Transformations of testosterone and related steroids by Botrytis cinerea

Botrytis cinerea (strain AM235) was used to investigate the transformations of testosterone and related steroids. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. Botrytis cinerea converts the examined substrates mainly to 7 alpha-hydroxy derivatives. 1-Dehydrotestosterone was also significantly hydroxylated at a 14 alpha-position.     

Effects of water extracts of a composted manure-straw mixture on the plant pathogen Botrytis cinerea

Manure-straw mixtures were composted and water extracts, made by incubating compost in water for 3 to 18 days, were assessed for antagonistic activity against Botrytis cinerea, using a range of tests. Extracts of all ages inhibited conidial germination on glass slides and reduced mycelial growth on agar. Mixing extracts of all ages with droplets of suspensions of B. cinerea conidia on detached Phaseolus bean leaves suppressed lesion development, but only 3- to 8-day-old extracts had an effect when sprayed onto leaves 2 days before inoculation. Extracts contained a large and varied microbial population of actinomycetes (0.3 to 2.4×10⁵ c.f.u.ml^–1), bacteria (1.5 to 5.6×10¹⁰ c.f.u.ml^–1), filamentous fungi (25.0 to 45.5 c.f.u. ml^–1) and yeasts (26.1 to 62.6 c.f.u.ml^–1). Eight- and 18-day-old extracts lost activity completely on filter sterilization or autoclaving. Weekly sprays of 8-day-old extracts onto lettuce in the glasshouse had no effect on the incidence of grey mould, but significantly reduced its severity and increased marketable yield. The use of compost extracts in biocontrol of plant diseases and their possible mode of action is discussed.M.P. McQuilken and J.M. Whipps are and J.M. Lynch was with the Microbiology and Crop Protection Department, Horticulture Research International, Littlehampton BN17 6LP, UK; J.M. Lynch is now with the School of Biological Sciences, University of Surrey, Guildford GU2 5XH, UK.     

Melanin in the extracellular matrix of germlings of Botrytis cinerea

Previous work on the composition of the extracellular matrix of germlings of the plant pathogenic fungus Botrytis cinerea demonstrated the presence of carbohydrate, protein, and simple lipids; which, together, comprised 50-60% of the dry weight. Here we show that most of the remaining mass of the extracellular matrix consists of a chemically inert dark pigment with the electron paramagnetic resonance characteristics of a melanin. Scanning electron micrographs of the purified pigment, and transmission electron micrographs of thin sections made using the pigment indicate that it has a filamentous structure. We conclude that melanin is an important component of the extracellular matrix of germlings of B. cinerea. This is the first report of a melanin present in the extracellular matrix of a plant pathogenic fungus.     

The biosynthetic pathway to abscisic acid via ionylideneethane in the fungus Botrytis cinerea

The biosynthetic pathway to abscisic acid (ABA) from isopentenyl diphosphate in the fungus, Botrytis cinerea, was investigated. Labeling experiments with (18)O2 and H2(18)O indicated that all oxygen atoms at C-1, -1, -1' and -4' of ABA were derived from molecular oxygen, and not from water. This finding was inconsistent not only with the known carotenoid pathway via oxidative cleavage of carotenoids, but also with the classical direct pathway via cyclization of farnesyl diphosphate. The fungus produced new C15-compounds, 2E,4E-alpha-ionylideneethane and 2Z,4E-alpha-ionylideneethane, along with 2E,4E,6E-allofarnesene and 2Z,4E,6E-allofarnesene, but did not apparently produce carotenoids except for a trace of phytoene. The C15-compounds labeled with 13C were converted to ABA by the fungus, and the incorporation ratio of 2Z,4E-alpha-ionylideneethane was higher than that of 2E,4E-alpha-ionylideneethane. From these results, it was concluded that farnesyl diphosphate was reduced at C-1, desaturated at C-4, and isomerized at C-2 to form 2Z,4E,6E-allofarnesene before being cyclized to 2Z,4E-alpha-ionylideneethane; the ionylideneethane was then oxidized to ABA with molecular oxygen. This direct pathway via ionylideneethane means that the biosynthetic pathway to fungal ABA, not only before but also after isopentenyl diphosphate, differs from that to ABA in plants, since plant ABA is biosynthesized using the non-mevalonate and carotenoid pathways.     

