Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source

Abstract

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.     

Optimization of medium components for high-molecular-weight hyaluronic acid production by <Emphasis Type="Italic">Streptococcus</Emphasis> sp. ID9102 via a statistical approach

Hyaluronic acid (HA), linear high-molecular-weight glycosaminoglycan produced from Streptococcus sp., has raised interest in the medical and cosmetics industries because of the various biological functions of HA. In this paper, we report on the optimization of medium components for HA production in Streptococcus sp. ID9102 (KCTC 11935BP) by two-step optimization (one-factor-at-a-time and taguchi orthogonal array design). In the first step, medium components, such as carbon, nitrogen, phosphate, and mineral sources, were selected for HA production in Streptococcus sp. ID9102 (KCTC 11935BP) using the one-factor-at-a-time method. In the second step, the concentration of the selected medium components was optimized using taguchi orthogonal array design. The design for medium optimization was developed and analyzed using MINITAB 14 software. In addition, the effect of amino acid and organic acid, such as glutamine, glutamate, and oxalic acid, was studied for HA production in Streptococcus sp. ID9102 (KCTC 11935BP). Through these processes, the optimum medium comprising 4% glucose, 0.75% yeast extract, 1.0% casein peptone, 0.25% K₂HPO₄, 0.05% MgCl₂, 0.5% NaCl, 0.04% glutamine, 0.06% glutamate, and 0.02% oxalic acid was determined. We were able to produce HA with a molecular weight of 5.9 × 10⁶ at a productivity of 6.94 g/l on pilot scale fermentation.     

Medium optimization for pyrroloquinoline quinone (PQQ) production by Methylobacillus sp. zju323 using response surface methodology and artificial neural network–genetic algorithm

Methylobacillus sp. zju323 was adopted to improve the biosynthesis of pyrroloquinoline quinone (PQQ) by systematic optimization of the fermentation medium. The Plackett–Burman design was implemented to screen for the key medium components for the PQQ production. CoCl₂?·?6H₂O, ρ-amino benzoic acid, and MgSO₄?·?7H₂O were found capable of enhancing the PQQ production most significantly. A five-level three-factor central composite design was used to investigate the direct and interactive effects of these variables. Both response surface methodology (RSM) and artificial neural network–genetic algorithm (ANN–GA) were used to predict the PQQ production and to optimize the medium composition. The results showed that the medium optimized by ANN–GA was better than that by RSM in maximizing PQQ production and the experimental PQQ concentration in the ANN–GA-optimized medium was improved by 44.3% compared with that in the unoptimized medium. Further study showed that this ANN–GA-optimized medium was also effective in improving PQQ production by fed-batch mode, reaching the highest PQQ accumulation of 232.0?mg/L, which was about 47.6% increase relative to that in the original medium. The present work provided an optimized medium and developed a fed-batch strategy which might be potentially applicable in industrial PQQ production.     

Production of tyrosinase by Auricularia auricula using low cost fermentation medium

Low cost fermentation media using agricultural by-products (wheat bran extract, rice bran extract and soybean meal extract) as a major nutrient source, were evaluated for the production of tyrosinase from the fungus Auricularia auricula in submerged culture. In single-factor experiments, three components (wheat bran extract, casein and CuSO₄) were chosen to further optimize medium composition using response surface methodology (RSM). The central composite experimental results showed the following optimum medium composition: wheat bran extract 36.0 %, casein 1.1 g/l and CuSO₄ 0.13 g/l. Under these conditions, the highest tyrosinase activity was 17.22 U/ml, which was 2.1 fold higher than that obtained using the non-optimized medium. The present study is the first to report the statistical optimization of medium composition for production of tyrosinase by A. auricula using cheaper wheat bran extract as a major nutrient source. These results might provide a reference for the development of a cost-effective medium for commercial production of tyrosinase.     

Optimization of riboflavin production by recombinant <Emphasis Type="Italic">Bacillus subtilis</Emphasis> RH44 using statistical designs

A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin by recombinant Bacillus subtilis RH44. In the first instance, the medium components were optimized in shake flask cultures. After preliminary experiments of nitrogen source selection, the two-level Plackett–Burman (PB) design was implemented to screen medium components that significantly influence riboflavin production. Among the 15 variables tested, glucose, NaNO₃, K₂HPO₄, ZnSO₄, and MnCl₂ were identified as the most significant factors (confidence levels above 95%) for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). The validity of the model developed was verified, and the optimum medium led to a maximum riboflavin concentration of 6.65 g/l, which was 44.3 and 76.4% higher than the improved medium and the basal medium, respectively. A glucose-limited fed-batch culture profile in a 5-l fermentor was consequently designed according to the above optimum medium in shake flasks. A final riboflavin concentration of 16.36 g/l was obtained in 48 h, which further verified the practicability of this optimum strategy.     

