A Novel Family 8 Xylanase : Functional and Physico-chemical Characterization

Xylanases are generally classified into glycosyl hydrolase families 10 and 11 and are found to frequently have an inverse relationship between their pI and molecular mass values. However, we have isolated a psychrophilic xylanase that belongs to family 8 and which has both a high pI and high molecular mass. This novel xylanase, isolated from the Antarctic bacteriumPseudoalteromonas haloplanktis, is not homologous to family 10 or 11 enzymes but has 20–30% identity with family 8 members. NMR analysis shows that this enzyme hydrolyzes with inversion of anomeric configuration, in contrast to other known xylanases which are retaining. No cellulase, chitosanase or lichenase activity was detected. It appears to be functionally similar to family 11 xylanases. It hydrolyzes xylan to principally xylotriose and xylotetraose and is most active on long chain xylo-oligosaccharides. Kinetic studies indicate that it has a large substrate binding cleft, containing at least six xylose-binding subsites. Typical psychrophilic characteristics of a high catalytic activity at low temperatures and low thermal stability are observed. An evolutionary tree of family 8 enzymes revealed the presence of six distinct clusters. Indeed classification in family 8 would suggest an (α/α)6fold, distinct from that of other currently known xylanases.


Summary
Xylanases are generally classified into glycosyl hydrolase families 10 and 11 and are found to frequently have an inverse relationship between their pI and molecular mass values. We however have isolated a psychrophilic xylanase which belongs to family 8 and which has both a high pI and high molecular mass. This novel xylanase, isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis, is not homologous to family 10 or 11 enzymes but has 20 -30% identity with family 8 members. NMR analysis shows that this enzyme hydrolyzes with inversion of anomeric configuration, in contrast to other known xylanases which are retaining. No cellulase, chitosanase or lichenase activity was detected. It appears to be functionally similar to family 11 xylanases. It hydrolyzes xylan to principally xylotriose and xylotetraose and is most active on long chain xylo-oligosaccharides. Kinetic studies indicate that it has a large substrate binding cleft, containing at least six xylose binding subsites. Typical psychrophilic characteristics of a high catalytic activity at low temperatures and low thermal stability are observed. An Depending on the source, the xylan backbone may contain a varying degree of glucuronosyl, 4-O-methyl-D-glucuronopyranosyl, α-L-arabinofuranosyl, acetyl, feruloyl and/or p-coumaroyl substituents. Unsubstituted linear xylans also exist and have been isolated from esparto grass and seaweed (2).
The wide diversity of xylan structures is paralleled by a large variety of xylanases with widely different hydrolytic activities, physicochemical properties and structures. In an attempt to contend with this diversity of enzymes Henrissat (3) introduced a classification system for glycoside hydrolases based on sequence homologies and designed to integrate both structural and mechanistic features. Currently 87 families have been identified and members of each family are believed to have evolved from a common ancestral sequence. Divergent evolution to acquire new substrate specificity has resulted in more than one-third of the families being polyspecific (4) and in different families having a related fold. As the structures of proteins are better conserved than their sequences, the grouping of several families in 'clans' has thus been introduced (5). In contrast, convergent evolution has resulted in enzymes with identical substrate and reaction specificity being found in totally unrelated families with disparate three-dimensional folds (6).
Endo-β-1,4-D-xylanases have been assigned to two distinct families, 10 (formerly F) and 11 (formerly G) (7)(8)(9). Indeed, this separation into two families is in agreement with an earlier classification by Wong et al. (10) who indicated that xylanases generally either have a high molecular mass (> 30Kda) and low pI or a low molecular mass (<30Kda) and high pI. It has been found that family 10 generally groups acidic high molecular mass enzymes while family 11 members are generally much smaller basic proteins, although acidic pI's have been observed for some enzymes of fungal origin (11). A significant difference in the structure and catalytic properties of the two families also occurs.
Family 10 members present an (α/β) 8 barrel fold (12), belong to clan GH-A, the 4/7 superfamily, (13) and have approximately 40% of the secondary structure in an α-helical form (14). Members of family 11 are approximately 3-5% α helical in nature, have a β-jelly roll fold conformation (15) and belong to clan GH-C. In relation to their catalytic properties, it has been found that family 10 xylanases typically have smaller substrate binding sites, lower substrate specificities (frequently having endoglucanase activity) and hydrolyze heteroxylans to a higher degree (16) as compared to family 11 xylanases (true xylanases). The common feature of the two families is in the mode of action. All xylanases characterized to date retain the anomeric configuration of the glycosidic oxygen following hydrolysis in which two conserved glutamates function as the catalytic nucleophile and acid/base catalyst (17).
In the present study we have isolated a novel cold-active xylanase which belongs to glycoside hydrolase family 8 (formerly D), a family which is mainly comprised of endoglucanases (EC 3.2.1.4), but also lichenases (EC 3.2.1.73) and chitosanases (EC 3.2.1.132) and which typically operate with inversion of anomeric configuration. The cloning, overexpression, purification and characterization of this novel xylanase as well as the evolutionary relationships within family 8 members are described. Catalytic properties including the specificity, action pattern and mode of action of this enzyme are also assessed. void was collected and immediately loaded on a S-Sepharose Fast Flow (Pharmacia) column (7 x 2.5cm), also equilibrated in the above mentioned buffer, and eluted with a linear NaCl gradient (0-100mM in 350ml).