A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.     

Direct fungicidal activities of C6-aldehydes are important constituents for defense responses in Arabidopsis against Botrytis cinerea

C6-aldehydes, such as (Z)-3-hexenal, (E)-2-hexenal, and n-hexanal, are volatile compounds formed by hydroperoxide lyase (HPL) and found in most terrestrial plants. They are fungicidal and bactericidal compounds, and are also signaling compounds to induce defense responses in plants. Transgenic plants having overexpressed or suppressed HPL activity (SH or ASH, respectively) showed lower or higher susceptibility against a necrotrophic fungal pathogen, Botrytis cinerea. In this study, we examined whether the modulated susceptibility was accountable to the direct fungicidal activity or to the signaling potency of C6-aldehydes. When wild-type Arabidopsis leaves were inoculated with B. cinerea, HPL expression was upregulated, and concomitantly, the amounts of C6-aldehydes increased. Higher amounts of C6-aldehydes found in inoculated SH plants inhibited growth of B. cinerea in vitro, while lower amounts found in ASH plants caused no inhibitory effect on the fungi. Thus, it was suggested that direct fungicidal activity of C6-aldehydes accounted for the modulated susceptibility. With SH plants higher amounts of camalexin could be found, but with the ASH plants no difference from wild-type plants could be found. Surplus amounts of C6-aldehydes could induce formation of camalexin as signaling compounds; however, this was not the case with wild-type and ASH plants. Accordingly, it could be assumed that direct fungicidal activity of C6-aldehydes were prominently responsible to the defense against B. cinerea but their signaling roles could be little responsible if any.     

A new member of the aldo-keto reductase family from the plant pathogen Xylella fastidiosa

The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of dl-glyceraldehyde (K(cat) 2.26s(-1), Km 8.20+/-0.98 mM) and 2-nitrobenzaldehyde (K(cat) 11.74 s(-1), Km 0.14+/-0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants.     

Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses.Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1, were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2, PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea, it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment.We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.     

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S

A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe³⁺ had an active effect on the enzyme obviously.     

A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris

Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L⁻¹ of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K_M values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I₅₀ = 1.4 M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research.     

Overexpression of OgPAE1 from wild rice confers fungal resistance against Botrytis cinerea

A full-length cDNA of the OgPAE1 gene encoding the alpha5 subunit of the 20S proteasome was isolated from wild rice (Oryza grandiglumis) treated by wounding or with a fungal elicitor. The deduced amino acid sequence of OgPAE1 comprises 237 amino acids (25.99 kDa), and shows 94.5% homology with Arabidopsis thaliana AtPAE1. Expression of OgPAE1 is regulated by defense-related signaling chemicals such as cantharidin, endothall and jasmonic acid. Overexpression of OgPAE1 in A. thaliana leads to resistance to the fungal pathogen Botrytis cinerea by lowering disease rate and size of necrotic lesions, and by less penetration and colonization of fungal hyphae. The results indicate that the 20S proteasome from wild rice is involved in the B. cinerea defense pathway via an as yet undetermined mechanism.     

Isolation and identification of the principal siderophore of the plant pathogenic fungusBotrytis cinerea

Summary The plant pathogenic hyphomyceteBotrytis cinerea has been shown to produce several trihydroxamate siderophores under conditions of low-iron stress. The total siderophores amounted to approximately 30 mg/l culture filtrate after 5 days of incubation in an asparagine/salt/glucose medium. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) on a reversed phase indicated that ferrirhodin is the predominant siderophore of this fungus. Chemical characterization of the principal siderophore by fast-atom-bombardment (FAB) mass spectrometry, nuclear magnetic resonance (¹H-NMR,¹³C-NMR) and comparison with a reference revealed the identity with ferrirhodin. NMR studies performed on desferrirhodin (desferrirhodin) in dimethylsulfoxide and water revealed the existence of two conformers in D₂O resulting from acis-trans isomerization of the hydroxamic acid groups. Comparative iron-uptake studies showed the following order of uptake inB. cinerea: ferrichrysin (100%), ferrirubin (57%), ferrirhodin (45%), hexahydroferrirhodin (45%), coprogen 6%. Concentration-dependent uptake of ferrirhodin resulted in saturation kinetics only in the low concentration range of 0–30 M (K _m = 2.5 M,V _max = 80 pmol min^–1 mg(^–1). A non-saturable, linear uptake was observed in the high concentration range of 30–80 M. The low concentration range appears to be the physiologically significant range, where siderophore-mediated iron transport inB. cinerea occurs.     

