[methods in enzymology] protein engineering volume 388 || engineering the thermotolerance and ph...

that these protocols may open a new avenue to study extensively enzymeregioselectivity on a wide range of ester substrates; an opportunity that hasbeen difficult to achieve thus far using DME due to the lack of relevantexperimental tools.

Acknowledgments

This work was supported by the Biotechnology Research and Development Corporation.

156 applications: optimization and screening [14]

[14] Engineering the Thermotolerance andpH Optimum of Family 11 Xylanases by

Site-Directed Mutagenesis

By Ossi Turunen, Janne Janis,Fred Fenel, and Matti Leisola

Introduction

The potential of using xylanases in industrial applications generated anintensive academic and industrial activity to hunt xylanases with desiredproperties from all kinds of natural sources. As a consequence, the genebanks contain presently over 100 sequences for family 11 xylanases. Widelyused commercial enzymes for biomass modification in animal feeding,baking, and pulp bleaching have come from mesophilic filamentous fungiTrichoderma reesei and Aspergillus niger.1,2 The mesophilic xylanases areuseful but not optimal in all industrial applications. These enzymes are easilyinactivated in the preparation of animal feed; furthermore, they are notvery active in hot alkaline conditions of pulp bleaching. The screeningactivities produced numerous thermophilic xylanases, of which only fewhave entered the markets. An alternative for screening new xylanases fromnature is to optimize by rational design or directed evolution an alreadyknown xylanase that has several advantages (e.g., good production system)but is missing an acceptable thermostability, alkalitolerance, or some otherkey properties.

1 L. Viikari, A. Kantelinen, J. Sundquist, and M. Linko, FEMS Microbiol. Rev. 13, 335

(1994).2 R. A. Prade, Biotechnol. Genet. Eng. Rev. 13, 101 (1996).

Copyright 2004, Elsevier Inc.All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 388 0076-6879/04 $35.00

[14] family 11 xylanases 157

Structural Features of Family 11 Xylanases

The sequence identities among family 11 xylanases range from 40 to98%. A large number of the conserved sites allow some variation, and thenumber of highly conserved sites is in the range of 20% of all the sites. Thesize of family 11 xylanases is quite small, containing approximately 190amino acid residues. The enzyme has been described to form a right-hand-like structure.3 About 120 of all the residues belong to � strands.4 The basicfold is formed by a double-layered � sheet that is twisted to form a largeactive site cleft. A conserved single � helix is located on the outer surface.Crystallographic analysis and molecular dynamics simulations revealed anopen–close movement of the active site.5 Two hinge regions are involved inthis movement. The substrate apparently promotes the closing of the activesite.5 It is evident that the engineering of stabilizing mutations should notdisturb the open–close movement of the active site.

Stabilization Strategies

The thermostability of mesophilic xylanases (especially T. reesei andBacillus circulans xylanases) has been increased considerably by designedmutations. T. reesei xylanase II (XYNII) is stable at 40–45

�, whereas

temperatures above 50�

cause conformational changes and the enzymeactivity is lost quite quickly.6–8 B. circulans xylanase is completely inacti-vated in 30 min at 57

�.9 There are several strategies how to design

mutations that could improve the thermostability.

Disulfide Bridges at Protein N Terminus

Xylanases contain naturally disulfide bridges at the � helix and cordregion (see structures with PDB-codes 1yna, 1PVX, 1BK1, and lukr).Disulfide bridges can also be introduced into different sites that do notnaturally contain a disulfide bridge in any of the family 11 xylanases. Such a

3 A. Torronen and J. Rouvinen, J. Biotechnol. 57, 137 (1997).4 N. Hakulinen, O. Turunen, J. Janis, M. Leisola, and J. Rouvinen, Eur. J. Biochem. 270, 1399

