Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.     

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.     

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.     

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.     

Production of xylanase from a newly isolated <Emphasis Type="Italic">Penicillium</Emphasis> sp. ZH-30

A new strain of Penicillium sp. ZH-30 that produces xylanase was isolated from soil. According to the morphology and comparison of internal transcribed spacer (ITS) rDNA gene sequence, the strain Penicillium sp. ZH-30 was identified as a strain of Penicillium oxalicum. When xylan or wheat bran was used as substrate at 30°C for 3 days under submerged cultivation, xylanase production was 5.3 and 13.3 U ml⁻¹, respectively. The temperature and pH for optimum activity were 50°C and 5.0–6.0, respectively.     

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     

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.     

Production and characterization of xylanase from a Streptomyces species grown on agricultural wastes

Alkali-treated corn stalk gave maximum xylanase production at supporting growth of Streptomyces HM-15. Xylanase was stable for 24 h over a pH range of 5.0 to 7.0, had optimal activity between 50 and 60°C and a halflife of 5 h at 60°C. Xylanase production and activity were inhibited by xylose.The authors are with Department of Biosciences, Sardar Patel University. Vallabh Vidyanagar-388120, Gujarat, India.     

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.     

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.     

Production of sterigmatocystin by Aspergillus versicolor isolated from roughage

A total of 69 samples of hay and straw collected during the winter period of 1984/85 were surveyed for their contamination by Aspergillus versicolor. The percentage of A. versicolor-positive samples was 14.5%. Nineteen A. versicolor strains mainly isolated from roughage were tested for the production of sterigmatocystin. All of the isolates examined were capable of producing different levels of sterigmatocystin on a cracked corn substrate. The majority of these strains were highly toxigenic; 53% of the isolates produced more than 500 mg/kg of sterigmatocystin. These findings suggest that corn is a very suitable substrate for sterigmatocystin production and that particularly in the surface layers of feed stocks and corn silos such toxigenic strains of A. versicolor can produce considerable growth and possibly sterigmatocystin, too.     

Production and characterization of a xylanase from Cyathus stercoreus

Cyathus stercoreus grown on wheat straw had a higher xylanase activity than when it was grown on rice husk or extracted hemicellulose. Inclusion of casein hydrolysate, Tween 80 and Mn²⁺ (at 0.02%, 0.2% and 0.075%, respectively) increased the production of extracellular xylanase. Optimal yield of xylanase (0.73 U/ml) was at pH 5.6 after 9 to 12 days at 30°C. The xylanase was stable at pH 4.5 to 7.5 for 2h but above 50°C its stability fell sharply.The authors are with the Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India;     

Synthesis of itaconic acid from agricultural waste using novel Aspergillus niveus

Abstract

Filamentous fungi from the genus Aspergillus are of high importance for the production of organic acids. Itaconic acid (IA) is considered as an important component for the production of synthetic fibers, resin, plastics, rubber, paints, coatings, adhesives, thickeners and binders. Aspergillus niveus MG183809 was isolated from the soil sample (wastewater unit) which was collected from Avadi, Chennai, India. In the present study, itaconic acid was successfully produced by isolated A. niveus by submerged batch fermentation. In the fermentation process, various low-cost substrates like corn starch, wheat flour and sweet potato were used for itaconic acid production. Further, the factor influencing parameters such as substrate concentration and incubation period were optimized. Maximum yield of itaconic acid (15.65?±?1.75?g/L) was achieved by using A. niveus from corn starch at a concentration of 120?g/L after 168?hr (pH 3.0). And also extraction of itaconic acid from the fermentation was performed with 91.96?±?1.57 degree of extraction.     

<Emphasis Type="Italic">Acinetobacter</Emphasis> sp. strain Ths,a novel psychrotolerant and alkalitolerant bacterium that utilizes hydrocarbon

A novel psychrotolerant, alkalitolerant bacterium, strain Ths, was isolated from a soil sample immersed in hot spring water containing hydrocarbons and grown on a chemically defined medium containing n-tetradecane as the sole carbon source. The isolate grew at 0 degrees C but not at temperatures higher than 45 degrees C; its optimum growth temperature was 27 degrees C. It grew in the pH range of 7-9. The strain utilized C(13)-C(30) n-alkane and fluorene at pH 9 and 4 degrees C. To our knowledge, this is the first report on the bacterium that utilizes a wide range of hydrocarbons at a high pH and a low temperature. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ths is closely related to genomic species 6 ATCC 17979 (99.1% similarity), genomic species BJ13/TU14 ATCC 17905 (97.8% similarity), genomic species 9 ATCC 9957 (97.6% similarity) belonging to the genus Acinetobacter and to Acinetobacter calcoaceticus JCM 6842(T) (97.5% similarity). DNA-DNA hybridization revealed that the isolate has 62, 25, 18 and 19% relatedness, respectively, to genomic species 6 ATCC 17979, genomic species BJ13/TU14 ATCC 17905, genomic species 9 ATCC 9957 and A. calcoaceticus, respectively.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

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.     

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml⁻¹ at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml⁻¹).     

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus

Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.