Ferulic Acid Esterase Activity from Schizophyllum commune

Schizophyllum commune produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase, p-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate.     

Studies on the extracellular xylanase activity of some thermophilic actinomycetes

Summary An agar plate-clearing assay was used to screen 37 thermophilic actinomycete strains for extracellular xylanase production. The xylanase activity in culture supernatants of strains representing Saccharomonospora viridis and three Thermomonospora spp. was characterised by measurement of reducing sugar released from oat spelt xylan and analysis of degradation products by thin-layer chromatography. In all four species, xylanase activity was optimal within the temperature range 60–75°C and between pH 5 and pH 8. While culture supernatants of Thermomonospora strains incubated at 70°C for 60 min retained >80% of their activity, that of S. viridis was almost, totally inactivated.All of the culture supernatants initially hydrolysed xylan to a mixture of oligomeric products, indicating that the main activity was of the endoxylanase type. Prolonged incubation for 24h resulted in the hydrolysis of xylan to d-xylose by T curvata and T. fusca preparations, indicating the additional presence of exoxylanase or -xylosidase activity. Xylanase production was induced by growth on xylan although low levels of activity were also detected in glucose-grown cultures. Thermomonospora curvata MT815 culture supernatant was the most active and produced d-xylose from milled wheat straw in yields approximately 10% of those from oat spelt xylan.     

Production of feruloyl esterases and xylanases by Talaromyces stipitatus and Humicola grisea var. thermoidea on industrial food processing by-products

Feruloyl esterase (FAE) and xylanase activities were detected in culture supernatants from Humicola grisea var. thermoidea and Talaromyces stipitatus grown on brewers' spent grain (BSG) and wheat bran (WB), two agro-industrial by-products. Maximum activities were detected from cultures of H. grisea grown at 150 rpm, with 16.9 U/ml and 9.1 U/ml of xylanase activity on BSG and WB, respectively. Maximum FAE activity was 0.47 U/ml and 0.33 U/ml on BSG and WB, respectively. Analysis of residual cell wall material after microbial growth shows the preferential solubilisation of arabinoxylan and cellulose, two main polysaccharides present in BSG and WB. The production of low-cost cell-wall-deconstructing enzymes on agro-industrial by-products could lead to the production of low-cost enzymes for use in the valorisation of food processing wastes.     

Purification and characterization of arabinofuranosidase from the corn endophyte Acremonium zeae

Acremonium zeae, one of the most prevalent fungal colonists of preharvest corn, possesses a suite of hemicellulolytic activities including xylanase, xylosidase, and arabinofuranosidase. Two enzymes with arabinofuranosidase activity were purified from cell-free culture supernatants of A. zeae grown on oat spelt xylan. A 47 kDa enzyme (AF47) was optimally active at 37 °C and pH 6.0, and had a specific activity for 4-nitrophenyl-α-L-arabinofuranoside (4NPA) of 6.2 U/mg. A 30 kDa enzyme (AF30) was optimally active at 50 °C and pH 4.5, and had a specific activity for 4NPA of 12.4 U/mg. AF47 hydrolyzed 4-nitrophenyl-β-D-xylopyranoside, 4-nitrophenyl-β-D-glucopyranoside, and 4-nitrophenyl-β-D-cellobioside, as well as producing reducing sugars from corn fiber, wheat, and oat spelt arabinoxylan. AF30 had little detectable activity on the 4-nitrophenyl substrates, except for 4NPA, but activity on arabinoxylans from corn fiber, wheat, and oat spelt was at least 7-fold higher than AF47, with specific activities of 109, 358, and 153 U/mg, respectively. A combination of the two enzymes released 61 and 88% of the total arabinose from corn fiber and wheat arabinoxylans. The arabinofuranosidases produced by A. zeae may have industrial application for the enzymatic hydrolysis of recalcitrant lignocellulosic feedstocks such as corn fiber and wheat straw.     

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.     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Xylanase IV, an Exoxylanase of Aeromonas caviae ME-1 Which Produces Xylotetraose as the Only Low-Molecular-Weight Oligosaccharide from Xylan

A novel xylanase (xylanase IV) which produces xylotetraose as the only low-molecular-weight oligosaccharide from oat spelt xylan was isolated from the culture medium of Aeromonas caviae ME-1. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the xylanase IV molecular weight was 41,000. Xylanase IV catalyzed the hydrolysis of oat spelt xylan, producing exclusively xylotetraose. The acid hydrolysate of the product gave d-xylose. The enzyme did not hydrolyze either p-nitrophenyl-(beta)-d-xyloside, small oligosaccharides (xylobiose and xylotetraose), or polysaccharides, such as starch, cellulose, carboxymethyl cellulose, laminarin, and (beta)-1,3-xylan.     

Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli

A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, lambda gtWES lambda B, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated lambda FSX101 and lambda FSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from lambda FSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-beta-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the beta-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of beta-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan.     

Optimisation of culture medium and conditions for alpha-l-Arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus

The culture medium for Rhodothermus marinus was optimised on a shake-flask scale by using statistical factorial designs for enhanced production of a highly thermostable alpha-L-arabinofuranosidase (AFase). The medium containing 3.6 g/l birch wood xylan and 8.2 g/l yeast extract yielded a maximum of 110 nkat/ml AFase activity together with 125 nkat/ml xylanase and 65 nkat/ml beta-xylosidase activity. In addition, low levels of beta-mannanase (30 nkat/ml), alpha-galactosidase (0.2 nkat/ml), beta-galactosidase (0.3 nkat/ml), endoglucanase (5 nkat/ml) and beta-glucosidase (30 nkat/ml) were detected in the culture filtrate. Among the various carbon sources tested, birchwood xylan was most effective for the formation of AFase and xylanase activities, followed by oat spelt and beechwood xylans, and xylan-rich lignocelluoses (e.g., starch-free sugar beet pulp and wheat bran). Constitutive levels of enzyme activities were detected when the bacterium was grown on other polysaccharides and low-molecular-weight carbohydrates. A fermentation in a 5-l fermenter (3-l working volume) using the optimised medium yielded 60 nkat/ml AFase associated with 65 nkat/ml xylanase and 35 nkat/ml beta-xylosidase activities. The crude AFase displayed optimal activity between pH 5.5 and 7 and at 85 degrees C. It had half-lives of 8.3 h at 85 degrees C and 17 min at 90 degrees C. It showed high stability between pH 5 and 9 (24 h at 65 degrees C). The combined use of AFase-rich xylanase and mannanase from R. marinus in the prebleaching of softwood kraft pulp gave a brightness increase of 1.8% ISO. To our knowledge, this is the first report on the production of a high AFase activity by an extreme thermophilic bacterium and this enzyme is the most thermostable AFase reported so far.     

Purification and characterization studies of a thermostable β-xylanase from <Emphasis Type="Italic">Aspergillus awamori</Emphasis>

This study presents data on the production, purification, and properties of a thermostable β-xylanase produced by an Aspergillus awamori 2B.361 U2/1 submerged culture using wheat bran as carbon source. Fractionation of the culture filtrate by membrane ultrafiltration followed by Sephacryl S-200 and Q-Sepharose chromatography allowed for the isolation of a homogeneous xylanase (PXII-1), which was 32.87 kDa according to MS analysis. The enzyme-specific activity towards soluble oat spelt xylan, which was found to be 490 IU/mg under optimum reaction conditions (50°C and pH 5.0–5.5), was 17-fold higher than that measured in the culture supernatant. Xylan reaction products were identified as xylobiose, xylotriose, and xylotetraose. K _m values (mg ml⁻¹) for soluble oat spelt and birchwood xylan were 11.8 and 9.45, respectively. Although PXII-1 showed 85% activity retention upon incubation at 50°C and pH 5.0 for 20 days, incubation at pH 7.0 resulted in 50% activity loss within 3 days. PXII-1 stability at pH 7.0 was improved in the presence of 20 mM cysteine, which allowed for 85% activity retention for 25 days. This study on the production in high yields of a remarkably thermostable xylanase is of significance due to the central role that this class of biocatalyst shares, along with cellulases, for the much needed enzymatic hydrolysis of biomass. Furthermore, stable xylanases are important for the manufacture of paper, animal feed, and xylooligosaccharides.     

Novel ferulic acid esterases are induced by growth of Aspergillus niger on sugar-beet pulp

 A ferulic acid esterase (FAE-III), which was induced by growth of Aspergillus niger CBS 120.49 on oat-spelts xylan, was capable of releasing ferulic acid from wheat bran but not from sugar-beet pulp (SBP) [Faulds CB, Williamson G (1994) Microbiology 140:779–787]. Growth of this strain on SBP gave low levels of ferulic acid esterase activity (using methyl ferulate as substrate). A similar growth with a different A. niger strain (CS 180) gave tenfold higher levels of esterase activity. Assaying culture filtrates obtained from A. niger CS 180 grown on SBP over a 3 to 10-day period against four simple phenolic methyl esters demonstrated that at least two esterases were produced, and, by comparison of substrate specificity, FAE-III was either absent or present only at low levels. Furthermore, immunodetection of proteins did not detect the presence of FAE-III in culture supernatants of SBP-grown cultures, whereas it did in cultures grown on oat-spelts xylan. These results show that SBP does not contain the inducer for FAE-III, but does induce novel esterases. When A. niger CS 180 cultures were grown on different carbon sources, esterase activity was induced on SBP, sugar-beet arabinan and oat-spelts xylan, but not on simple sugars or de-esterified sugar-beet pectin. Further, SBP-grown cultures co-inoculated with arabinanase, galactanase or xylanase did not exhibit increased levels of extracellular FAE activity or an earlier appearance of esterase activity, although there was an increase in esterase activity with added polygalacturonase. These results show that novel esterases are induced by growth of A. niger on SBP. Received: 11 September 1995/Received revision: 5 December 1995/Accepted: 11 December 1995     