Use of some essential oils as post-harvest botanical fungicides in the management of grey mould of grapes caused by Botrytis cinerea

During screening of twenty six essential oils against Botrytis cinerea, the essential oils of the ten plants viz. Chenopodium ambrosioides, Eucalyptus citriodora, Eupatorium cannabinum, Lawsonia inermis, Ocimum canum, O. gratissimum, O. sanctum, Prunus persica, Zingiber cassumunar and Z. officinale were found to exhibit absolute fungitoxic activity (100% growth inhibition). The essential oils of O. sanctum, P. persica and Z. officinale were selected for further investigation because these oils showed lower Minimum Inhibitory Concentration (MIC) as compared to the other fungitoxic oils. The selected oils were subsequently standardized through physico-chemical and fungitoxic properties. The MIC values of O. sanctum, P. persica and Z. officinale were found to be 200, 100 and 100 ppm (mg/l) respectively. The oils showed fungistatic nature at their respective MIC. The oils were thermostable, and exhibited a wide range of fungitoxicity against 15 other post-harvest fungal pathogens. The oils had the potency to withstand high inoculum density. The antifungal potency of oils was found to be greater in comparison to some prevalent synthetic fungicides. Practical applicability of the essential oils was observed in control of grey mould of grapes caused by B. cinerea during storage. The O. sanctum- and P. persica-oil-treated grapes showed enhancement of storage life up to 5 and 4 days respectively. The storage life of Z. officinale-oil-treated grapes was found to be enhanced up to 6 days. The oils did not exhibit any phytotoxic effect on the fruit peel. Therefore, the oils could be recommended as a potential source of ecofriendly botanical fungicide, after long term and wide ranging trials.     

Local and systemic protection of poinsettia (Euphorbia pulcherrima Willd.) against Botrytis cinerea Pers. induced by benzothiadiazole

Benzothiadiazole (BTH) was found to be highly effective in increasing resistance of two poinsettia cultivars — ‘Coco White’ and ‘Malibu Red’, moderately susceptible to the fungus Botrytis cinerea. BTH applied at a concentration of 0.3 mM on the discs cut out from the leaves of these poinsettia cultivars reduced disease symptoms by more than 60 % in comparison to the control discs treated with water and exposed to infection. It was also observed that the applied inducer at a concentration of 0.03 and 0.3 mM had a favourable influence on the increase of poinsettia systemic resistance of SAR type (systemic acquired resistance). The effectiveness of BTH was much less when disease development was examined on detached leaves (a 20 % reduction of lesion area) in comparison with a pronounced inhibition of grey mould development on intact leaves of previously induced plants (a 80 % protection of intact plants). Benzothiadiazole in the concentration range from 0.03 to 1.4 mM added to in vitro agar medium was not found to have an inhibitory influence on Botrytis cinerea mycelium growth and sporulation.     

Botrytis cinerea induces the formation of free radicals in fruits ofCapsicum annuum at positions remote from the site of infection

Summary Free radical adducts of the spin trap -(4-pyridyl-1-oxide)-N-tert-butylnitrone have been observed by electron paramagnetic resonance spectroscopy in detached fruits ofCapsicum annuum investigated 5 days after infection withBotrytis cinerea.The spectra of these adducts were at a maximum within the soft rot lesion, but they could also be detected at distances up to 50 mm from the edge of the lesion in samples following main vascular bundles. At distances greater than 40 mm, the spectrum of the ascorbate radical was also seen, and at greater distances from the lesion it was the only radical detected. With samples taken from parenchyma tissue adjacent to the vascular bundles there was little adduct formation and the ascorbate radical could be detected, albeit with reduced intensity compared to healthy tissue, at distances as small as 10 mm from the edge of the lesion. This observation of chemical changes at considerable distances from the infected tissue is in contrast to previous observations on the behaviour of other markers of oxidative stress (e.g., 4-hydroxynonenal, malondialdehyde, single-peak free radical, and Fe(III) (g = 4.27) electron paramagnetic resonance signals), where their levels decreased rapidly outside of the soft rot.     

