High stability and low competitive inhibition of thermophilic Thermopolyspora flexuosa GH10 xylanase in biomass-dissolving ionic liquids

Thermophilic Thermopolyspora flexuosa GH10 xylanase (TfXYN10A) was studied in the presence of biomass-dissolving hydrophilic ionic liquids (ILs) [EMIM]OAc, [EMIM]DMP and [DBNH]OAc. The temperature optimum of TfXYN10A with insoluble xylan in the pulp was at 65–70 °C, with solubilised 1 % xylan at 70–75 °C and with 3 % xylan at 75–80 °C. Therefore, the amount of soluble substrate affects the enzyme activity at high temperatures. The experiments with ILs were done with 1 % substrate. TfXYN10A can partially hydrolyse soluble xylan even in the presence of 40 % (v/v) ILs. Although ILs decrease the apparent temperature optimum, a surprising finding was that at the inactivating temperatures (80–90 °C), especially [EMIM]OAc increases the stability of TfXYN10A indicating that the binding of IL molecules strengthens the protein structure. Earlier kinetic studies showed an increased K _m with ILs, indicating that ILs function as competitive inhibitors. TfXYN10A showed low increase of K _m, which was 2-, 3- and 4-fold with 15 % [EMIM]OAc, [DBNH]OAc and [EMIM]DMP, respectively. One reason for the low competitive inhibition could be the high affinity to the substrate (low K _m). Xylanases with low K _m (~1 mg/mL) appear to show higher tolerance to ILs than xylanases with higher K _m (~2 mg/mL). Capillary electrophoresis showed that TfXYN10A hydrolyses xylan to the end-products in 15–35 % ILs practically as completely as without IL, also indicating good binding of the short substrate molecules by TfXYN10A despite of major apparent IL binding sites above the catalytic residues. Substrate binding interactions in the active site appear to explain the high tolerance of TfXYN10A to ILs.

     

Purification and properties of the xylanases from the termite Macrotermes bellicosus and its symbiotic fungus Termitomyces sp.

Four xylanases were purified, two from the termite Macrotermes bellicosus workers (X1T and X2T) and two from its symbiotic fungus Termitomyces sp. (X1Mc and X2Mc). The analysis of the step required for the purification of X1T and X1Mc and the comparison of their different properties suggested that xylanases X1T and X1Mc were the same enzyme, X1. The determination of the reducing sugars by TLC revealed that X1 was an endoxylanase (EC 3.2.1.8) and X2T and X2Mc were exoxylanases (EC 3.2.1.37). The apparent molecular weights of the three xylanases, determined by SDS-polyacrylamide gel electrophoresis, were 36 kDa for X1, 56 kDa for X2T and 22.5 kDa for X2Mc. The optimal pH of the three xylanases was 5.5, and K_m values determined with birchwood xylan as substrate were 0.2% for X1, 0.1% for X2T and 0.3% for X2Mc, showing a high affinity for this substrate. The three enzymes differed also by their thermal stability.     

Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis

Three sulfated polysaccharide fractions (F1, F2, and F3) were isolated from Porphyra haitanesis, an important economic alga in China, through anion-exchange column chromatography and their in vitro antioxidant activities were investigated in this study. Galactose was the main sugar unit of the three fractions. The analytical results indicated that polysaccharide fractions from P. haitanesis had similar chemical components to porphyran from other species, but differed in their high sulfate content. The sulfate content of F1, F2 and F3 was 17.4%, 20.5% and 33.5% respectively. All three polysaccharide fractions showed antioxidant activities. They had strong scavenging effect on superoxide radical, and much weaker effect on hydroxyl free radical. Lipid peroxide in rat liver microsome was significantly inhibited, and H₂O₂ induced hemolysis of rat erythrocyte was partly inhibited by F1, F2 and F3. Among them, F3 showed strongest scavenging effect on superoxide radical; F2 had strongest effect on hydroxyl radical and lipid peroxide.     

Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b ₅₅₄ (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b ₅₅₆ (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth. Received: 21 August 1996 / Accepted: 24 October 1996     

A xylan-degrading strain of<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>: isolation and characterization of the xylanase activity

Two strains (O and X₂) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O and X₂, grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100°C, and exhibited an optimal temperature and pH of 90°C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.Communicated by G. Antranikian     

The antenna system of Rhodospirillum rubrum. Detection of macromolecular constituents not stainable by Coomassie brilliant blue in solubilized preparations of the B880 complex

A solubilized preparation of the major Rhodospirillum rubrum antenna complex (B880) was obtained by a described procedure and its polypeptide composition was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only two polypeptides of molecular weights close to 7000 were detected after staining the gels with Coomassie brilliant blue. However, several other constituents could be visualized by silver staining or by an immunochemical method. When the preparation was chromatographed on Sephacryl, some of the resulting fractions exhibited the characteristic B880 absorption spectrum and contained only the two proteins that were detectable with Coomassie brilliant blue. In those fractions the A ₂₈₀/A ₈₈₀ratio was 0.4, which indicated a significant improvement of the bacteriochlorophyll to protein ratio over the unchromatographed preparation (A ₂₈₀/A ₈₈₀=0.7). Other chromatography fractions lacked bacteriochlorophyll and contained a carotenoid which seemed to be bound to protein. The macromolecular constituents present in these latter fractions differed from those associated to the purified B880 complex in their electrophoretic moblities and/or in their staining properties. That suggested the possible existence of a carotenoprotein that did not result from the B880 complex upon loss of bacteriochlorophyll.     

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000     

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Summary Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-¹⁴C-glucose or UDP-¹⁴C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl₂ was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl₂ the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10M UDP-glucose in the presence of 3 mM MnCl₂ was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 M UDP-xylose in the presence of 3 mM MnCl₂, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-¹⁴C-glucose, and fractions enriched in Golgi apparatus, both (13)- and (14)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (13)-glucosyl. With the substrate, UDP-¹⁴C-xylose, mostly (14)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.Abbreviations ATP adenosine-5-triphosphate - CR crude fraction from downward centrifugation - FL purified fraction from flotation centrifugation - GC gas chromatography - GC-RPC gas chromatography-radiogas proportional counting - IDP inosine-5-disphosphate - NPA naphthylphthalamic acid - UDP uridine-5-diphosphate - TEM transmission electron microscopy     

Extracellular xylanases from two pathogenic races of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">ciceris</Emphasis>: enzyme production in culture and purification and characterization of a major isoform as an alkaline endo-β-(1,4)-xylanase of low molecular weight

Fusarium oxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpea, comprises eight pathogenic races and two pathotypes. Races 0 and 5, representative of the least virulent yellowing pathotype and the most virulent wilt pathotype, respectively, produced extracellular xylanases when grown on minimal medium supplemented with either 1% commercial birchwood xylan or 0.3% chickpea cell walls. The pattern of extracellular proteins analysed by denaturing polyacrylamide gel electrophoresis in the two media presented some minor but distinctive differences between fungal races. By preparative isoelectrofocusing, the xylanase activity in cell wall-culture filtrates could be resolved into basic and neutral fractions with pI values around to 10 and 8, respectively, whereas the xylan-culture filtrates contained an additional acidic fraction of pI around 4. A common major xylanase was purified 7-fold to homogeneity by cation-exchange chromatography and chromatofocusing. The purified xylanase has a molecular weight of 21.6 kDa, optimum pH and temperature of 5.5 and 55 °C, respectively, pI in the range of 8.2 to 9.0, and K_m and V_max values of 2.24 mg ml^–1 (birchwood xylan as substrate) and 1200 nkat mg^–1 protein (72 U mg^–1 protein), respectively. The enzyme has an endo mode of action, hydrolysing xylan to xylobiose and higher short-chain xylooligosaccharides without forming free xylose.     

