Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor

AIM: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor. METHODS AND RESULTS: Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0.1% KH2PO4, 0.5% NH4Cl, 0.24% (NH4)2HPO4, 0.01% MgSO4.7H2O and 1% emulsified palm oil, at 45 degrees C and pH 7.0, agitated at 250 rev min(-1) with 6-h-old inoculum (2% v/v) for 20 h. A fourfold enhancement in lipase production (50 U ml(-1)) and an approximately three fold increase in specific activity (160 U mg(-1)) by B. cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture. The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation. The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables. CONCLUSIONS: The maximum lipase production was 50 U ml(-1), with specific activity 160 U mg(-1) protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks. Further, the model predicted reduction in time for lipase production with reduction in total carbon supply. SIGNIFICANCE AND IMPACT OF THE STUDY: Statistical optimization allows quick optimization of a large number of variables. It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.     

Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal-0.77% (w/v); (NH(4))(2)SO(4)-0.1m; KH(2)PO(4)-0.05 m; rice bran oil-2% (v/v); CaCl(2)-0.05 m; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35°C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6-fold increase in lipase production was achieved. Partial purification by (NH(4))(2)SO(4) precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40°C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.     

A glycerol-inducible thermostable lipase from Bacillus sp.: medium optimization by a Plackett-Burman design and by response surface methodology

The production of a neutral lipase from a Bacillus sp. was improved tremendously (193-fold) following media optimization involving both the "one-at-a-time" and the statistical designing approaches. The present lipase was poorly induced by oils, instead its production was induced in the presence of sugars and sugar alcohols, mainly galactose, lactose, glycerol, and mannitol. A high inoculum density of 15% v/v (A550 = 0.8) led to maximum lipase production. Interestingly, the enzyme induction was growth independent, a property very different from most of the lipases investigated to date. The optimal composition of the growth medium to achieve maximum lipase production was determined to be as follows: NH4Cl, 35 g x L(-1); glycerol, 10 mL x L(-1); K2HPO4, 3 g x L(-1); KH2PO4, 1 g x L(-1); MgSO4.7H2O, 0.1 g x L(-1); glucose, 2 g x L(-1); MgCl2, 0.6 mmol x L(-1), with 15% inoculum density and an incubation period of 24 h. About 62 U x mL(-1) of enzyme production was achieved in the optimized medium.     

Maximizing production of Penicillium cyclopium partial acylglycerol lipase

Penicillium cyclopium partial acylglycerol lipase production was maximized in shaken batch culture. The effect of inoculum size and substrate concentration on the lipase activity released in the culture medium was visualized using a surface response methodology based on a Doehlert experimental design. The main advantage of this approach is the low number of experiments required to construct a predictive model of the experimental domain. Substrate percentage (corn steep, w/v) ranged from 0.1% to 1.9% and inoculum from 100 spores/ml to 3,200 spores/ml. We determined that an optimal set of experimental conditions for high lipase production was 1.0% substrate and 3,200 spores/ml, with initial pH 5.0, temperature 25 degrees C and shaking speed 120 rpm. Between the conditions giving the minimum and the maximum lipase production, we observed a three-fold increase in both the predicted and the measured values.     

Lipase production by Trichosporon fermentans WU-C12, a newly isolated yeast

From the soil samples of various locations, 245 strains of microorganisms were isolated by the enrichment culture method using olive oil as a carbon source. Of these microorganisms one deuteromycotinous yeast was the best producer of extracellular lipase, and the strain WU-C12 was identified as Trichosporon fermentans from the morphological and taxonomical properties. When cultivated at 30°C for 4 d in the medium containing 8% (w/v) corn steep and 3% (v/v) olive oil as sources of nitrogen and carbon, T. fermentans WU-C12 produced 126 U/ml of extracellular lipase. When 3% (v/v) tung oil was used instead of 3% (v/v) olive oil, 146 U/ml of the lipase was produced. Although lipase production decreased to 40 U/ml by the addition of 2% (w/v) glucose to the corn steep-olive oil medium, the strain WU-C12 produced 34 U/ml of lipase in the medium containing 2% (w/v) glucose instead of 3% (v/v) olive oil. On the other hand, T. fermentans WU-C12 could grow and produce lipase in the medium containing n-paraffin as a carbon source.     