Inhibition of Astrocyte Glutamine Production by α-Ketoisocaproic Acid

Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used ¹⁵NH₄Cl as a metabolic tracer to measure the production of both [5-¹⁵N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-¹⁵N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [¹⁵N]-glutamate to [2-¹⁵N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-¹⁵N]glutamine and stimulated the formation of [2-¹⁵N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-¹⁵N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-¹⁵N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-¹⁵N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of ¹⁴CO₂ from [U-¹⁴C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K_m of glutamine synthetase for glutamate, which we determined to be ～7 mM.     

Coproduction of menaquinone-7 and nattokinase by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> using soybean curd residue as a renewable substrate combined with a dissolved oxygen control strategy

Numerous physiological functions of menaquinone-7 (MK-7) act to reduce vascular calcification, suggesting that MK-7 may be a potential therapy for Alzheimer’s and Parkinson’s disease, and in this study, we attempted to increase the concentration of MK-7 synthesized by Bacillus subtilis natto, a standard nattokinase (NK) producing strain. Different Bacillus subtilis isolates demonstrated positive correlations between MK-7 and NK concentrations. Response surface methodology (RSM) was employed to optimize a culture medium for the simultaneous production of these molecules; the optimized medium contained the following components (%, w/v): soybean curd residue, 12.2; soya peptone, 5.7; lactose, 2.6; and K₂HPO₄, 0.6. The fermentation process was subsequently optimized based on online feedback control of fermentation process parameters. The dissolved oxygen (DO) concentration played an important role in the production of MK-7 and NK. With increased DO concentrations, the cell growth rate and NK activity increased. In contrast, at low DO concentrations, the concentration of MK-7 rapidly increased during the late fermentation stage. Thus, in this study, the production of MK-7 and NK by Bacillus subtilis was accomplished using soybean curd residue through medium optimization and DO control. This novel coproduction strategy was developed by controlling the aeration rate during the fermentation process. The concentrations of MK-7 and NK achieved in this study reached 91.25 mg/L and 2675.73 U/mL, respectively.     

Statistical Optimization of Medium and Fermentation Conditions of Recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> for the Production of Xylanase

Recombinant xylanase (rPcXynC) from Pichia pastoris was produced on large-scale by optimizing production-medium composition using statistical experimental methods. Production medium was optimized through the use of statistical methods such as one factor at a time (OFAT), Plackett-Burman design, fractional factorial design (FFD), steepest ascent method (SAM), and response surface methodology (RSM). The optimum medium composition was established to be (g/L); wheat bran 11.62, yeast extract 30, Tween 60.5, DL-β-Phenylalanine 0.5, Thiamine 0.5, FeSO₄ 0.01, KH₂PO₄ 0.66, and KHSO₄ 0.09. The optimum medium composition yielded 3,051 mU/mL of xylanase activity which was three times higher than that obtained from the initial medium composition. Finally, fermentation conditions were examined using the optimized production medium in a laboratory bioreactor. The optimal fermentation conditions were found to be 25ºC, pH 6, 170 rpm and 1 vvm with intermittent feeding of methanol (67.5 mL) and the xylanase activity was 3,683 mU/mL. In repeated-batch fermentation using optimized production medium and fermentation condition, the xylanase activity was 3,680 mU/mL at the first cycle of 96 h harvesting time using 90% of the culture solution. The activity was similarly maintained until the last cycle of 264 h.     

Optimization of fermentation condition for antibiotic production by Xenorhabdus nematophila with response surface methodology

Aims: To evaluate the influence of environmental parameters on the production of antibiotics (xenocoumacins and nematophin) by Xenorhabdus nematophila and enhance the antibiotic activity. Methods and Results: Response surface methodology (RSM) was employed to study the effects of five parameters (the initial pH, medium volume in flask, rotary speed, temperature and inoculation volume) on the production of antibiotics in flask cultures by X. nematophila YL001. A 2^5?1‐factorial central composite design was chosen to explain the combined effects of the five parameters and to design a minimum number of experiments. The experimental results and software‐predicted values of production of antibiotics were comparable. The statistical analysis of the results showed that, in the range studied, medium volume in flask, rotary speed, temperature and inoculation volume had a significant effect (P < 0·05) on the production of antibiotics at their individual level, medium volume in flask and rotary speed showed a significant influence at interactive level and were most significant at individual level. The maximum antibiotic activity was achieved at the initial pH 7·64, medium volume in 250 ml flask 25 ml, rotary speed of 220 rev min^?1, temperature 27·8°C and inoculation volume of 15·0%. Maximum antibiotic activity of 331·7 U ml^?1 was achieved under the optimized condition. Conclusions: As far as known, there are no reports of production of antibiotic from X. nematophila by engineering the condition of fermentation using RSM. The results strongly support the use of RSM for fermentation condition optimization. The optimization of the environmental parameters resulted not only in a 43·4% higher antibiotic activity than unoptimized conditions but also in a reduced amount of the experiments. The chosen method of optimization of fermentation condition was efficient, relatively simple and time and material saving. Significance and Impact of the Study: This study should contribute towards improving the antibiotics activity of X. nematophila. Integrated into a broader study of the impact of environmental factors on the production of antibiotic, this work should help to build more rational control strategy, possibly involving scale‐up of production of antibiotics by X. nematophila.     

Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a′ using response surface methodology

The novel exopolysaccharide bioflocculant HBF-3 is produced by Halomonas sp. V3a′, which is a mutant strain of the deep-sea bacterium Halomonas sp. V3a. Response surface methodology (RSM) was employed to optimize the production medium for increasing HBF-3 production. Using a Plackett–Burman experimental design to aid in the first step of optimization, edible glucose, MgSO₄·7H₂O, and NH₄Cl were found to be significant factors affecting HBF-3 production. To determine the optimal concentration of each significant variable, a central composite design was employed. Based on response surface and canonical analysis, the optimum concentrations of the critical components were obtained as follows: edible glucose, 16.14 g/l; MgSO₄·7H₂O, 2.73 g/l; and NH₄Cl, 1.97 g/l. HBF-3 production obtained by using the optimized medium was 4.52 g/l, which was in close agreement with the predicted value of 4.55 g/l. By scaling up fermentation from flask to fermenter, HBF-3 production was further increased to 5.58 g/l.     

Optimisation of medium and culture conditions for the production of antifungal substances to Colletotrichum musae by Trametes elegans SR06

An endophytic fungus SR06 was isolated from a leaf of Amomum villosum Lour., which had a high antagonistic effect on Colletotrichum musae with an inhibition ratio of 41.20%. The antifungal substances could be secreted into fermentation broth, which had a high inhibitory activity. Strain SR06 was identified as Trametes elegans according to internal transcribed spacer sequence analysis. Response surface methodology (RSM) was used to optimise the process parameters of antifungal substances production. Using the Plackett–Burman design, three variables (glucose, yeast extract and MgSO₄·7H₂O) exerted significant effects on antifungal substances production. Then RSM experiments were conducted to further optimise the three variables. The optimal medium components were 26.45?g/L glucose, 10?g/L peptone, 14.96?g/L yeast extract and 1.49?g/L MgSO₄·7H₂O, and the optimal initial pH was 6.0, with a culture temperature of 28°C and a shaking speed of 180?rpm. Under the optimised conditions, a significant improvement in the production of antifungal substances by T. elegans SR06 was accomplished, and the inhibition zone diameter was up to 29.2?mm after culturing for 7d. The average control efficacy of the fermentation supernatant of SR06 against C. musae was 51.29% on banana fruits, which was significantly higher than that of the fungicide carbendazim.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Optimization of medium composition for butyric acid production by Clostridium thermobutyricum using response surface methodology

The optimal medium for butyric acid production by Clostridium thermobutyricum in a shake flask culture was studied using statistical experimental design and analysis. The optimal composition of the fermentation medium for maximum butyric acid yield, as determined on the basis of a three-level four-factor Box-Behnken design (BBD), was obtained by response surface methodology (RSM). The high correlation between the predicted and observed values indicated the validity of the model. A maximum butyric acid yield of 12.05 g/l was obtained at K₂HPO₄ 7.2 g/l, 34.9 g/l glucose, 20 g/l yeast extract, and 15 g/l acetate, which compared well to the predicated production of 12.13 g/l.     

Development of a novel solid-state fermentation strategy for the production of poly-3-hydroxybutyrate using polyurethane foams by Bacillus sphaericus NII 0838

The extensive use of synthetic plastics has caused serious waste disposal problems in our environment. Poly-3-hydroxybutyrates (PHB) are eco-friendly bacterial polyesters which are produced under unbalanced nutrient conditions. Few reports are available on PHB production by solid state fermentation (SSF). We have developed a novel SSF bioprocess in which polyurethane foam (PUF) is used as a physical inert support for the production of PHB by Bacillus sphaericus NII 0838. Media engineering for optimal PHB production was carried out using response surface methodology (RSM) adopting a Box–Behnken design. The factors optimized by RSM were inoculum size, pH and (NH₄)₂SO₄ concentration. Under optimized conditions—6.5 % inoculum size, 1.7 % (w/v) (NH₄)₂SO₄ and pH 9.0—PHB production and biomass were 0.169?±?0.03 and 0.4?±?0.002 g/g PUF, respectively. This is the first report on PHB production by SSF using PUF as an inert support. Our results demonstrate that SSF can be used as an alternative strategy for the production of PHB.     

