Kinetics of improved extracellular beta-d-fructofuranosidase fructohydrolase production by a derepressed Saccharomyces cerevisiae

AIMS: beta-d-fructofuranosidase fructohydrolase (FFH, EC 3.2.1.26) is an enzyme which hydrolyses the alpha-1,4 glycosidic bonds of sucrose and releases monosaccharides. The present study deals with the kinetics of improved extracellular FFH production by Saccharomyces cerevisiae in batch culture. MATERIALS AND RESULTS: Strains of S. cerevisiae can show increased FFH activity when grown on chemically defined medium. In the present study, wild-culture S. cerevisiae GCB-IV was mutated by treatment with ethyl methane sulfonate (EMS). Among six yeast mutants, EMS-II was found to be the highest FFH-producing strain (51.46 +/- 2.4 U ml(-1)). Maximum FFH production (78.46 +/- 3.2 U ml(-1)) was obtained 48 h after incubation by this 2-deoxy-d-glucose (2dg)-resistant mutant (76.20 mg ml(-1) protein). The optimal concentration of sucrose, incubation period and initial pH were 30.0 g l(-1), 28 degrees C and 6.5, respectively. The mutant EMS-II showed improvement in FFH production when 5.0 g l(-1) urea was added as a sole nitrogen source into SAPY medium. Values for Q(p) (1.802 +/- 0.2 U ml(-1) h(-1)) and Y(p/s) (3.460 +/- 1.1 U g(-1)) of EMS-II were significantly improved over the other yeast strains. CONCLUSION: The E(a) value (40.28 +/- 3.5 kJ mol(-1)) of EMS-II was significant (P 相似文献   

Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design

In the present study, eight different strains of Geotrichum candidum were isolated and screened for an extracellular exo-inulinase production using chemically enriched sucrose–mineral media. The isolate (Zool-3i) with a better enzyme activity (1.38 IU/ml) was subjected to induced mutagenesis using methyl methane sulphonate (MMS) and a mutant with an enzyme activity of 32.06 IU/ml was obtained. Further exposure to ethyl methane sulphonate (EMS) and ultraviolet (UV) radiations yielded a mutant exhibiting an improved activity of 39.34 IU/ml. The potential mutant was cultured overnight and plated on 5fc–YPR agar medium and thus made resistant against 5-flourocytocine. Over 50-fold enhancement in enzyme production (71.85 IU/ml) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g/L), pH (6.0), inoculum size (2.0%, 16 h old) and urea (0.2%) were identified using Plackett–Burman design. On the basis of kinetic variables, notably Q_p (0.723 U/g/h), Y_p/s (2.036 U/g) and q_p (0.091 U/g cells/h), the mutant MEU-5fc-6 was found to be a hyper producer of exo-inulinase (HS, LSD 0.045, p ? 0.05).     

β-Fructofuranosidase production by repeated batch fermentation with immobilized <Emphasis Type="Italic">Aspergillus japonicus</Emphasis>

The fungus Aspergillus japonicus ATCC 20236 was immobilized in vegetal fiber and used in repeated batch fermentations of sucrose (200 g/l) for the production of β-fructofuranosidases (FFase). The assays were performed during eight consecutive cycles that were completed in a total period of 216 h. After each 24-h cycle of fermentation (except for the first cycle, which lasted 48 h), the fermented broth was replaced by fresh medium, and the FFase activity was determined in the replaced medium. The average value of FFase activity was a constant 40.6 U/ml at the end of the initial seven cycles, but had decreased by 22% at the end of the eighth cycle. Concurrent with these high and constant FFase values, the hydrolyzing activity of this enzyme increased during the cycles, while the transfructosylating activity decreased. As a consequence, the maximum production of fructooligosaccharides of 134.60 g/l observed in the initial 30 h of fermentation (first cycle) had gradually decreased by the end of the subsequent cycles, reaching approximately 23% of this value during cycles 4–8. Based on these results, we conclude that the present immobilization system has a great potential for application in a semi-continuous process for the production of FFase, but further studies are necessary to maintain the FFase transfructosylation activity at high levels during the overall process.     

