Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.     

Degradation of textile dyes mediated by plant peroxidases

The peroxidase enzyme from the plants Ipomea palmata (1.003 IU/g of leaf) and Saccharum spontaneum (3.6 IU/g of leaf) can be used as an alternative to the commercial source of horseradish and soybean peroxidase enzyme for the decolorization of textile dyes, mainly azo dyes. Eight textiles dyes currently used by the industry and seven other dyes were selected for decolorization studies at 25–200 mg/L levels using these plant enzymes. The enzymes were purified prior to use by ammonium sulfate precipitation, and ion exchange and gel permeation chromatographic techniques. Peroxidase of S. spontaneum leaf (specific activity of 0.23 IU/mg) could completely degrade Supranol Green and Procion Green HE-4BD (100%) dyes within 1 h, whereas Direct Blue, Procion Brilliant Blue H-7G and Chrysoidine were degraded >70% in 1 h. Peroxidase of Ipomea (I. palmata leaf; specific activity of 0.827 U/mg) degraded 50 mg/L of the dyes Methyl Orange (26%), Crystal Violet (36%), and Supranol Green (68%) in 2–4 h and Brilliant Green 54%), Direct Blue (15%), and Chrysoidine (44%) at the 25 mg/L level in 1 to 2 h of treatment. The Saccharum peroxidase was immobilized on a hydrophobic matrix. Four textile dyes, Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green, at an initial concentration of 50 mg/L were completely degraded within 8 h by the enzyme immobilized on the modified polyethylene matrix. The immobilized enzyme was used in a batch reactor for the degradation of Procion Green HE-4BD and the reusability was studied for 15 cycles, and the halflife was found to be 60 h.     

Xylanase production by Aspergillus awamori in solid-state fermentation and influence of different nitrogen sources

The use of purified xylan as a substrate for bioconversion into xylanases increases the cost of enzyme production. Consequently, there have been attempts to develop a bioprocess to produce such enzymes using different lignocellulosic residues. Filamentous fungi have been widely used to produce hydrolytic enzymes for industrial applications, including xylanases, whose levels in fungi are generally much higher than those in yeast and bacteria. Considering the industrial importance of xylanases, the present study evaluated the use of milled sugarcane bagasse, without any pretreatment, as a carbon source. Also, the effect of different nitrogen sources and the C∶N ratio on xylanase production by Aspergillus awamori were investigated, in experiments carried out in solid-state fermentation. High extracellular xylanolytic activity was observed on cultivation of A. awamori on milled sugarcane bagasse and organic nitrogen sources (45 IU/mL for endoxylanase and 3.5 IU/mL for β-xylosidase). Endoxylanase and β-xylosidase activities were higher when sodium nitrate was used as the nitrogen source, when compared with peptone, urea, and ammonium sulfate at the optimized C∶N ratio of 10∶1. The use of yeast extract as a supplement to the these nitrogen sources resulted in considerable improvementin the production of xylanases, showing the importance of this organic nitrogen source on A. awamori metabolism.     

Evaluation of Antineoplastic Activity of Extracellular Asparaginase Produced by Isolated <Emphasis Type="Italic">Bacillus circulans</Emphasis>

l-Asparaginase is an important component in the treatment of acute lymphoblastic leukemia in children. Its antineoplastic activity toward malignant cells is due to their characteristic nature in slow synthesis of l-asparagine (Asn), which causes starvation for this amino acid, while normal cells are protected from Asn starvation due to their ability to produce this amino acid. The relative selectivity with regard to the metabolism of malignant cells forces to look for novel asparaginase with little glutaminase-producing systems compared to existing enzyme. In this investigation, the role of the extracellular asparaginase enzyme produced by an isolated bacterial strain was studied. Biochemical characterization denoted that this isolated bacterial strain belongs to the Bacillus circulans species. The strain was tested for l-asparaginase production, and it was observed that, under an optimized environment, this isolate produces a maximum of 85 IU ml⁻¹ within 24-h incubation. This enzyme showed less (60%) glutaminase activity compared to commercial Erwinia sp. l-asparaginase. The partially purified enzyme showed an approximate molecular weight of 140 kDa. This enzyme potency in terms of antineoplastic activity was analyzed against the cancer cells, CCRF-CEM. Flow cytometry experiments indicated an increase of sub-G1 cell population when the cells were treated with l-asparaginase.     

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.     

