Effects of conditions ofPenicillium funiculosum G-15 cultivation on production of extracellular glucose oxidase

Effects of conditions ofPenicillium funiculosum G-15 cultivation on the production of extracellular glucose oxidase were studied. The data showed that surface and submerged methods of cultivation can be used for obtaining a glucose oxidizing enzyme. The optimum conditions for submerged cultivation (25°C., initial pH 5.0, and aeration of 3 1/1 per min) and surface cultivation (temperature 25°C and initial pH 4.0) providing the maximum levels of glucose oxidase synthesis were determined.     

A comparative study on the production of exopolysaccharides between two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures

AIMS: The present study comparatively investigates the optimal culture conditions for the production of exopolysaccharides (EPS) and cordycepin during submerged mycelial culture of two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis. METHODS AND RESULTS: Fermentations were performed in flasks and in 5-l stirred-tank fermenters. In the case of C. militaris, the highest mycelial biomass (22.9 g l(-1)) and EPS production (5 g l(-1)) were achieved in a medium of 40 g l(-1) sucrose, 5 g l(-1) corn steep powder at 30 degrees C, and an initial pH 8.0. The optimum culture conditions for C. sinensis was shown to be (in g l(-1)) 20 sucrose, 25 corn steep powder, 0.78 CaCl2, 1.73 MgSO4.7H2O at 20 degrees C, and an initial pH 4.0, where the maximum mycelial biomass and EPS were 20.9 and 4.1 g l(-1) respectively. Cordycepin, another bioactive metabolite, was excreted at low levels during the early fermentation period (maximum 38.8 mg l(-1) in C. militaris; 18.2 mg l(-1) in C. sinensis). CONCLUSIONS: The two fungi showed different nutritional and environmental requirements in their submerged cultures. Overall, the concentrations of mycelial biomass, EPS and cordycepin achieved in submerged culture of C. militaris were higher than those of C. sinensis. SIGNIFICANCE AND IMPACT OF THE STUDY: C. militaris and C. sinensis are representative insect-born fungi which have been longstanding and widely used as traditional medicines in eastern Asia. Comparative studies between two fungi are currently not available and this is the first report on the optimum medium composition for submerged culture of C. sinensis.     

Optimization of culture conditions for production of the anti-tubercular alkaloid hirsutellone A by Trichoderma gelatinosum BCC 7579

AIMS: This work aimed to optimize the culture conditions for production of a novel and potent anti-tubercular alkaloid, hirsutellone A, by the saprophytic soil fungus Trichoderma gelatinosum BCC 7579. METHODS AND RESULTS: The fungus was initially cultured in shake flasks at 25 degrees C in the potato dextrose broth (PDB) supplemented with various carbon and nitrogen sources and mineral salts to select suitable medium for mycelial growth and hirsutellone A production. Cultivation conditions were further optimized by adjusting initial pH and changing temperature levels to maximize the production of hirsutellone A. The optimal condition that increased the production of hirsutellone A from 19.04 mg l(-1), obtained from basal condition, to 610.55 mg l(-1) and reduced the cultivation time from 40 to 6 days was to cultivate in a shaker at 200 rev min(-1) at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with initial pH of 7. Production of hirsutellone A in larger-scale using a 5-l batch fermenter was also completed yielding 958 mg l(-1) of hirsutellone A within 6 days. CONCLUSIONS: The suitable culture conditions for hirsutellone A production by T. gelatinosum BCC 7579 was the cultivation in 5-l fermenter at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with an initial pH of 7. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of hirsutellone A in a fermenter to obtain a high yield and reduce an incubation period will become very useful in anti-tubercular drug development process in the future.     

Production of beta-fructofuranosidase with transfructosylating activity for fructooligosaccharides synthesis by Aspergillus japonicus NTU-1249.

