Niger Cellulase Research Articles

Enhanced Elution of Sperm from Cotton Swabs Via Enzymatic Digestion for Rape Kit Analysis*

This report describes development of a method for enhanced cell elution from cotton swabs. The method exploits an enzyme mixture for digestion of the cotton to remove intact cells, and can be utilized in conjunction with or to circumvent conventional differential extraction (DE). Samples digested with Aspergillus niger cellulase yielded sperm cell recoveries (18+/-3.5%) similar to conventional DE buffer (23+/-7.8%) while providing intact epithelial cells. Storage time affected the concentration of enzyme required for optimal sperm cell recovery, with longer times requiring increased cellulase concentrations. Cellulase from A. niger yielded a twofold enhancement in sperm cell elution over buffer alone, and preliminary testing of higher activity cellulases from Trichoderma reesei and Trichoderma viride showed even greater enhancement. These results indicate that cellulose-digesting enzymes enhance the release of sperm and epithelial cells from a cotton swab over buffer alone, providing for efficient DNA analysis.

Physical and kinetic properties of the family 3 beta-glucosidase from Aspergillus niger which is important for cellulose breakdown.

A beta-glucosidase (BGS) purified from Aspergillus niger cellulase powder (obtained from Sigma, St. Louis, MO, USA) was characterized. Electrophoresis, size exclusion chromatography, and dynamic light scattering indicated that the enzyme is a dimer of approximately 200 kDa. Five of the seven N-glycosylated oligosaccharides attached to BGS were composed of D-mannoses attached to a beta(1-4)-N-acetyl-glucosamine-beta-(1-4)-fucose-alpha-(1-6)-N-acetylglucosamine core. The other two were similar, but the cores of these did not have the D-fucose. The enzyme is a retaining glycosidase, and it also has a distinct preference for the beta-configuration at the reducing end of cellobiose. BGS is thermostable up to 65 degrees C but is sensitive to freezing and thawing. The extinction coefficient of BGS was found to be 1.8 cm(-1) mg(-1). All substrates assayed resulted in Eadie-Hofstee plots that were curved at high substrate concentrations. TLC of the reaction products showed that the substrates themselves act as acceptors when present at high concentrations. The transglucosidic activity rate is different from the hydrolytic activity rate and this causes the curvature at high substrate concentrations. The enzyme produces gentiobiose when D-glucose is the acceptor. pH optima of the Vmax(h) with pNPGlc, oNPGlc, and cellobiose were between pH 4 and 4.5, and the Km values decreased at pH values between 3 and 5. Inhibition experiments indicated that the enzyme is specific for glucosyl substrates and suggested that D-gluconolactone is a transition state analog. Studies with cello-oligosaccharides and 3,4-dinitrophenyl-cellobiose showed that BGS is an exo-hydrolase having at least five glucose subsites and that it cleaves from the nonreducing end. The properties of a family 3 beta-glucosidase (BG3) sequenced by Dan et al. [Dan, S., Marton, I., Dekel, M., Bravdo, B-A., He, S., Withers, S. G., and Shoseyov, O. (2000) J. Biol. Chem. 275: 4973-4980] was also studied and was shown to have very similar properties to those of BGS. Sequence analysis of a portion of BGS verified that these are the same enzymes.

High-performance hydrolysis of cellulose using mixed cellulase species and ultrasonication pretreatment

Effective approaches to a practical high-performance enzymatic hydrolysis of cellulose were investigated using a ultrasonic irradiation as a pretreatment and a combination of cellulases from Trichoderma viride and Aspergillus niger. Their efficacy on enhancement of reactivity was discussed based on the kinetic parameters, i.e. Michaelis constant K m, maximum reaction rate V max and initial reaction rate, correlating with ultrasonic conditions and enzyme combination, respectively. A relatively high-weight fraction of A. niger cellulase in the mixed-enzyme system afforded rapid initiation of the hydrolysis reaction, while a kinetic analysis revealed that a T. viride cellulase weight fraction of ca. 0.3 was optimal in enhancing V max without increasing K m. Pretreatment of the cellulose fibers with ultrasonic irradiation prior to initiating the enzyme reaction further improved the reaction rate. Enhancement of V max by ultrasonication was effective in greater weight fraction of T. viride used system. The correlation between the ultrasonic irradiation time required to obtain a desired initial reaction rate and the irradiation power employed was examined, and it was found that the time needed for effective degradation could be markedly reduced by increasing the irradiation power. This finding is considered to support the possibility of applying ultrasonication pretreatment in a practical cellulose saccharification process.