Cloning of the Cold-Adapted Xylanase Gene.
The psychrophilic xylanase gene was isolated and sequenced using a procedure similar to that described (20), (21). Degenerate primers based on the amino-terminal sequence (5'-GCIT TYAAYAAYAAYCC-3') and an internal peptide sequence determined after CNBr cleavage (see

Overexpression of the Cold-Adapted Xylanase.
The xylanase gene, including its signal sequence, was PCR amplified using Pwo polymerase

Physico-chemical Characterization.
The 20,585Da mesophilic Family 11 xylanase (Xyl1) from Streptomyces sp. S38 was obtained from J. Georis (22). The temperature optimum was determined by monitoring activity over 5 minutes at pH 6.
The stereoselectivity of hydrolysis was analyzed essentially as described (25), 0.8% Nothogenia erinacea xylan, isolated as described in (26) and 43.5µM of the enzyme, lyophilized and redissolved twice in D 2 O were used. Proton NMR spectra were recorded with a Bruker Digital NMR Avance 500 spectrometer at 25°C using a 5mm sample tube. Spectra were recorded immediately after mixing and at 15 minute intervals for 1 hour.
Activity on birchwood xylan, beechwood xylan, oat spelt xylan, CM-cellulose (Fluka), microcrystalline cellulose (Aldrich), cellobiose, arabinogalactan (from larchwood), lichenane (from Cetraria Islandica), laminarin (from Laminaria digitata) and starch (from potato) was determined by the DNS method at 25°C with a final substrate concentration of 3%. Due to interaction of the chitosan with the citrate-acetate buffer and insolubility at pH 6.5, the assay for chitosanase activity was carried out in potassium hydrogen phthalate buffer at pH 6.0. Activity was measured at 25°C by monitoring the decrease in viscosity over 30 hours with a Brookfield LVDV viscometer. Chitoclear TM low viscosity (3% acetylation) and FC222B (17% acetylation) chitosan (Primex Ingredients) were examined, appropriate controls were included in all cases.
xylopyranoside and pNp-α-D-xylopyranoside was examined at 25°C at a final concentration of 5mM.
To determine the extent of hydrolysis of birchwood, oat spelt and Palmaria palmata xylan, 25nM enzyme was incubated at 25°C, pH 6.5 with a 3% substrate solution containing 0.01% sodium azide (Merck), samples were removed at various time points, boiled for 4 minutes, filtered through a 0.45µm Millex membrane (Millipore) and analyzed for reducing sugars by the DNS method.
Products of enzymatic hydrolysis were analyzed using a high performance anion exchange Carboxy-terminal sequencing was performed on a Procise 494C Sequencer (PE Biosystems) with 1nmol enzyme as described (31). The molecular mass was determined by nanoelectrospray ionization spectrometry on a Q-TOF mass spectrometer (Micromass) with 20pmol of enzyme.

Construction of Evolutionary Tree.
Family 8 members were identified from the carbohydrate-active enzymes server (http://afmb.cnrs-mrs.fr/CAZY/) and by a protein database (SWALL) fasta33-t search (32) with the isolated psychrophilic xylanase sequence. Surplus or additional regions and domains were removed, leaving essentially the catalytic core, and the tree was constructed with the Drawtree program (33) by application of the neighbor-joining method (34) to multiple alignments from the CLUSTAL W program (35).