Production of amylase by newly isolated moderate halophile,Halobacillus sp. strain MA-2

Production of extracellular amylase was demonstrated under stress conditions of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately halophilic bacterium of spore-forming Halobacillus sp. strain MA-2 in medium containing starch, peptone, beef extract, and NaCl. The maximum amylase production was secreted in the presence of 15% (w/v) Na(2)SO(4) (3.2 U ml(-1)). The isolate was capable of producing amylase in the presence of NaCl, NaCH(3)COOH, or KCl, with the results NaCl>NaCH(3)COOH>KCl. Maximum amylase activity was exhibited in the medium containing 5% (w/v) NaCl (2.4 U ml(-1)). Various carbon sources induced enzyme production. The potential of different carbohydrates in the amylase production was in the order: dextrin>starch>maltose>lactose>glucose>sucrose. In the presence of sodium arsenate (100 mM), maximum production of the enzyme was observed at 3.0 U ml(-1). Copper sulfate (0.1 mM) decreased the amylase production considerately, while lead nitrate had no significant enhancement on amylase production (p<0.05). The pH, temperature, and aeration optima for enzyme production were 7.8, 30 degrees C, and 200 rpm, respectively, while the optimum pH and temperature for enzyme activity was 7.5-8.5 and 50 degrees C, respectively.     

Effect of culture conditions on lipase production by Fusarium solani in batch fermentation

Lipase (Glycerol ester hydrolase EC 3.1.1.3.) from a Brazilian strain of Fusarium solani FSI has been investigated. The effect of different carbon sources and trace elements added to basal medium was observed with the aim of improving enzyme production. Lipase specific activity was highest (0.45 U mg(-1)) for sesame oil. When this medium was supplemented with trace elements using olive oil, corn oil and sesame oil the lipase specific activity increased to 0.86, 1.89 and 1.64 U mg(-1), respectively, after 96 h cultivation without any considerable biomass increase. The Km of this lipase using pNPP (p-nitrophenylpalmitate) as substrate, was 1.8 mM with a Vmax of 1.7 micromol min(-1) mg protein(-1). Lipase activity increased in the presence of increasing concentrations of hexane and toluene. In contrast, incubation of this enzyme with water-soluble solvents decreased its activity after 10% concentration (v/v) of the solvent. The lipase activity was stable below 35 degrees C but above this temperature activity losses were observed.     

Enhanced production of exocellular glucansucrase from Leuconostoc dextranicum NRRL B-1146 using response surface method

Statistically-based experimental designs were applied to optimize the fermentation for the production of glucosyltransferase by Leuconostoc dextranicum NRRL B-1146. Eleven medium components were examined for their significance on enzyme production using Plackett-Burman factorial design. Tween 80, sucrose and K2HPO4 significantly improved the enzyme production process. The combined effect of these nutrients on glucansucrase production were studied using a 2 2 full-factorial central composite design, a second-order polynomial was established to identify the relationship between the enzyme output and the three medium components. The optimal concentration of variables for maximum glucansucrase production were Tween 80 (0.55%, v/v); sucrose (5.6%, w/v) and K2HPO4 (1%, w/v). The maximum enzyme activity by predicted model was 6.53 U/ml that was in perfect agreement with the actual experimental value (6.40 U/ml).     

Effect of growth conditions on the production of manganese peroxidase by three strains of Bjerkandera adusta

We were looking for a strain of Bjerkandera adusta that produces high titres of manganese peroxidase under optimal conditions for large-scale enzyme purification. We have chosen two strains from the University of Alberta Microfungus Collection and Herbarium, UAMH 7308 and 8258, and compared the effects of growth conditions and medium composition on enzyme production with the well-characterized strain BOS55 (ATCC 90940). Of four types of cereal bran examined, rice bran at 3% (w/v) in 60 mM phosphate buffer pH 6 supported the highest levels of enzyme production. Using 100 mL medium in 500-mL Erlenmeyer flasks, maximum enzyme levels in the culture supernatant occurred after about 10 days of growth; 5.5 U x mL(-1) for UAMH 7308, 4.4 U x mL(-1) for UAMH 8258, and 1.7 U x mL(-1) for BOS55, where units are expressed as micromoles of Mn-malonate formed per minute. Growth as submerged cultures in 10-L stirred tank reactors produced 3.5 U x mL(-1) of manganese peroxidase (MnP) by UAMH 8258 and 2.5 U x mL(-1) of MnP by 7308, while enzyme production by BOS55 was not successful in stirred tank reactors but could be scaled up in 2-L shake flasks containing 400 mL rice bran or glucose-malt-yeast extract (GMY)-Mn-glycolate medium to produce MnP levels of 1.7 U x mL(-1). These results show that the two strains of B. adusta, UAMH 7308 and 8258, can produce between two and three times the manganese peroxidase level of B. adusta BOS55, that they are good candidates for scale up of enzyme production, and that the rice bran medium supports higher levels of enzyme production than most previously described media.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Isolation and screening of Streptomyces sp. Al-Dhabi-49 from the environment of Saudi Arabia with concomitant production of lipase and protease in submerged fermentation