(2003).5 J. Muilu, A. Torronen, M. Perakyla, and J. Rouvinen, Proteins 31, 434 (1998).6 M. Tenkanen, J. Puls, and K. Poutanen, Enzyme Microb. Technol. 14, 566 (1992).7 J. Janis, J. Rouvinen, M. Leisola, O. Turunen, and P. Vainiotalo, Biochem. J. 356, 453

(2001).8 O. Turunen, K. Etuaho, F. Fenel, J. Vehmaanpera, X. Wu, J. Rouvinen, and M. Leisola,

J. Biotechnol. 88, 37 (2001).9 W. W. Wakarchuk, W. L. Sung, R. L. Campbell, A. Cunningham, D. C. Watson, and

M. Yaguchi, Protein Eng. 7, 1379 (1994).


place is the N-terminal region.9,10 A disulfide bridge between positions2 and 28 in T. reesei XYNII increased the thermostability about 15

�, both

in the absence and in the presence of the substrate.10 A disulfide bridgecross-linking protein N and C termini also has a significant stabilizingeffect.9

Disulfide Bridges at a Helix

The single � helix is another region important for the thermostability offamily 11 xylanases. Disulfide bridges have been introduced into severalpositions of the � helix, with the disulfide bridge in the N terminus of the� helix being most effective.9,11 These disulfide bridges increased theresistance to heat-induced denaturation, but not the activity at hightemperatures (apparent temperature optimum).

Single Amino Acid Mutations

A large number of stabilizing amino acid changes (other than disulfidebridges) have been reported for family 11 xylanases.8,12–18 Single aminoacid mutations have been done at the N-terminal region, � helix, and otherregions. Furthermore, an N-terminal extension has been reported to have astabilizing effect; correspondingly, an N-terminal deletion can be destabi-lizing.13,19 Comparison of crystal structures from thermophilic and meso-philic organisms indicated that Thr/Ser and Arg/Lys ratios are higher inthermostable family 11 xylanases.4

A combination of various mutations has often a cumulative effect onthe thermostability of family 11 xylanases. When mutations with only asmall stabilizing effect were combined with the disulfide bridge engineeredinto the � helix, a considerable increase in thermostability was achieved.8

10 F. Fenel, M. Leisola, J. Janis, and O. Turunen, J. Biotechnol. 108, 137 (2004).11 H. Xiong, F. Fenel, M. Leisola, and O. Turunen, submitted for publication.12 A. Arase, T. Yomo, I. Urabe, Y. Hata, Y. Katsube, and H. Okada, FEBS Lett. 316, 123

(1993).13 W. L. Sung, M. Yaguchi, and K.Ishikawa, U. S. Patent Number 5,866,408 (1998).14 J. Georis, F. de Lemos Esteves, J. Lamotte-Brasseur, V. Bougnet, B. Devreese, F. Giannotta,

B. Granier, and J.-M. Frere, Protein Sci. 9, 466 (2000).15 W. L. Sung and J. S. Tolan, Patent WO00/29587 (2000).16 W. L. Sung, Patent WO0192487 (2001).17 O. Turunen, M. Vuorio, F. Fenel, and M. Leisola, Protein Eng. 15, 141 (2002).18 J.-M. G. Daran, H. H. Menke, J. P. van den Hombergh, and J. M. van den Laar, Patent

EP1184460 (2002).19 D. D. Morris, M. D. Gibbs, C. W. Chin, M. H. Koh, K. K. Y. Wong, R. W. Allison, P. J.

Nelson, and P. L. Bergquist, Appl. Environ. Microb. 64, 1759 (1998).


However, many thermostable family 11 xylanases achieve their highthermostability without disulfide bridges.

