Extracellular Lipase Production Research Articles

Screening and optimization of submerged fermentation of lipolytic Aspergillus oryzae

Lipases are enzymes commonly used in industry. This study describes the production of lipolytic enzyme via a newly isolated strain of Aspergillus oryzae under a submerged fermentation process. Five strains of A. oryzae were isolated from oil-contaminated soil and water as well as dead decaying organic matter. Qualitative screening revealed that A. oryzae RBM4 strain was a lipase producer, and the process was optimized for enhanced production. Incubation time, incubation temperature, initial pH, use of agricultural by-products, nitrogen sources, and substrates were tested. The results revealed that initial pH 5.5(12.7 U/mL/min) in 72 h (19.39 U/mL/min) at 30 °C (27.40 U/mL/min) sorghum (35.66 U/mL/min), NaNO3 (17% more than blank), yeast extract (47.95 U/mL/min), and Shan ghee (58.12 U/mL/min) were the best conditions. Extracellular lipase production was increased up to 78% by applying all the above conditions.

Use of low-cost substrates for cost-effective production of extracellular and cell-bound lipases by a newly isolated yeast Dipodascus capitatus A4C

Abstract As the enzyme production and its application is a costly process mainly because of the inducible production, this study aimed to screen the basic components of media for the production of cost-effective lipases. A newly isolated yeast strain, namely Dipodascus capitatus A4C is a specific yeast that could produce lipases in both extracellular and cell-bound forms. Various low-cost substrates were applied as single or mixed carbon sources for D. capitatus A4C. These included non-edible vegetable oil (Jatropha curcas oil), crude glycerol, and molasses. The suitable cost-effective carbon source for extracellular lipase production was 2% J. curcas oil, which gave high extracellular lipase activity of 2670 U/L at 96 h. While the suitable low-cost carbon source for cell-bound lipase production was 2% molasses, which gave high cell-bound lipase activity of 373 U/L at 48 h. The present work emphasized that the lipolytic activity of D. capitatus A4C can be improved with a compatible use of low-cost carbon sources as co-substrates.

Improved germination efficiency of Salicornia ramosissima seeds inoculated with Bacillus aryabhattai SP1016‐20

Saline agriculture and the crop cultivation of halophytes represent an alternative for the reclamation of salinized soils and for the management of irrigation water. Halotolerant plant growth promoting bacteria with biocontrol effect, as an alternative to commercial fungicides, may contribute to improve crop productivity while mitigating saline stress effects. The objective of this work was to isolate autochthonous rhizobacteria with biocontrol features, to be used as germination enhancers and plant‐growth promoting agents in the crop cultivation of Salicornia ramosissima. A set of isolates obtained from the rhizosphere of S. ramosissima was characterised in terms of Gram, motility, salt tolerance and biocontrol traits (hydrogen cyanide production, antifungal activity and production of extracellular lipases and proteases). One Gram‐positive motile isolate that tested positive for all biocontrol traits was identified by 16S rRNA gene sequencing as Bacillus aryabhattai. The inoculation of S. ramosissima seeds with B. aryabhattai SP1016‐20 reduced the negative effect of salinity on the germination efficiency. At the highest tested salinity (30 g/L NaCl) the final germination efficiency of inoculated seeds doubled in relation to non‐inoculated controls. Although the mechanisms involved in the biocontrol effect were not defined in the current work, the results highlight the potential of Bacillus aryabhattai SP1016‐20 as a plant‐growth promoting agent for the crop cultivation of Salicornia and contribute to the strengthening of the scientific basis of biosaline agriculture and plant growth promoting rhizobacteria‐assisted crop cultivation of halophytes in saline soils and estuarine sediments.

Production, Extraction and Characterization of Lipases from the Antarctic Yeast Guehomyces pullulans

The production of extracellular lipases from the Antarctic yeast Guehomyces pullulans is induced using an olive oil medium as an inductor substrate and a first characterization of its enzyme, using the protein extract obtained from the medium, is described. For this, the effect of pH and temperature on the lipase activity are evaluated and the enzyme kinetic for the lipase is determined. Lipase production was 0.27 U/mL, a high value compared to lipolytic activities in non-optimized media. However, this value can be increased by optimizing the culture medium. The lipase of G. pullulans has maximum activity at pH 8.0 and 40°C (thermal stability 40-50°C). Regarding the kinetic parameters, a KM = 3.7×10-4 M was obtained, a value located in the range of industrial lipases. In addition, its kinetics presented the phenomenon of interfacial activation. The results presented in this work show the biotechnological potential of the lipase due its biochemical properties and are useful for later work directed to study other factors that affect the enzyme activity and potential biotechnological applications of the Guehomyces pullulans lipase.