Identification and Growth Characteristics of Isolated Strain.
The xylanase producing strain was identified as the Gram negative bacterium Pseudoalteromonas haloplanktis with a Microbial Identification Score of 0.391 and no alternative being proposed. Figure 1 shows that this bacterium has typical cold-adapted characteristics as xylanase production and stability are much higher at 4°C than at 28°C. Furthermore, the optical density at 28°C drops rapidly to zero, indicating cell lysis at this temperature.
Optimal growth occurs in marine broth containing high concentrations of marine salts (20g/l) while optimal xylanase production was obtained with high concentrations (1.5%) of xylan, birchwood being more suitable than oat spelt due to foaming induced by the less soluble oat spelt xylan at high concentrations. Low quantities of xylanase activity were detected in the absence of xylan in the production medium indicating that it is produced constitutively by the isolate. This organism is however unable to utilize xylan as a carbon source, as witnessed from its inability to grow on xylan supplemented minimal media.

Purification of Wild-Type Xylanase.
A simple two step purification protocol taking advantage of the high pI (pH 9.5) of the extracellular enzyme resulted in an 11 fold purification with a final yield of 55%, equivalent to 1.9 mg of pure xylanase per litre of culture. Only one xylanase was detected for this organism. Low concentrations of salts (10mM NaCl) were used throughout the purification procedure so as to prevent interaction between the enzyme and the xylan used for production. SDS-PAGE and mass spectrometry indicated that the enzyme was >98% pure with a molecular mass of 45,982 Da.

Cloning and Sequence Analysis of Recombinant Xylanase.
PCR screening allowed isolation of one clone containing a fragment of approximately 11Kb. An open reading frame of 1278bp encoding a protein of 426 amino acids was identified (Table I)

Overexpression and Purification of Recombinant Xylanase.
Cloning of the xylanase gene, including its proper signal sequence, in a pET22b(+) vector in E.coli and cultivation in Terrific broth resulted in production of approximately 85mg of the enzyme per liter. The protein was purified with a procedure similar to that used for the wild-type enzyme, however, due to the absence of added xylan, no salt was required. An additional gel filtration step was also included, resulting in a final yield of 58mg/l equivalent to a recovery of 68%.

Physicochemical Characterization.
The enzyme has both a high molecular weight and a high pI (Table I). It has a wide pH activity range with maximum activity occurring between pH 5.3 and 8. While acknowledging the limitations of the test utilized for determination of the kinetic parameters, it can be seen that the apparent K m and k cat of the cold xylanase at 25°C are relatively high (2). Heavy metals such as Hg 2+ , Cu 2+ , Zn 2+ and Ni 2+ were found to be inhibitory to activity, whereas Mg 2+ , Ca 2+ , Na + , K + , PO 4 3and Clas well as chelating agents (EDTA, EGTA) had no effect.
The thermostability and thermodependence of activity of the isolated xylanase are compared to that of a mesophilic xylanase in figures 3 and 4. The cold enzyme shows a shift in apparent optimal activity of approximately 25°C towards low temperatures. Activity at 5°C is 60% of the maximum, compared to the mesophilic xylanase where activity at this temperature is less than 5% of the maximum. It can also be seen that the isolated xylanase is much less stable than the mesophilic xylanase with a 10°C lower melting temperature (52.6°C versus 63.1°C) and a 12 times shorter half life at 55°C (1.9 versus 23 minutes). glucose chains. Of note is the inability of this enzyme to cleave aryl-β-glycosides of X 1 , X 2 or X 3 , under the conditions used, in agreement with the inability of the enzyme to hydrolyze X 2 or X 3 (Table II). Indeed activity on X 4 is also negligible, with minute quantities of X 3 + X 1 being produced only on prolonged incubation (24hours) with high enzyme and substrate concentrations (0.5µM and 10mM, respectively). The activity of the enzyme on X 5 is extremely low, giving principally X 3 + X 2 , minute quantities of X 4 + X 1 are also formed but formation is too low to allow accurate determination of its kinetic parameters and can be taken as negligible. The catalytic efficiency on X 6 is approximately 46 fold higher than on X 5 , with X 3 being preferentially formed as well as low amounts of X 4 + X 2 . At no point were xylo-oligomer products larger than the original substrates detected, demonstrating the absence of transglycosylation reactions and in good agreement with the NMR analysis which showed that this enzyme hydrolyzes with inversion of the anomeric configuration (results not shown). In this study, on addition of enzyme to substrate a rapid increase and subsequent decrease of a doublet at 5.18 ppm, assigned to the α configuration of the anomeric proton, is observed. The resonance of the β configuration at 4.56 ppm slowly increases due to mutarotation of the initially formed α-anomeric proton until equilibration of the α and β forms is eventually reached.

