Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

Effect of cultivation pH and agitation rate on growth and xylanase production by Aspergillus oryzae in spent sulphite liquor

The effects of cultivation pH and agitation rate on growth and extracellular xylanase production by Aspergillus oryzae NRRL 3485 were investigated in bioreactor cultures using spent sulphite liquor (SSL) and oats spelts xylan as respective carbon substrates. Xylanase production by this fungus was greatly affected by the culture pH, with pH 7.5 resulting in a high extracellular xylanase activity in the SSL-based medium as well as in a complex medium with xylan as carbon substrate. This effect, therefore, was not solely due to growth inhibition at the lower pH values by the acetic acid in the SSL. The xylanase activity in the SSL medium peaked at 199 U ml(-1) at pH 7.5 with a corresponding maximum specific growth rate of 0.39 h(-1). By contrast, the maximum extracellular beta-xylosidase activity pf 0.36 U ml(-1) was recorded at pH 4.0. Three low molecular weight xylanase isozymes were secreted at all pH values within the range of pH 4-8, whereas cellulase activity on both carbon substrates was negligible. Impeller tip velocities within the range of 1.56-3.12 m s(-1) had no marked effect, either on the xylanase activity, or on the maximum volumetric rate of xylanase production. These results also demonstrated that SSL constituted a suitable carbon feedstock as well as inducer for xylanase production in aerobic submerged culture by this strain of A. oryzae.     

Identification of three distinct Clostridium thermocellum xylanase genes by molecular cloning

Three genes coding for xylanase synthesis in Clostridium thermocellum were cloned and expressed in Escherichia coli. Genomic DNA from Clostridium thermocellum was digested to completion with HindIII, BamHI, and SalI. The fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Two of the genes encoded for xylanases which depolymerized xylans but were unable to extensively convert these substrates to reducing sugar. The third gene encoded for an enzyme that extensively hydrolyzed xylan. The insert containing the latter gene was subjected to extensive mapping and was found to encode for a xylanase with a molecular weight of approximately 25,000. The protein product of the cloned gene was obtained in a relatively pure form by heat treatment, ion exchange and gel permeation steps. The enzyme was quite stable to high temperatures with a half-life of 24 h at 70°C.Issued as National Research Council of Canada No. 30545     

海枣曲霉木聚糖酶的纯化及末端序列研究

海枣曲霉麸曲经水浸提、硫酸铵盐析、凝胶过滤、离子交换层析及ＨＰＬＣ分子排阻层析制备了ＰＡＧＥ,ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ酶谱及ＨＰＬＣ纯的木聚糖酶。     

海枣曲霉木聚糖酶降解寡聚木糖的特性

利用滤纸层析或ＡｃｒｙｌｅｘＰ－２凝胶过滤从落叶松木聚糖硫酸水解液中分离纯化子木二糖至木五糖。采用硅胶薄层层析分析底物和产物的方法研究了海枣霉木聚糖酶降解寡聚木糖的特点。此酶作用于寡糖的最适ＰＨ为５．０,终产物为Ｘ和Ｘ２。酶作用于Ｘ３、Ｘ４及Ｘ５的相对初速度分别为１、３４和４００,Ｘ２几乎不被酶解,推断该酶的底物结合部位至少具有５个亚位点,在高底物浓度,低酶量,远离最适ＰＨ以及在反应初期都能检测到     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Abstract Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase ( M _r 31000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 °C, respectively. pH stability ranged from 5 to 9.5 and the t_{1 / 2} at 50 °C was 490 min. It had a K _m of 4.88 mg ml⁻¹and a V _max of 588 μmol min⁻¹ mg⁻¹. The activity was inhibited (95%) by AlCl₃ (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.     

Purification and characterization of xylanase from Aspergillus ficuum AF-98

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH₄)₂SO₄, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7 U/ mg protein) was a monomeric protein with a molecular mass of 35.0 kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45 °C and 5.0, respectively. The xylanase was activated by Cu²⁺ up to 115.8% of activity, and was strongly inhibited by Hg²⁺, Pb²⁺ up to 52.8% and 89%, respectively. The xylanase exhibited K_m and V_max values of 3.267 mg/mL, 18.38 M/min/mg for beechwood xylan and 3.747 mg/mL, 11.1 M/min/mg for birchwood xylan, respectively.     