Thermostabilization may increase the rigidity of the enzymes. It is oftenimportant for industrial applications that the specific activity does notdecrease. The determination of kinetic parameters revealed that an exten-sive stabilization of T. reesei XYNII simultaneously at the protein Nterminus and the � helix did not affect enzyme activity negatively, asindicated by unchanged Km and Vmax values when compared to the wildtype.11

Modification of pH-Dependent Properties

Several factors have an influence on the pH-dependent properties ofxylanases. The ionization states of the nucleophile and acid/base gluta-mates control the pH-dependent activity profile.20 The activity at low pHcan be increased by modifying the environment of the acid/base catalyst(Glu-177 in T. reesei XYNII and Glu-172 in B. circulans xylanase). Themutation of nearby Asn to Asp (N35D in B. circulans xylanase) loweredthe pH optimum considerably and explains the low pH optimum of acidicxylanases.20 In addition, acidic xylanases appear to have a higher numberof acidic and a lower number of basic amino acids on the protein surface.

The reduced catalytic activity of xylanases at alkaline conditions ap-pears to involve pH-induced unfolding and ionization or deprotonation ofkey amino acid residues.21 In principle, the pH optimum can be shiftedtoward alkaline pH by increasing the pKa value of the acid/base glutamate.The short distance effects on the pKa value of a residue are important indetermining the pH activity profile of xylanases.22 A histidine residuelocated near the acid/base catalyst in the active site cleft is involved inmaintaining the high pH optimum in family 10 xylanases.23 The introduc-tion of arginines into the Ser/Thr surface17 and modification of the proteinN terminus13 have improved the activity of T. reesei XYNII at alkaline pH.Furthermore, amino acid substitutions that do not involve charged aminoacid residues have increased the activity of xylanase at alkaline pH.24

Bacillus agaradhaerens is a bacterium that grows at very alkaline sources,

20 M. D. Joshi, G. Sidhu, I. Pot, G. D. Brayer, S. G. Withers, and L. P. McIntosh, J. Mol. Biol.

299, 255 (2000).21 D. Nath and M. Rao, Enzyme Microb. Technol. 28, 397 (2001).22 M. D. Joshi, G. Sidhu, J. E. Nielsen, G. D. Brayer, S. G. Withers, and L. P. McIntosh,

Biochemistry 40, 10115 (2001).23 M. Roberge, F. Shareck, R. Morosoli, D. Kluepfel, and C. Dupont, Protein Eng. 11, 399

(2001).24 Y.-L. Chen, T.-Y. Tang, and K.-J. Cheng, Can. J. Microbiol. 47, 1088 (2001).


such as soda lakes. The B. agaradhaerens xylanase shows a high alkalist-ability, but curiously a low pH optimum (pH 5.6) and at pH 9 the enzymeactivity is even close to zero.25 In conclusion, factors controlling the pH-dependent activity of xylanases are only partially known and require muchfurther research. This is a relevant challenge in engineering the pH activityprofile of xylanases for industrial purposes.

Engineering of Arginines

We tested how the engineering of additional arginines affects thethermostability of T. reesei XYNII. The increase of arginines on differentsides of the enzyme did not increase the thermostability. Instead, theintroduction of five arginines into the Ser/Thr surface had a considerablethermostabilizing effect.17 The Ser/Thr surface is part of the outer � sheetlayer on the protein surface and contains a large number of serines andthreonines. Figures 1 and 2 show results obtained for an arginine mutant ofT. reesei XYNII.17 The five engineered arginines increased the apparenttemperature optimum by 5

�(Fig. 1) and the pH-dependent activity profile

shifted clearly to alkaline pH (Fig. 2). The shift was seen in both the acidicand the alkaline side of the bell-shaped pH-dependent activity profile.However, the stabilizing effect was enigmatic because the half-lifemeasured in the absence of the substrate decreased as the number ofarginines increased on the Ser/Thr surface. A reason for this could be thatthe electrostatic repulsion caused by a high positive net charge was desta-bilizing in the absence of the substrate. Thus, the stabilizing effect ofarginines was seen only in the presence of the substrate. The half-lifeincreased four-fold in the presence of the substrate due to five arginineson the Ser/Thr surface. In conclusion, the efficient use of arginines asprotein stabilizers requires further research: the location on the proteinsurface and the local environment need special attention.