Functional Characterization
In relation to the xylan substrates, it can be seen that Palmaria palmata xylan is the most efficiently (figure 5) and extensively (figure 6) hydrolyzed while birchwood xylan is more efficiently but less extensively hydrolyzed than oat spelt xylan. Analysis of the products of hydrolysis ( figure 7) indicates that for all substrates tested X 3 , X 4 , X 5 , X 6 and higher xylodextrins are initially formed. As the hydrolysis progresses, X 4 and in particular X 3 accumulate while X 5 and X 6 are slowly degraded, eventually, after prolonged digestion (up to 21 days in some cases), giving rise to large quantities of X 3 and X 4 plus low quantities of X 1 , X 2 and X 5 . In all cases, varying quantities of mixed linkage or substituted compounds were also detected, these depending on the structure of the substrate used. Due to lack of suitable standards, all compounds could not be identified. Acidic compounds produced from birchwood and oat spelt xylan are probably aldouronic acids with arabinose substituted xylo-oligomers of varying lengths also being liberated from oat spelt xylan. In the case of Palmaria palmata xylan, a mixed linkage IsoX 4 and most probably IsoX 5 and IsoX 6 are produced.

Discussion
The isolated Pseudoalteromonas haloplanktis strain displays characteristics typical for a cold adapted micro-organism. Optimal biomass production occurs at 4°C, in accord with the description of a psychrophile as any organism capable of growing close to 0°C (39). At temperatures higher than that of the natural environment higher growth rates do occur but cell development, as measured by cell biomass, and enzyme production and secretion, as measured by xylanase levels in the culture supernatant, are markedly reduced. This is in agreement with previous studies (40) which suggested that this may be due to alterations in the secretory pathway, membrane fluidity and/or protein synthesis mechanisms. When compared to the mesophilic xylanase from Streptomyces sp. S38 the enzyme displays a lower apparent optimum temperature, a lower thermal stability of activity as well as a lower conformational stability. Such an influence of temperature points to the enzymes adaptation to its cold habitat. Indeed it has been proposed that psychrophilic enzymes have a high flexibility which results in improved activity at low temperatures and concomitantly a decreased stability (41). The enzyme has a drastically improved turnover rate at low temperatures (0 -40°C), which is probably its main adaptation strategy. The apparent K m of the enzyme on soluble birchwood xylan is relatively high, as typically K m values of between 0.5 -5 mg/ml are found (2) and suggests that this parameter is not optimized for the cold adapted xylanase. It should be noted however that high K m values for xylanases from Acrophiulophora nainana (42), Trichoderma reesei (43) and Streptomyces T7 (44) have been reported. Relatively high k cat and K m values have also been found for other extracellular cold-adapted enzymes where optimization of k cat was the only relevant parameter and indeed is to be expected for enzymes that normally operate in high concentrations of substrate e.g. digestive enzymes or enzymes from organisms growing on organic debris (39).
The production of only one xylanase by this organism, its inability to utilize xylan as a source of carbon, as well as the digestion products released by the xylanase would suggest that this enzyme does not partake in production of sugars for cellular metabolism by this micro-organism.
In the Antarctic environment, sources of xylan and of other polysaccharides are extremely limited, with the main source being the cell walls of green and red algae belonging to microphytobenthos (45). It is probable that the xylanase is used to loosen the cell wall structure of algae, thereby allowing better access to the cellulose in the cell walls as well as to the storage polysaccharides.
The xylanase of this study is unique, it has a high molecular weight, in common with most family 10 xylanases yet it also has a high pI, typical for family 11 enzymes. The deduced amino-acid sequence has no isology with currently identified xylanases of families 10 or 11, however it does have low similarity with endoglucanases, lichenases and chitosanases of family 8. As the fold of a particular family is generally conserved among its members this indicates that the coldadapted xylanase has an (α/α) 6 like fold (46), widely different to that of all other known xylanases. In addition, xylanases typically catalyze hydrolysis via a double displacement mechanism in which the anomeric configuration is retained while family 8 enzymes are believed to catalyze hydrolysis with inversion of configuration. Endoglucanase C from Clostridium cellulolyticum has been shown to invert the anomeric configuration (47) and in conjunction with the opinion that members of a given family have the same stereoselectivity (25) we have shown that the cold adapted xylanase also hydrolyzes with inversion of configuration.