Extra Cellular Lipase Production, Purification and Characterization by Using Bacillus Subtilis in Submerged State Fermentation

Extra Cellular Lipase Production, Purification and Characterization by Using Bacillus Subtilis in Submerged State Fermentation

Optimization of culture conditions for extracellular fungal lipase production by submerged fermentation process

The present study aimed to optimize culture conditions for optimal growth and production of extracellular lipase. Lipolytic fungal strain named as S3St2 previously isolated from a petrol pump soil sample of Newai Town was used for optimization study. Among the tested carbohydrate carbon sources, polysaccharide-starch exhibited maximum lipase production (21.25±0.70 IU/ml/min) with highest specific activity (1.47±0.06 U/mg). Lipase activity and specific activity were higher with mustard oil 1 % (v/v) among all lipidic carbon sources. Among inorganic nitrogen source, potassium nitrate was found best inducer of lipase activity, malt extract supported the fungus growth (dry weight of cell pellets was 0.467 g) and exhibited maximum lipase activity among all organic nitrogen sources. Lipase activity was optimum at pH 8.0, indicates alkalophillic nature of production media supports the growth of fungus. Higher lipase activity (27.92±0.87 IU/ml/min) was detected at 28ºC. The incubation time of 5 days was found optimum for maximum lipase production (31.51±0.21 IU/ml/min).

Optimization of culture conditions for enhanced lipase production by an indigenous Bacillus aryabhattai SE3-PB using response surface methodology

Lipases are enzymes that hydrolyze fats into fatty acids and glycerol at the water–lipid interface and are also involved in a variety of bioconversion reactions in non-aqueous and micro-aqueous environments. In this study, we optimized the culture conditions for extracellular lipase production by an indigenous lipase-producing bacterial strain isolated from lipid-rich wastewater, using response surface methodology. The studied isolate was identified as Bacillus aryabhattai SE3-PB by polymerase chain reaction and analysis of 16S rDNA. Sunflower oil was found to induce maximum lipase production. Face centered central composite design revealed that temperature (40 °C), pH (7.6), inoculum volume (2.8%, v/v), agitation (193 rpm) and inducer oil concentration (2%, v/v) significantly influenced lipase production at the respective optimum conditions. The coincidence of observed lipase production (264.02 ± 1.94 U/mL) with predicted lipase yield (265.82 U/mL) coupled with a high correlation coefficient (R2 = 0.9919, P < 0.01) confirmed the validity of the model. A 7.2-fold increase in lipase production was obtained in the optimized medium compared to the basal medium. These findings provide the first report on lipase production and optimization by B. aryabhattai SE3-PB and suggest a rational choice of optimum processing conditions for commercial lipase production by B. aryabhattai SE3-PB.

Statistical enhancement of lipase extracellular production byBacillus stratosphericusPSP8 in a batch submerged fermentation process

The aim of this study was to isolate and identify lipolytic bacteria. Perform a statistical stepwise physicochemical optimization for maximum production of extracellular lipase and its validation in a bioreactor. Several lipolytic bacteria were isolated from petroleum hydrocarbon-polluted soil. The strain expressing the highest lipase activity (47Uml-1 ) was genetically identified as Gram-positive Bacillus stratosphericus PSP8 (NCBI GenBank accession no. MH120423). The response surface methodology (RSM)-central composite face centre (CCF) design of experiments was performed based on the preselected levels of the studied parameters obtained from the performed one-factor-at-a-time sequential experiments. A second-order polynomial model was predicted and improved the lipase production by approximately 1·6-fold. Preliminary scaling up of the validated optimized process was carried out in a batch 10-l stirred tank bioreactor, applying the optimum predicted operating conditions; pH 6·98, 34·8°C, 2·2×106 cells per ml, 200revmin-1 , 4·82gl-1 tributyrine concentration, 1% sucrose and 0·1% yeast extract. This yielded 89Uml-1 at the late log phase of bacterial growth (48h). Logistic kinetic model effectively characterized the submerged fermentation process, and the maximum specific growth and lipase production rates were estimated to be 0·338 and 0·164h-1 respectively. The mesophilic and neutrophilic B. stratosphericus PSP8 isolated from petroleum hydrocarbon-contaminated soil is a proper source of lipase. The closeness of the predicted response with that of the experimental value and the enhancement of lipase productivity in fermenter scale by approximately 1·9-fold, showed that statistically optimized design can be used in order to improve the lipase production to meet the increasing demand. The RSM-CCF statistical optimization is useful for optimizing a large number of variables and studying their interactive effects on extracellular lipase production.

