Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile

An endo-beta-1,4-xylanase (1,4-beta-D-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70 degrees C. Thin-layer chromatography (TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a beta-(1-->4)-beta(1-->3)-xylan). Also, the enzyme releases an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves preferentially the internal glycosidic bonds of xylooligosaccharides, [1-3H]-xylooligosaccharides and xylan. The enzyme also hydrolyses the 4-methylumbelliferyl glycosides of beta-xylobiose and beta-xylotriose at the second glycosidic bond adjacent to the aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of omega-epoxyalkyl glycosides of D-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes belonging to family 11.     

Purification and characterization of Thermobifida fusca xylanase 10B

Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 degrees C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl alpha-D-arabinofuranoside, p-nitrophenyl-beta-D-xylobioside, p-nitrophenyl-beta-D-xyloside, and p-nitrophenyl-beta-D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.     

Purification and properties of three endo-beta-1,4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps*(2)

In the presence of xylan, Streptomyces sp. strain S38 secretes three xylanases (Xyl1, Xyl2, and Xyl3) that were purified to protein homogeneity and characterized. When used in bleach boosting tests on kraft hardwood and softwood, Xyl1, a family-11 enzyme, was more effective than Xyl2 and Xyl3 that belonged to family-10. Xyl1 was fully responsible for the biodelignification potential of the culture supernatants with a minimal effective amount of 10 IU per gram of dry pulp for both softwood and hardwood pulp. Complete conventional CEDED bleaching sequences showed that enzymatic pretreatment (20 IU/g dry pulp) could result in active chlorine savings of 8.6 and 4.9 kg/ton of dry pulp with hardwood and softwood, respectively. The purified enzymes were totally devoid of cellulase activity on CM-cellulose and their activities were optimal at about 60 degrees C and pH 6. Moreover, the V(max) value of Xyl1 at 50 degrees C measured on birchwood xylan (5,700 μmoles/min/mg prot.) was significantly higher than those of Xyl2 and Xyl3 whereas their K(m) values were similar. Their half-lives at 50 degrees C were larger than 16 h but sharply decreased at 60 degrees C where the family-11 Xyl1 was less stable (t(1/2)(60 degrees C) = 10 min) than both family-10 enzymes Xyl2 (t(1/2)(60 degrees C) = 30 min) and Xyl3 (t(1/2)(60 degrees C) = 70 min).     

Enzymatic properties of a neutral endo-1,3(4)-beta-xylanase Xyl II from Bacillus subtilis

A Bacillus sp. CCMI 966, characterised as Bacillus subtilis, has a duplication time of about 24 min. It produces at least two extracellular xylanases, Xyl I and Xyl II. The extracellular xylanase activity seems to be strongly correlated with the biomass growth profile. The Xyl II isoenzyme was purified by ammonium sulphate precipitation and anionic exchange chromatography, with a purification factor of 8.3. The molecular weight of the isoenzyme was estimated by SDS-PAGE revealing that Xyl II is a multimeric enzyme with a catalytic subunit of about 20 kDa. Under non-denaturing conditions, a molecular weight of about 340 kDa was obtained by native PAGE gel and of 20 kDa by gel filtration chromatography. The enzyme showed an optimum pH and temperature of 6.0 at 60 degrees C. Xyl II was stable at 40 degrees C for 180 min at pH 6.0. The specificity of Xyl II for different substrates was evaluated. Xyl II presents a higher affinity towards OSX, with a K(m) of 1.56 g l(-1) and showed the ability to hydrolyse laminarin, with a K(m) of 1.02 g l(-1). Xylotetraose is the main product of xylan degradation. The Xyl II ability for binding to cellulose and/or xylan was also studied.     

Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1.

An alkaliphilic Bacillus sp. strain, 41M-1, isolated from soil produced multiple xylanases extracellularly. One of these xylanases was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The moleculr mass of this enzyme (xylanase J) was 36 kDa, and the isoelectric point was pH 5.3. Xylanase J was most active at pH 9.0. The optimum temperature for the activity at pH 9.0 was around 50 degrees C. The enzyme was stable up to 55 degrees C at pH 9.0 for 30 min. Xylanase J was completely inhibited by the Hg2+ion and N-bromosuccinimide. The predominant products of xylan hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that the enzyme was an endoxylanase. The apparent Km and Vmax values on xylan were 3.3 mg/ml and 1,100 micromol-1 mg-1, respectively. Xylanase J showed high sequence homology with the xylanases from Bacillus pumilus and Clostridium acetobutylicum in the N-terminal region. Xylanase J acted on neither crystalline cellulose nor carboxymethyl cellulose, indicating a possible application of the enzyme in biobleaching processes.     

