Purification and properties of three cellobiases from Aspergillus niger A20

Three cellobiases, here called cellobiase A, B, and C, from the culture filtrate of Aspergillus niger A20, were purified by precipitation with ammonium sulphate, gel filtration through Sephadex G-75, and column chromatography of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified enzymes were estimated by SDS-gelelectrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A, B, and C, respectively. The enzymes were active at pH 4.5 and 55–60°C. The pattern of their aminoacid compositions showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine. The apparent K_m values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co²⁺ and Mg²⁺ ions stimulated cell obiase A. The purified enzymes hydrolyzed cellobiose and aryl-β-d-glucosides, but they had no action on sucrose, maltose, and cellulose. The three cellobiases catalyzed transglycosylate reaction, and the major product formed from cellobiose was tetramer of glucose.     

Production of citric acid using immobilized conidia of Aspergillus niger

Conidia of Aspergillus niger were immobilized in calcium alginate gel for the production of citric acid. First, the type of the preactivation medium, together with the preactivation period, was investigated. It was found that A. niger requires a 2-d preactivation period at a 0.05 g/L NH₄NO₃ concentration. Second, preactivated cells were used to determine the effects of nitrogen concentration and the flow rate of oxygen and air on the production of citric acid. Maximum citric acid production was attained with medium containing 0.01 g/L of NH₄NO₃. The rate of citric acid production in the nitrogenous medium was 33% higher when oxygen was used instead of air during the production phase. This corresponds to an increase of 85% when compared to production when neither oxygen nor air was fed into the system. In the nonnitrogenous medium citric acid concentration remained similar regardless of the use of air or oxygen. However, in the nonnitrogenous production medium, citric acid production was not influenced considerably when oxygen was used instead of air. The advantage of using immobilized cells is that production is achieved easily in the continuous system. Therefore, citric acid production was also tested using a packed-bed bioreactor, and an increase in productivity by a factor of 22 was achieved compared to the batch system.     

Microencapsulated mycelium-bound tannase from Aspergillus niger

Microencapsulated Aspergillus niger with mycelium-bound tannase activity was employed to investigate the esterification of propyl gallate from gallic acid and propanol in organic solvents. The effects of various organic solvents (log P:−1.0 to 6.6) on the enzymatic reactions showed that benzene (log P:2.0) was the suitable solvent, for which the conversion reached 26.8%. The optimum catalyst concentration and water concentration was found at 25 capsules in 10 mL of benzene and 0.04 g of water/capsule. The external mass transfer effect could be eliminated at stirring speeds of 180 rpm or higher. Both substrates 1-propanol and gallic acid had significant inhibition effects on the tannase activity. Maximum molar conversion (36.2%) was achieved with 9.1% (v/v) 1-propanol and 8 mM gallic acid and decreased with increasing amounts of substrates.     

Mutagenesis and analysis of mold Aspergillus niger for extracellular glucose oxidase production using sugarcane molasses

Aspergillus niger ORS-4.410, a mutant of A. niger ORS-4, was generated by repeated ultraviolet (UV) irradiation. Analysis of the UV treatment dose on wild-type (WT) A. niger ORS-4, conidial survival, and frequency of mutation showed that the maximum frequency of positive mutants (25.5%) was obtained with a 57% conidial survival rate after the second stage of UV irradiation. The level of glucose oxidase (GOX) production from mutant A. niger ORS-4.410 thus obtained was 149% higher than that for WT strain A. niger ORS-4 under liquid culture conditions using hexacyanoferrate (HCF)-treated sugarcane molasses (TM) as a cheaper carbohydrate source. When subcultured monthly for 24 mo, the mutant strain had consistent levels of GOX production (2.62±0.51 U/mL). Mutant A. niger ORS-4.410 was markedly different from the parent strain morphologically and was found to grow abundantly on sugarcane molasses. The mutant strain showed 3.43-fold increases in GOX levels (2.62±0.51 U/mL) using HCF-TM compared with the crude form of cane molasses (0.762±0.158 U/mL). The results reported herein were obtained while the author was working at the Department of Biotechnology, Indian Institute of Technology, Roorkee-247667, India.     

Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger

Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant laccase from A. nigher harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties similar to those of native T. versicolor laccase A.     

Influence of Tween 80 on lipid metabolism of an Aspergillus niger strain

Addition of 0.1% of nonionic surface-active Tween 80 to a medium optimized for pectolytic enzyme production of Aspergillus niger increased the amount of enzymes excreted by 70%. In the presence of Tween 80 the amount of sterol esters and triacylglycerols was increased. During the course of cultivation the amounts of precursors for ergosterol biosynthesis diminished with an increase of ergosterol. A. niger incorporated cholesterol from the medium, partly converting it to cholesterol esters. Sterol esters were formed only with selected fatty acids. Oleic acid, the hydrophobic part of Tween 80, was mainly incorporated in phospholipids and glycolipids.     

Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation

This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Optimization of glucoamylase production by Aspergillus niger in solid-state fermentation

Glucoamylase production by Aspergillus niger in solid-state fermentation was optimized using factorial design and response surface techniques. The variables evaluated were pH and bed thickness in tray, having as response enzyme production and productivity. The bed thickness in tray was the most significant variable for both responses. The highest values for glucoamylase production occurred using pH 4.5 and bed thickness in the inferior limits at 2.0–4.2 cm. For productivity, the optimal conditions were at pH 4.5 as well and bed thickness from 4.4 to 7.5 cm. The optimal conditions for glucoamylase production while obtaining high activity without loss of productivity were pH 4.5 and bed thickness in tray from 4.0 to 4.5 cm, which resulted in an enzyme production of 695 U/g and productivity of 5791 U/h.     

Enzyme production and profile by Aspergillus niger during solid substrate fermentation using palm kernel cake as substrate

The oil palm sector is one of the major plantation industries in Malaysia. Palm kernel cake is a byproduct of extracted palm kernel oil. Mostly palm kernel cake is wasted or is mixed with other nutrients and used as animal feed, especially for ruminant animals. Recently, palm kernel cake has been identified as an important ingredient for the formulation of animal feed, and it is also exported especially to Europe, South Korea, and Japan. It can barely be consumed by nonruminant (monogastric) animals owing to the high percentages of hemicellulose and cellulose contents. Palm kernel cake must undergo suitable pretreatment in order to decrease the percentage of hemicellulose and cellulose. One of the methods employed in this study is fermentation with microorganisms, particularly fungi, to partially degrade the hemicellulose and cellulose content. This work focused on the production of enzymes by Aspergillus niger and profiling using palm kernel cake as carbon source.     

Fungal morphology in submerged cultures and its relation to glucose oxidase excretion by recombinant Aspergillus niger

The effect of culture conditions such as medium composition and shear stress on the fungal pellet morphology in shake-flask cultures and its relation to glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL 3 (GOD 3–18) was investigated. It was shown that culture conditions resulting in the formation of smaller fungal pellets with an increased mycelial density result in higher yields of exocellular GOD. The pellets obtained in shake-flask cultures showed distinct layers of mycelial density with only the thin outer layer consisting of a dense mycelial network. The performance of the recombinant strain and the process of pellet formation was also analyzed during batch cultivation in a stirred-tank bioreactor. It was shown that the process of pellet formation occurred in two steps: (1) aggregation of free spores to spore clusters with subsequent germination and formation of small aggregates surrounded by a loose hyphal network, and (2) aggregation of the primary aggregates to the final full-size pellets. The fungal pellets formed during bioreactor cultivation were smaller, did not show large differences in mycelial density, and were more efficient with respect to the production of exocellular GOD. The decreasing pellet size also correlated with an increased mycelial density, indicating an improvement of the transport of nutrients to the inner parts of the pellet.     

Effect of span 20 concentration on oxalic acid production from post-refining fatty acids by Aspergillus niger XP

Submerged fermentation experiments were carried out to study the stimulating effects of the surfactant Span 20 on the growth of Aspergillus niger XP mutant and oxalic acid production from the post-refining fatty acids. Span 20 concentration of 0.75 g dm⁻³ was found to be the most suitable for oxalic acid production from fatty acids. Using this dose and a fermentation medium containing 30 g dm⁻³ of post-refining fatty acids, the oxalic acid production, oxalate yield, and overall oxalate productivity were the highest. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.     

Invertase production on solid-state fermentation by Aspergillus niger strains improved by parasexual recombination

Invertase production by Aspergillus niger grown by solid-state fermentation was found to be higher than by conventional submerged fermentation. The haploid mutant strains Aw96-3 and Aw96-4 showed better productivity of various enzymes, as compared to wild-type parental strain A. niger C28B25. Here we use parasexual crosses of those mutants to increase further the productivity of invertase in solid-state fermentation. We isolated both a diploid (DAR2) and an autodiploid (AD96-4) strain, which were able to grow in minimal medium after mutation complementation of previously isolated haploid auxotrophic strains. Invertase production was measured in solid-state fermentation cultures, using polyurethane foam as an inert support for fungal growth. Water activity value (Aw) was adjusted to 0.96, since low Aw values are characteristic in some solid-state fermentation processes. Such diploid strains showed invertase productivity levels 5–18 times higher than levels achieved by the corresponding haploid strains. For instance, values for C28B25, Aw96-3, Aw96-4, DAR2, and AD96-4 were 441, 254, 62, 1324, and 2677 IU/(L·h), respectively. These results showed that genetic recombination, achieved through parasexual crosses in A. niger, results in improved strains with potential applications for solid-state fermentation processes.     

