An alkaline active xylanase: Insights into mechanisms of high pH catalytic adaptation

The alkaliphilic bacterium, Bacillus halodurans S7, produces an alkaline active xylanase (EC 3.2.1.8), which differs from many other xylanases in being operationally stable under alkaline conditions as well as at elevated temperature. Compared to non-alkaline active xylanases, this enzyme has a high percent composition of acidic amino acids which results in high ratio of negatively to positively charged residues. A positive correlation was observed between the charge ratio and the pH optima of xylanases. The recombinant xylanase was crystallized using a hanging drop diffusion method. The crystals belong to the space group P2₁2₁2₁ and the structure was determined at a resolution of 2.1 Å. The enzyme has the common eight-fold TIM-barrel structure of family 10 xylanases; however, unlike non-alkaline active xylanases, it has a highly negatively charged surface and a deeper active site cleft. Mutational analysis of non-conserved amino acids which are close to the acid/base residue has shown that Val169, Ile170 and Asp171 are important to hydrolyze xylan at high pH. Unlike the wild type xylanase which has optimum pH at 9–9.5, the triple mutant xylanase (V169A, I170F and D171N), which was constructed using sequence information of alkaline sensitive xylanses was optimally active around pH 7. Compared to non-alkaline active xylanases, the alkaline active xylanases have highly acidic surfaces and fewer solvent exposed alkali labile residues. Based on these results obtained from sequence, structural and mutational analysis, the possible mechanisms of high pH stability and catalysis are discussed. This will provide useful information to understand the mechanism of high pH adaptation and engineering of enzymes that can be operationally stable at high pH.     

In vitro mutagenesis of a xylanase from the extreme thermophile Caldocellum saccharolyticum

Summary Six mutant xylanases were obtained by in vitro mutagenesis of a xylanase gene from the extremely thermophilic bacterium Caldocellum saccharolyticum. The temperature stability of all enzymes was affected by mutation to various degrees and one of the xylanases had an altered temperature optimum. The mutations had no effect on the pH optimum. The C. saccharolyticum xylanase showed strong homology to several thermophilic and mesophilic xylanases, and comparison of primary sequences allowed the localization of probable active sites and residues involved in thermostability. Offprint requests to: P. L. Bergquist     

Specific characterization of substrate and inhibitor binding sites of a glycosyl hydrolase family 11 xylanase from Aspergillus niger

The importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase from Aspergillus niger was evaluated using site-directed mutagenesis. Ten mutant proteins were heterologously produced in Pichia pastoris, and their biochemical properties and kinetic parameters were determined. The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced. The low specific activities of Y6A and Y89A were entirely accounted for by a change in k(cat) and K(m), respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K(m) and decreased k(cat). Tyr(6), Tyr(10), Tyr(89), Tyr(164), and Trp(172) are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A. niger xylanase in complex with the modeled xylobiose. All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity. Only N117A lost its sensitivity to xylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism. The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase. The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration. A close inspection of the three-dimensional structure of A. niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases.     

Acidophilic adaptation of family 11 endo-beta-1,4-xylanases: modeling and mutational analysis

Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo-beta-1,4-xylanases. Its three-dimensional structure has been solved at 2.0 A and its optimum temperature and pH for enzymatic activity are 60 degrees C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and "alkaline" xylanases whereas the introduced Tyr 20 probably modifies the pKa of the acid-base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis.     

Directed evolution for engineering pH profile of endoglucanase III from Trichoderma reesei

The potential of cellulase has been revealed not only in biomass conversion but also in various industrial processes, including food, textiles, laundry, pulp, and paper. Due to the need for alkali-tolerant cellulase with high specific activity at alkaline pH, for example, for application in detergent industry an error-prone PCR approach was employed for enhancing the alkali-tolerant ability of endoglucanase III (EG III) from Trichoderma reesei by error-prone PCR. One mutant (N321T) which exhibited an optimal activity at pH 5.4, corresponded to a basic shift of 0.6 pH unit compared to the wild-type enzyme, was selected and characterized. In addition, two site-directed mutations, N321D and N321H, were designed to study the role of residue at position 321. As expected, the N321D mutation changed enzyme's optimal activity to pH 4.0, resulting in a large decrease in the specific activity. However, the N321H mutated enzyme was active over a broader pH range compared to the wild type, with no much change in the specific activity. These properties suggest that the residue at position 321 is important amino acid residue in determining the pH activity profile of the EG III from T. reesei.     