In this study, Streptomyces sp. Al-Dhabi-49 was isolated from the soil sample of Saudi Arabian environment for the simultaneous production of lipase and protease in submerged fermentation. The process parameters were optimized to enhance enzymes production. The production of protease and lipase was found to be maximum after 5 days of incubation (139.2 ± 2.1 U/ml, 253 ± 4.4 U/ml). Proteolytic enzyme increases with the increase in pH up to 9.0 (147.2 ± 3.6 U/ml) and enzyme production depleted significantly at higher pH values. In the case of lipase, production was maximum in the culture medium containing pH 8.0 (166 ± 1.3 U/ml). The maximum production of protease was observed at 40 °C (174 ± 12.1 U/ml) by Streptomyces sp. Lipase activity was found to be optimum at the range of temperatures (30–50 °C) and maximum production was achieved at 35 °C (168 ± 7.8 U/ml). Among the evaluated carbon sources, maltose significantly influenced on protease production (218 ± 12.8 U/ml). Lipase production was maximum when Streptomyces sp. was cultured in the presence of glucose (162 ± 10.8U/ml). Among various concentrations of peptone, 1.0% (w/v) significantly enhanced protease production. The lipase production was very high in the culture medium containing malt extract as nitrogen source (86 ± 10.2 U/ml). Protease production was maximum in the presence of Ca²⁺ as ionic source (212 ± 3.8 U/ml) and lipase production was enhanced by the addition of Mg²⁺ with the fermentation medium (163.7 ± 6.2 U/ml).     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method

Rhizopus chinensis CCTCC M201021 was a versatile strain capable of producing whole-cell lipase with synthetic activity in submerged fermentation. In order to improve the production of whole-cell lipase and study the culture conditions systematically, the combination of taguchi method and response surface methodology was performed. Taguchi method was used for the initial optimization, and eight factors viz., maltose, olive oil, peptone, K2HPO4, agitation, inoculum size, fermentation volume and pH were selected for this study. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25 x 10(8) spores/L, olive oil 2.367% (w/v), fermentation volume 18 mL/250 mL flask, peptone 4.06% (w/v). Subsequent experimental trails confirmed the validity of the model. These optimal culture conditions in the shake flask led to a lipase yield of 13875 U/L, which 120% increased compare with the non-optimized conditions.     

Cellobiase from Termitomyces clypeatus: activity and secretion in presence of glycosylation inhibitors

In presence of the glycosylation inhibitors, 2-deoxy-d-glucose (1 mg/ml), tunicamycin (30 μg/ml), 1-deoxynojirimycin (30 μg/ml) and d-glucono-δ-lactone (1 mg/ml), total cellobiase activity, in the extracellular, intracellular and cell bound fractions, of the fungus Termitomyces clypeatus grown in 20 ml cellobiose medium (1%, w/v) increased by 50-, 1.8-, 2.4-, 1.3-fold, respectively, with respect to control medium (16.3 U). The inhibitors also stimulated secretion of 95% of the total protein in culture medium, except d-glucono-δ-lactone which released 60% of the total protein. 2-Deoxy-d-glucose (1 mg/ml) led to production of extracellular cellobiase up to 40 U/ml, whereas in absence of the inhibitors only 0.59 U/ml enzyme was detected.     

Optimization of Alkaline Protease Production from Bacillus sp. by Response Surface Methodology

High yields (1939 U/ml) of an alkaline protease were obtained in batch fermentation of a Bacillus sp. using a response surface methodology. The interaction of four variables, viz., starch, peptone, incubation time, and inoculum density, suggested inoculum density to be an insignificant variable. However, incubation time had a profound effect on protease yields at all the concentrations of carbon and nitrogen used. The response surface raised and flattened with increase in time of incubation, and maximum protease production up to 1939 U/ml was obtained after 96 h of incubation. The model equation obtained was validated experimentally at maximum starch (15 mg/ml) and peptone (7.5 mg/ml) concentration with increased incubation time up to 144 h in the presence of minimum inoculum density (1%). An overall 2.6-fold increase in protease production was obtained as compared with mean observed response (750 U/ml) at zero level of all variables. Received: 19 July 2001 / Accepted: 15 August 2001     