Role of Transglycosylation Products in the Expression of Multiple Xylanases in <Emphasis Type="Italic">Myceliophthora</Emphasis> sp. IMI 387099

This study reports the regulation of multiple xylanases produced by Myceliophthora sp. IMI 387099. Fructose was found to positively regulate the expression of multiple xylanase when used as sole carbon source. The xylanases (EX₁ and EX₂) of acidic pI were expressed in the presence of simple sugars (glucose, arabinose, and xylose), whereas xylanase of both acidic as well as basic pI (EX_1, EX_2, EX₃, and EX₅) were expressed in the presence of fructose, xylan, and combination of xylan and alcohol. The combination of fructose and xylan also led to expression of an additional xylanase (EX₄). The positional isomer (iso-X4) was found to be the key transglycosylation product when cultures were grown in the presence of fructose and xylan. In the presence of alcohols, the higher expression of xylanase was ascribed to the synergistic effect of alkyl glycoside and other transglycosylation products present in the culture extracts.     

Degradation of Muskmelon Cell Wall by the Xylanases of Colletotrichum lagenarium

Xylose-rich polysaccharide fractions were isolated from cell walls of muskmelon (Cucumis melo L. cv. Cantaloup Charentais) stems. A xylan with a (1 → 4) linked backbone has been characterized as a component of these fractions. Two xylanases excreted by the fungus pathogen Colletotrichum lagenarium were able to degrade this substrate both after extraction and when it was included in the crude cell walls. The results suggest that the xylanases are involved in the parasitism of this pathogen.     

Purification and characterization of a high molecular weight endoxylanase from the solid-state culture of an alkali-tolerant Aspergillus fumigatus MKU1

Summary A high molecular weight endoxylanase (XylF2) from the solid state culture of Aspergillus fumigatus MKU1 was purified to homogeneity by a combination of tube gel electrophoresis and electroelution methods. The purity was demonstrated by SDS-PAGE and the molecular mass of the XylF2 was found to be 66 kDa. The optimal pH and temperature for activity were 5.0 and 90 °C, respectively. The apparent K _m and V _max values of XylF2 with oat spelt xylan as substrate were 1.6 mg/ml and 3.25 mmol/min/mg protein respectively. The enzyme showed high activity towards oat spelt xylan while negligible activity was observed on carboxymethylcellulose. The activity of XylF2 was strongly inhibited by Hg²⁺, Ni²⁺, Zn²⁺, SDS and N-bromosuccinimide and stimulated by l-cysteine and iodoacetamide. The hydrolysis of oat spelt xylan by XylF2 released only xylo-oligosaccharides.     

Purification and characterization of two different xylanases from the thermophilic actinomycete Microtetraspora flexuosa SIIX

Two endoxylanases were isolated from the xylanolytic enzyme system of the thermophilic actinomycete Microtetraspora flexuosa SIIX, and purified by ammonium sulfate fractionation, DEAE-Sepharose chromatography, gel filtration on Sephacryl S 200 and fast protein liquid chromatography on Q-Sepharose. The molecular masses of xylanase I and II were 26.3 and 16.8 kDa, and isoelectric points were 8.4 and 9.45, respectively. optimal enzyme activities were obtained at 80° C and pH 6.0. The thermostability of both xylanases was greatly diminished during purification but could be restored by preincubation of the purified enzymes in the presence of xylan. The half-lives at 80° C were approximately 25 min. The kinetic constants of xylanases I and II determined with Remazol-brilliant-blue xylan were V_max of 1537 and 353 mol·min^-1·mg protein^-1 and K _m values of 2.44 and 1.07 mg·ml^-1, respectively. Purified xylanases utilized xylan as well as small oligosaccharides such as xylotriose as substrate. They did not exhibit xylobiase or debranching activities. The predominant products of arabinoxylan hydrolysis were xylobiose and xylotriose, the latter being hydrolysed to xylobiose and xylose upon further incubation. In addition, fragments containing arabinose side chains accumulated. The xylanases did not act on crystalline or amorphous cellulose indicating a possible application in biobleaching processes.     

