Economical glucoamylase production by alginate-immobilized Thermomucor indicae-seudaticae in cane molasses medium

AIMS: The present investigation is aimed at assessing the suitability of cane molasses as a cheaper carbon and energy source for glucoamylase production using alginate-immobilized Thermomucor indicae-seudaticae. METHODS AND RESULTS: The culture variables for glucoamylase production were optimized by 'one-variable-at-a-time' strategy and response surface methodology (RSM). A high glucoamylase titre was attained when 40 alginate beads (c. 5x10(6) immobilized spores) were used to inoculate 50 ml of cane molasses (8%) medium in 250-ml Erlenmeyer flasks. Response surface optimization of fermentation parameters (cane molasses 7%, inoculum level 44 alginate beads per 50 ml of medium and ammonium nitrate 0.25%) resulted in 1.8-fold higher glucoamylase production (27 U ml(-1)) than that in the unoptimized medium (15 U ml(-1)). Enzyme production was also sustainable in 22 l of laboratory air-lift bioreactor. CONCLUSIONS: Cane molasses served as an excellent carbon and energy source for the economical production of glucoamylase, which was almost comparable with that in sucrose yeast-extract broth. The statistical model developed using RSM allowed determination of optimum levels of the variables for improving glucoamylase production. SIGNIFICANCE AND IMPACT OF THE STUDY: The cost of glucoamylase produced in cane molasses supplemented with ammonium nitrate was considerably lower (euro1.43 per million U) than in synthetic medium containing sucrose and yeast-extract (euro35.66 per million U). The reduction in fermentation time in air-lift bioreactor with sustainable glucoamylase titres suggested the feasibility of scale up of the process.     

Optimization of key process variables for enhanced hydrogen production by Enterobacter aerogenes using statistical methods

The individual and mutual effects of glucose concentration, temperature and pH on the hydrogen production by Enterobacter aerogenes were investigated in a batch system. A Box-Behnken design and response surface methodology (RSM) were employed to determine the optimum condition for enhanced hydrogen production. The hydrogen production rate was investigated by simultaneously changing the three independent variables, which all had significant influences on the hydrogen production rate. The maximum hydrogen production rate of 425.8 ml H(2)(g dry cell h)(-1) was obtained under the optimum condition of glucose concentration 118.06 mM, temperature 38 degrees C and pH 6.13. The experimental results showed that the RSM with the Box-Behnken design was a useful tool for achieving high rate of hydrogen production by E. aerogenes.     

Purification and kinetics of a raw starch-hydrolyzing,thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae

The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degrees C and pH 7.0, with a t(1/2) of 12 h at 60 degrees C and 7 h at 80 degrees C. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degrees C (K(m) 0.50 mg mL(-1) and V(max) 109 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.40 and V(max) 143 micromol mg(-1) protein min(-1)). The experimental K(m) and V(max) values are in agreement with the predicted values at 50 degrees C (K(m) 0.45 mg mL(-1) and V(max) 111.11 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.36 mg mL(-1)and V(max) 142.85 micromol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degrees C, and thereafter, inactivation. The enzyme was strongly stimulated by Co(2+), Fe(2+), Ag(2+), and Ca(2+), slightly stimulated by Cu(2+) and Mg(2+), and inhibited by Hg(2+), Zn(2+), Ni(2+), and Mn(2+). Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K(m) 2.4 mg mL(-1), V(max) 50 micromol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification.     

Optimization of a culture medium for xylanase production by Bacillus sp. using statistical experimental designs

The concentrations of oat spelt xylan, casein hydrolysate and NH4Cl in the culture medium for production of xylanase from Bacillus sp. I-1018 were optimized by means of response surface methods. The path of steepest ascent was used to approach the optimal region of the medium composition. The optimum composition of the nutrient medium was then easily determined by using a central composite design and was found to be 3.16g/l of xylan, 1.94g/l casein hydrolysate, 0.8g/l of NH4Cl. The xylanase production was increased by 135% when the strain was grown in the optimized medium compared to initial medium.     

Optimization of a culture medium for xylanase production by Bacillus sp. using statistical experimental designs

The concentrations of oat spelt xylan, casein hydrolysate and NH4Cl in the culture medium for production of xylanase from Bacillus sp. I-1018 were optimized by means of response surface methods. The path of steepest ascent was used to approach the optimal region of the medium composition. The optimum composition of the nutrient medium was then easily determined by using a central composite design and was found to be 3.16g/l of xylan, 1.94g/l casein hydrolysate, 0.8g/l of NH4Cl. The xylanase production was increased by 135% when the strain was grown in the optimized medium compared to initial medium.     