Structure-based mutagenesis of Penicillium griseofulvum xylanase using computational design

Penicillium griseofulvum xylanase (PgXynA) belongs to family 11 glycoside hydrolase. It exhibits unique amino acid features but its three-dimensional structure is not known. Based upon the X-ray structure of Penicillium funiculosum xylanase (PfXynC), we generated a three-dimensional model of PgXynA by homology modeling. The native structure of PgXynA displayed the overall beta-jelly roll folding common to family 11 xylanases with two large beta-pleated sheets and a single alpha-helix that form a structure resembling a partially closed right hand. Although many features of PgXynA were very similar to previously described enzymes from this family, crucial differences were observed in the loop forming the "thumb" and at the edge of the binding cleft. The robustness of the xylanase was challenged by extensive in silico-based mutagenesis analysis targeting mutations retaining stereochemical and energetical control of the protein folding. On the basis of structural alignments, modeled three-dimensional structure, in silico mutations and docking analysis, we targeted several positions for the replacement of amino acids by site-directed mutagenesis to change substrate and inhibitor specificity, alter pH profile and improve overall catalytic activity. We demonstrated the crucial role played by Ser44(PgXynA) and Ser129(PgXynA), two residues unique to PgXynA, in conferring distinct specificity to P. griseofulvum xylanase. We showed that the pH optimum of PgXynA could be shifted by -1 to +0.5 units by mutating Ser44(PgXynA) to Asp and Asn, respectively. The S44D and S44N mutants showed only slight alteration in K(m) and V(max) whereas a S44A mutant lost both pH-dependence profile and activity. We were able to produce PgXynA S129G mutants with acquired sensitivity to the Xylanase Inhibitor Protein, XIP-I. The replacement of Gln121(PgXynA), located at the start of the thumb, into an Arg residue resulted in an enzyme that possessed a higher catalytic activity.     

Structural basis of the properties of an industrially relevant thermophilic xylanase

A thermophilic xylanase from Bacillusstrain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60°C and relatively stable at high temperatures, with a temperature optimum of 75°C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of BacillusD3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or “sticky patches” between pairs of molecules. These “sticky patches” on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase. Proteins 29:77–86, 1997. © 1997 Wiley-Liss, Inc.     

High secretory production of an alkaliphilic actinomycete xylanase and functional roles of some important residues

Using native signal peptide, an alkaliphilic actinomycete xylanase XynK was overexpressed in Escherichia coli and secreted into the culture medium completely. At its optimum catalytic temperature of 55 °C, the cellulose-free xylanase exhibits high activity and stability at pH 7.0–11.0. In comparison with the well-studied actinomycete xylanase from Streptomyces lividans, as an alkaliphilic xylanase, XynK exhibited different biochemical and catalytic characteristics. With the aid of site-directed mutagenesis, some residues were demonstrated to be important to the activity, stability, or substrate binding of the enzyme. The pH stability of mutants H131S and W135A both decreased obviously under high pH values. Combined with their K _m parameters and homology model analysis, His131 was proposed to be important to both substrate binding and enzyme catalyzing, whereas Trp135 significantly influenced enzyme stability. Good stability under alkaline condition, as well as high secretory expression implies good potentials of the alkaline xylanase in various industrial applications. In addition, results from site-directed mutagenesis provide useful information for further pH stability mechanisms investigation.     

Improving the alkalophilic performances of the Xyl1 xylanase from Streptomyces sp. S38: structural comparison and mutational analysis