Aflatoxigenicity in Aspergillus: molecular genetics, phylogenetic relationships and evolutionary implications

Aflatoxins (AFs) are toxic and carcinogenic secondary metabolites produced by isolates of Aspergillus section Flavi as well as a number of Aspergillus isolates that are classified outside of section Flavi. Characterization of the AF and sterigmatocystin (ST) gene clusters and analysis of factors governing regulation of their biosynthesis has resulted in these two mycotoxins being the most extensively studied of fungal secondary metabolites. This wealth of information has allowed the determination of the molecular basis for non-production of AF in natural isolates of A. flavus and domesticated strains of A. oryzae. This review provides an overview of the molecular analysis of the AF and ST gene clusters as well as new information on an AF gene cluster identified in the non-section Flavi isolate, Aspergillus ochraceoroseus. Additionally, molecular phylogenetic analysis using AF biosynthetic gene sequences as well as ribosomal DNA internal transcribed spacer (ITS) sequences between various section Flavi and non-section Flavi species has enabled determination of the probable evolutionary history of the AF and ST gene clusters. A model for the evolution of the AF and ST gene clusters as well as possible biological roles for AF are discussed.     

Enzymatic bleaching of kraft pulp by xylanase from Aspergillus sydowii SBS 45

Crude xylanase from Aspergillus sydowii SBS 45 was tested for enzymatic bleaching of kraft (Decker) pulp. After optimization of three parameters, consistency of pulp, retention time and enzyme dose, considerable increase in the release of UV and visible absorbance spectra of materials and reducing sugars was observed, which clearly indicated the action of xylanase on pulp. Final brightness of pulp was increased from 29.42 to 70.42% and kappa number was reduced from 15.93 to 1.61, when 25 U of xylanase was given with a retention time of 5 h and at a consistency of 10%. When 10 U g⁻¹ xylanase was given, 14.3% elemental chlorine and 14.3% H₂O₂ could be reduced and when 25 U g⁻¹ xylanase was given 14.3% elemental chlorine and 28.6% H ₂O₂ could be reduced thereby retaining the brightness at control level.     

Cloning and molecular characterisation of the amdR controlled gatA gene of Aspergillus nidulans

Summary The gamma-amino-n-butyrate transaminase gene (gatA) of Aspergillus nidulans is one of several genes under positive control by the regulatory gene amdR (also called intA). The gatA gene has been cloned from a cosmid library by complementation of a gatA mutation. The sequence of a 2.6 kb genomic fragment containing gatA has been determined. An open reading frame of 1497 bp within this sequences is interrupted by three putative introns and predicts a protein of 55 kDa. Northern analysis confirms control of gatA RNA levels by amdR and also indicates that gatA is not strongly regulated by areA-mediated nitrogen metabolite repression. A. nidulans transformants containing multiple copies of a plasmid carrying an 88 bp fragment from the 5 untranscribed region of gatA grew poorly on substrates whose utilisation is dependent on genes controlled by amdR. This indicated titration of limiting amounts of the amdR gene product by this 88 bp fragment. Comparison of this sequence with the 5 region of the coregulated gene, amdS, reveals probable sites of action for the amdR protein.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

Regulation of gene expression by pH of the growth medium in Aspergillus nidulans

Summary In the fungus Aspergillus nidulans the levels of a number of enzymes whose location is at least in part extracellular (e.g. acid phosphatase, alkaline phosphatase, phosphodiesterase) and of certain permeases (e.g. that for -amino-n-butyrate) are controlled by the pH of the growth medium. For example, at acidic pH, levels of acid phosphatase are high and those of alkaline phosphatase are low whereas at alkaline pH the reverse is true. Mutations in five genes, palA, B, C, E and F, mimic the effects of growth at acid pH whereas mutations in pacC mimic the effects of growth at alkaline pH. palA, B, C, E and F mutations result in an intracellular pH (pH_in) which is more alkaline than that of the wild type whereas pacC mutations result in a pH_in more acidic than that of the wild type. This indicates that these mutations exert their primary effects on the regulation of gene expression by pH rather than on the pH homeostatic mechanism but that the expression of at least some component(s) of the pH homeostatic mechanism is subject to the pH regulatory system. It is suggested that pacC might be a wide domain regulatory gene whose product acts positively in some cases (e.g. acid phosphatase) and negatively in others (e.g. alkaline phosphatase). The products of palA, B, C, E and F are proposed to be involved in a metabolic pathway leading to synthesis of an effector molecule able to prevent the (positive and negative) action of the pacC product.These genes are, to our knowledge, the first examples of genes involved in the regulation of extracellular enzyme and permease synthesis by the pH of the growth medium to be described in any organism.     

Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan

The mature peptide of Aspergillus niger xylanase A (AnxA) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant AnxA (reAnxA) was secreted into culture medium. After 96-h 0.25% methanol induction, the activity of reAnxA in the culture supernatant reached the peak, 175 U/mg, which was 1.9 times as high as that of the native AnxA (92 U/mg). Studies on enzymatic properties showed that the optimum temperature and optimum pH of reAnxA were 50 degrees C and 5.0, respectively. The reAnxA was very stable in a wide pH range of 3.0-8.0. After incubation at the pH 3.0-8.0, 25 degrees C for 1h, all the residual activities of reAnxA were over 80%. The K(m) and k(cat) values for reAnxA were 4.8 mg/ml and 123.2s(-1), respectively. HPLC analysis showed that xylotriose was the main hydrolysis product of birchwood xylan and bran insoluble xylan by reAnxA.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Optimized expression of an acid xylanase from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its biochemical characterization

The xylanase gene xyn II from Aspergillus usamii E001 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K and integrated into the genome of a methylotrophic yeast, P. pastoris GS115, by electroporation. His⁺ transformants were screened for on the basis of their resistance to G418 and activity assay. A transformant, P. pastoris GSC12, which showed resistance to over 6 mg G418/ml and highest xylanase activity was selected. Recombinant xylanase was secreted by P. pastoris GSC12 24 h after methanol induction of shake-flask cultures, and reached a final yield of 3139. About 68 U/mg 120 h after the induction. The molecular mass of this xylanase was estimated to be 21 kDa by SDS-PAGE. The optimum pH and temperature were 4.2 and 50 °C, respectively. Xylanase was stable below 50 °C and within pH 3.0–7.0. Its activity was increased by EDTA and Co²⁺ ion and strongly inhibited by Mn²⁺, Li⁺ and Ag⁺ ions. The K _m and V _max values with birchwood xylan as the substrate were found to be 5.56 mg/ml and 216 μmol/mg/min, respectively. This is the first report on expression and characterization of xylanase from A. usamii in P. pastoris. The hydrolysis products consisted of xylooligosaccharides together with a small amount of xylose. This property made the enzyme attractive for industrial purposes, as relatively pure xylooligosaccharides could be obtained.     

Optimization of biobleaching of paper pulp in an expanded bed bioreactor with immobilized alkali stable xylanase by using response surface methodology

Purified alkali stable xylanase from Aspergillus fischeri was immobilized on polystyrene beads using diazotization method. An expanded bed bioreactor was developed with these immobilized beads to biobleach the paper pulp in continuous mode. Response surface methodology was applied to optimize the biobleaching conditions. Temperature (degrees C), flow rate of pulp (ml/min) and concentration of the pulp (%) were selected as variables in this study. Optimal conditions for biobleaching process were reaction temperature 60 degrees C, flow rate of 2 ml/min and 5% (w/v) of pulp. The kappa number reduced from 66 in the unbleached pulp to 20 (reduction of 87%). This system proves to be a better option for the conventional chlorine based pulp bleaching.     

Regulation of the xylanase gene, cgxA, from Chaetomium gracile by transcriptional factors, XlnR and AnRP

The roles of XlnR and AnRP in regulating the expression of the xylanase gene, cgxA, from Chaetomium gracile were investigated using Aspergillus nidulansas an intermediate host. The XlnR consensus binding sequence –GGCTAA– in the promoter region was functional in vivo. The cgxA gene was induced when xylan was used as a carbon source but this inducibility was abolished when the XlnR binding sequence was mutated. Furthermore, the induction by xylan was increased when the AnRP binding sequence –TTGACAAAT– was mutated. Electrophoretic mobility shift assays using partially purified AnRP and an Aspergillus oryzae XlnR fusion protein, MalE-AoXlnR, provided evidence that the binding of the two proteins to their respective sites in the cgxA promoter region was mutually exclusive.