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.     

Breeding by protoplast fusion for glucoamylase production

Summary Protoplast fusion was carried out between two strains ofAspergillus niger 8-2, a fast growing culture and poor producer of glucoamylase enzyme andA.niger 8-7, a slow growing culture and good producer of the enzyme. The nonconidiating fused mass in presence of benomyl, produced fast growing segregants showing various combinations of the two parental gene markers. Some of the segregants produced up to 68% more glucoamylase than the better yielding parent 8-7.     

Medium optimization for hen egg white lysozyme production by recombinant Aspergillus niger using statistical methods

Statistics-based experimental design was used to investigate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl2.2H2O) on hen's egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2(5-1) fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other factors were not important within the levels tested. The method of steepest ascent was used to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g L-1, peptone 34 g L-1, ammonium sulfate 11.9 g L-1, yeast extract 0.5 g L-1, and CaCl2.2H2O 0.5 g L-1. This medium was projected to produce, theoretically, 212 mg L-1 lysozyme. Using this medium, an experimental maximum lysozyme concentration of 209+/-18 mg L-1 verified the applied methodology.     

产腈水解酶基因工程菌的产酶诱导条件及培养基优化

本文通过对产酶诱导条件及发酵培养基进行优化,成功提高了产腈水解酶基因工程菌E. coli BL21(DE3)-pETNYNit的产酶水平。研究结果显示,最佳发酵培养基为：葡萄糖0.2%、甘油0.7%(v/v)、蛋白胨1.2%、酵母膏0.8%、NaCl 0.3%、(NH4)2SO40.3%、NH4Cl 0.13%、Na2 HPO4·12H2 O 1.04%、KH2 PO40.39%、MgSO4·7H2 O 0.03%,pH 7.2。最佳产酶诱导条件为：发酵4 h时加入0.5 mmol/L IPTG,然后在28℃、240 r/min下诱导腈水解酶基因表达14 h~16 h。采用优化方案,重组菌产酶水平可提升至0.9~1×105 U,与野生菌株的产酶水平相比,提高幅度超过50%。同时重组菌培养仅需24 h,培养周期缩短超过50 h。     

Maximum glucoamylase production by a temperature-sensitive mutant of Saccharomyces cerevisiae in batch culture

In order to maximize the glucoamylase production by recombinant Saccharomyces cerevisiae in batch culture, first a temperature-controlled expression system for a foreign gene in S. cerevisiae was constructed. A temperature-sensitive pho80 mutant of S. cerevisiae for the PHO regulatory system, YKU131, was used for this purpose. A DNA fragment bearing the promoter of the PHO84 gene, which encodes an inorganic phosphate (P_i) transporter of S. cerevisiae and is derepressed by P_i starvation, was used as promoter. The glucoamylase gene connected with the PHO84 promoter was ligated into a YEp13 vector, designated pKU122. When the temperature-sensitive pho80 ^ts mutant harboring the plasmid pKU122 is cultivated at a lower temperature, the expression of glucoamylase gene is repressed, but at a higher temperature it is expressed. Next the effect of temperature on the specific growth rate, μ, and specific production rate, ρ, was investigated. Maximum values of ρ and ρ at various temperatures were at 30°C and 34°C, respectively. The optimal cultivation temperature strategy for maximum production of glucoamylase by this recombinant strain in batch culture was then determined by the Maximum principle using the relationships of μ and ρ to the cultivation temperature. Finally, the optimal strategy was experimentally realized by changing the cultivation temperature from Tμ (30°C) to Tρ (34°C) at the switching time, t_s. Received 18 September 1997/ Accepted in revised form 07 January 1998     

Optimization of culture medium for lactosucrose ( G-beta-D-galactosylsucrose) Production by Sterigmatomyces elviae mutant using statistical analysis