Unexpected affinity of polysaccharides and its application in separation of enzymes on fluidized beds

Fluidized bed chromatography is emerging as a powerful tool in bioseparation of proteins/enzymes. In this work, alginate and chitosan beads have been used as affinity media in fluidized mode to selectively capture α-amylases and Aspergillus niger cellulase, respectively. Fairly high yields (>80%), with homogeneity at the level of single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, were achieved in all these cases. The main novel features are unexpected affinity of the polysaccharides towards the target enzymes; such macroaffinity ligands avoid any costly chemical steps for affinity ligands; and possibility of easy scale up in fluidized bed mode and single step purification protocols.

Hydrolysis of rice hull by crosslinked Aspergillus niger cellulase

A.niger cellulase was crosslinked by glutaraldehyde to obtain a heat-stable enzyme preparation for rice hull cellulose hydrolysis. Under optimized crosslinking conditions of 0.12 M glutaraldehyde, pH 7.0, temperature 40°C and at 45 min of crosslinking, a preparation having 15% more activity than free enzyme was obtained which also had considerable improvement in heat stability at 65°C and 70°C. Whereas the free enzyme lost 80% of its activity in 4 h at 65°C, the crosslinked preparation lost only 30% activity. The crosslinked preparation hydrolyzed cellulosic biomass more effectively giving 2.2 mg/ml glucose and 52% corresponding saccharification in 4 h at 65°C as compared to 14% saccharification by free enzyme under similar conditions.

Exploiting unusual affinity of usual polysaccharides for separation of enzymes on fluidized beds

Two polysaccharides, alginate and chitosan, showed unusual affinity and bound alpha-amylase (from various sources) and Aspergillus niger cellulase, respectively. The beads prepared from these polymers were successfully used for the purification of the respective enzymes by fluidized bed affinity chromatography. alpha-amylase from wheat germ could be purified by 58-fold with about 90% recovery of activity. Aspergillus niger cellulase, on the other hand, was purified by 30-fold with 80% recovery of enzyme activity. Both purified preparations show single band on SDS-PAGE.

Saccharification of used paper with different cellulases

Various used paper materials have been exposed to the action of cellulases from Penicillium funiculosum, Trichoderma reesei, Trichoderma viride and Aspergillus niger. A 2 h incubation period showed cellulase from T. viride the most active except for office paper that was maximally degraded by A. niger cellulase. Cellulase mixtures increased saccharification while sequential treatment with cellulases from T. reesei and P. funiculosum increased biodegradation at values between 15% and 190%. The maximum increase of saccharification (190%) was obtained when T. reesei cellulase initiated the sequential treatment of newspaper relative to the sole action of P. funiculosum cellulase on this non-pretreated and pretreated material.

Comparison of batch, packed bed and expanded bed purification of A. niger cellulase using cellulose beads

Rigid macroporous cross-linked cellulose beads were prepared and used as a useful affinity medium for purification of A. niger cellulase from commercial preparation, in batch; packed bed and expanded bed modes. The beads bound 99% activity in both packed bed and expanded bed modes and upto 91% activity could be recovered by washing the adsorbent with 1 M phosphate buffer, pH 7.0. While batch adsorption and elution gave only 4-fold purification, packed bed operation gave 14-fold purification and expanded bed, the highest, 36-fold purification.

Protection of Aspergillus niger cellulases by urea during growth on glucose or glycerol supplemented media.

The cellulase enzymes of Aspergillus niger were found to undergo catabolite repression in the presence of glucose and glycerol accompanied by sudden drop in pH of the fermentation medium below 2.0. This sudden drop in pH caused inactivation of cellulolytic enzymes produced by Aspergillus niger. The supplementation of nitrogen sources, especially urea, protects A. niger cellulases from inactivation caused by a sudden drop in pH, since urea helped to maintain the pH of the fermentation medium between 3.5 and 4.5. The role of urea in the protection of cellulase was more prominent when it was used in combination with glycerol (5%).

Saccharification of cellulosic substrates byAspergillus niger cellulase

Chemical pre-treatment enhanced saccharification of agricultural lignocellulosic residues usingAspergillus niger cellulase by between 16 and 38%. Maximum saccharification (76%) of alkali-treated bagasse was with 0.5% substrate over 48 h using 0.3 U enzyme/ml.