Microbial beta-fructofuranosidases with transfructosylating activity can catalyze the transfructosylation of sucrose and synthesize fructooligosaccharides. Aspergillus japonicus NTU-1249 isolated from natural habitat was found to produce a significant amount of beta-fructofuranosidase with high transfructosylating activity and to have the potential for industrial production of fructooligosaccharides. In order to improve it's enzyme productivity, the medium composition and the cultivation conditions for A. japonicus NTU-1249 were studied. A. japonicus NTU-1249 can produce 83.5 units of transfructosylating activity per ml broth when cultivated in a shaking flask at 28 degrees C for 72 hours with a modified medium containing 80 g/l sucrose, 15 g/l soybean flour, 5 g/l yeast extract and 5 g/l NaCl at an initial pH of 6.0. The enzyme productivity was also optimized by submerged cultivation in a 5-litre jar fermentor with aeration at 1.5 vvm and agitation at 500 rpm. Under these operating conditions, the productivity of transfructosylating activity increased to 185.6 U/ml. Furthermore, the transfructosylating activity was improved to 256.1 U/ml in 1,000-litre pilot-scale fermentor. Enzymatic synthesis of fructooligosaccharides by beta-fructofuranosidase from A. japonicus NTU-1249 was performed in batch type by adding 5.6 units of transfructosylating activity per gram of sucrose to a 50% (w/v) sucrose solution at pH 5.0 and 50 degrees C. The yield of fructooligosaccharides was about 60% after reaction for 24 hours, and the syrup produced contained 29.8% (w/v) fructooligosaccharides, 15.2% (w/v) glucose and 5.0% (w/v) sucrose.     

Glucose oxidase ofAspergillus niger

The authors studied the effect of the various components of synthetic nutrient medium on glucose oxidase production in submerged cultivation ofAspergillus niger. It was found that the optimal glucose concentration was 3.5–6%. The only suitable source of nitrogen was nitrate nitrogen. If the medium contained ammonia nitrogen, glucose oxidase was not formed. The addition of citric acid to the medium very effectively stimulated theQ _{O 2} of the mycelium. Calcium added in the form of calcium nitrate had the same effect. A decrease in the Mg²⁺ ion concentration raised the activity of the enzyme, while inhibiting growth of the mycelium. If the initial pH was less than 4, glucose oxidase production was inhibited and did not start until the pH rose in the course of fermentation. Differences in the initial pH affected not only production of the enzyme, but also the formation of acids and the morphological appearance of the submerged mycelium. On the basis of the findings the synthetic medium for submerged cultivation ofAspergillus niger was modified, resulting in a 50–100% increase in glucose oxidase production as compared with the original medium.     

胆红素氧化酶产酶菌株的分离及最佳产酶条件的研究

A bilirubin oxidase (EC 1.3.3.5) producing strain, Mv 2.1089, was isolated from several strains of Myrothecium verrucaria by dilution method. The optimum conditions of enzyme production were investigated and the results were as follows: the suitable medium was cultured at 25 degrees C on a rotating shaker glucose and peptone, at pH 6.0. The strain was cultured at 25 degrees C on a rotating shaker (150 r/min) for 96 h. Bilirubin oxidase with 0.5-1.5 u/ml was obtained in the culture medium.     

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.     

Optimization of culture conditions for production of antimalarial menisporopsin A by the seed fungus Menisporopsis theobromae BCC 4162

AIMS: The aim of this work was to optimize the production of a novel antimaralial menisporopsin A by the seed fungus Menisporopsis theobromae BCC 4162. METHODS AND RESULTS: Fungal cultures were grown in shake flasks at 25 degrees C in the basal medium with varying carbon and nitrogen sources, aeration rates and initial pH levels. The optimal carbon and nitrogen sources that improved the production of menisporopsin A were 1% fructose and 2.5% meat extract respectively. The production was further enhanced when the culture incubated on a shaker at 200 rev min(-1) with an initial pH of 8. The yield of menisporopsin A cultured under the optimized conditions was increased from 348.30 (obtained from basal medium) to 889.02 mg l(-1), and the cultivation time was reduced from 28 to only 4 days. As a result, the productivity of menisporopsin A was greatly enhanced to 222.26 mg l(-1) day(-1) which is 18-fold higher than that of basal conditions. Larger scale production in a fermenter was also achieved, yielding menisporopsin A at a maximal level of 594.32 mg l(-1) in 4 days. CONCLUSIONS: The optimized culture conditions for menisporopsin A production by M. theobromae BCC 4162 was the cultivation under shaking or agitation at 25 degrees C in fructose-meat extract medium with an initial pH of 8. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of menisporopsin A in a fermenter with a relatively short incubation period could be valuable for further utilization for chemical structure modification and derivatization.     