Experimental Procedures

Planning of Mutations

The protein databank contains a large number of resolved three-dimensional structures of family 11 xylanases.4 The available structuralinformation is essential in understanding the structure–function relation-ships. Structural and sequence comparisons in the large xylanase family are

25 D. K. Y. Poon, P. Webster, S. G. Withers, and L. P. McIntosh, Carbohydr. Res. 338, 415

(2003).

Fig. 1. Effect of engineered arginines on the temperature-dependent activity of T. reesei

XYNII. E. coli culture broth was used as the source for enzymes. Enzyme activity was

measured by the DNS assay using a 10-min incubation time in 50 mM citrate-phosphate

buffer (pH 5). Relative activity values were calculated from absorbance (A540) values

obtained from the enzyme assay. The arginine mutant (ST5) contains five arginines on the Ser/

Thr surface. W.T., wild-type XYNII. Reproduced in modified form with permission from

Turunen et al.,17 � Oxford University Press.

Fig. 2. Effect of engineered arginines on the pH-dependent activity profile of T. reesei

XYNII. Xylanase activity was measured at 50�

in each pH. Otherwise the enzyme assay was

done in the same way as in Fig. 1. The arginine mutant was ST5 as in Fig. 1. Reproduced in

modified form with permission from Turunen et al.,17 � Oxford University Press.


used to plan mutations that would change the properties of the enzyme in adesired manner. In planning mutations, we have used mainly Swiss-PdbViewer (http://us.expasy.org/spdbv)26 as a molecular graphical tool to

26 N. Guex and M. C. Peitsch, Electrophoresis 18, 2714 (1997).

http://us.expasy.org/spdbv


examine the xylanase structure (e.g., lxyp). The program allows the intro-duction of mutations into the known or modeled xylanase structures. Theeffects of the mutations can be evaluated first by the score values calculatedby the program and by inspecting the local interactions of the substitutedresidues and also by using the torsion command of the program. Modelingby other programs may be useful in estimating the conformation of themutated residue. Crystallographic B values and molecular dynamics simu-lations may give useful information about the mobility of different proteinregions. Optimization of the hydrogen-bond network and calculationof theoretical pKa values can be done by WHATIF.27 The alignment ofxylanase protein sequences was done mainly by ClustalX (or ClustalW).

Site-Directed Mutagenesis

Mutations are generated by polymerase chain reaction (PCR) in whichthe mutations are introduced into the oligonucleotide primers.8 The two(forward and reverse) primers overlap fully and contain the mutated codonin a middle position, and the primers are designed so that the Tm of theprimers is at least 78

�. The PCR conditions are basically those of

the QuikChange mutagenesis system (Stratagene, La Jolla, CA). Thestandard PCR reaction mixture contains 5–50 ng template DNA,0.08 mM dNTP, 0.2 mM of each primer, and 10� PFU reaction buffer(Stratagene). After a 5-min heating at 95

�, 1.0 �l of PFU Turbo polymerase

(Stratagene) is added to the hot PCR reaction mixture. One cycleis typically as follows: 50 s at 95

�/50 s at 60

�/1.0 min/kb of plasmid at 68

�.

The number of cycles is between 12 and 18, and after the last cycle thereis a 7-min extension at 68

�. After the PCR, the parent DNA is digested

by adding 1.0 �l of the DpnI restriction enzyme (New England Biolabs,Beverly, MA) and incubating for 1–2 h at 37

�. Competent Escherichia

coli XL1-Blue cells are used in the transformation. E. coli clones con-taining mutant xylanases are grown at 37

�overnight on LB agar plates

containing 0.1–0.2% birchwood xylan (Sigma, Steinheim, Germany), whichis coupled to Remazol brilliant blue (Sigma) according to Biely et al.28

Xylanase activity is indicated by white halos around the positive colonies.

