Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and beta-xylosidase while maintaining low protease production

A growth medium was developed for maximal production in batch culture of extracellular xylanase and beta-xylosidase by Aspergillus awamori CMI 142717 and a mutant (AANTG 43) derived from the wild-type strain. The optimum pH for the production of xylanase and beta-xylosidase was 4.0. The best temperature of xylanase production was 30 degrees C; 35 degrees C was optimal for beta-xylosidase. Protease production was never completely suppressed under any of the conditions tested. However, protease titre was 3.5-fold less than the control in medium in which proteose peptone and yeast extract were omitted: the level of xylanase was not affected (8.6 U mL(-1)) but beta-xylosidase titre was increased 4.7-fold to 1.5 U mL(-1). When corn steep liquor was used as the sole nitrogen source, xylanse and beta-xylosidase titres were further increased by 1.5- and 1.9-fold, respectively. Of the carbon sources investigated, ball-milled oat straw or oat spelt xylan produced the highest titres of xylanse and beta-xylosidase. None of the soluble carbon sources investigated produced the high titres of xylanase or beta-xylosidase induced by either oat straw for xylanse and beta-xylosidase was 2% and the optimum spore inoculum was between 10(6) and 10(7) spores/mL(-1) final concentration. The level of xylanse activity obtained in the culture filtrates of the mutant was a remarkable 820 U mL(-1) when the reducing sugar released was measured by the dinitrosalicylic acid method. This enzyme titre would appear to be the highest reported so far. The xylanases system contained the correct balance of enzymes to effect extensive hydrolysis of oat spelt xylan. The protease titre was very low.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain.

We isolated a thermophilic bacterium that produces both xylanase and beta-xylosidase. Based on taxonomical research, this bacterium was identified as Bacillus stearothermophilus. Each extracellular enzyme was separated by hydrophobic chromatography by using a Toyopearl HW-65 column, followed by gel filtration with a Sephacryl S-200 column. Each enzyme in the culture was further purified to homogeneity (62-fold for xylanase and 72-fold for beta-xylosidase) by using a fast protein liquid chromatography system with a Mono Q HR 5/5 column. The optimum temperatures were 60 degrees C for xylanase and 70 degrees C for beta-xylosidase. The isoelectric points and molecular masses were 5.1 and 39.5 kDa for xylanase and 4.2 and 150 kDa for beta-xylosidase, respectively. Heat treatment at 60 degrees C for 1 h did not cause inhibition of the activities of these enzymes. The action of the two enzymes on xylan gave only xylose.     

A role of xylanase,alpha-L-arabinofuranosidase,and xylosidase in xylan degradation

Renewable natural resources such as xylans are abundant in many agricultural wastes. Penicillium sp. AHT-1 is a strong producer of xylanolytic enzymes. The sequential activities of its xylanase, alpha-L-arabinofuranosidase, and beta-xylosidase on model hemicellulose oat-spelt xylan was investigated. Optimum production of the enzymes was found in culture containing oat-spelt xylan at 30 degrees C and initial pH 7.0 after 6 days. The enzymes were partially purified by ammonium sulphate fractionation and anion-exchange chromatography on DEAE-Toyopearl 650 S. The apparent molecular mass was 21 kDa, and the protein displayed an "endo" mode of action. The xylanase exhibited glycotansferase activity. It synthesized higher oligosaccharides from the initial substrates, and xylotriose was the shortest unit of substrate transglycosylated. Xylanolytic enzymes (enzyme mixture) produced by this Penicillium sp. interacted cooperatively and sequentially in the hydrolysis of oat-spelt xylan in the following order: alpha-L-arabinofuranosidase --> xylanase --> beta-xylosidase. All three enzymes exhibited optimal activity under the same conditions (temperature, pH, cultivation), indicating that they alone are sufficient to completely depolymerize the test xylan. Results indicate that the xylanolytic enzyme mixture of Penicillium sp. AHT-1 could be useful for bioconversion of xylan-rich plant wastes to value-added products.     

Optimization of xylanase production from Aspergillus niger for biobleaching of eucalyptus pulp

A crude endo-xylanase produced by Aspergillus niger BCC14405 was investigated for its potential in pre-bleaching of chemical pulp from eucalyptus. The optimal fermentation conditions on the basis of optimization using response surface methodology included cultivation in a complex medium comprising wheat bran, rice bran, and soybean meal supplemented with yeast extract, glucose, peptone, and lactose with a starting pH of 6.0 for 7 d. This resulted in production of 89.5 IU/mL of xylanase with minor cellulase activity. Proteomic analysis using LC/MS/MS revealed that the crude enzyme was a composite of hemicellulolytic enzymes, including endo-β-1,4-xylanase and other hemicellulolytic enzymes attacking arabinoxylan and mannan. Pretreatment of the pulp at a xylanase dosage of 10 IU/g increased the brightness ceiling after the C-Eop-H bleaching step up to 3.0% using a chlorine charge with a C-factor of 0.16-0.20. Xylanase treatment also led to reduction in chlorine charge of at least 20%, with an acceptable brightness level. The enzyme pretreatment resulted in a slight increase in pulp viscosity, suggesting an increase in relative cellulose content. The crude enzyme was potent in the enzyme-aided bleaching of chemical pulp in an environmentally friendly pulping process.     