Stimulatory effect of ferulic acid on the production of extracellular xylanolytic enzymes by Aspergillus kawachii

Production of extracellular beta-1,4-xylanase, alpha-L-arabinofuranosidase, feruloyl esterase, and acetyl xylan esterase from Aspergillus kawachii was higher in a culture supplemented with ferulic acid than in a counterpart. Culture supernatant grown on oat spelt xylan supplemented with ferulic acid exhibited an increase in ferulic acid-releasing activity from insoluble arabinoxylan relative as compared to that from the ferulic acid-free culture.     

Construction of the trifunctional enzyme associating the Thermoanaerobacter ethanolicus xylosidase-arabinosidase with the Thermomyces lanuginosus xylanase for degradation of arabinoxylan

The trifunctional enzyme (XAR–XYN) associating the Thermoanaerobacter ethanolicus xylosidase-arabinosidase (XAR) with the Thermomyces lanuginosus xylanase (XYN) was produced in E. coli to study the effect of the physical association of the fusion partners on the enzymatic efficiency. Recombinant XAR, XYN and XAR–XYN were purified to homogeneity and characterized. The optimal pH and temperature of the XAR–XYN were found to be similar to those of the XAR and XYN, except for less temperature optimum of α-arabinosidase activity. Its pH and xylanase activity exhibited more stable than those of the XAR and XYN. Finally, the XAR–XYN was tested for degradation of oat spelt xylan and wheat bran, the XAR–XYN was found to be more facile than the corresponding free enzyme degradation of wheat bran but provided little or no advantage on purified xylan. Furthermore cooperation within a trifunctional enzyme containing linker SAGSSAAGSGSG between each partner was achieved, leading to a trifunctional enzyme with enhanced enzymatic efficiency on arabinoxylan.     

Effect of Carbon Source on Production of α-L-Arabinofuranosidase by Aureobasidium pullulans

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-L-arabinofuranosidase (α-L-AFase) when grown in liquid culture on sugar beet arabinan, wheat arabinoxylan, L-arabinose, L-arabitol, xylose, xylitol, oat spelt xylan, corn fiber, or arabinogalactan. L-Arabinose was most effective for production of both whole-broth and extracellular α-L-AFase activity, followed by L-arabitol. Oat spelt xylan, sugar beet arabinan, xylose, xylitol, and wheat arabinoxylan were intermediate in their ability to support α-L-AFase production. Lower amounts of enzyme activity were detected in corn fiber- and arabinogalactan-grown cultures. Received: 16 April 1998 / Accepted: 17 June 1998     

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum.

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.     

A non-cellulase producing strain of Bacillus subtilis and its potential use in pulp biobleaching

A newly isolated strain of Bacillus subtilis produced -mannanase when cultivated in a medium containing either locust bean gum, konjac mannan or guar gum as a sole carbon source. In contrast, xylanase was produced only when oat spelt xylan or wheat bran was used as a carbon source. The culture supernatant, which contained both -mannanase and xylanase, was used to biobleach crude paper pulp to 50% gain in brightness.     

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     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13

Two distinct xylanase genes (designated xynA and xynB) were subcloned in pUC13 from non-homologous restriction fragments of Ruminococcus flavefaciens 17 DNA originally isolated in lambda EMBL3. The products of the two genes showed similar pH optima for hydrolysis of oat spelt xylan (around 5.5) and had little or no activity against carboxymethylcellulose. Trace activities against p-nitrophenyl (pNP) cellobioside and pNP-xyloside were detected in clones containing xynA, but not in one harbouring xynB. The xylanase associated with clones carrying xynA produced mainly xylobiose and xylose from xylan and did not give hydrolysis of xylobiose, while that encoded by xynB produced mainly xylobiose and higher xylo-oligosaccharides from xylan. There was evidence of increased expression, at the RNA level, of these two genes, and of another cloned region encoding multiple activities including xylanase, in R. flavefaciens 17 grown with xylan, as compared with cellobiose, as energy source. Total cell-associated xylanase and beta-xylosidase activities, and supernatant xylanase activity, were shown to be similarly induced in xylan-grown R. flavefaciens, 17.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.