Expression Vectors

In E. coli cells, xylanases are secreted into the periplasmic place. In ourwork, we have used either �-amylase or pectate lyase secretion signalsequence.8 Enough xylanase leaks from the periplasmic place into the

27 J. E. Nielsen and G. Vriend, Proteins 43, 403 (2001).28 P. Biely, D. Mislovicova, and R. Toman, Anal. Biochem. 144, 142 (1985).


culture medium for purification and characterization of the enzyme.T. reesei XYNII is produced in E. coli XL1-Blue (Strategene) or E. coliRv308 (ATCC 31608) strains using the pALK143 expression construct(ROAL, Rajamaki, Finland) or pKKtac vector (VTT, Espoo, Finland).The low-copy plasmid pWSK29 has been used to construct pALK143,which contains the T. reesei xylanase II cDNA, including 11 amino acidsfrom the prosequence, and the Bacillus amyloliquefaciens �-amylase signalsequence under the B. amyloliquefaciens �-amylase promoter. The xyla-nase is produced constitutively from pALK143. pKKtac has beenconstructed from the pKK233 vector (Amersham-Pharmacia Biotech, Up-psala, Sweden) and contains the tac promoter for induction by 1 mM IPTGand lacIq repressor. This construct has an Erwinia carotovora pectate lyasesignal sequence, which we fused with the mature XYNII protein withoutprosequence. Higher quantities of xylanases are usually produced from thepKKtac vector. The prosequence present in pALK143 is not fully removedin some of the mutants.29

Purification of Xylanases

Recombinant xylanases are produced in E. coli grown in Luria brothsupplemented with ampicillin using shake flasks at 30 or 37

�(shaking 200–

250 rpm). When xylanases are produced from the pKKtac vector, E. colicells are grown for overnight, diluted in �2 � 10�2 dilution, and grown for3–4 h at 30

�(OD600 ¼ 0.5); 1 mM IPTG is then added overnight to induce

xylanase production (growth at 30�). The xylanase is precipitated from the

clarified culture broth by 65% saturated ammonium sulfate. The resultingpellet is dissolved in 0.01 M citrate-phosphate buffer (pH 4) and desaltedby a PD10 column (Amersham-Pharmacia Biotech) or dialysis (molecularweight cutoff 6000–8000).

Protein purification is carried out by the BioPilot system (Amersham-Pharmacia Biotech).8 The desalted solution containing xylanase is loadedonto a CM-Sepharose fast flow (Amersham-Pharmacia Biotech) column(1.6 � 11 cm) equilibrated with 0.01 M citrate buffer (pH 4). Elution isdone with a linear sodium chloride gradient (0–0.25 M) at a flow rate of5 ml min�1. Further purification is carried out by hydrophobic interactionchromatography using a phenyl-Sepharose fast flow (Amersham-Pharmacia Biotech) column (1.6 � 10 cm) equilibrated with 1.5 M ammo-nium sulfate in 0.01 M citrate buffer (pH 4). The enzyme is washed outfrom the column by decreasing the concentration of ammonium sulfate.

29 J. Janis, O. Turunen, M. Leisola, P. J. Derrick, J. Rouvinen, and P. Vainiotalo, submitted

for publication.


The protein concentration is measured spectrophotometrically by absor-bance at 280 nm (A280) using the molar extinction coefficient 54,050determined for T. reesei XYNII.8

Enzyme Assay and Characterization of Thermostability andOther Properties

Xylanase activity is determined using the DNS assay to measure theamount of reducing sugars liberated from 1% birchwood xylan.30 Theactivity determination in standard conditions for T. reesei XYNII is carriedout at pH 5–6 and 50

�, with a reaction time of 10 min. Citrate-phosphate

buffer (50 mM) is used in the xylanase assays at pH 4–7.5 and 50 mM Tris–HCl at pH 7.5–9. Bovine serum albumin (BSA; 0.1 mg/ml) or even inacti-vated E. coli culture broth can be used as a stabilizer in activity andthermostability measurements. When purified enzymes are used inthe thermostability assays, a stabilizer is recommended to be includedin the diluted enzyme solutions because diluted pure xylanase may disap-pear quite quickly from the solution (considerable amounts may be lostin 30 min). Several properties of xylanases can even be tested using non-purified enzymes; these include apparent temperature optimum, pH-dependent activity profile, stability against thermal inactivation, half-life,and pH-dependent stability. Determination of kinetic (Km and Vmax) pa-rameters, differential scanning calorimetry (DSC), and mass spectrometryare done with purified proteins.