Isolation of a ?-glucosidase binding and activating polysaccharide from cell walls of Trichoderma reesei

The extracellular β-glucosidase from the filamentous fungus Trichoderma reesei QM 9414 is mainly bound to the cell wall of the fungus and only partially released into the medium. Isolation of the cell walls and its hydrolysis by enzymatic treatment with Aspergillus niger cellulase released β-glucosidase, which appeared tightly associated with a cell wall polysaccharide. This polysaccharide was purified by gel filtration and ion exchange chromatography and was shown to consist of mannose, galactose, glucose, galacturonic acid and glucuronic acid. It was devoid of protein and phosphate. It reassociated both with extracellular β-glucosidase as well as β-glucosidase released from the fungus' cell wall. Addition of the polysaccharide to the β-glucosidase in vitro increased the enzyme's activity against 4-nitrophenyl-β-glucoside twofold. These findings suggest, that the isolated polysaccharide functions as an “anchor glycan” for the β-glucosidase in Trichoderma reesei.

Termitomyces: A New Source of Potent Cellulases

The association of certain species of termites with fungi is a very complex phenomenon. Previously, it was demonstrated the importance of a basidiomycete fungus belonging to the genus Termitomyces as a source of potent cellulases. In this paper, the saccharifying activities of Termitomyces conidiophores against various oligo- and polysaccharides were compared. In addition, partial purification of the cellulase complex of conidiophores was achieved by using a Sephadex column G-IOO. The fungal cellulase activity was also compared with five commercial cellulases namely: A. niger cellulase (from Calbiochem comp.), Drisellase-NIO (Kyomo Hakko Comp.), Trichoderma-SPI22 (The Novo Comp.), Trichoderma-4 (Natick, US Army) and Onozuka SSPI500 (Kink Yakult Comp.). Cellulase( s) activities were measured against cotton wool (CJ, filter paper (FP), carboxymethylcellulose (CMC) and cellobios (Bglucosidase). Results showed that the activity of Termitomyces cellulase was far more superior than others when the substrates were filter paper and CMC. However, the cellulolytic activity on cotton wool was intermediate.

Hydrolysis of cellulose by a mixture of Trichoderma reesei cellobiohydrolase and Aspergillus niger endoglucanase

Two endoglucanase-containing fractions were separated from Aspergillus niger cellulase by gel filtration and fast protein liquid chromatofocusing (FPLC). They possessed no ability to bind to or hydrolyze insoluble microcrystalline cellulose (Avicel) but were active toward soluble carboxymethylcellulose. No synergism was observed between Trichoderma reesei cellobiohydrolase I and either endoglucanase from A. niger. These findings may indicate that the role of the endoglucanase component of cellulase in insoluble microcrystalline cellulose hydrolysis is dependent upon its ability to be adsorbed upon the substrate.

Enzymic saccharification of some pretreated agricultural wastes

Summary Cellulosic wastes, artichoke leaves and stalks, sugar-cane bagasse and fennel seeds after extraction of essential oils were treated with various concentrations of peracetic acid at 100 °C, 60 °C and room temperature several times, washed with water and ethanol and air dried. The degree of enzymatic solubilization of each treated cellulosic waste was measured with Aspergillus niger cellulase (Endo-1,4-B-Glucanase; 1,4-(1,3; 1,4)-β-D-glucan 4-glucanohydrolase; EC 3. 2.1.4). Artichoke waste and sugar-cane bagasse were solubilized more effectively by the enzymethan fennel waste. Data are presented describing the effect of time, enzyme and substrate concentration on the rate of enzymic hydrolysis. Infrared spectra of the treated and untreated cellulosic materials were recorded.

Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography

Eight chemically modified cellulose supports were tested for their ability to absorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography.

Hydration of cellobial by exo- and endo-type cellulases: evidence for catalytic flexibility of glycosylases.

