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.     

Lipase production of Aspergillus oryzae

Aspergillus oryzae produced a small amount of lipase (0.05–0.8 U/wet-g of solid medium) in solid cultures, in contrast to the larger amount (0.46 U/ml) in a shake-flask culture in a modified GYP medium containing 2% glucose, 1% yeast extract and 2% Polypepton. Optimum conditions of lipase production in the submerged culture of A. oryzae were determined in terms of pH, composition of medium, and temperature. In a shake-flask culture at 28°C, the maximum amount of lipase increased to 0.78 U/ml upon the addition of 3% soybean oil to the modified GYP medium. In a jar fermentor culture, 30 U/ml lipase activity was obtained after 72 h at 28°C under appropriate conditions. Lipase production was greatly influenced by the culture temperature, and the optimum temperature for lipase production was about 24°C with a narrow temperature range, which was 10 degrees lower than that for the growth. In the submerged cultures, two kinds of lipase at least exhibiting different substrate specificities were also suggested.     

Production of a Pseudomonas lipase in n-alkane substrate and its isolation using an improved ammonium sulfate precipitation technique

Among the various lipidic and non-lipidic substances, normal alkanes within the chain lengths of C-12 to C-20 served as the best carbon substrates for the production of extracellular lipase by Pseudomonas species G6. Maximum lipase production of 25 U/ml of the culture broth was obtained by using n-hexadecane as the sole carbon substrate. The optimum pH of 8 and temperature of 34 + 1 degrees C were demonstrated for the production of lipase in n-hexadecane substrate. The optimum concentration of iron, which played a critical role on the lipase production, was found to be 0.25 mg/l. Lipase production could be enhanced to nearly 2.4-fold by using tributyrin at a concentration of 0.05% (v/v) in the culture medium. High recovery of the lipase protein (83%) from the culture broth was achieved by treating the culture supernatant with Silicone 21 Defoamer followed by ammonium sulfate (60% saturation) fractionation.     

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.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

多拷贝毕赤酵母重组菌表达GCL摇瓶优化和高密度发酵

目的:构建高效表达白地霉脂肪酶的毕赤酵母重组菌株,并对筛选得到的菌株进行摇瓶发酵条件优化和分批补料高密度发酵工艺研究。方法:将诱导型表达载体pPIC9K-gcl电转化至毕赤酵母GS115。通过橄榄油-罗丹明B平板和摇瓶发酵筛选高脂肪酶活力的重组菌株,运用基于TaqMan探针的实时荧光定量PCR 法确定其拷贝数,并对菌株进行摇瓶发酵条件优化。在此基础上,研究重组菌在3L 发酵罐中的高密度发酵工艺。结果:筛选得到一株具有3 个白地霉脂肪酶基因拷贝的菌株GS115/pPIC9K-gcl 78#,初始酶活力为220 U/ml。当摇瓶发酵条件为甲醇诱导96 h,每24 h甲醇添加量1 %,接种量2 %,培养基初始pH 7.0,500 ml摇瓶装液量50 ml,甲醇诱导温度25℃ 时酶活力达735 U/ml。3L 发酵罐高密度发酵176.5 h,酶活力达到3360 U/ml,总蛋白含量达到4.30 g/L,且发酵过程中细胞活性一直保持在96 % 以上。结论:基因拷贝数与重组菌株的产酶水平呈正相关,摇瓶优化可显著提高重组菌株的产酶能力,为白地霉脂肪酶的工业化生产奠定了技术基础。     

Lipase production by Aspergillus terreus using mustard seed oil cake as a carbon source

An extracellular lipase-producing fungus was isolated from the garden soil of the Post Graduate Department of Botany, Utkal University, Bhubaneswar, Odisha, India and identified as Aspergillus terreus. The A. terreus strain isolated was found to be capable of producing lipase in both solid state culture and liquid static surface culture. Experiments aimed at evaluating and improving the production of lipase and at studying the culture conditions revealed that of the many different materials tested as substrates, mustard oil cake (MoC) was the best substrate for extracellular lipase production. A correlation was found between the lipase production profile and biomass development. In a study aimed at continuing this line of research, we have investigated the influence of various culture conditions, such as environmental (i.e. temperature and pH), nutritional (i.e. carbon, nitrogen, metal ions, vitamins, combined agro-wastes and growth regulators) and other factors (inoculum size and initial moisture content) on the production of lipase by A. terreus in solid state and liquid static surface cultures. We observed that optimum lipase biosynthesis occurred under the following conditions: initial pH of 6.0, 30 °C, a 96-h incubation, lactose and ammonium persulphate as the carbon and nitrogen source respectively and 80 % moisture content. Changes in the vitamins (vitamin C, riboflavin, folic acid and vitamin E) and growth regulators (gibberellic acid, kinetin, 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid) did not support enhanced lipase production. MoC and neem oil cake (NoC) added to the media at a ratio of 9:1 respectively, supported maximum lipase production. Based on these results, we concluded that controlling the various culture conditions, supplementing MoC as a substrate and nutrient source modification of the medium can spectacularly enhance lipase biosynthesis by A. terreus.     