Glucoamylase production by solid-state fermentation using rice flake manufacturing waste products as substrate

Glucoamylase production has been investigated by solid-state fermentation of agro-industrial wastes generated during the processing of paddy to rice flakes (categorized as coarse, medium and fine waste), along with wheat bran and rice powder by a local soil isolate Aspergillus sp. HA-2. Highest enzyme production was obtained with wheat bran (264 +/- 0.64 U/gds) followed by coarse waste (211.5 +/- 1.44 U/gds) and medium waste (192.1 +/- 1.15 U/gds) using 10(6) spores/ml as inoculum at 28 +/- 2 degrees C, pH 5. A combination of wheat bran and coarse waste (1:1) gave enzyme yield as compared to wheat bran alone. Media supplementation with carbon source (0.04 g/gds) as sucrose in wheat bran and glucose in coarse and medium waste increased enzyme production to 271.2 +/- 0.92, 220.2 +/- 0.75 and 208.2 +/- 1.99 U/gds respectively. Organic nitrogen supplementation (yeast extract and peptone, 0.02 g/gds) showed a higher enzyme production compared to inorganic source. Optimum enzyme activity was observed at 55 degrees C, pH 5. Enzyme activity was enhanced in the presence of calcium whereas presence of EDTA gave reverse effect.     

Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis

In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.     

Optimization of medium for lipase production by Acinetobacter haemolyticus from healthy human skin

A lipase producing Acinetobacter haemolyticus TA106 was isolated from healthy human skin of tribal population. The maximum activity of 55 U/ml was observed after medium optimization using the "one variable at a time" and the statistical approaches. The optimal composition of the medium was determined as (% w/v or v/v): tryptone--1, yeast extract--0.5, sodium chloride-1, olive oil-1, Tween-80 1, manganese sulphate--5 mM, sucrose--1, pH-7. It was found that maximum production occurred in late log phase, i.e., after 72 h and at 200 rpm. From factorial design and statistical analysis, it was found that pH, temperature, salt, inoculum density and aeration significantly affected the lipase production. It was also noted that inoculum density of 3% (v/v), sucrose (1% w/v) and manganese sulphate (5 mM) displayed maximum lipase activity of 55 U/ml by conventional as well as statistical method. Optimization studies also indicated the increase in specific activity from 0.2 U/mg to 6.7 U/mg.     

Production of glutaminase (E.C.3.2.1.5) from Zygosaccharomyces rouxii: statistical optimization using response surface methodology

A face centered central composite design was employed to investigate the interactive effects of four variables, viz. concentrations of sucrose, yeast extract, sodium chloride, and glutamine, identified earlier by one-factor-at-a-time approach, on glutaminase production by Zygosaccharomyces rouxii. A significant influence of yeast extract on glutaminase production was noted. Response surface methodology (RSM) showed that a medium containing (g/l) sucrose, 17.8; yeast extract, 48.0; glutamine, 5.0 and sodium chloride, 55.6 to be optimum for the production of glutaminase. This medium was projected to produce, theoretically, an enzyme activity of 149.98 U/l and a specific activity of 0.488 U/mg protein. The applied methodology was validated using this optimized media and enzyme activity 155.89+/-1.68 U/l and specific activity of 0.468+/-0.088 U/mg protein was obtained. Further, this optimization strategy combined with an increase in inoculum enhanced the enzyme activity and specific activity by 2.94 and 3.58 fold, respectively, as compared to the unoptimized media.     

Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054

The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h(-1) for S. cerevisiae TMB 3399 to 0.14 h(-1) for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h(-1). All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose(-1) and 0.001, 0.10, and 0.16 g ethanol g biomass(-1) h(-1) for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein(-1), was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant.     

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.     

Production of fructosyltransferase by <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. ATCC 20524 in batch and two-step batch cultures

A comparison of fructosyltransferase (EC 2.4.1.9) production by Aureobasidium sp. ATCC 20524 in batch and two step batch cultures was investigated in a 1-l stirred tank reactor using a sucrose supply of 200 g/l. Results showed that the innovative cultivation in two step of Aureobasidium sp. produced more fructosyltransferase (FFase) than the single batch culture at the same sucrose concentration with a maximal enzyme production of 523 U/ml, which was 80.5% higher than the one obtained in the batch culture. The production of fructooligosaccharides (FOSs) was also analyzed; their concentration reached a maximum value of 160 g/l the first day in the two-step culture and 127 g/l in the single-batch mode. The use of the two-step batch culture with Aureobasidium sp. ATCC 20524 in allowing the microorganism to grow up prior to the induction of sucrose (second step), proved to be a powerful method for producing fructosyltransferase and FOSs.     

