Laboratory of Bioprocess Engineering, Helsinki University of Technology, PO Box 6100, 02015-HUT, Finland
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Abstract |
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Keywords: endo-1,4-ß-xylanase/family 11/pH-dependent activity/protein surface arginines/thermostability
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Introduction |
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Endo-1,4-ß-xylanases hydrolyze ß-1,4-linked xylopyranose chains found in xylan, one of the most abundant polysaccharide fibers in nature. Xylanases are used for biomass modification in pulp bleaching, baking and manufacture of animal feed (Viikari et al., 1994; Prade, 1996
). In family 11 xylanases (EC 3.2.1.8.; previously family G), two large ß-pleated sheets and one
-helix form a structure that resembles a partly-closed right hand (Törrönen and Rouvinen, 1997
). The stability of family 11 xylanases has been improved by several mutations at different positions in the protein structure (Arase et al., 1993
; Wakarchuk et al., 1994
; Sung et al., 1998
; Georis et al., 2000
; Sung and Tolan, 2000
; Turunenet al., 2001
). In this study, we used Trichoderma reesei xylanase II (XYNII) (Tenkanen et al., 1992
), which shows optimal enzyme activity at pH 56, as a model to study how a systematic increase of arginines on the protein surface affects the pH optimum, thermostability and enzyme activity at high temperature.
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Materials and methods |
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We used two clones of T.reesei xylanase II (XYNII). The first was the pALK143 expression construct (ROAL, Rajamäki, Finland; Turunen et al., 2001), in which XYNII was expressed under the Bacillus amyloliquefaciens -amylase promoter. Eleven amino acids of the T.reesei XYNII prosequence were present in this construct (Saarelainen et al., 1993
). The xylanase expressed from pALK143 was secreted into the medium under the control of the
-amylase signal sequence. Secondly, XYNII was expressed under the pKKtac vector (VTT, Espoo, Finland), in which protein secretion is governed by the pectate lyase (pelB) signal sequence (induction by 1 mM IPTG). The wild-type XYNII was expressed in higher quantities from pKKtac than from pALK143. The R series mutations were introduced to XYNII via the pKKtac vector and the ST series mutations via pALK143.
Generation of XYNII mutants
In planning the mutations, Swiss-PdbViewer (http://www.expasy.ch/spdbv/) (Guex and Peitsch, 1997) was used as a tool to examine the XYNII structure (1xyp). The mutations were generated by polymerase chain reaction (PCR), in which the mutations were introduced into the oligonucleotide primers as described elsewhere (Turunen et al., 2001
). The mutant clones were grown on agar plates containing xylan (birchwood xylan; Sigma, Steinheim, Germany) coupled to Remazol-brilliant blue, in which the xylanase activity is indicated by white halos around the positive colonies (Biely et al., 1985
).
Enzyme assay
Xylanase activity was determined by measuring the amount of reducing sugars liberated from 1% birchwood xylan (Bailey et al., 1992). The activity determination in standard conditions was carried out at pH 5 and 50°C, with a reaction time of 10 min. Citrate-phosphate buffer (50 mM) was used in the xylanase assays at pH 3.07.5. Properties of the mutant xylanases were tested using the Escherichia coli culture broth as the source of xylanases. When the xylanase activity was very low in the E.coli culture broth, the enzyme was concentrated by 65% ammonium sulfate precipitation followed by dialysis against 2550 mM citrate-phosphate buffer, pH 6. Inactivated E.coli culture broth or citrate-phosphate buffer was used to dilute the enzyme for activity and stability measurements. Bovine serum albumin (0.1 mg/ml) was included in the time-dependent hydrolysis experiments (Figure 5
).
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Results |
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The sites on the protein surface to be replaced by arginines were mainly selected on the basis that the site in question is occupied by a charged amino acid (often arginine) in some members of the family 11 xylanases. Two mutation series were performed: the R series and the ST series (Table I). The mutations in the R series were located on different sides of the protein, whereas the mutations in the ST series were located only on the Ser/Thr surface (Figure 1
). The Ser/Thr surface of the wild-type XYNII does not contain any charged residues. The wild-type XYNII contains altogether six arginines and four lysines.
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The engineered arginines in the R series did not increase the thermostability, except for the mutation K58R (mutant R1), which increased the half-life of XYNII at 55°C (pH 6) from ~5 min to 1020 min. R1 was more stable than wild-type XYNII and the other mutants over a wide pH range (pH 59) (results not shown). A surprising finding was that the other R series mutants (R2R6) generally had a similar stability than the wild-type XYNII (results not shown). Introduction of several arginines on different sides of XYNII made the pH-dependent activity profile narrower (Figure 2). The R series mutants had essentially the same temperature optimum (maximum 12°C elevation in R4R6) than the wild-type XYNII (not shown).
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When the arginines were introduced into the Ser/Thr surface (Figure 1), the effect was much different from that in the R series mutants. The thermostability in the absence of substrate decreased with an increasing number of arginines (Table II
). When four to five arginines were introduced into the Ser/Thr surface, the half-life decreased from ~60 to ~715 min at 50°C, whereas one or two arginines on the Ser/Thr surface had little effect on the stability.
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The half-lives in the presence of the substrate could be determined from the time-dependent hydrolysis curves (Figure 5). The results (Table II
) showed that the stability of the five-arginine mutants (ST5 and ST6) at 60 and 65°C was four to five times higher than that of the wild-type XYNII.