Thermostabilities measured for xylanases are approximations and aredependent on conditions. However, the obtained values can be used tomake comparisons of stability and other properties. The stability againstthermal inactivation is assayed usually so that the enzyme is heated at aseries of different temperatures for a chosen time (e.g., 10 or 30 min), andafter rapid cooling the remaining activity is measured at a low temperature(e.g., 50

�for T. reesei XYNII). The temperature at which 50% of the

activity is left (T50) can be compared to the melting point values (Tm)obtained by other methods, such as DSC and hydrogen/deuterium (H/D)exchange analysis with mass spectrometry. However, there can be differ-ences in these values that are dependent on experimental conditions onone side, and on the other side, the enzyme activity can be lost at a lowertemperature than where the total unfolding of the protein occurs. Anotherapproach to assay thermostability is that the enzyme is incubated at a giventemperature, samples are removed at various time intervals, and then theremaining activity is measured at the standard assay temperature. Half-live

30 M. J. Bailey, P. Biely, and K. Poutanen, J. Biotech. 23, 257 (1992).


values are calculated from the latter graphs obtained for time-dependentinactivation of the enzyme activity. These methods can be used to assessthe irreversible inactivation at given conditions, but they do not detectreversible inactivation. Mutations that increase the apparent temperatureoptimum may reveal a site for reversible unfolding in the enzyme structure.

In xylanases, the substrate appears to increase the thermostability. Themeasurement of the enzyme activity as a function of time (productivityassay) can be used to calculate the half life of a rough enzyme in thepresence of substrate.17 Arrhenius activation energy can be calculated fromthe slope of the temperature-dependent activity profile using the equation

ln k ¼ ln A � Ea=RTð Þ (1)

A plot of ln k versus 1/T gives a straight line with slope �Ea/R, fromwhich the Arrhenius activation energy (Ea) can be calculated.31 k is therate constant for the reaction, A is the Arrhenius constant, R is the idealgas constant, and T is the absolute temperature. Our most stable T. reeseiXYNII disulfide bridge mutants did not show any significant differences inthe activation energy to the wild-type value (�50 kJ gmol�1), whereas theintroduction of five arginines into the Ser/Thr surface increased theactivation energy by 40%.11,17

Use of Mass Spectrometry to Assay Thermostability

Hydrogen/deuterium exchange is a sensitive mass spectrometric appli-cation for conformational analyses of proteins.7,32,33 H/D exchange hasbeen used to probe thermally induced structural changes in proteins. Therate at which hydrogens are replaced with deuterons in solution is acombination of intrinsic exchange rates, hydrogen bonding, and solventaccessibility. Whereas H/D exchange rates of the amino acid side chainhydrogens are typically too fast in a timescale for mass spectrometricdetection, the exchange of amide hydrogens is detectable when solutionconditions are adjusted carefully. However, in a tightly folded proteinstructure, H/D exchange rates of hydrogens buried into a hydrophobicprotein core or ones involved in hydrogen bonding can be a few magni-tudes lower than the corresponding rates of the surface hydrogens that areexposed readily to the solvent. Conformational changes and unfolding ofthe protein structure expose buried hydrogens to solvent, which can be

31 P. M. Doran, ‘‘Bioprocess Engineering Principles,’’ p. 262. Academic Press, New York,

2000.32 V. Katta and B. T. Chait, J. Am. Chem. Soc. 115, 6317 (1993).33 A. N. Hoofnagle, K. A. Resing, and N. G. Ahn, Annu. Rev. Biophys. Biomol. Struct. 32, 1

(2003).


detected from the change of the H/D exchange rate. H/D exchange canprovide information about the melting point, protein rigidity, degree ofexchangeable hydrogens, and stability of protein variants present in theprotein sample.