Database searches have identified two further xylanases with homology to the investigated xylanase and which can also be included as members of family 8. This shows that the cold xylanase is not a unique case and supports the suggestion that the current view, in which xylanases are restricted to families 10 and 11, be revised to include family 8. In addition, database searches also indicate that xylanases have been reported which can be classified into families 5 and 43, however apart from xylanase A from Erwinia chrysanthemi (8), information, in particular on functional characteristics, are minimal.
Although the enzyme shares isology with endoglucanases, lichenases and chitosanases, it is a true xylanase. Highest activity was found on xylan from Palmaria palmata, a linear mixed linkage (β1,3) (β1,4) seaweed xylan and indeed this might be expected as the main source of xylan in the natural environment of this enzyme is probably of algal origin. Our study also shows that the degree of substitution of the substrate influences the activity of the enzyme, with birchwood xylan being the least extensively hydrolyzed due to its high degree of substitution, 15 -30% (48,49), and thus greater steric hindrance. Analysis of the products released from these substrates shows that the cold enzyme generates products similar to those from family 11 enzymes but larger than those from family 10 enzymes. In addition, the cold enzyme liberates IsoX 4 as the shortest mixed linkage fragment from Palmaria palmata xylan, in agreement with that shown for family 11 xylanases (16), and on the basis of chromatographic mobility this is suggested to be Xylβ1,3-Xylβ1,4-Xylβ1,4-Xyl. This indicates that the cold-adapted xylanase can attack the β1,4 linkage that precedes (non-reducing end) a β1,3 linkage but can only cleave the β1,4 linkage two xylosyl residues distal from the reducing end of a β1,3 bond.
The activity on xylo-oligosaccharides was also investigated and it can be seen that here again the enzyme seems to be functionally similar to family 11 xylanases. Based on the catalytic efficiency of X 3 production, it appears that X 6 is the smallest chain length broken rapidly by the enzyme. It is more active on higher xylo-oligosaccharides and appears to have a substratebinding site of at least six subsites, with the catalytic site in the middle. In contrast to all tested xylanases (16) but in consonance with the family 8 endoglucanase from Clostridium thermocellum (50) the cold-adapted enzyme was not active on aryl-β-glycosides of X 2 or X 3 .
This, in conjunction with its inactivity on X 2 and X 3 as well as its slow cleavage of X 4 and X 5 suggests that, analogous to the family 8 endoglucanase from Clostridium thermocellum (38), the substrate residue at subsite -1 adapts a distorted boat conformation with the energy for this distortion being obtained from that released on substrate binding. It is possible that high subsite occupancy (at least 5 subsites) is required to provide sufficient energy for the substrate to adapt the distorted conformation. However, the fact that principally only X 3 is produced from X 6 indicates that it is also possible that like family 10 and 11 xylanases (51) the subsites adjacent to the catalytic site (i.e. -1 and +1) have a negative affinity for monomer units but unlike other xylanases the -2 or +2 subsites, or perhaps even both subsites, may also have a negative or weak affinity.
The cold-adapted enzyme is thus unique among xylanases, in particular at the level of its primary and probably also tertiary structures, it exhibits similarities in its catalytic function to family 11 enzymes while retaining individuality in its stereoselectivity and probably also in its specificity site structure. Further studies, in particular of its three dimensional structure, should give further information on the structurefunction relationship of this novel enzyme. 2.4515.00).
The nucleotide sequence reported in this paper has been submitted to the EMBL Nucleotide Sequence Database with accession number AJ427921.     Baseline subtracted DSC data have been normalized for protein concentration.