The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F₁ progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.     

Dual Antagonism of Aldehydes and Epiphytic Bacteria from Strawberry Leaf Surfaces against the Pathogenic Fungus Botrytis cinerea in vitro

Dual culture experiments were conducted in vitro to evaluate the potential combined biological effect of epiphytic bacteria and plant volatiles formed during fatty acids degradation on the pathogenic fungus Botrytis cinerea. The aliphatic aldehydes hexanal, (E)-2-hexenal, (Z)-3-hexenal and (E)-2-nonenal showed an enhancing effect on the antagonistic interaction between the epiphytic bacteria Pseudomonas lurida, Pseudomonas rhizosphaerae, Pseudomonas parafulva, and Bacillus megaterium against the pathogenic fungus. The unsaturated aldehydes were found to be the most potent with the minimum effective concentration being 1 ppm. Increasing volatile concentrations led to the inhibition of Botrytis cinerea growth with concomitant increase of colony diameters of epiphytic bacteria. Especially (E)-2-nonenal showed a stronger inhibitory effect on different strains of the plant pathogenic fungus Botrytis cinerea than on the epiphytic bacteria. These results suggest that co-application of antagonistic bacteria with natural plant volatiles can enhance the effectiveness of the biocontrol agents against B. cinerea.     

Inhibitors of polygalacturonase and laccase of Botrytis cinerea and their application to the control of this fungus

EDTA, calcium chloride, and two siderophores (rhodotorulic acid produced by Rhodotorula glutinis BNM 0524, and enterochelin from the bacterium Rahnella aquatilis BNM 0523) were evaluated as possible inhibitors of polygalacturonase (PG) and laccase (LC) from Botrytis cinerea. The aim was to apply them to the control of this pathogen, taking into account the fact that these enzymes are related to the invasion and installation of the fungus in the host. Two B. cinerea Pers.:Fr strains (BNM 0527 and BNM 0528) were used. Enzyme activities were measured in the supernatant of 7-day-old cultures. EDTA, calcium chloride, rhodotorulic acid, or enterochelin were added in the reaction mixture. Laccase activity from two strains was more affected by enterochelin (70-80% inhibition) than by the other compounds, while polygalacturonase was more inhibited (45% inhibition) by calcium chloride. The inhibitors were added to the growth medium and after 7 days of culture, the activities of the enzymes were measured in the supernatants. The production of PG and LC in both strains was lower when enterochelin or calcium chloride was added. In the third step, when the inhibitors were tested on apple, all them provided both effects, preventive and curative, against infections caused by B. cinerea, with EDTA and rhodotorulic acid exhibiting more preventive effects while calcium chloride and enterochelin provided more control of pre-existing infections (curative effect), coinciding with their ability to inhibit the production of polygalacturonase and laccase.     

Cloning and characterization of two endoxylanases from the cereal phytopathogen Fusarium graminearum and their inhibition profile against endoxylanase inhibitors from wheat

Two genes encoding family 11 endo-beta-1,4-xylanases (XylA, XylB) from Fusarium graminearum were cloned and expressed in Escherichia coli. The amount of active endoxylanase in the cytoplasmic soluble fraction was considerably improved by varying different expression parameters, including host strain and temperature during induction. Both recombinant endoxylanases showed a temperature optimum around 35 degrees C and neutral pH optima (around pH 7 and 8 for XylB and XylA, respectively). For the first time this allowed one to test endoxylanases of a phytopathogenic organism for inhibition by proteinaceous endoxylanase inhibitors TAXI and XIP. Whereas XylA and XylB were inhibited by TAXI-I, no inhibition activity could be detected upon incubation with XIP-I. The insensitivity of both F. graminearum endoxylanases towards XIP is surprising, since the latter is typically active against endoxylanases produced by (aerobic) fungi. As F. graminearum is an important phytopathogen, these findings have implications for the role of endoxylanase inhibitors in plant defence.     

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus

Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.