GpdA-promoter-controlled production of glucose oxidase by recombinant aspergillus niger using nonglucose carbon sources

The gpdA-promoter-controlled exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) during growth on glucose and nonglucose carbon sources was investigated. Screening of various carbon substrates in shake-flask cultures revealed that exocellular GOD activities were not only obtained on glucose but also during growth on mannose, fructose, and xylose. The performance of A. niger NRRL-3 (GOD3-18) using glucose, fructose, or xylose as carbon substrate was compared in more detail in bioreactor cultures. These studies revealed that gpdA-promoter-controlled GOD synthesis was strictly coupled to cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid GOD-catalyzed transformation of glucose into gluconic acid, a carbon source not supporting further cell growth and GOD production, resulted in low biomass yields and, therefore, reduced the advantageous properties of glucose. The total (endo- and exocellular) specific GOD activities were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization of xylose resulted in total specific GOD activities nearly as high as reached during growth on glucose. Also, the portion of GOD excreted into the culture fluid reached similar high levels (≅ 90%) by using either glucose or xylose as substrate, whereas growth on fructose resulted in a more pelleted morphology with more than half the total GOD activity retained in the fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and, consequently, the highest total volumetric GOD activity. These results show that xylose is the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular GOD.     

Solid-state fermentation with Aspergillus niger for cellobiase production

Aspergillus niger NRRL3 was cultivated in a moist wheat bran and ground corncob solid medium supplemented with inorganic minerals for the production of cellobiase (β-1,4-glucosidase, EC 3.2.1.21). With this method, A. niger NRRL3 was able to produce a high concentration of cellobiase (215 IU/gofsolid substrate) after 96 h of incubation. Temperature and moisture content affected final cellobiase titers. The best conditions for cell obiase production from solid substrate by A. niger NRRL3 were determined to be 70% moisture and 35°C.     

Optimization of ammonium nitrate concentration in single-stage continuous cultures of Aspergillus niger with biomass retention

The effect of ammonium nitrate concentration in the citric acid biosynthesis by Aspergillus niger NC-12 in single-stage continuous cultures with biomass retention was investigated. Experiments were carried out in a BIOMER laboratory fermenter with 5 dm³ working volume. At the initial stage of each cultivation, the substrate in the bioreactor contained 1.5 g NH₄NO₃ dm⁻³. After 120 h onwards, the bioreactor was fed continuously at a constant dilution rate of 0.009 h⁻¹. NH₄NO₃ concentration in the feed was varied from one culture to another, ranging between 0.5 g dm⁻³ and 2.5 g dm⁻³. Promising results were obtained when NH₄NO₃ concentration of 1.5 g dm⁻³ was used. The observed concentration of citric acid (c _P) and yield of citric acid with respect to the introduced sucrose (Y _P/S) were 117.88 g dm⁻³ and 78.59 %, respectively. The efficiency coefficient of citric acid biosynthesis (K _ef) was very high, amounting to 83.38. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.     

Biotransformation of (−)β-pinene by Aspergillus niger ATCC 9642

The main objective of this work was to investigate the biotransformations of (−)α-pinene, (−)β-pinene, and (+) limonene by Aspergillus niger ATCC 9642. The culture conditions involved—concentration of cosolvent (EtOH), substrate applied, and sequential addition of substrates—were investigated. Adaptation of the precultures with small amounts of substrate was also studied. The experiments were performed in conical flasks with liquid cultures. This strain of A. niger was able to convert only (−)β-pinene into α-terpineol. An optimum conversion of (−)β-pinene into α-terpineol of about 4% was obtained when the substrate was applied as a diluted solution in EtOH and sequential addition of substrate was used.     

Purification and properties of carnitine acetyltransferase from citric acid producingAspergillus niger

Carnitine acetyltransferase was purified from the citric acid producingA. niger mycelium with a protein band showing a relative molecular weight of 77,000 and a pH optimum of 7.3. TheK _m values for the purified enzyme for acetyl-CoA and for carnitine were 0.1 mM and 1 mM, respectively. Carnitine acetyltransferase was located both in the mitochondria and in the cytosol. Both mitochondrial and cytosolic enzyme were purified using ammonium sulfate precipitation, Mono Q and Superose 12 separation. Regarding the localization, except for maximum velocity, there were no differences observed in substrate specificity and inhibition. Inhibition of the enzyme with micromolar concentrations of Cu²⁺ could contribute to a greater citric acid biosynthesis. Carnitine acetyltransferase can be considered as an enzyme necessary for the transport of acetyl groups through mitochondrial membrane in both directions.     

Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.     

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.     

Culture conditions dictate protease and tannase production in submerged and solid-state cultures of Aspergillus niger Aa-20

Undesirable protease production by Aspergillus niger Aa-20 in submerged culture and solid-state culture was evaluated using different concentrations of tannic acid as sole carbon source in a model system designed for tannase production. Protease production was found to be dependent on the culture system used (submerged culture or solid-state culture) and on the initial tannic acid concentration. Expression of protease activity in submerged culture was higher (up to 10 times) than activity obtained in solid-state culture, using identical culture medium composition. In submerged culture, the lowest final protease activity (0.13 IU) was obtained with the highest tannic acid concentration, while in solid-state culture protease activity was not affected by changes in initial substrate concentration. Absence of detectable proteolytic activity in solid-state culture is related to high production of tannase enzyme. Hence, the use of solid-state culture for fungal enzyme production may allow for higher and more stable enzyme titers present in culture extracts.