Selection and adaptation of Saccharomyces cerevisae to increased ethanol tolerance and production

A total of 24 yeast strains were tested for their capacity to produce ethanol, and of these, 8 were characterized by the best ethanol yields (73.11-8 1.78%). The most active mutant Saccharomyce s cerevisiae ER-A, resistant to ethanol stress, was characterized by high resistance to acidic (pH 1.0 and 2.0), oxidative (1 and 2% of H2O2), and high temperature (45 and 52 degrees C) stresses. During cultivation under all stress conditions, the mutants showed a considerably increased viability ranging widely from about 1.04 to 3.94-fold in comparison with the parent strain S. cerevisiae ER. At an initial sucrose concentration of 150 g/l in basal medium A containing yeast extract and mineral salts, at 300C and within 72 h, the most active strain, S. cerevisiae ER-A, reached an ethanol concentration of 80 g/1, ethanol productivity of 1.1 g/Il/h, and an ethanol yield (% of theoretical) of 99.13. Those values were significantly higher in comparison with parent strain (ethanol concentration 71 g/1 and productivity of 0,99 g/l/h). The present study seems to confirm the high effectiveness of selection of ethanol-resistant yeast strains by adaptation to high ethanol concentrations, for increased ethanol production.     

Production of an alkaline proteinase fromConidiobolus coronatus and its use to resolvedl-phenylalanine anddl-phenylglycine

An isolate ofConidiobolus coronatus (NCIM 1238) showed high proteinase activity (20.1 U/ml) and productivity (600 U/l.h) when 1% (w/v) glucose or sucrose was used as the carbon source in shake flasks. Addition of organic nitrogen sources, casein (2%), soybean flour (4%), liver extract (2%) or Edamin-S (2%), enhanced growth and proteinase production up to three-fold and seven-fold, respectively. The system was successfully run up to 6 l in a laboratory fermenter with a productivity of 600 U/l.h. The proteinase was successfully used to resolve the recemic mixtures ofdl-phenylalanine anddl-phenylglycine and thus could replace the currently used subtilisin.     

ENHANCED β-GALACTOSIDASE PRODUCTION OF Aspergillus oryzae MUTATED BY UV AND LiCl

In order to breed a high-yield β-galactosidase-producing strain, Aspergillus oryzae was used as the parent strain and mutagenized with ultraviolet (UV) and UV plus lithium chloride (LiCl), respectively. After being mutagenized by UV, the β-galactosidase activity of mutant UV-15-20 reached 114.08 U/mL, which revealed a 49.22% increase compared with the original strain. A mutant UV-LiCl-38 with high β-galactosidase activity (121.42U/mL) was obtained after compound mutagenesis of UV and LiCl; the β-galactosidase activity of this mutant was 58.82% higher than that of the parent strain. Subculture testing indicated that UV-15-20 and UV-LiCl-38 had good hereditary stability and may be ideal strains for the production of β-galactosidase. Additionally, it was demonstrated that compound mutagenesis with UV and LiCl is an effective mutation method for breeding industrially interesting strains.     

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.     

Beta-fructofuranosidase production by 2-deoxyglucose resistant mutants of aspergillus niger in submerged and solid-state fermentation

Aspergillus niger produces extracellular beta-fructofuranosidase under submerged (SmF) and solid state fermentation (SSF) conditions. After UV mutagenesis of conidiospores of A. niger, 2-deoxyglucose (10 g/l) resistant mutants were isolated on Czapek's minimal medium containing glycerol as a carbon source and the mutants were examined for improved production of beta-fructofuranosidase in SmF and SSF conditions. One of such mutant DGRA-1 overproduced beta-fructofuranosidase in both SmF and SSF conditions. In SmF, the mutant DGRA-1 showed higher beta-fructofuranosidase productivity (110.8 U/l/hr) than the wild type (48.3 U/l/hr). While in SSF the same strain produced 322 U/l/hr of beta-fructofuranosidase, 2 times higher than that of wild type (154.2 U/l/hr). In SmF, both wild type and mutants produced relatively low level of beta-fructofuranosidase in medium containing sucrose with glucose than from the sucrose medium. However in SSF, the DGRA-1 mutant grown in sucrose and sucrose+ glucose did not show any difference with respect to beta-fructofuranosidase production. These results indicate that the catabolite repression of beta-fructofuranosidase synthesis is observed in SmF whereas in SSF such regulation was not prominent.     

