Xylanolytic enzyme production by anAspergillus niger isolate

Production of xylanolytic enzymes by anAspergillus niger CCMI 850 isolate was investigated in batch cultures. The effect of the composition of a fermentation medium that did not include chemical inducers, on β-xylanase, β-xylosidase, α-l-arabinofuranosidase, and total cellulase activity was studied. With 4% xylan as the carbon source, about 65 U/mL of β-xylanase was obtained, whereas the total cellulase activity was undetectable, under the specified conditions. This β-xylanase activity represents the highest reported for a wild-type strain ofA. niger. The effect of pH and temperature on the activity of β-xylanase was studied. Partial characterization of the β-xylanase showed that with insoluble birchwood as substrate theK _m andV _max were 0.3 mM and 19 μmol/min, respectively. Aspects of using the crude β-xylanase preparation for applications in the pulp and paper industry were discussed.     

Synthesis of neoproteoglycans using the transglycosylation reaction as a reverse reaction of endo-glycosidases

A method for the synthesis of carbohydrate chains (glycosaminoglycans) and their coupling to peptides was investigated using proteoglycans. Glycosidases generally catalyze a hydrolytic reaction, but can also mediate the reverse reaction, which in this case is a transglycosylation. In the transglycosylation reaction of bovine testicular hyaluronidase, which is an endoglycosidase, glycosaminoglycans (hyaluronan and chondroitin sulfates) release disaccharide (uronic acid-N-acetylhexosamine) moieties from non-reducing terminal sites, and then the liberated disaccharides are transferred immediately to the non-reducing termini of other glycosaminoglycan chains. Using such continuous reactions, it is possible to synthesize glycosaminoglycan chains according to a specific design. It then becomes possible to transfer glycosaminoglycan chains synthesized on a peptide to other peptides using the transglycosylation reaction of endo-β-xylosidase acting on the linkage region between a peptide and glycosaminoglycan chains of proteoglycans. We believe this approach will open a new field for the synthesis of homogeneous proteoglycans or their corresponding analogues.(Communicated by Takao SEKIYA, M.J.A.).     

Xylanase production byAspergillus tamarii

Aspergillus tamarii has been found to grow well and to produce high cellulase-free xylanase activity when growing on corn cob powder as the principal substrate. Maximum xylanase production (285-350 U/mL) was obtained when the strain was grown in media supplemented with high corn cob concentration (5-8%, w/v) for 5 d. The presence of constitutive levels of xylanase was detected in cultures with glucose as the carbon source. Zymogram analysis for detection of xylanase activity after electrophoresis in polyacrylamide gels has shown thatA. tamarii produces at least two xylanases under the conditions utilized. The hydrolysis patterns of xylan demonstrated that the xylanases were endoenzymes, yielding mainly xylobiose, xylotriose, and higher xylooligosaccharides with traces of xylose.     

β-Xylosidase recovery by reversed micelles

β-Xylosidase, an enzyme produced by Penicillium janthinellum fungus, was prepurified by fractionated precipitation with ethanol and extracted by reversed micelles of N-benzyl-N-dodecyl-N-bis(2-hydroxyethyl) ammonium chloride (BDBAC) cationic surfactant. A 2^5-1 fractional factorial design was employed to evaluate the influence of the following factors on the enzyme extraction: pH (A), conductivity (B), surfactant concentration (C), cosolvent concentration (D) and temperature (E). A statistical analysis of the results revealed that, of the five variables studied, pH, surfactant concentration, and temperature had significant main effects (p<0.05) on the recovery of β-xylosidase (Y). However, the interactions between pH and temperature, conductivity and cosolvent concentration, conductivity and temperature, BDBAC concentration and temperature also had significant influences. A first-order model (R ²=0.98) expressed by the equation Y=7.73−5.55A−0.67B+3.49C−0.41D+5.26E+2.05AB−3.26AC+0.96AD−7.07AE−3.93BD−2.19BE+0.99CD+0.78CE was proposed to represent the enzyme recovery as a function of the effects that were really significant. This model predicts a recovery value of about 40%, which is similar to that obtained experimentally (39.5%).     

Kinetic method for the study of xylan hydrolysis by xylan hydrolases

Summary The Somogyi-Nelson colorimetric method was used in a new manner more suitable for evaluating the kinetics of the enzyme hydrolysis of xylan catalyzed by xylan hydrolases. The values of the Michaelis parameters (Km=5.56 g l^–1 andV=2.94 · 10^–5 M s^–1) were determined.

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Eine kinetische Methode zur Untersuchung der Hydrolyse von Xylan durch Xylan-Hydrolase

Zusammenfassung Die kolorimetrische Methode nach Somogyi-Nelson wurde nach einem neuen Verfahren zur Verfolgung der Kinetik der hydrolytischen Spaltung von Xylan, katalysiert durch Xylan-Hydrolasen vonAspergillus oryzae, angewandt. Es wurden die Michaelis-Parameter (Km=5.56 g l^–1 undV=2.94 · 10^–5 M s^–1) bestimmt.

     

Talaromyces thermophilus β-d-Xylosidase: Purification, Characterization and Xylobiose Synthesis

When grown on wheat bran as the only carbon source, the filamentous fungus Talaromyces thermophilus produces large amounts of beta-xylosidase activity. The presence of glucose drastically decreases the beta-xylosidase production level. The beta-xylosidase of T. thermophilus was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration (high-performance liquid chromatography). The molecular mass of the enzyme was estimated to be 97 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The enzyme activity was optimum at 50 degrees C and pH 7. The apparent Michaelis constant K(m) of the beta-xylosidase was 2.37 mM for p-nitrophenyl-beta-D-xylopyranoside, with a V(max) of 0.049 micromol min(-1) per milligram protein. Enzyme activity was inhibited by Cu(2+), Hg(2+), and Zn(2+) and activated by Ca(2+), Mn(2+), and Co(+) at a concentration of 5 mM. At high xylose concentration, this enzyme catalyses the condensation reaction leading to xylobiose production.     

Screening of variables in β-xylosidase recovery using cetyl trimethyl ammonium bromide reversed micelles

β-Xylosidase recovery by micelles using cetyl trimethyl ammonium bromide (CTAB) cationic surfactant was verified under different experimental conditions. A 2⁵⁻¹ fractional factorial design with center points was employed to verify the influence of the following factors on enzyme extraction: pH (x ₁), CTAB concentration (x ₂), electrical conductivity (x ₃), hexanol concentration (x ₄), and butanol concentration (x ₅). Statistical analysis of the results shows that of the fivevariables studied only hexanol and electrical conductivity did not have significant effects on the recovery of β-xylosidase. The other factors had significant effects in increasing order: (x ₁)>(x ₂)>(x ₅). The model predicts a recovery value of about 45%, which is similar to that obtained experimentally (43.5%).