Development of fermentation process for PLA-degrading enzyme production by a new thermophilic Actinomadura sp. T16-1

The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-degrading Actinomadura sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at 50°C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2 % KH₂PO₄, and 0.02% MgSO₄ · 7H₂O. The optimal concentration of PLA film and gelatin obtained by response surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238% (w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter.     

Improvement of 1,3-dihydroxyacetone production from Gluconobacter oxydans by ion beam implantation

Improvement of dihydroxyacetone (DHA) production by mutagenesis of ion beam implantation and medium optimization using response-surface methodology (RSM) were investigated in this work. More than 1000 mutant strains were selected through a mutagenesis method using N(+) ions implantation with a dose of 60?×?(2.6?×?10(13)) ions/cm(2) and energy of 10?keV. Several high-yield mutant strains were showed the potent application for DHA production and the genetically stable mutant strain G. oxydans ZJB09113 was selected for optimization of cultivation condition by RSM. The optimal medium for DHA fermentation is composed (in g/L) of yeast extract 4.88, CaCO(3) 2.00, and glycerol 52.86?mL/L (initial pH 4.89). The maximal DHA concentration of 40.0?g/L was achieved after 24?hr of shaken flask fermentation at 30°C with 150?rpm, and 196.3% increase in DHA production in comparison with unoptimized conditions.     

Optimization of antifungal lipopeptide production from Bacillus sp. BH072 by response surface methodology

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH₄Cl, urea, and ammonium citrate); and metal ions (Zn²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ca²⁺, and K⁺). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg²⁺ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg²⁺. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.     

Improved production of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. ECU1011 acetyl esterase by medium design and fed-batch fermentation

We optimized culture medium and batch-fed fermentation conditions to enhance production of an acetyl esterase from Pseudomonas sp. ECU1011 (PSAE). This enzyme enantioselectively deacetylates α-acetoxyphenylacetic acid. The medium was redesigned by single-factor and statistical optimization. The addition of ZnSO₄ enhanced enzyme production by 37%. Yeast extract concentration was directly associated with the enzyme production. The fermentation was scaled up in a 5-l fermenter with the optimized medium, and the correlations between enzyme production and dissolved oxygen, pH, and feeding strategy were investigated. The fermentation process was highly oxygen-demanding, pH sensitive and mandelic acid-inducible. The fermentation pH was controlled at 7.5 by a pH and dissolved oxygen feedback strategy. Feeding mandelic acid as both a pH regulator and an enzyme inducer increased the enzyme production by 23%. The results of the medium redesign experiments were confirmed and explained in fed-batch culture experiments. Mathematical models describing the fermentation processes indicated that the enzyme production was strongly associated with cell growth. The optimized pH and dissolved oxygen stat fed-batch process resulted high volumetric production of PSAE (4166 U/l, 7.2-fold higher than the initial) without enantioselectivity decline. This process has potential applications for industrial production of chiral mandelic acid or its derivatives.     

Response surface methodology for optimization of medium components in submerged culture of Aspergillus flavus for enhanced heparinase production

Aims: Aim of the study was to develop a medium for optimal heparinase production with a strain of Aspergillus flavus (MTCC‐8654) by using a multidimensional statistical approach. Methods and Results: Statistical optimization of intracellular heparinase production by A. flavus, a new isolate, was investigated. Plackett–Burman design was used to evaluate the affect of medium constituents on heparinase yield. The experimental results showed that the production of heparinase was dependent upon heparin, the inducer; chitin, structurally similar to heparin and NH₄NO_3, the nitrogen source. A central composite design was applied to derive a statistical model for optimizing the composition of the fermentation medium for the production of heparinase enzyme. The optimum fermentation medium consisted of (g l^?1) Mannitol, 8·0; NH₄NO₃, 2·5; K₂HPO₄, 2·5; Na₂HPO₄, 2·5; MgSO₄.7H₂O, 0·5; Chitin, 17·1; Heparin, 0·6; trace salt solution (NaMoO₄.2H₂O, CoCl₂.6H₂O, CuSO₄.5H₂O, FeSO₄.7H₂O, CaCl₂), 10^?4 mol l^?1. Conclusions: A 2·37‐fold increase in heparinase production was achieved in economic and effective manner by the application of statistical designs in medium optimization. Significance and Impact of the Study: Heparinase production was doubled by statistical optimization in a cost‐effective manner. This heparinase can find application in pharmaceutical industry and for the generation of low‐molecular‐weight heparins, active as antithrombotic and antitumour agents.     

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.