Optimization of extracellular lipase production by Geotrichum sp. using factorial design

Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp. The experiment included a 2(4) central composite rotatable design (CCRD) and four others 2(3) CCRD. According to the responses from the experimental designs, the effects of each variable were calculated and the interactions between them were determined. The response surface methodology was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30 degrees C. The optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.1-2.5%, corn steep liquor 13-15% and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum composition was ammonium nitrate 0.8-1%, corn steep liquor 13-15% and olive oil 0.6%, leading to an activity of 17 U/ml.     

Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125

Response surface methodology (RSM) was employed to optimize culture medium for production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was used to evaluate the effects of different components in the culture medium. Soybean oil, soybean powder and K₂HPO₄ have significant influences on the lipase production. The concentrations of three factors were optimized subsequently using central composite designs and response surface analysis. The optimized condition allowed the production of lipase to be increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l fermenter reached 9600 IU/ml.     

单因子-响应面法优化白地霉Y162产脂肪酶条件

对白地霉Y162液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适碳源为橄榄油,氮源为黄豆粉和NH4Cl,无机盐为BaCl2和MgCl2。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的橄榄油、BaCl2和NH4Cl三个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,得出橄榄油、BaCl2和NH4Cl最佳浓度分别为2.35％,0.36％,1.35％。优化后液体发酵液中脂肪酶活力提高到31.85 U/mL,比初始酶活力14.16 U/mL提高了2.25倍,表明单因子-响应面结合法可显著优化白地霉Y162液体发酵产脂肪酶条件。     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。     

Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of citric acid in submerged culture. For screening of fermentation medium composition significantly influencing citric acid production, the two-level Plackett-Burman design was used. Under our experimental conditions, beet molasses and corn steep liquor were found to be the major factors of the acid production. A near optimum medium formulation was obtained using this method with increased citric acid yield by five-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box-Behnken design was applied. A polynomial model was created to correlate the relationship between the three variables (beet molasses, corn steep liquor and inoculum concentration) and citric acid yield. Estimated optimum composition for the production of citric acid is as follows pretreated beet molasses, 240.1g/l; corn steep liquor, 10.5g/l; and spores concentration, 10(8)spores/ml. The optimum citric acid yield was 87.81% which is 14 times than the basal medium. The five level central composite design was used for outlining the optimum values of the fermentation factors initial pH, aeration rate and temperature on citric acid production. Estimated optimum values for the production of citric acid are as follows initial pH 4.0; aeration rate, 6500ml/min and fermentation temperature, 31.5 degrees C.     

Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

Abstract

The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7?days at 25?°C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ～50?kDa and ～54?kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40?°C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25?°C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10?°C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation.     

The effect of cultural conditions on the production of lipase by Acremonium strictum

Summary The optimum cultural conditions for the production of lipase byA. strictum under stationary condition are: period of incubation, 7 days; temperature, 30°C; xylose at a concentration of 2% (w/v) and 3.5% (w/v) soyabean meal as carbon and nitrogen sources respectively. Incorporation of 1% (v/v) of Tween 80 in culture medium enhanced enzyme production while the presence of fatty acids reduced both fungal growth and lipase production. The enzyme showed broad substrate specificity.     

Aflatoxin B1, zearalenone and deoxynivalenol production by Aspergillus parasiticus and Fusarium graminearum in interactive cultures on irradiated corn kernels