A novel thermostable xylanase from Thermomonospora sp.: influence of additives on thermostability

An alkalothermophilic Thermomonospora sp. producing high levels of xylanase was isolated from self-heating compost. The culture produced 125 IU/ml of xylanase when grown in shake flasks at pH 9 and 50 degrees C for 96 h. The culture filtrate also contained cellulase (23 IU/ml), mannanase (1 IU/ml) and beta-xylosidase (0.1 IU/ml) activities. The xylanase was active at a broad range of pH (5-9) and temperature (40-90 degrees C). The optimum pH and temperature were 7 and 70 degrees C, respectively. The enzyme was stable in the pH range 5-8 and was thermostable with half-lives of 8 and 4 h at 60 degrees C and 70 degrees C, respectively, but only 9 min at 80 degrees C. The effects of a variety of compounds to enhance the stability of xylanase at 80 degrees C was studied. Addition of sorbitol, mannitol and glycerol increased the thermostability of xylanase in proportion to the number of hydroxyl groups per polyol molecule. Glycine also offered protection against thermoinactivation. Xylan, trehalose, gelatin and trehalose-gelatin mixture had marginal effect on the thermostability of xylanase at 80 degrees C.     

A novel cold-tolerant Clostridium strain PXYL1 isolated from a psychrophilic cattle manure digester that secretes thermolabile xylanase and cellulase

A Clostridium strain PXYL1 was isolated from a cold-adapted cattle manure biogas digester at 15 degrees C. It could grow at temperatures as low as 5 degrees C up to 50 degrees C with highest specific growth rate at 20 degrees C and is a psychrotroph. It produced extracellular hydrolytic enzymes namely xylanase, endoglucanase, beta-xylosidase, beta-glucosidase and filter paper cellulase, all of which had maximal activity at 20 degrees C. The induction of xylanase was highest on birch wood xylan (37 IU(mg protein)(-1)) compared with xylose (1.11 IU(mg protein)(-1)), cellobiose (1.43 IU(mg protein)(-1)) and glucose (no activity). The xylanase was thermolabile with a half-life of 30 min at 40 degrees C and 8 min at 50 degrees C but stable for over 2 h at 20 degrees C. The crude enzyme released reducing sugars (1.25 g l(-1)) from finger millet flour at 20 degrees C, while commercial food-grade xylanases showed no hydrolysis at this temperature. This is the first report of a Clostridium strain growing at 20 degrees C and producing an array of xylanolytic and cellulolytic enzymes, possessing low temperature optima of 20 degrees C, which may facilitate degradation of plant fibre under low-temperature conditions.     

alpha-L-Arabinofuranosidase from Streptomyces sp. PC22: purification, characterization and its synergistic action with xylanolytic enzymes in the degradation of xylan and agricultural residues

alpha-l-Arabinofuranosidase was purified from culture filtrates of the thermoalkaliphilic Streptomyces sp. PC22 to about 108-fold purity by (NH(4))(2)SO(4) precipitation followed by column chromatography. Its approximate molecular weight was 404kDa, with a subunit mass of approximately 79kDa. The evaluated K(m) and V(max) values with p-nitrophenyl-alpha-l-arabinofuranoside as substrate were 0.23mM and 124 U.mg(-1), respectively. The purified enzyme was optimally active at 65 degrees C and pH 6.0 and showed a mild but significant synergistic effect in combination with other xylanolytic enzymes, including xylanase, beta-xylosidase and acetyl esterase, on the degradation of oat-spelt xylan, corn cob and corn husk substrates with a 1.25, 1.32 and 1.21-fold increase in the amount of reducing sugar released, respectively, compared to the expected (additive) amounts for the individual enzymes acting alone. Sequential reactions using two xylan-backbone degrading enzymes (xylanase/beta-xylosidase) and two debranching enzymes (alpha-l-arabinofuranosidase/acetyl esterase) were also determined. The highest degree of synergy was obtained in sequential reactions with the debranching enzyme digestion preceding the xylan-backbone degrading enzymes.     