New insight has been obtained into the catalytic capabilities of cellulase. Essentially homogeneous preparations of exo- (or Avicelase-) type and endo- (or CMCase-) type cellulases from Irpex lacteus and Aspergillus niger, respectively, were shown to hydrate the enolic bond of cellobial to form 2-deoxycellobiose. The A. niger enzyme also synthesized a small amount of a 2-deoxycellobiosyl-transfer product from cellobial. By use of digests conducted in deuterated buffer and 1H NMR spectra for product analysis, both cellulases were found to protonate (deuterate) the double bond of cellobial from below the si face of the D-glucal moiety, i.e., from a direction opposite that assumed for protonation of the beta-D-glycosidic linkages of cellulose and cellodextrins. The exo enzyme, which hydrolyzes the latter substrates primarily to cellobiose, rapidly catalyzed cellobial hydration to produce the beta-anomer of beta-D-glucopyranosyl(1----4)-2-deoxy-D-glucose-2(e)-d. The A. niger cellulase produced the same 2-deoxycellobiose-d from cellobial, though too slowly for its configuration to be determined. However, evidence was obtained for the formation of a beta-2-deoxycellobiosyl-d-D-glucose-transfer product by the enzyme. Thus, it is likely that all of the observed reactions with cellobial represent trans additions at the double bond. In any case, the anomeric configuration of products is created de novo. Separate mechanisms are described for the reaction of cellobial hydration and for the stereochemically different reaction of cellulose hydrolysis catalyzed by the present enzymes, assuming an arrangement of their catalytic groups analogous to that found in lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnical utilization of wood carbohydrates after steaming pretreatment

Birch wood was used as raw material to study the effect of steaming pretreatment on the characteristics, enzymatic hydrolysis and fermentation of cellulose and hemicellulose. The cellulose remained undissolved in the fibres after steaming, but the degree of polymerization decreased and the surface area increased with increasing steaming temperature. The yield in enzymatic hydrolysis with T. reesei and A. niger cellulases increased from 40 to 75% of theoretical when the pretreatment temperature was increased from 170 to 210°C at a residence time of 10 minutes. The glucose released was fermented to ethanol by yeast without interference of toxic compounds. After steaming, the hemicellulose was mainly in the form of xylo-oligomers. The average chain length decreased with increasing temperature. Only the monomeric sugars were fermented to ethanol by Fusarium oxysporum. After steaming at 210°C toxic decomposition products inhibited the fermentation completely. In aerobic conditions also the xylo-oligomers were metabolized.

Cellulase immobilized on a soluble polymer

Aspergillus niger cellulase was imobilized on cyanogen bromide activated dextran of varying molecular weights. The effect of different concentrations of cyanogen bromide used for the activation process was also studied. About 50% conjugation and 70% retention activity was achieved in the immobilized cellulase. The pH activity of immobilized enzyme was unchanged, but exhibited more stable activity at acidic pH than the free enzyme. Higher resistance to heat inactivation was also observed.

Comparison of forage digestibility in vitro with enzymic solubility

Two methods of determining the enzymatic solubility of organic matter were compared with digestibility evaluated in vitro (IVOMD). The first method (OMSA) used pepsin and Trichoderma viride cellulase, whereas the other (OMSB) entailed boiling with HCl prior to enzymatic attack by low-cost Aspergillus niger cellulase. The relationship between digestibility in vitro and acid detergent fibre (ADF) content was also considered. Determinations were made in 6 successive runs on 19 samples of grasses and 13 of legumes. On the basis of 3 standard samples (whose digestibility in vivo was known and which were included in each batch), significant differences among runs were found with the in vitro method but not with the enzymatic methods. Repeatability, expressed as a standard deviation, was 1.29 units of digestibility for IVOMD, 0.87 for OMSA and 0.80 for OMSB. The IVOMD was more closely correlated with OMSA ( r = 0.96∗∗∗) than with OMSB ( r = 0.91∗∗∗) or with ADF content ( r = −0.88∗∗∗). The 2 enzymatic methods were also shown to be closely correlated; ADF content correlated best with OMSB. Reference samples with known digestibility in vivo indicate that OMSA and IVOMD underestimate organic matter digestibility, especially for less digestible samples, and OMSB overestimates digestibility more or less constantly. The OMSA of legumes was greater than that of grasses for the same digestibility in vitro. No differences in solubility between botanical families were revealed by OMSB.

The monosaccharide composition of cell wall material in cassava tuber (Manihot utilissima).

The monosaccharide composition of cell wall material (CWM) in the cassava tuber and the contents of the other constituents were determined for more advanced industrial utilization. Starch, 80% ethanol–soluble sugar, uronic acid, lignin, ash, and CWM contents in the cassava tuber 86.1, 2.4, 3.4, 0.5, 0.9, and 4.5%, respectively. Rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose contents in CWM were 1.9, 1.2, 2.6, 4.2, 2.0, 12.8, and 52.7%, respectively. Then, the degradation pattern of CWM by enzymatic and sequential acid hydrolysis was studied. Aspergillus niger cellulase preparation was the most effective, and 57.1 % of CWM was degraded by the enzyme preparation. On the other hand, about 50% of the hemicellulose part was extracted from CWM by hot water only.