Rapid evaluation of 1-kestose producing β-fructofuranosidases from Aspergillus species and enhancement of 1-kestose production using a PgsA surface-display system

1-Kestose is a key prebiotic fructooligosaccharide (FOS) sugar. Some β-fructofuranosidases (FFases) have high transfructosylation activity, which is useful for manufacturing FOS. Therefore, obtaining FFases that produce 1-kestose efficiently is important. Here, we established a rapid FFase evaluation method using Escherichia coli that display different FFases fused to a PgsA anchor protein from Bacillus subtilis. E. coli cell suspensions expressing the PgsA-FFase fusion efficiently produce FOS from sucrose. Using this screening technique, we found that the E. coli transformant expressing Aspergillus kawachii FFase (AkFFase) produced a larger amount of 1-kestose than those expressing FFases from A. oryzae and A. terreus. Saturation mutagenesis of AkFFase was performed, and the mutant G85W was obtained. The E. coli transformant expressing AkFFase G85W markedly increased production of 1-kestose. Our results indicate that the surface display technique using PgsA is useful for screening of FFases, and AkFFase G85W is likely to be suitable for 1-kestose production.

Abbreviations: AkFFase: Aspergillus kawachii FFase; AoFFase: Aspergillus oryzae FFase; AtFFase: Aspergillus terreus FFase; FFase: β-fructofuranosidase; FOS: fructooligosaccharide; fructosylnystose: 1^F-β-fructofuranosylnystose     

Screening and optimization of nutritional factors for higher dextransucrase production by Leuconostocmesenteroides NRRL B-640 using statistical approach

To improve dextransucrase production from Leuconostocmesenteroides NRRL B-640 culture medium was screened and optimized using the statistical design techniques of Plackett-Burman and response surface methodology (RSM). Plackett-Burman design with six variables viz. sucrose, yeast extract, K2HPO4, peptone, beef extract and Tween 80 was performed to screen the nutrients that were significantly affecting dextransucrase production. The variables sucrose, K2HPO4, yeast extract and beef extract showed above 90% confidence levels for dextransucrase production and were considered as significant factors for optimization using response surface methodology. 2(4)-central composite design was used for RSM optimization. The experimental results were fitted to a second-order polynomial model which gave a coefficient of determination R2=0.95. The optimized composition of 30g/l sucrose, 18.9g/l yeast extract, 19.4g/l K2HPO4 and 15g/l beef extract gave an experimental value of dextransucrase activity of 10.7U/ml which corresponded well with the predicted value of 10.9U/ml by the model.     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 +/- 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-guanidine (MNNG). Out of 3,2-deoxy-D-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 +/- 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 x 10(-5) M MgSO4 x 5H2O into the fermentation medium reduced the Fe2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 +/- 5 g/l).     

Mutant selection of <Emphasis Type="Italic">Hahella chejuensis</Emphasis> KCTC 2396 and statistical optimization of medium components for prodigiosin yield-up

Prodigiosin is a natural red pigment with algicidal activity against Cochlodinium polykrikoides, a major harmful red-tide microalga. To increase the yield of prodigiosin, a mutant of Hahella chejuenesis KCTC 2396, assigned M3349, was developed by an antibiotic mutagenesis using chloramphenicol. When cultured in Sucrose-based Marine Broth medium (SMB), M3349 could produce prodigiosin at 1.628+/-0.06 g/L, while wild type producing at 0.658+/-0.12 g/L under the same conditions. To increase the yield of prodigiosin production by M3349, significant medium components were determined using a two-level Plackett-Burman statistical design technique. Among fourteen components included in SMB medium, NaCl, Na2SiO3, MgCl2, H3BO3, Na2HPO4, Na2SO4, and CaCl2 were determined to be important for prodigiosin production. The medium formulation was finally optimized using a Box-Behnken design as follows: sucrose 10.0, peptone 8.0, yeast extract 2.0, NaCl 10.0, Na2SO4 12.0, CaCl2 1.8, MgCl2 0.7 g/L; and H3BO3 22.0, Na2HPO4 20.0, Na2SiO3 8.0 mg/L. The predicted maximum yield of prodigiosin in the optimized medium was 2.43 g/L by the Box-Behnken design, while the practical production was 2.60+/-0.176 g/L, which was 3.9 times higher than wild type with SMB Medium (0.658 g/L).