Purification and characterization of an extracellular β-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis

The purification of beta-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Neocallimastix frontalis was performed by ammonium sulphate precipitation, ion exchange chromatography, gel filtration and preparative isoelectric focusing. The enzyme had a molecular mass of 180,000 Da, an isoelectric point at pH 4.35 and catalysed the hydrolysis of p-nitrophenyl-beta-D-xylopyranoside optimally at pH 6.5 and 35 degrees C with a Km of 0.33 mg ml-1. The enzymatic activity was strongly increased by the presence of Ca2+, Mn2+, Zn2+, Co2+ or Mg2+ and completely inhibited by Hg2+ and p-chloromercuribenzoate. The purified protein also had a low level of xylanase activity.     

酸性木聚糖酶产生菌的筛选及产酶条件

从１５０株真菌中筛选到８株产木聚糖酶活力在１００Ｕ／ｍＬ以上的菌株,其中活力最高的为黑曲霉（编号１４９）（Ａｓｐｅｒｇｉｌｕｓｎｉｇｅｒ）。该菌株产酶较适培养基为：麸皮半纤维素４％,ＮａＮＯ３１％,麸皮１％,用不加（ＮＨ４）２ＳＯ４和尿素的Ｍａｎｄｅｌｓ氏营养盐液配制。２８℃～３０℃振荡培养６０ｈ,酶活力最高可达３７５．２Ｕ／ｍＬ。该酶最适作用ｐＨ为４６,在ｐＨ３～１１之间基本稳定。该菌株发酵液中含有木聚糖酶（相对活力１００）外还有淀粉酶（１８）,甘露聚糖酶（０９８）,β木糖苷酶（０９４）和纤维素酶（０１７）。     

Properties of thermostable hemicellulolytic enzymes from Thermomonospora strain 29 grown in solid state fermentation on coffee processing solid waste

During decaffeination of Coffee Processing Plant Solid Wastes (CPSW) by actinomycetes, Thermomonospora, Strain 29 exhibited high titers of cellulase and xylanase. This organism, originally isolated on soybean seed coat was grown in solid state fermentation on CPSW supplemented with mineral salts. Enzymes recovered were arabinosidase, xylanase, and beta-D-xylosidase. Higher activity of the former two enzymes was in the extracellular broth, whereas the beta-D-xylosidase activity was highest in the cell fraction. The enzymes were characterized after precipitation with (NH(4))(2)SO(4), dialysis, and gel filtration. Production of all three enzymes was inhibited by monomeric sugars and sugar alcohols but not by arabinoxylan, xylans, or xylan containing water insoluble carbohydrates. The optimum pH for the activity was 6.5, 7.0, and 7.5 for beta-xylosidase, xylanase and arabinosidase (alpha-L-arabinofuranosidase, alpha-arabinosidase, alpha-L-arabinosidase) respectively. These enzymes were stable in the pH range of 6.5 to 8.0. All three enzymes were thermostable up to 80 degrees C. At 55 degrees C, arabinosidase had the longest half life of 120 h. However, at 40 degrees C, xylanase had the longest half life (504 h). At either temperature, beta-D-xylosidase had the shortest half life. The molecular weights (kDa), and Kms (mM) were estimated to be 95, 0.27; 45, 12.4; and 106, 0.67 for arbinosidase, xylanase, and beta-xylosidase respectively. Step wise addition of the three enzymes showed higher saccharification of lignocellulosics.     

Hydrolysis of xylan at high temperature by co-action of the xylanase from Anoxybacillus flavithermus BC and the beta-xylosidase/alpha-arabinosidase from Sulfolobus solfataricus Oalpha

AIMS: It is evaluated the effectiveness of the combined action of two highly thermostable enzymes for the hydrolysis of xylans at high temperature in order to produce D-xylose. METHODS AND RESULTS: Xylans from different sources were hydrolyzed at high degree at 70 degrees C by co-action of a xylanase from the thermophilic bacterium Anoxybacillus flavithermus BC and the novel beta-xylosidase/alpha-arabinosidase from the hyperthermophilic crenarchaeon Sulfolobus solfataricus Oalpha. Beechwood xylan was the best substrate among the xylans tested giving, by incubation only with xylanase, 32.8 % hydrolysis after 4 h. The addition of the beta-xylosidase/alpha-arabinosidase significantly improved the rate of hydrolysis, yielding 63.6% conversion after 4 h incubation, and the main sugar identified was xylose. CONCLUSIONS: This study demonstrates that a significant degree of xylan degradation was reached at high temperature by co-action of the two enzymes. Xylose was obtained as a final product in considerable yield. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the xylan represents the second most abundant polysaccharide in nature, it still doesn't have significant utilization for the difficulties encountered in its hydrolysis. Its successful hydrolysis to xylose in only one stage process could make of it a cheap sugar source and could have an enormous economic potential for the conversion of plant biomass into fuels and chemicals.     