Effect of Cultural Conditions and Media Constituents on Production of Penicillin V Acylase and CTAB Treatment to Enhance Whole-Cell Enzyme Activity of <Emphasis Type="Italic">Rhodotorula aurantiaca</Emphasis> (NCIM 3425)

Penicillin V acylase (PVA) is a pharmaceutically important enzyme as it plays a vital role in the manufacture of semi-synthetic β-lactam antibiotics. Rhodotorula aurantiaca (NCIM 3425) produced high levels of intracellular penicillin V acylase after 18 h at pH 8.0 and temperature 27 °C. Fructose was the best carbon source for PVA production, whereas tryptone was the best nitrogen source to produce the enzyme up to 170 and 1,088 IU/l of culture, respectively. Additionally, the cell-bound PVA activity was enhanced on treatment with cationic detergent. Whole-cell activity was found to be doubled (204%) on treatment of 0.01 g dry weight of cells with 50 μg/ml solution of N-cetyl-N,N,N-trimethylammoniumbromide at pH 8.0 for 1 h at room temperature. Atomic force microscopy images of permeabilized cells show perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Kinetics and bioenergetics of Spirulina platensis cultivation by fed-batch addition of urea as nitrogen source

The cyanobacterium Spirulina platensis was cultivated in bench-scale miniponds on bicarbonate/carbonate solutions using urea as nitrogen source. To minimize limitation and inhibition phenomena, urea was supplied semicontinuously using exponentially increasing feeding rates. The average growth rates obtained alternately varying the total mass of urea added per unit reactor volume (275<m _T<725 mg/L) and the total feeding time (9<t _T<15 d) clearly evidenced nitrogen limitation for m _T<500 mg/L and excess nitrogen inhibition above this threshold. The time behavior of the specific growth rate at variable urea feeding patterns allowed estimation of the time-dependent Gibbsenergy dissipation for cell growth under the actual depletion conditions of fed-batch cultivations. Comparison of the yield of growth on Gibbs energy obtained using either urea or KNO₃ pointed to the preference of S. platensis for the former nitrogen source, likely owing to more favorable bioenergetic conditions.     

l-Asparaginase II of Saccharomyces cerevisiae

The activity profile of the periplasmic asparaginase of Saccharomyces cerevisiae was determined during cell growth in an ure2 mutant; in an ure2 transformed with a plasmid containing the gene URE2 and, for comparison, in the strain D273-10B. Cells were cultivated in media presenting variable quantitative and qualitative nitrogen availability and the enzyme activity was evaluated in fresh and in nitrogen-starved cells. Nitrogen affected the asparaginase II level in fresh and starved cells of all strains. In the best condition, enzyme was produced by the wild-type cells at the late log-phase in the glucose/ammonium medium with a carbon to nitrogen ratio 4.3:1. Upon starvation, the activity doubled. The overall profile of the transformed strain was similar to that of the wild-type strain. In the ure2 mutant, highenzyme levels were observed during growth, as expected. However the activity level, upon starvation, in proline grown cells, increased sixfold, suggesting that in addition to the Ure2p-Gln3p system, another system regulates asparaginase II biosynthesis.     

Evaluation of inoculum of Candida guilliermondii grown in presence of glucose on xylose reductase and xylitol dehydrogenase activities and xylitol production during batch fermentation of sugarcane bagasse hydrolysate

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mg_prot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mg_prot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L·h]) after 48 h of fermentation.     

Enzymatic synthesis of oligosaccharides, alkyl and terpenyl glucosides, by recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II

The biosynthetic activity of yeast Pichia etchellsii β-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate: 1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent. Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethylacetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.     

Microbial production of polyhydroxyalkanoates by bacteria isolated from oil wastes

A Gram-positive coccus-shaped bacterium capable of synthesizing higher relative molecular weight (M _r), polyhydroxybutyrate (PHB) was isolated from sesame oil and identified as Staphylococcus epidermidis (by Microbial ID, Inc., Newark, NJ). The experiment was conducted by shake flask fermentation culture using media containing fructose. Cell growth up to a dry mass of 2.5 g/L and PHB accumulation up to 15.02% of cell dry wt was observed. Apart from using single carbohydrate as a sole carbon source, various industrial food wastes including sesame oil, ice cream, malt, and soya wastes were investigated as nutrients for S. epidermidis to reduce the cost of the carbon source. As a result, we found that by using malt wastes as nutrient for cell growth, PHB accumulation of S. epidermidis was much better than using other wastes as nutrient source. The final dried cell mass and PHB production using malt wastes were 1.76 g/L and 6.93% polymer/cells (grams/gram), and 3.5 g/L and 3.31% polymer/cells (grams/gram) in shake flask culture and in fermentor culture, respectively. The bacterial polymer was characterized by ¹H-nuclear magnetic resonance (NMR), ¹³C-NMR, Fourier transform infrared, and differential scanning calorimetry. The results show that with different industrial food wastes as carbon and energy sources, the same biopolymer (PHB) was obtained. However, the use of sesame oil as the carbon source resulted in the accumulation of PHB with a higher melting point than that produced from other food wastes as carbon sources by this organism under similar experimental conditions.     

