthetic catalytic Fv fragment. J Mol Biol1999, 291:1129-1134.

34. Wirtz P, Steipe B: Intrabody construction and expression III:engineering hyperstable VH domains. Protein Sci 1999, 8:2245-2250.

35. Nikolova PV, Henckel J, Lane DP, Fersht AR: Semirational design ofactive tumor suppressor p53 DNA binding domain with enhancedstability. Proc Natl Acad Sci USA 1998, 95:14675-14680.

36. Wang Q, Buckle AM, Foster NW, Johnson CM, Fersht AR: Design of• highly stable functional GroEL minichaperones. Protein Sci 1999,

8:2186-2193.An amino acid sequence alignment of 130 homologous chaperonin 60 pro-teins was used to identify amino acids of an E. coli GroEL minichaperonethat occur with a frequency of <35% or <20% at their respective position inthe alignment. When each of these residues was replaced individually withthe most frequently occurring residue(s), stability differences relative to wildtype of +1.55 to –1.78 kcal/mol were observed, and 13 out of 34 (for aminoacids with a frequency of <35%; 38% success rate) and 13 out of 18 aminoacid substitutions (<20%; 72% success rate) were actually found to be sta-bilising. Only five of the stabilising amino acid substitutions could have alsobeen predicted by comparison of E. coli GroEL with the sequences of thefive thermophilic chaperonin 60 proteins known at the time. When six of thestabilising amino acid substitutions were combined in a multiple mutant, theenergetic effects were approximately additive, yielding an increase in Tm ofup to 18.6°C. Finally, a small-to-large stabilising mutation, Ala223Thr, wasidentified that is one of the rare examples where the introduction of a polarhydroxyl group is accompanied by stabilisation of a hydrophobic core.

37. Wang Q, Buckle AM, Fersht AR: Stabilization of GroELminichaperones by core and surface mutations. J Mol Biol 2000,298:917-926.

374 Protein technologies and commercial enzymes

38. Lehmann M, Kostrewa D, Wyss M, Brugger R, D’Arcy A, •• Pasamontes L, van Loon APGM: From DNA sequence to improved

functionality: using protein sequence comparisons to rapidly designa thermostable consensus phytase. Protein Eng 2000, 13:49-57.

Whereas Steipe’s and Fersht’s groups only tested their versions of the ‘consen-sus concept’ on individual residues, the authors of this paper were the first to applythe consensus approach over the entire sequence of a protein. A consensusamino acid sequence was calculated from an alignment of 13 homologous fungalphytases (myo-inositol hexakisphosphate phosphohydrolases), followed by con-struction of a synthetic gene and recombinant expression. Astonishingly enough,the resulting consensus phytase-1 was 15–26°C more thermostable than all itsparents but displayed catalytic properties closely resembling those of its parents.

39. Cherry JR, Lamsa MH, Schneider P, Vind J, Svendsen A, Jones A,• Pedersen AH: Directed evolution of a fungal peroxidase. Nat

Biotechnol 1999, 17:379-384.To increase the stability of a haem peroxidase under laundry conditions (i.e.,at pH 10.5, 50°C and 5–10 mM peroxide concentration), rational and direct-ed evolution approaches were merged. Four rationally designed amino acidsubstitutions were randomly (re-) combined with several thermostabilisingmutations obtained by directed evolution to yield a sevenfold mutant with174 times the thermal stability and 100 times the oxidative stability of wildtype. These impressive improvements were compromised, however, by a 20-fold lower specific activity at pH 10.5.

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