Production of laccase by a newly isolated strain of Trametes modesta

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by Trametes modesta were evaluated using the one-factor-at-a-time method. The final optimisation was done using a central composite design resulting in a four-fold increase of the laccase activity to 178 nkat ml(-1). Response-surface analysis showed that 7.34 g l(-1) wheat bran, 0.87 g l(-1) glucose, 2.9 g l(-1) yeast extract, 0.25 g l(-1) ammonium chloride, an initial pH of 6.95 and an incubation temperature of 30.26 degrees C were the optimal conditions for laccase production. Laccase produced by T. modesta was fully active at pH 4 and at 50 degrees C. The laccase was very stable at pH 4.5 and at 40 degrees C but half-lives decreased to 120 and 125 min at higher temperature (60 degrees C) and lower pH (pH 3).     

Optimization of conditions for cultivation of the basidiomycete Coriolus hirsutus--producer of extracellular laccase

The effects of various factors on the biosynthesis of extracellular laccase (EC 1.14.18.1) by the basidiomycete Coriolus hirsutus (Wulf.: Fr.) Quel. no. 072 during submerged cultivation were examined. Optimal parameters for cultivation in a fermenter of 10 l were determined: temperature, 28 degrees C; stirrer rotation speed, 160 rpm; and the inoculum volume, 15% of the working volume of the fermenter. The filtrate contained peroxidase, laccase, and phenol oxidase activities and displayed a high thermal stability.     

Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738

AIMS: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture. METHODS AND RESULTS: The optimal temperatures for mycelial biomass and EPS production were 20 degrees C and 25 degrees C, respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium composition for EPS production was as follows: 6% (w/v) sucrose, 1% (w/v) polypeptone, and 0.05% (w/v) K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture (pH 6.0), suggesting that larger and more compact pellets were desirable for polysaccharide production (0.91 g g(-1) cell d(-1). CONCLUSIONS: Under the optimized culture conditions (with pH control at 6), the maximum concentration of biomass and EPS were 12.7 g l(-1) and 7.3 g l(-1), respectively, in a 5-l stirred-tank fermenter. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effect of pH on fungal morphology and rheology presented in this study can be widely applied to other mushroom fermentation processes.     

Bioconversion of starch processing waste to Phellinus linteus mycelium in solid-state cultivation

The objective of the experiment was to use starch processing waste as an alternative growth medium for cultivation of mycelia of the mushroom Phellinus linteus and to find an optimum condition under solid-state cultivation. Response surface analysis along with a central composite design was successfully applied to approximate the simultaneous effects of the substrate concentration (16-36 g l(-1)), pH (4.5-6.5), and temperature (25-35 degrees C) on the mycelial growth rate. In the model, pH and temperature significantly affected the mycelial growth but substrate concentration did not. The optimal substrate concentration, pH, and temperature for maximizing growth rate of P. linteus mycelia were found to be 16.5 g l(-1), pH 6.0, and 29.7 degrees C, respectively. Subsequent verification of these levels agreed with model predictions and the maximum mycelial growth rate at these conditions was 6.1 +/- 0.8 mm day(-1). Therefore, the results of the experiments suggest that starch processing waste could be utilized as a growth substrate for the cultivation of the mushroom mycelia of P. linteus, enhancing the usefulness of this byproduct of the starch manufacturing industry. This approach is likely to be useful for establishing similar parameters for the cultivation of other fungi.     

Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Effect of submerged culture conditions on exopolysaccharides production by Armillaria luteo-virens Sacc QH and kinetic modeling

This work aimed to develop the submerged cultivation conditions for improved exopolysaccharides (EPS) production by Armillaria luteo-virens Sacc. The effects of culture temperature, aeration rate, inoculum level, initial pH, and additives on EPS formation and mycelial growth are investigated. The aeration rate, initial pH, and inoculum level significantly affected EPS production under the submerged cultivation. The developed conditions were as follows: cultivation temperature 23 °C, initial pH 5.0, aeration rate 0.5 vvm, 0.5% Tween 80, inoculum level 5% (v/v), and shaking speed 120 r/min. Under the developed conditions, the highest EPS production was 13.01 g/L at 5 days culture time. EPS production was examined in a 5 L bioreactor, and an unstructured kinetic model for EPS formation was well developed. The verified investigations in the large-scale cultivation system showed that the developed models are able to predict the submerged cultivation process of EPS formation. Current results revealed that the submerged cultivation conditions can be utilized to control EPS production, and the unstructured models developed are suitable for explaining EPS production by A. luteo-virens Sacc QH in a large-scale cultivation bioreactor.     

Optimization of submerged culture condition for the production of mycelial biomass and exopolysaccharides by Agrocybe cylindracea

The optimization of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) from Agrocybe cylindracea ASI-9002 using the statistically based experimental design in a shake flask culture. Both maximum mycelial biomass and EPS were observed at 25 degrees C. The optimal initial pH for the production of mycelial biomass and EPS were found to be pH 4.0 and pH 6.0, respectively. Subsequently, optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth was as follows: maltose 80 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 1.4 g/l, and CaCl2 1.1 g/l; for EPS production: maltose 60 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 0.9 g/l, and CaCl2 1.1 g/l. Under the optimal culture condition, the maximum EPS concentration achieved in a 5-l stirred-tank bioreactor indicated 3.0 g/l, which is about three times higher than that at the basal medium.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Statistical optimization of glucose oxidase production from Aspergillus niger NRC9 under submerged fermentation using response surface methodology

Response surface methodology (RSM), employing the fractional factorial design (FFD) was used to optimize the fermentation medium for the production of glucose oxidase (GOD) from a marine isolate (NRC9) of Aspergillus niger under submerged fermentation. The design was employed by selecting glucose, CaCO_3, ammonium phosphate and MgSO₄ concentrations as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum concentrations (g/l) glucose, 100; CaCO₃, 25; (NH₄)₂HPO₄, 1.8 and 0.4 of MgSO₄, resulted in an improvement of GOD production (170?±?0.88 U/ml) as compared to the initial level (109.81?±?1.38 U/ml) after four days of incubation at 200 rpm and 30 °C, whereas its predicted value obtained by the quadratic model was 164.36 U/ml. Analysis of variance (ANOVA) showed a high coefficient of determination value (R ²) of 0.967, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is the first report on production of glucose oxidase from a marine fungal isolate, Aspergillus niger NRC9, using statistical experimental design and response surface methodology in optimization of its production under submerged fermentation.     

毛头鬼伞子实体中甾类化合物的结构鉴定及其抑制肿瘤细胞增殖活性的研究

从毛头鬼伞子实体中分离得到4个甾类化合物,通过波谱分析,分别鉴定为麦角甾醇(1)、啤酒甾醇(2)、麦角甾醇葡萄糖甙(3)和tuberoside(4)。4个化合物均为首次从毛头鬼伞中得到。通过体外细胞毒性筛选试验,结果表明化合物4有较强的抑制人乳腺癌细胞MCF-7和狗肾细胞MDCK增殖的活性,其抑制增殖的IC50值分别为10.9μg/mL(18.4μmol/L)和5.8μg/mL(9.8μmol/L)。化合物3对MCF-7和MDCK的抑制作用则较弱,当其浓度为10.0μg/mL(17.9μmol/L)时,对MCF-7和MDCK的增殖抑制率分别为12.5%和7.5%。