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.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

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.     

Nitrogen regulation of Saccharomyces cerevisiae invertase

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE 2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure 2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure 2 mutant cells from both phases. When these cells were trans-formed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cells was not observed. These results suggested that the URE2 protein plays a role in invertase activity.     

Efficient production of mannan-degrading enzymes by the basidiomyceteSclerotium rolfsii

Sclerotium rolfsii CBS 191.62 was cultivated on a number of carbon (C) sources, including mono- and disaccharides, as well as on polysaccharides, to study the formation of different mannan-degrading enzyme activities. Highest levels of mannanase activity were obtained when α-cellulose-based media were used for growth, but formation of mannanase could not be enhanced by employing galactomannan as the only carbon source. Although both xylanase and cellulase formation was almost completely repressed whenS. rolfsii was grown on more readily metabolizable carbohydrates, including glucose or mannose, considerable amounts of mannanase activity were secreted under these growth conditions. Enhanced mannanase production only commenced when glucose was depleted in the medium. The maximal mannanase activity of 240 IU/mL obtained in a laboratory fermentation is remarkable. Mannanase activity formed under these derepressed conditions could be mainly attributed to one major, acidic mannanase isoenzyme with a pI value of 2.75.     

Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications

Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Trisacetate buffer, pH 7.3, at 4°C had a t _1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.     

A biotechnological process involving filamentous fungi to produce natural crystalline vanillin from maize bran

A new process involving the filamentous fungi Aspergillus niger and Pycnoporus cinnabarinus has been designed for the release of ferulic acid by enzymic degradation of a cheap and natural agricultural byproduct (autoclaved maize bran) and its biotransformation into vanillic acid and/or vanillin with a limited number of steps. On the one hand, the potentialities of A. niger I-1472 to produce high levels of polysaccharide-degrading enzymes including feruloyl esterases and to transform ferulic acid into vanillic acid were successfully combined for the release of free ferulic acid from autoclaved maize bran. Then vanillic acid was recovered and efficiently transformed into vanillin by P. cinnabarinus MUCL 39533, since 767 mg/L of biotechnologic vanillin could be produced in the presence of cellobiose and XAD-2 resin. On the other hand, 3-d-old high-density cultures of P. cinnabarinus MUCL39533 could be fed with the autoclaved fraction of maize bran as a ferulic acid source and a. niger I-1472 culture filtrate as an extracellular enzyme source. Under these conditions, P. cinnabarinus MUCL39533 was shown to directly biotransform free ferulic acid released from the autoclaved maize bran by A. niger I-1472 enzymes into 584 mg/L of vanillin. These processes, involving physical, enzymic, and fungal treatments, permitted us to produce crystallin vanillin from autoclaved maize bran without any purification step.     

Ion/Molekülreaktionen negativ geladener KomplexeSchiffscher Basen mit potentiellen Ligandmolekülen in der Gasphase

Ion/molecule reactions of four coordinateSchiff base complexes under negative ion chemical ionization conditions have been studied. The complex metal ions consisted of cobalt(II), nickel(II) and copper(II).Schiff base ligands with different donor strengths were employed. The gas mixtures used contained 90% methane and 10% of the gases O₂, NO or CO. The spectra showed intense molecular negative ions, formed by secondary electron capture processes. Secondary ions were formed via ion/molecule reactions between the parent molecular negative ion and added gas molecules to giveMLX ^–,X=O₂, NO, CO;L=Schiff base ligand,M=Co(II) or Ni(II). Consistent with former investigations, secondary ion formation was not found for the copper compounds. Influence of the central metal ion as well as the ligand donor strength on the ion/molecule reactions are discussed. From the results obtained a mechanism of the secondary ion formation is suggested.