In this study, the optimization of culture medium using a Sterigmatomyces elviae mutant was investigated using statistical analysis to increase the cell mass and lactosucrose ((4)G-beta-D-galactosylsucrose) production. In basal medium, the cell mass and lactosucrose production were 4.12 g/l and 140.91 g/l, respectively. However, because of the low cell mass and lactosucrose production, optimization of culture medium was carried out to increase the cell mass and lactosucrose production. Culture media were optimized by the S. elviae mutant using analysis of variance (ANOVA) and response surface methodology (RSM). Central composite designs using RSM were utilized in this investigation. Quadratic models were obtained for cell mass and lactosucrose production. In the case of cell mass, optimal components of the medium were as follows: sucrose 1.13%, yeast extract 0.99%, bactopeptone 2.96%, and ammonium sulfate 0.40%. The predicted maximum value of cell mass was about 5.20 g/l and its experimental value was 5.08 g/l. In the case of lactosucrose production, optimal components of the medium were as follows: sucrose 0.96%, yeast extract 1.2%, bactopeptone 3.0%, and ammonium sulfate 0.48%. Then, the predicted maximum value of lactosucrose production was about 194.12 g/l and the corresponding experimental value was about 183.78 g/l. Therefore, by culturing using predicted conditions, the real cell mass and lactosucrose production increased to 23.3% and 30.42%, respectively.     

Optimization of mouse embryo culture media using simplex methods

Culture media were developed for pronuclear-stage mouse embryos using simplex optimization, which has the benefit of being able to optimize several components simultaneously. Initially, several different media were generated. All media contained the same components, yet each medium was characterized by having a different component at a high concentration. The simplex procedure identified 4 components (NaCl, pyruvate, KH2PO4 and glucose) which at high concentrations were detrimental to embryo development, compared to the other components tested. For example, all embryos cultured in a medium with high NaCl blocked at the 2-cell stage. The optimization method then adjusted each medium by lowering the concentration of the component or removing it entirely, which resulted in a significant increase in development. In an experiment comparing 8 media generated from the simplex optimization, along with 7 other media, removal of KH2PO4 resulted in the largest increase in development; 88% of embryos were greater than or equal to 4 cells on Day 3 after hCG, and 53% developed into blastocysts by Day 5. Another experiment compared 4 of the best media generated from the simplex optimization. In 3 out of the 4 media, 90% or more of the embryos were greater than or equal to 4 cells on Day 3. In 3 of the media, approximately 60% or more of the embryos developed into blastocysts. The simplex optimization procedure is an efficient method for developing culture media and determining requirements for development in vitro.     

Optimization of medium constituents for improved chitinase production by Paenibacillus sp. D1 using statistical approach

Aims:  Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1.
Methods and Results:  Urea, K₂HPO₄, chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett–Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l⁻¹): urea, 0·33; K₂HPO₄, 1·17; MgSO₄, 0·3; yeast extract, 0·65 and chitin, 3·75. This statistical optimization approach led to the production of 93·2 ± 0·58 U ml⁻¹ of chitinase.
Conclusions:  The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K₂HPO₄, chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2·56-fold increase in chitinase production.
Significance and Impact of the Study:  The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.     

Nutritional regulation of actinomycin-D production by a new isolate of Streptomyces sindenensis using statistical methods

Production of actinomycin-D, by an isolate, S. sindenensis, was optimized by statistical methods. Fructose peptone and NaNO3 were found to be critical for antibiotic production. In the second step, their concentrations were optimized with Central Composite Design and Response Surface Methodology. Fructose, peptone and NaNO3 at 2.55, 0.309 and 0.114% respectively gave approximately 261% higher yield (289 mg/l). Cultivation in fermentor at 600 rpm agitation and 1.5 vvm aeration with optimized medium gave 3.56 folds higher yield (365 mg/l) as compared to the yields in shake flasks using normal production medium (80 mg/l).     

Optimization of culture conditions for production of yeast biomass using bamboo wastewater by response surface methodology

Response surface methodology (RSM) was applied to optimize culture conditions for the growth of Candida utilis with bamboo wastewater. A significant influence of initial pH, fermentation time and yeast extract on biomass of C. utilis was evaluated by Plackett–Burman design (PBD). These factors were further optimized using a central composite design (CCD) and RSM. A combination of initial pH 6.1, fermentation time 69 h and yeast extract 1.17 g/L was optimum for maximum biomass of C. utilis. A 1.7-fold enhancement of biomass of C. utilis was gained after optimization in shake-flask cultivation. The biomass of C. utilis reached 19.17 g/L in 3 L fermentor.     