The Arrhenius activation energy was determined from temperature-dependent activity curves (temperature range 4055°C) (Doran, 2000). The activation energy clearly increased when five arginines were introduced into the Ser/Thr surface (Table III
). The level of increase was ~40%. The four to six arginines in the R series increased the Arrhenius activation energy only by 1020% (not shown). The higher Arrhenius activation energy indicates that the catalytic activity was decreased at these temperatures in the mutant enzymes compared to the wild-type XYNII. The temperature-dependent activity profiles show that elevation of the temperature increased the activity of ST mutants more than that of the wild-type XYNII (Figure 4
).
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Discussion |
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In addition to their effects on protein stability, electrostatic interactions also have a role in the adaptation of proteins to functioning at low or high pH. Some acidic xylanases (PDB codes: 1BK1, 1ukr), which function optimally at low pH have an increased number of acidic residues and tend to avoid basic residues on the Ser/Thr surface (Krengel and Dijkstra, 1996; Fushinobu et al., 1998
). There is a similar correlation between the number of acidic residues and low pH optimum in pepsin, which also functions in the acidic pH range (Sielecki et al., 1990
). Sequence comparisons of bacterial serine proteases revealed that adaptation to highly alkaline conditions involves a decrease in the number of acidic amino acids and lysine and an increase in arginine and neutral hydrophilic amino acids (Masui et al., 1994
; Shirai et al., 1997
). In particular, the Arg/Lys ratio increased with increasing activity at alkaline pH (Masui et al., 1994
). In a subtilisin-family of serine proteases, the arginine residues were localized to a hemisphere of the protein structure (Shirai et al., 1997
).
The family 11 xylanases can be active at acidic or alkaline pH even without charged amino acids on the Ser/Thr surface. The acidic T.reesei xylanase I (PDB code: 1xyn) and the xylanase (1QH7) from a strictly alkaliphilic B.agaradhaerens do not contain any charged amino acids on the Ser/Thr surface (Törrönen and Rouvinen, 1995; Sabini et al., 1999
). However, the catalytic core of B.agaradhaerens xylanase contains many ion pairs, including two arginines (altogether eight) and five lysines (altogether nine) more than that of T.reesei XYNII, which could be involved in the functioning at high alkaline pH.
In our study, we tested how a systematic increase in the amount of arginines on the protein surface affects the stability and functional properties of T.reesei xylanase II (XYNII). The T.reesei XYNII has low thermostability and is inactivated rapidly above 50°C, a process that involves conformational changes (Jänis et al., 2001; Turunen et al., 2001
). In the R series mutants, six arginines were introduced into different sides of XYNII, and in the ST series five arginines were introduced into the Ser/Thr surface, which forms a major part of the outer surface of the double-layered ß-sheet and contains a large number of serines and threonines (Figure 1
). The active site is located in a large canyon on the inner side of the partly closed structure formed of the double-layered ß-sheet. Two active site glutamates are responsible for the catalytic activity, one functioning as a nucleophile and the other as an acid/base catalyst (for review see Törrönen and Rouvinen, 1997
).
The introduction of arginines into different sides of the protein surface in the R series did not have any significant effect on the thermostability. The major effect was that the optimal region of the pH-dependent activity became narrower than in the wild-type XYNII. The introduction of arginines into the Ser/Thr surface resulted in a shift of the pH profile to alkaline pH by 0.51.0 pH units. A clear shift in the pH-dependent activity was observed when four or five arginines were introduced into the Ser/Thr surface. These results indicate that the Ser/Thr surface of family 11 xylanases has a role in determining the functional properties of the catalytic site. However, an increase in the number of arginines is not in itself sufficient to cause an acidic enzyme to function in highly alkaline conditions.
The effect of the Ser/Thr surface arginines on thermostability was complex. These arginines decreased the thermostability in the absence of the substrate. However, in the presence of the substrate the 5-fold arginine mutations on the Ser/Thr surface considerably increased the enzyme activity at elevated temperature. The hydrolysis experiments performed as a function of time showed that the presence of five arginines on the Ser/Thr surface increased the thermostability. The half-life in the presence of substrate was increased four to five times after the introduction of five arginines. Thus, when the destabilizing effect of the mutations was neutralized by the substrate, a clear stabilizing effect of the Ser/Thr arginines was revealed. The increased apparent temperature optimum appears to be mainly a result of the increased thermostability.
The location of arginines on the Ser/Thr surface appeared not to be critical, since the same effect was seen when the fifth arginine was located at two different sites. The dramatic effect of the fifth arginine indicates that the formed positive net charge on the Ser/Thr surface could be mainly responsible for the effects on enzyme properties. The engineering of arginines into the Ser/Thr surface did not create any ion pairs. In B.agaradhaerens xylanase (1QH7), the number of basic residues is high in other regions of the protein, indicating that the Ser/Thr surface is not the only region suitable for the introduction of arginines. In conclusion, the straightforward increasing of arginines on different sides of the protein surface may not alter enzyme properties in a desired way. It is evident from our study that local introduction of arginines on a specific protein surface can be a more successful approach.
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Notes |
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Acknowledgments |
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References |
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Received March 30, 2001; revised October 10, 2001; accepted November 1, 2001.