We used H/D exchange for measuring conformational stability on theheat-induced unfolding of T. reesei XYNII and its mutants having disulfidebridges and other stabilizing mutations.7,29 We were also able to measurethe thermostability of protein variants caused by the incomplete removal ofprosequence, resulting in a difference of even 5

�in the Tm values.29 A

typical experimental procedure is as follows. Purified protein in water orbuffered solution is diluted with a fully deuterated solvent (typically deu-terium oxide, D2O). A sample tube is placed into a heating block for apredefined time, after which the exchange reaction is quenched either byacid or by placing the tube in an ice bath (0

�). Deuterium incorporation

versus incubation temperature is measured immediately using mass spec-trometry. If a heat-induced conformational change takes place at a certaintemperature, this can be monitored by the increase in the H/D exchangerate. If bimodal distribution in deuterium incorporation is detected, i.e.,two conformers with distinct exchange rates, relative abundances can becalculated from the spectral peak intensities. Therefore, both qualitativeand quantitative aspects of the heat-induced conformational changes canbe analyzed. When H/D exchange ESI MS spectra was determined forT. reesei XYNII exposed to heat in D2O solvent in the temperature regionof 54–60

�, two distinct protein conformers were detected based on their

Fig. 3. H/D exchange results for T. reesei XYNII. Deuterium incorporation (A) of folded

(r) and partly unfolded (&) conformers and relative abundance (B) of the partly unfolded

conformer (&) as a function of incubation temperature. Data are plotted based on ESI MS

spectra of XYNII after 20-min incubations in D2O. Reproduced with permission from Janis

et al.,7 � Biochemical Society.

[15] oxidative resistance 167

different exchange rates. One represents the folded (M), whereas the otherrepresents the partly unfolded (M0) conformer. Average deuterium incor-porations of M and M0, as well as the relative abundance of the M0 as afunction of the incubation temperature, are presented in Fig. 3.

Conclusions

Because of commercial interests, the stabilization of mesophilic xyla-nases has been studied quite thoroughly by mutagenesis. The thermosta-bility of these xylanases has been improved by over 20

�. However, the

stabilizing mutations found thus far may not be applicable to all family 11xylanases. A few studies have also focused on the alkalitolerance and someprogress has been achieved in this area, but we do not know yet how far inalkaline pH the pH optimum of family 11 xylanases can be shifted. Thebasis for the stability of most thermostable family 11 xylanases is not fullyunderstood yet. There may also be a need to engineer the functionalproperties (e.g., pH optimum) of highly thermostable xylanases, but aproblem may appear that a well-optimized protein structure does not easilytolerate amino acid substitutions.

[15] Screening for Oxidative Resistance

By Joel R. Cherry and Michael H. Lamsa

Introduction

The oxidation of amino acid side chains in proteins has long beenrecognized as a primary pathway for functional inactivation. Whether aprotein drug targeted for intracellular action or an enzyme formulatedinto laundry detergent, activated oxygen species are generated that canmodify protein side chains, significantly altering their hydrophobicity,charge, and size. These modifications in turn render many proteins inactiveand more susceptible to aggregation or proteolysis. In addition to the well-known targets of oxidation that include methionine, cysteine, tryptophan,tyrosine, and histidine, other side chains, including phenylalanine,glutamate, leucine, valine, proline, lysine, and glycine, have also beenshown to be oxidized by a variety of mechanisms.1 While removal of alloxidizable amino acids from a protein is impractical, only a small fraction

1 R. T. Dean, S. Fu, R. Stocker, and M. J. Davies, Biochem. J. 324, 1 (1997).

Copyright 2004, Elsevier Inc.All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 388 0076-6879/04 $35.00

[methods in enzymology] protein engineering volume 388 || engineering the thermotolerance and ph...

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