Characterization of Xylanolytic Enzymes in Clostridium cellulovorans: Expression of Xylanase Activity Dependent on Growth Substrates

Xylanase activity of Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium, was characterized. Most of the activity was secreted into the growth medium when the bacterium was grown on xylan. Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions. Each of these fractions contained at least two major and three minor xylanase activities. In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis. The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000. High alpha-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions. Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose- and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan. These results strongly indicated that C. cellulovorans is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate.     

Action of different types of endoxylanases on eucalyptus xylan in situ

Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields.

     

Hydrolysis of xylans by a thermostable hybrid xylanase expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli-expressed a hybrid xylanase, Btx, encoded by a designed hybrid xylanase gene btx was purified. The molecular mass of the enzyme was estimated to be 22 kDa. The K _m and k _cat values for Btx were 1.9 mg/ml and 140 s⁻¹, respectively. It hydrolyzed xylan principally to xylobiose and xylotriose, and was functionally similar to family 11 xylanases. As some differences were found in the hydrolytic products between birchwood xylan and wheat bran insoluble xylan, the xylan binding domains in xylanase Btx must have different effects on soluble and insoluble xylan.     

Degradation and Utilization of Xylans by the Rumen AnaerobePrevotella bryantii(formerlyP. ruminicolasubsp.brevis) B14

Freshly harvested whole cells from cultures ofP. bryantiiB₁4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantiiB₁4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation withP. bryantiishowed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobeRuminococcus flavefaciensgave little increase in overall pentose utilization suggesting that externalP. bryantiixylanases are as effective as the clonedR. flavefaciensenzymes in releasing products that can be utilised byP. bryantiicells. The xylanase system ofP. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

Polysaccharide breakdown by mixed populations of human faecal bacteria

Measurements of polysaccharide-degrading activity in different fractions of human faeces showed that bacterial polysaccharidases and glycosidases were primarily associated with the washed bacterial fractions. Amylase, pectinase and xylanase were the major polysaccharide-hydrolysing enzymes detected, whilst α-L-arabinofuranosidase, β-D-xylosidase, β-D-galactosidase and β-D-glucosidase were the most active glycosidases. Starch and 3 non-starch polysaccharides (NSP; pectin, xylan and arabinogalactan) were fermented by mixed populations of human faecal bacteria in batch culture. Detailed carbohydrate analysis demonstrated that starch and pectin were the most rapidly degraded substrates and that arabinogalactan and the relatively insoluble polysaccharide xylan were broken down more slowly. Free sugars and oligosaccharides did not accumulate in culture media with any polysaccharide tested. Time-course measurements of polysaccharide remaining in the batch culture fermentations showed that the arabinose side chains of pectin, xylan and arabinogalactan were co-utilised with the backbone sugars. In these cultures, polysaccharide-degrading activity was mainly cell-associated, but extracellular polysaccharidase activity increased as the fermentations progressed. Molar ratios of acetate, propionate and butyrate produced in these experiments were dependent upon the polysaccharide substrate tested. Molar ratios of acetate, propionate and butyrate in the starch, arabinogalactan, xylan and pectin fermentations were 50:22:29, 50:42:8, 82:15:3, and 84:14:2, respectively. The presence of starch did not inhibit the breakdown of arabinogalactan, xylan or pectin by faecal bacterial, providing evidence that multicomponent substrate utilisation occurs when complex populations of faecal bacteria are provided with mixed polysaccharide substrates.     

Multicomponent cellulase production by Cellulomonas biazotea NCIM‐2550 and its applications for cellulosic biohydrogen production

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM‐2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl₂ as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H₂ energy was examined with seven H₂‐producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H₂ production performance with the hydrolysate of SCB and xylan. The cumulative H₂ production and H₂ yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H₂/g reducing sugar (or 1.91 mmol H₂/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H₂ production and H₂ yield of 87.02 mL/L and 5.03 mmol H₂/g reducing sugar (or 4.01 mmol H₂/g cellulose), respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552 U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn²⁺, Sn²⁺, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl β-d-xylopyranoside. The apparent K _m value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3 ± 0.236 and 60.2 ± 6.788 mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.