Endo-beta-1,4-xylanases of the family 11 glycosyl-hydrolases are catalytically active over a wide range of pH. Xyl1 from Streptomyces sp. S38 belongs to this family, and its optimum pH for enzymatic activity is 6. Xyn11 from Bacillus agaradhaerens and XylJ from Bacillus sp. 41M-1 share 85% sequence identity and have been described as highly alkalophilic enzymes. In an attempt to better understand the alkalophilic adaptation of xylanases, the three-dimensional structures of Xyn11 and Xyl1 were compared. This comparison highlighted an increased number of salt-bridges and the presence of more charged residues in the catalytic cleft as well as an eight-residue-longer loop in the alkalophilic xylanase Xyn11. Some of these charges were introduced in the structure of Xyl1 by site-directed mutagenesis with substitutions Y16D, S18E, G50R, N92D, A135Q, E139K, and Y186E. Furthermore, the eight additional loop residues of Xyn11 were introduced in the homologous loop of Xyl1. In addition, the coding sequence of the XylJ catalytic domain was synthesized by recursive PCR, expressed in a Streptomyces host, purified, and characterized together with the Xyl1 mutants. The Y186E substitution inactivated Xyl1, but the activity was restored when this mutation was combined with the G50R or S18E substitutions. Interestingly, the E139K mutation raised the optimum pH of Xyl1 from 6 to 7.5 but had no effect when combined with the N92D substitution. Modeling studies identified the possible formation of an interaction between the introduced lysine and the substrate, which could be eliminated by the formation of a putative salt-bridge in the N92D/E139K mutant.     

Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase

Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes. The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase. The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus. The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values. This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase. The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.     

脱乙酰壳聚糖固定化宇佐美曲霉木聚糖酶及固定化酶的性质和应用(英文)

研究壳聚糖吸附和戊二醛交联对木聚糖酶固定化条件 .将酶液加入到经醋酸溶液处理过的脱乙酰壳聚糖的pH 4 8的悬液中 ,加入浓度为 0 3%～ 0 4 %的戊二醛溶液 ,室温下 ,8h后得到固定化酶 .固定化酶的半失活温度比游离酶高 ,由 5 1℃升至 71℃ ,Km 值由游离酶的 1 2mg ml增加到1 5mg ml ,最适反应温度也由 5 5℃增加到 71℃ ,而最适反应pH由 4 6下降到 3 8.该固定化木聚糖酶可用于制造低聚木糖 .经过 10次连续应用实验后 ,该固定化酶的活力保持 81%     

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10128 micromol glucose x min(-1) x (mg protein)(-1) at pH 6.0. It was more thermostable at 60 degrees C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.     

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Improvement of enzyme function by engineering pH dependence of enzymatic activity is of importance for industrial application of Bacillus circulans xylanases. Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima. We selected non-conserved mutant sites within 8 Å from the catalytic residues, to see whether these residues have some role in modulating pK_as of the catalytic residues. We hypothesized that the non-conserved residues which may not have any role in enzyme catalysis might perturb pK_as of the catalytic residues. Change in pK_a of a titratable group due to change in electrostatic potential of a mutation was calculated and the change in pH optimum was predicted from the change in pK_a of the catalytic residues. Our strategy is proved to be useful in selection of promising mutants to shift the pH optimum of the xylanases towards desired side.     

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Improvement of enzyme function by engineering pH dependence of enzymatic activity is of importance for industrial application of Bacillus circulans xylanases. Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima. We selected non-conserved mutant sites within 8 Å from the catalytic residues, to see whether these residues have some role in modulating pK_as of the catalytic residues. We hypothesized that the non-conserved residues which may not have any role in enzyme catalysis might perturb pK_as of the catalytic residues. Change in pK_a of a titratable group due to change in electrostatic potential of a mutation was calculated and the change in pH optimum was predicted from the change in pK_a of the catalytic residues. Our strategy is proved to be useful in selection of promising mutants to shift the pH optimum of the xylanases towards desired side.     

Single mutations of residues outside the active center of the xylanase Xys1 Delta from Streptomyces halstedii JM8 affect its activity

Mutagenesis of the xylanase Xys1 of Streptomyces halstedii JM8 has been done by error prone PCR. Mutants with modified hydrolytic activity were isolated, the recombinant variant proteins purified and the catalytic activities of each one determined and compared with the wild type enzyme. Two of the isolated single point mutants, m1 (G133D) and m8 (N148D), showed 22-25% increase in specific activity towards xylan compared to wild type xylanase. Two other mutants, m5a (D175A) and m7 (T160A), showed a significant reduction in specific activity of 40-50% with respect to the wild type enzyme. These residues are mainly located in the beta alpha-loops of the xylanase, the region showing the main structural divergences within family 10 of xylanases. This study shows the usefulness of random mutagenesis to point out some key residues not directly involved in the active center, but in which mutation produces subtle structural rearrangements affecting the enzymatic function.     