Purification and characterization of xylanases from<Emphasis Type="Italic">Aspergillus giganteus</Emphasis>

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50 degrees C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50 degrees C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.     

Purification of the alkaliphilic xylanases from Myceliophthora sp. IMI 387099 using cellulose-binding domain as an affinity tag

Ten xylanase isoforms produced by Myceliophthora sp. were characterized for their ability to bind to avicel. Three of the xylanases showing differential affinity for avicel were purified by column chromatography. The purified xylanase Xyl IIa, IIb and IIc showed molecular mass of 47, 41 and 30 kDa and pI of ∼3.5, 4.8 and 5.2, respectively. Xyl IIa was optimally active at pH 8.0 and temperature 70 °C, while Xyl IIb and IIc were optimally active at pH 9.0 and 60 °C and 7.0 and 80 °C, respectively. Xyl IIa and Xyl IIb showed higher stability under alkaline conditions (pH 9.0) and retained 80% of the original activity upto 1 h and 3 h respectively, at 50 °C. All three purified iso-xylanases showed enhanced activities in presence of Na⁺, Mg²⁺, Mn²⁺ and K⁺ ions, whereas, Zn²⁺ and Cu²⁺ showed negative effect on Xyl IIa. The activity of Xyl IIa increased in presence of reducing agents DTT and mercaptoethanol, however, SDS showed inhibitory effect. Kinetic studies showed that Xyl IIb and IIc degrade rye arabinoxylan, much more efficiently than oat spelt xylan, whereas, Xyl IIa showed much higher Kcat/Km value for birch wood xylan as compared to oat spelt xylan. The purified xylanases were apparently classified in family 10.     

Purification and characterization of endo-xylanases from Aspergillus niger. I. Two isozymes active on xylan backbones near branch points

Two endo-xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were purified to homogeneity from a crude Aspergillus niger pentosanase preparation by Ultrogel AcA 54 gel permeation chromatography, SP-Sephadex C-25 cation exchange chromatography at pH 4.5, Sephadex G-50 gel permeation chromatography, and a second SP-Sephadex C-25 step, this one at pH 5.8. The two xylanases hydrolyzed soluble xylan more rapidly than insoluble branched xylan, but attacked each substance to an equal extent. Their low activity on a linear xylooligosaccharide mixture and absence of activity on insoluble xylan freed of branches suggest that the xylanases require a branch point nearby for significant attack. No xylose or L-arabinose was produced, the major products of low molecular weight being tri- and pentasaccharides and smaller amounts of di-, tetra-, and hexasaccharides. There was low activity on untreated and crystalline cellulose and on carboxymethylcellulose and no activity on other polysaccharides tested. These two xylanases had molecular weights of ca. 1.3 x 10(4) and similar amino acid profiles, high in acidic and low in sulfur-containing residues. Isoelectric points were 8.6 for I and 9.0 for II. Optimum pH values for activity were 6.0 and 5.5, respectively. In a 20-min assay at pH 5.5, each was most active at 45 degrees C, with activation energies up to 40 degrees C of 30.4 and 38.8 kJ/ mol, respectively. Optimum pH levels for stability were 5.0 and 6.0, with half-lives at 60 degrees C and those pHs of 20 and 75 min, respectively.     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株BacilluspumilusWL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为2 6 0kD ,pI值9 5 ,以燕麦木聚糖为底物时的表观Km 值为16 6mg mL ,Vmax值为12 6 3μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7 2至8 0之间,是耐碱性木聚糖酶;最适作用温度为4 5℃～5 5℃,在37℃、4 5℃以下时该酶热稳定性均较好;5 0℃保温时,该酶活力的半衰期大约为2h ,在超过5 0℃的环境下,该酶的热稳定较差,5 5℃和6 0℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL_11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL_11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL_11主要合成内切型木聚     

Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca²⁺, Mn²⁺, Mg²⁺, Ba²⁺, Li²⁺, NaN₃ and isopropanol was observed. The K_m and V_max values determined for Xyl 1 with 4-O-methyl-d-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.Communicated by W.D. Grant     

Xylanases from Cellulomonas flavigena: purification and characterization

Three xylanases (Xyl1, Xyl2 and Xyl3) were purified and characterized from the culture supernatant of Cellulomonas flavigena grown on sugar cane bagasse. The enzymes were purified by affinity chromatography and gel filtration and had masses of 63 kDa, 17 kDa and 35 kDa, respectively, as measured by SDS-PAGE. All enzymes were active against 4-O-methyl-D-glucuronoxylan and xylan but had no cellulase activity with CM-cellulose, an important characteristics in biobleaching processes. © Rapid Science Ltd. 1998     

Thermostable xylanase from Marasmius sp.: purification and characterization

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as 90 degrees C. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of 90 degrees C. When using xylan from birchwood as substrate, it exhibits Km and Vmax values of 2.6 +/- 0.6 mg/ml and 428 +/- 26 U/mg, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to 70 degrees C. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at 70 degrees C for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.     

一种来源于Leifsonia shinshuensis DICP 16菌体的b-D-木糖苷酶的分离纯化及其性质

采用盐析、DE 52、Q-Sepharose Fast Flow阴离子交换层析、Toyopearl Butyl 650C疏水层析以及Sephacryl S-300 HR凝胶过滤层析联用的方法, 从Leifsonia shinshuensis DICP 16菌体中纯化出一种b-木糖苷酶。分离后该酶在SDS-PAGE 上呈单一蛋白质条带, 通过SDS-PAGE和凝胶过滤层析法, 测得该酶是一个由两个分子量约为91 kD的相同亚基组成的同源二聚体。其水解对硝基苯酚木糖苷(pNPX)的最适反应温度为55°C, pH值为7.0。该木糖苷酶在45°C以下, pH 6.0~11.0之间具有很好的稳定性。在45°C, pH值为7.0的条件下, 水解pNPX的Km, Vmax分别为1.04 mmol/L, 0.095 mmol/(min·mg)。研究不同的金属离子对该酶的活性影响, 发现Fe2+和Cu2+是很强的抑制剂。通过对天然木糖苷化合物的水解测试, 发现该酶可以水解人参皂苷Rb3的木糖基, 产生人参皂苷Rd, 却不能水解紫杉烷木糖苷的木糖基。     

Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate

A novel extra-cellular lipase from Bacillus coagulans MTCC-6375 was purified 76.4-fold by DEAE anion exchange and Octyl Sepharose chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 103 kDa. The lipase was optimally active at 45 degrees C and retained approximately 50% of its original activity after 20 min of incubation at 55 degrees C. The enzyme was optimally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+, and Fe3+ at 1mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions resulted in a maximal increase in lipase activity but Zn2+ and Co2+ ions showed an antagonistic effect on this enzyme. EDTA at 150 mM concentration inhibited the activity of lipase but Hg2+ or Al3+ (10mM) restored most of the activity of EDTA-quenched lipase. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was more specific to p-nitrophenyl esters of 8 (pNPC) and 16 (pNPP) carbon chain length esters. The lipase had a Vmax and Km of 0.44 mmol mg(-1)min(-1) and 28 mM for hydrolysis of pNPP, and 0.7 mmol mg(-1)min(-1) and 32 mM for hydrolysis of pNPC, respectively.     

Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis>

We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a K _m value of 1.95 mg/ml, while Xyl53 had a K _m value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55°C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.     