Optimal design for the maximization of Penicillium cyclopium lipase production

Penicillium cyclopium triacylglycerol lipase production was maximized in stationary batch culture. We used a surface response methodology based on a Doehlert experimental design to study the effect on the lipase activity released in the culture medium of the three most important factors: substrate concentration, pH and inoculum. Besides reducing the number of experiments required for optimization, this technique allowed us to quantify the lipase activity in any part of the experimental domain.We determined an optimal set of conditions for high lipase production: 1% substrate (corn steep), pH 5.5 and an inoculum of 10(4) spores/ml. Between conditions giving the minimum and the maximum lipase production, we observed a nine-fold increase of both the predicted and measured values.     

Effective enhancement of polylactic acid-degrading enzyme production by Amycolatopsis sp. strain SCM_MK2-4 using statistical and one-factor-at-a-time approaches

This study aims to find the optimal medium and conditions for polylactic acid (PLA)-degrading enzyme production by Amycolatopsis sp. SCM_MK2-4. Screening of the most effective components in the enzyme production medium by Plackett–Burman design revealed that the silk cocoon and PLA film were the most significant variables enhancing the PLA-degrading enzyme production. After an response surface methodology, a maximum amount of PLA-degrading enzyme activity at 0.74?U?mL^?1 was predicted and successfully validated at 95% after 0.39% (w/v) silk cocoon and 1.62% (w/v) PLA film were applied to the basal medium. The optimal initial pH value, temperature, and inoculum size were evaluated by a method considering one-factor-at-a-time. The values were recorded at an initial pH in the range of 7.5–9.0, a temperature of 30–32°C, and an inoculum size of 4–10%. The highest activity of approximately 0.95?U?mL^?1 was achieved after 4 days of cultivation using the optimized medium and under optimized conditions in a shake flask. Upscaling to the use of a 3-L stirred tank fermenter was found to be successful with a PLA-degrading activity of 5.53?U?mL^?1; which represents a 51-fold increase in the activity compared with that obtained from the nonoptimized medium and conditions in the shake flask.     

Production and extraction of extracellular lipase from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales)

Lipases are important cuticle degrading enzymes involved in the infection process of entomopathogens by hydrolysing the ester bonds of lipoproteins, fats and waxes present in the insect integument. Production of extracellular lipase by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 was investigated using different combinations of basal medium components. The effect of different vegetable oils added to a basal medium at different concentrations to improve enzyme production was evaluated. Maximum lipase activity (125.33±2.96 U/mL) as well as maximum biomass production (22.36±0.99 mg/mL) was observed for olive oil when used at a concentration of 2% (v/v) of the basal medium. In the presence of surfactants, the highest lipase activity occurred when SDS and Tween 80 were added at the time of fungal inoculation. SDS proved to be the best surfactant having 110.66±3.52 U/mL lipase activity. The effects of the divalent metal ions (iron and magnesium) on lipase activity were also studied. Iron inhibited, whereas magnesium slightly increased lipase activity. The optimum pH for lipase production was 5.7 while 32°C proved to be the best temperature for lipase production.     

Short communication: Production of l-glutamate oxidase by Streptomyces sp. N1 in submerged fermentation

In submerged fermentation of Streptomyces sp. N1 in a shake flask, glucose (3% w/v) and (NH₄)₂SO₄ (0.6% w/v) were found to be suitable for extracellular l-glutamate oxidase (GluOx) (EC.1.4.3.11) production. GluOx production was higher with the addition of further KCl or MgCl₂ to the medium within the range of 0 to 0.12% (w/v). The effect of inoculum type, that is, spore inoculation or mycelium inoculation on GluOx biosynthesis was also investigated, and the maximum GluOx production obtained was 2.7 U/ml after 33h fermentation with mycelium inoculation. The results demonstrated a much higher GluOx production and productivity compared with those reported previously.     

Statistical optimization of production conditions of alkaline pectin lyase from Bacillus cereus using response surface methodology

Alkaline pectin lyase finds applications in the degumming and retting of plant fibres, textile industry and pectic wastewater treatment where it degrades highly methylesterified pectin without prior action of any other pectinase. Response surface methodology (RSM) has been frequently utilized for the optimization of production process of industrially important enzymes from microbes. In the present work, fermentation conditions for the production of pectin lyase from Bacillus cereus were optimized using the factorial and central composite design of RSM. The cubic order polynomial regression model was found to be adequate and significant with a determination coefficient R² of 0.9505 (p?相似文献