Xylanase production by a new alkali-tolerant isolate of Bacillus

The xylanolytic system of an alkali-tolerant Bacillus sp. consists of several xylanases ranging from 22 to 120 kDa and pI values from 7.0 to 9.0. Crude xylanase retained 72% of initial activity after 5 h at pH 9.0 and 45°C. Xylanase production was induced by xylose and xylan and was maximum at 42°C and pH 7.8. Crude xylanase released xylotriose and xylotetraose as main products of xylan hydrolysis. Xylose was not detected. © Rapid Science Ltd. 1998     

中子与X-光衍射相结合解析木聚糖酶突变体N44D的晶体结构

木聚糖酶（Xylanase）是降解木质纤维素中半纤维素的特定酶,在酶法生产生物能源的过程中有重要应用. 木质纤维素在降解时需要用酸或碱预处理,而木聚糖酶反应的最适pH值为中性. 因此,获得在酸或碱性条件下酶活力仍然很高的木聚糖酶,是生物能源生产中的重要课题. 木聚糖酶活性中心的天冬酰胺突变为天冬氨酸（N44D）后,木聚糖酶的最适pH值从5.7下调到4.6,酶活提高20%. 本课题首次获得了分辨率为2.20A的木聚糖酶突变体N44D的晶体在291 K的中子衍射数据. 同时,本课题分别获得了分辨率为1.70A和1.07A的在291 K和100 K的X-光衍射晶体数据. 通过解析以上数据,本实验获得了木聚糖酶中几乎所有原子的空间位置. 以上结果将有助于在原子水平研究这种突变是如何影响酶反应最适pH值,并进一步为酶蛋白的改性提供了结构生物学的依据.     

Shifting the optimum pH of Bacillus circulans xylanase towards acidic side by introducing arginine

Electrostatic interactions are important in protein folding, binding, flexibility, stability and function. The pH at which the enzyme is maximally active is determined by the pK_as of the active site residues, which are modulated by several factors including the change in electrostatics in its vicinity. As the acidic xylanases are important in food and animal feed industries, electrostatic interactions are introduced in Bacillus circulans xylanase to shift their pH optima towards the acidic side. Arg substitutions are made to modulate the pKas of the active site residues. Neutral residues are substituted by Arg in such a way that the substituted residue can make direct interaction with the catalytic residues. However, the mutations with other titratable residues (Asp, Arg, Lys, His, Tyr, and Ser) present in between the catalytic sites and the substituted sites are avoided. Site directed mutagenesis was conducted to confirm the strategy. The results show the shift in pH optima of the mutants towards the acidic side by 0.5–1.5 unit. Molecular dynamics simulation of the mutant V37R reveals that the decrease in activity is due to the increase in distance between the substrate oxygen atoms and catalytic glutamates.     

Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase

The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pKa 4.6) and the acid/base catalyst Glu172 (pKa 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an approximately 20% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pKa values of 3.5 and 5.8. Based on 13C-NMR titrations, the predominant pKa values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pKa, is protonated, while Glu78, with the higher pKa, is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only approximately 1% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 A) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis.     

A single domain thermophilic xylanase can bind insoluble xylan: evidence for surface aromatic clusters.

A clone expressing xylanase activity in Escherichia coli has been selected from a genomic plasmid library of the thermophilic Bacillus strain D3. Subcloning from the 9-kb insert located the xylanase activity to a 2.7-kb HindII/BamHI fragment. The DNA sequence of this clone revealed an ORF of 367 codons encoding a single domain type-F or family 10 enzyme, which was designated as XynA. Purification of the enzyme following over-expression in E. coli produced an enzyme of 42 kDa with a temperature optimum of 75 degrees C which can efficiently bind and hydrolyse insoluble xylan. The pH optimum of the enzyme is 6.5, but it is active over a broad pH range. A homology model of the xylanase has been constructed which reveals a series of surface aromatic residues which form hydrophobic clusters. This unusual structural feature is strikingly similar to the situation observed in the structure determined for the type-G xylanase from the Bacillus D3 strain and may constitute a common evolutionary mechanism imposed on different structural frameworks by which these xylanases may bind potential substrates and exhibit thermostability.