Purification and properties of two endo-1,4-beta-xylanases from Irpex lacteus (Polyporus tulipiferae)

Two different endo-1,4-beta-xylanases [1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8], named Xylanases I and III, were purified to homogeneity by gel filtration and ion exchange column chromatography from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The purified enzymes were found to be homogeneous on polyacrylamide disc electrophoresis and their specific activities toward xylan were increased approximately 28.7 and 19.8 times, respectively. The activities of each enzyme were considerably inhibited by Hg2+, Ag+, and Mn2+. Their molecular weights were estimated to be approximately 38,000 and 62,000 by gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, respectively. Their carbohydrate contents were 2.5% and 8.0% as glucose, and their amino acid composition patterns resembled each other, showing high contents of acidic amino acids, serine, threonine, alanine, and glycine. Both enzymes were most active at pH 6.0 but Xylanase I was more stable as to pH. Their optimum temperatures were 60 degrees C and 70 degrees C, respectively. Xylanase I split up to 34.5% of larchwood xylan whereas Xylanase III split only 18.9% of it. The products with the former were mainly xylose (X1), xylobiose (X2), and xylotriose (X3), whereas X2 and X3 were the main products with the latter. Both enzymes did not hydrolyze X2. Xylanase I produced almost equal quantities of X1 and X2 from X3, while Xylanase III did not attack this substrate. Both enzymes showed no activity toward glycans, other than xylan, such as starch, pachyman and Avicel (microcrystalline cellulose), except the almost one twentieth activity of Xylanase III toward sodium carboxymethyl cellulose (CMC).     

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.     

Purification and some properties of five endo-1,4-beta-D-xylanases and a beta-D-xylosidase produced by a strain of Aspergillus niger.

Five different xylanases and a beta-D-xylosidase in the culture medium of Aspergillus niger have been purified to homogeneity from 13- to 52-fold by a procedure of gel and hydroxylapatite chromatography. The strain was isolated from soil of the African equatorial forest. Gel chromatography of the purified enzymes indicated that three of the xylanases have molecular weights of 31,000 and the other two xylanases have molecular weights of 50,000. beta-D-Xylosidase has a molecular weight of 78,000. The pH curves of the xylanases were quite diverse and showed pH optima ranging from 4.0 to 6.5. Characteristic action patterns were obtained for each of the purified xylanases by gel chromatography of the xylan digests on Bio-Gel P-2. The enzymes degraded arabinoxylan by an endomechanism, producing L-arabinose, D-xylose, xylobiose, and a mixture of branched arabinose-xylose and D-xylose oligosaccharides. All xylanases seemed to be capable of liberating L-arabinose from either arabinoxylan or the arabinose-xylose oligosaccharides. Branched arabinose-containing D-xylose oligosaccharides were slowly hydrolyzed, so that these sugars accumulate in the digest. Two xylanases showed relatively broad substrate specificity and were able to degrade also crystalline cellulose. beta-D-Xylosidase showed optimal activity at pH 6.7 to 7.0 and at 42 degrees C. The Km for o-nitrophenyl-beta-D-xylopyranoside was 0.22 mM and xylotriose was hydrolyzed more rapidly than xylobiose.     

Functional characterization of a novel xylanase from a corn strain of Erwinia chrysanthemi

A beta-1,4-xylan hydrolase (xylanase A) produced by Erwinia chrysanthemi D1 isolated from corn was analyzed with respect to its secondary structure and enzymatic function. The pH and temperature optima for the enzyme were found to be pH 6.0 and 35 degrees C, with a secondary structure under those conditions that consists of approximately 10 to 15% alpha-helices. The enzyme was still active at temperatures higher than 40 degrees C and at pHs of up to 9.0. The loss of enzymatic activity at temperatures above 45 degrees C was accompanied by significant loss of secondary structure. The enzyme was most active on xylan substrates with low ratios of xylose to 4-O-methyl-D-glucuronic acid and appears to require two 4-O-methyl-D-glucuronic acid residues for substrate recognition and/or cleavage of a beta-1,4-xylosidic bond. The enzyme hydrolyzed sweetgum xylan, generating products with a 4-O-methyl-glucuronic acid-substituted xylose residue one position from the nonreducing terminus of the oligoxyloside product. No internal cleavages of the xylan backbone between substituted xylose residues were observed, giving the enzyme a unique mode of action in the hydrolysis compared to all other xylanases that have been described. Given the size of the oligoxyloside products generated by the enzyme during depolymerization of xylan substrates, the function of the enzyme may be to render substrate available for other depolymerizing enzymes instead of producing oligoxylosides for cellular metabolism and may serve to produce elicitors during the initiation of the infectious process.