The influence of inoculum size in the production of aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) was determined when Aspergillus parasiticus NRRL 3000 and Fusarium graminearum ITEM 124 were cultured alone and in pairs on irradiated corn kernels at 28 °C and 0.97 water activity (aw). The highest levels of AFB1 produced by A. parasiticus were produced at the lowest levels of the inoculum (103 spores/ml). No significant differences were observed in ZEN and DON production at any inoculum level during the experimental period. When A. parasiticus was co-inoculated with F. graminearum both to the same inocula (106 spores/ml), AFB1 inhibition percentage were 60, 72 and 56% at 10, 20 and 35 days of incubation respectively, while at 106 spores/ml the percentages of inhibition were 34, 84 and 93% at 10, 20 and 35 days. In the mixture cultures A. parasiticus 103 × F. graminearum 106 spores/ml the percentage of inhibition of AFB1 oscillated in 99% during all the incubation. In the interaction A. parasiticus 106 spores/ml × F. graminearum 103 spores/ml the accumulation of AFB1 decreased in 80, 94 and 86% at 10, 20 and 35 days of incubation respectively. In single culture F. graminearum was inoculated with 10³ or 106 spores/ml and the highest levels of ZEN and DON were detected at 35 days of incubation. The levels oscillated in 538–622 μg/kg for ZEN and 870–834 μg/kg for DON respectively. In paired cultures there were no significant differences in the levels regardless of the spore concentrations during the incubation time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Lipase from marine strain using cooked sunflower oil waste: production optimization and application for hydrolysis and thermodynamic studies

The marine strain Pseudomonas otitidis was isolated to hydrolyze the cooked sunflower oil (CSO) followed by the production of lipase. The optimum culture conditions for the maximum lipase production were determined using Plackett–Burman design and response surface methodology. The maximum lipase production, 1,980 U/ml was achieved at the optimum culture conditions. After purification, an 8.4-fold purity of lipase with specific activity of 5,647 U/mg protein and molecular mass of 39 kDa was obtained. The purified lipase was stable at pH 5.0–9.0 and temperature 30–80 °C. Ca²⁺ and Triton X-100 showed stimulatory effect on the lipase activity. The purified lipase was highly stable in the non-polar solvents. The functional groups of the lipase were determined by Fourier transform-infrared (FT-IR) spectroscopy. The purified lipase showed higher hydrolytic activity towards CSO over the other cooked oil wastes. About 92.3 % of the CSO hydrolysis was observed by the lipase at the optimum time 3 h, pH 7.5 and temperature 35 °C. The hydrolysis of CSO obeyed pseudo first order rate kinetic model. The thermodynamic properties of the lipase hydrolysis were studied using the classical Van’t Hoff equation. The hydrolysis of CSO was confirmed by FT-IR studies.     

Galactomyces geotrichum Y25产脂肪酶条件的优化

应用响应面法对Galactomyces geotrichumY25液体发酵产脂肪酶的条件进行了优化。首先采用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出黄豆粉、玉米浆和发酵时间3个对产酶影响显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面设计对显著因素进行优化,得出黄豆粉、玉米浆最佳质量分数分别为2.51%、2.12%,最佳发酵时间101.95 h。优化后液体发酵液中脂肪酶活力提高到34.65 U/mL,比初始酶活力9.6 U/mL提高了3.61倍。表明响应面法可显著优化Galactomyces geotrichumY25液体发酵产脂肪酶条件。     

In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace's insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml.     

Lipase production of Staphylococcus carnosus in a dialysis fermentor

Summary In a newly constructed one-vessel dialysis fermentor, a strain of Staphylococcus carnosus TM300 carrying the lipase secretion plasmid pLipPS1 was used to investigate exoenzyme and biomass production. The bacterial culture grows in an inner compartment of 21 volume, separated from a 101 nutrient broth compartment by a conventional dialysis membrane. In order to avoid substrate depletion and to prolong the growth phase, a highly concentrated nutrient broth was used. The biomass production reached 60 g cell dry weight/l. The increase in extracellular lipase concentration was directly coupled with the increase of cell mass and reached a value of 230 mg/l culture supernatant. Harvesting the cells in the late growth phase, the lipase content was about 30% of the total exoproteins in the supernatant.     

Conversion of olive pomace oil to cocoa butter-like fat in a packed-bed enzyme reactor

Refined olive pomace oil (ROPO) was utilized as a source oil for production of cocoa butter-like fat. Immobilized sn-1,3 specific lipase catalyzed acidolysis of ROPO with palmitic (PA) and stearic (SA) acids was performed in a laboratory scale packed-bed reactor. Effect of reactor conditions on product formation was studied at various substrate mole ratios (ROPO:PA:SA; 1:1:1, 1:1:3, 1:3:3, 1:2:6), enzyme loads (10%, 20%, 40%), substrate flow rates (1.5, 4.5, 7.5, 15 ml/min) and solvent amounts (150, 400 ml). The highest yield (10.9% POP, 19.7% POS and 11.2% SOS) was obtained at 40% enzyme load, 1:2:6 substrate mole ratio, 45 degrees C, 7.5 ml/min substrate flow rate, 150 ml solvent and 3h reaction time. The melting profile and SFC of the product were comparable to those of CB. Polarized light microscope (PLM) images showed no drastic changes in polymorphic behavior between CB and product.