Application of thermostable xylanase of Dictyoglomus sp. in enzymatic treatment of kraft pulps

Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improved pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90°C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80°C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.Correspondence to: M. Rättö     

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.     

Enzymic saccharification of sugarcane bagasse pretreated by autohydrolysis-steam explosion

Pretreatment of bagasse by autohydrolysis at 200 degrees C for 4 min and explosive defibration resulted in the solubilization of 90% of the hemicellulose (a heteroxylan) and in the production of a pulp that was highly susceptible to hydrolysis by cellulases from Trichoderma reesei C-30 and QM 9414, and by a comercial preparation, Meicelase. Saccharification yields of 50% resulted after 24 h at 50 degrees C (pH 5.0) in enzymic digests containing 10% (w/v) bagasse pulps and 20 filter paper cellulase units (FPU). Saccharifications could be increased to more than 80% at 24 h by the addition of exogenous beta-glucosidase from Aspergillus niger. The crystallinity of cellulose in bagasse remained unchanged following autohydrolysis-explosion and did not appear to hinder the rate or extent of hydrolysis of cellulose. Autohydrolysis-exploded pulps extracted with alkali or ethanol to remove lignin resulted in lowere conversions of cellulose (28-36% after 25 h) than unextracted pulps. Alkali extracted pulps arising from autohydrolysis times of more than 10 min at 200 degrees C were less susceptible to enzymic hydrolysis than unextracted pulps and alkali-extracted pulps arising from short autohydrolysis times (e.g., 2 min at 200 degrees C). Autohydrolysis-explosion was as effective a pretreatment method as 0.25M NaOH (70 degrees C/2 h) both yielded pulps that resulted in high cellulose conversions with T. reesei cellulase preparations and Meicelase. Supplementation of T. reesei C-30 cellulose preparations with A. niger beta-glucosidases was effective in promoting the conversion of cellulose into glucose. A ration of FPU to beta-glucosidase of 1:1.25 was the minimum requirement to achieve more than 80% conversion of cellulose into glucose within 24 h. Other factors which influenced the extent of saccharification of autohydrolysis-exploded bagasse pulps were the enzyme-substrate ratio, the substrate concentration, and the saccharification mode.     

Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by<Emphasis Type="Italic">Mucor indicus</Emphasis> MTCC 6333: Purification and characterization of phytase

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.     

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.     

Mode of action and properties of xylanase and beta-Xylosidase from Neurospora crassa

Extracellular beta-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from culture filtrates of Neurospora crassa was purified to homogeneity by preparative isoelectric focusing followed by gel electrophoresis. The molecular weight of the purified xylosidase was 83,000 D and the K(m) on p-nitrophenyl-beta-D-xyloside was 0.047mM. The homogeneous xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) and beta-xylosidase showed differences in their mode of action towards xylooligosaccharides. The degree of hydrolysis of D-xylan by xylanase of N. crassa was 18%. Supplementation of beta-xylosidase from the same organism resulted in 48% hydrolysis. The synergistic effect was more pronounced, with the hydrolysis of 68%, when a homogeneous preparation of beta-xylosidase from Sclerotium rolfsii was added to the saccharification system.     

Isolation of two beta-xylosidase genes of Bacillus pumilus and comparison of their gene products

The chromosomal DNA fragments of Bacillus pumilus IPO, a potent xylan-hydrolyzing bacterium, were ligated to a vector plasmid, pBR322, and used to transform Escherichia coli C600 cells. Two hybrid plasmids, pOXD28 and pOXN29, were found to enable the transformants to produce beta-xylosidase. The former was found to contain a 2.6-MDa Bg/II fragment and the latter, a 7.7-MDa PstI fragment, both coding beta-xylosidase, but xylanase is coded only on the latter hybrid plasmid. The DNAs inserted in both plasmids originated from the B. pumilus chromosome, but from different regions, as shown by Southern hybridization and the analysis of restriction fragments. beta-Xylosidases I and II, coded on pOXN29 and pOXD28 respectively, were purified to homogeneous preparations and compared. Both were dimer enzymes consisting of 65000-70000-Da subunits. Specific activity and the Km value of beta-xylosidase I to p-nitrophenyl beta-D-xyloside as substrate were respectively 100 and 1/40 times those of beta-xylosidase II. The mobilities of beta-xylosidases I and II on polyacrylamide gel electrophoresis were also different. beta-Xylosidase I, the gene of which is located near the xylanase gene on pOXN29, can convert xylooligosaccharides to xylose, but beta-xylosidase II had little activity on xylobiose. These results suggest that beta-xylosidase I is the main enzyme for xylan hydrolysis in B. pumilus.