Optimization of protease production by Streptomyces sp. A6 using statistical approach for reclamation of shellfish waste

Protease producing Streptomyces sp. A6 was isolated from intertidal zone of the coast of Diu (Gujarat, India). Plackett–Burman method was applied to identify important factors (shrimp waste, FeCl₃, ZnSO₄ and pH) influencing protease production by Streptomyces sp. A6. Further optimization was done by response surface methodology using central composite design. The concentrations of medium components for higher protease production as optimized using the above approach were (g l^?1): Shrimp waste, 14; FeCl₃, 0.035; ZnSO₄, 0.065 and pH, 8.0. This statistical optimization approach led to production of 129.02 ± 2.03 U ml^?1 of protease which was 4.96 fold higher compared to that obtained using the unoptimized medium. The protease production was scaled to 3 l in a 5-l bench fermenter using optimized medium which further increased the production by 63.4%. Deproteinization and chitin recovery obtained at the end of fermentation was 85.12 ± 4.7 and 70.58 ± 1.33%, respectively. The present study is the first report on statistical optimization of medium components for production of protease by Streptomyces species using cheaper raw material such as shrimp waste. The study also explored the possibility Streptomyces sp. A6 for reclamation of shrimp wastes.     

Optimization of acetic acid production from synthesis gas by chemolithotrophic bacterium--Clostridium aceticum using statistical approach

Efforts in optimizing reducing agents, cysteine-HCl.H2O and sodium sulfide in order to attain satisfactory responses during acetic acid fermentation have been carried out in this study. Cysteine-HCl.H2O each with five concentrations (0.00-0.50 g/L) was optimized one at a time and followed by sodium sulfide component (0.00-0.50 g/L). Response surface methodology (RSM) was used to determine the optimum concentrations of cysteine-HCl.H2O and sodium sulfide. The statistical analysis showed that the amount of cells produced and efficiency in CO conversion were not affected by sodium sulfide concentration. However, sodium sulfide is required as it does influence the acetic acid production. The optimum reducing agents for acetic acid fermentation was at 0.30 g/L cysteine-HCl.H2O and sodium sulfide respectively and when operated for 60 h cultivation time resulted in 1.28 g/L acetic acid production and 100% CO conversion.     

Optimization of culture medium for cordycepin production using Cordyceps militaris mutant obtained by ion beam irradiation

The effect of medium components on cordycepin production by Cordyceps militaris mutant obtained by ion beam irradiation was investigated. According to the response surface analysis using a central composite design for the prospective mutant G81-3, the predicted optimal concentrations of glucose as the carbon source and the yeast extract as the nitrogen source were 86.2 g/l and 93.8 g/l, respectively, and 6.84 g/l cordycepin was obtained. To date, this is the highest value for cordycepin production. The optimal concentrations of glucose and yeast extract for cordycepin production of the mutant was much higher than that of control (wild strain) and the cordycepin production was 2.79 times higher. Therefore, this new mutant will be a promising strain for future higher cordycepin production at industrial levels.     

Optimization of culture conditions for hydrogen production by Ethanoligenens harbinense B49 using response surface methodology

The design of an optimum and cost-efficient medium for high-level production of hydrogen by Ethanoligenens harbinense B49 was attempted by using response surface methodology (RSM). Based on the Plackett-Burman design, Fe(2+) and Mg(2+) were selected as the most critical nutrient salts. Subsequently, the optimum combination of the selected factors and the sole carbon source glucose were investigated by the Box-Behnken design. Results showed that the maximum hydrogen yield of 2.21 mol/mol glucose was predicted when the concentrations of glucose, Fe(2+) and Mg(2+) were 14.57 g/L, 177.28 mg/L and 691.98 mg/L, respectively. The results were further verified by triplicate experiments. The batch reactors were operated under an optimized condition of the respective glucose, Fe(2+) and Mg(2+) concentration of 14.5 g/L, 180 mg/L and 690 mg/L, the initial pH of 6.0 and experimental temperature of 35+/-1(o)C. Without further pH adjustment, the maximum hydrogen yield of 2.20 mol/mol glucose was obtained based on the optimized medium with further verified the practicability of this optimum strategy.     

Optimization of reaction variables for sucrose monoester production using lipase in a solvent free system by Taguchi's method

Taguchi's method was attempted successfully to optimize the reaction variables and their ranges for sucrose monoester production. Optimal conditions determined by Taguchi's method were 35°C, 200rpm, capric acid (40mmol) and sucrose (3mmol), 24h, 0.5g lipase, 0.72g Ba(OH)2 ·8H2O, 0.72g Ba(OH)2·1H2O. A product yield of 21% was obtained. © Rapid Science Ltd. 1998