IDENTIFICATION AND OPTIMIZATION OF LIPASE PRODUCING BACTERIA FROM PALM OIL CONTAMINATED WASTE

Bacteria isolated from semi-solid waste, SS2B1 exhibited a greater zone of clearance, 9mm with higher lipase activity. SS2B1 isolate demonstrated a Gram-positive and rod shape arrangement under microscope observation belong to Bacillus sp based on biochemical characteristics. The effect of carbon source, nitrogen source, medium pH and temperature for the lipase production was studied. The lipase production was maximum (0.1228 µg/ml/min) at pH7, temperature 37 0 C by the lipase producing bacteria SS2B1, Bacillus sp. Increased enzymatic production was obtained when the organisms were cultured in medium supplemented with 1% tryptone and palm oil as substrate with 53.58% optimization process. The results of the present study demonstrate that the Bacillus sp. is ideal for extracellular lipase production at industrial level such as detergent, leather and fine chemical industries.

Natural substrates for extracellular lipase production from Pseudomonas aeruginosa HE05 isolated from diesel oil contaminated soil

Natural substrates for extracellular lipase production from Pseudomonas aeruginosa HE05 isolated from diesel oil contaminated soil

Production and Biotechnological Application of Extracellular Alkalophilic Lipase from Marine Macroalga-Associated Shewanella algae to Produce Enriched C20-22 n-3 Polyunsaturated Fatty Acid Concentrate.

An extracellular alkalophiliclipase was partially purified from heterotrophic Shewanella algae (KX 272637) associated with marine macroalgae Padina gymnospora. The enzyme possessed a molecular mass of 20kD, and was purified 60-fold with a specific activity of 36.33U/mg. The enzyme exhibited Vmax and Km of 1000mM/mg/min and 157mM, respectively, with an optimum activity at 55°C and pH10.0. The catalytic activity of the enzyme was improved by Ca2+ and Mg2+ ions, and the enzyme showed a good tolerance towards organic solvents, such as methanol, isopropanol, and ethanol. The purified lipase hydrolyzed the refined liver oil fromleafscale gulper shark Centrophorus squamosus, yielding a total C20-22 n-3 PUFA concentration of 34.99% with EPA+DHA accounting the major share (34% TFA), after 3h of hydrolysis. This study recognized the industrial applicability of the thermostable and alkalophilic lipase from marine macroalga-associated bacterium Shewanella algae to produce enriched C20-22 n-3 polyunsaturated fatty acid concentrate.

Utilization of animal fat waste as carbon source by carotenogenic yeasts – a screening study

Abstract Red yeast strains are ubiquitous microorganisms which accumulate substantial amounts of lipids and lipid-soluble metabolites. Red yeasts utilize many waste substrates of different origin. In this work red yeasts strains (Rhodotorula, Sporobolomyces, Cystofilobasidium) were used for screening of growth and metabolic activity. As a carbon source waste animal fat and its hydrolysis products were used. Hydrolysis of animal fat was tested in alkaline as well as acidic conditions. As the substrate glucose (control), glycerol, crude animal fat, acid fat hydrolyzate and hydrolysate: glucose 1:2 were used. Screening of growth and metabolic activity of red yeasts was performed by flow cytometry. Extracellular lipase production was monitored as adaptation mechanism. Carotenoids, ergosterol and ubiquinone were quantified by HPLC/PDA/MS/ESI and the biomass was evaluated gravimetrically. All tested strains utilized fat hydrolysate and produced red coloured biomass. Cultivation in media containing non-hydrolysed fat led to strain specific induction of extracellular lipase. Amount of lipid metabolites produced by individual strains was depended on glycerol content in medium. The highest increase of lipase production was observed in Cystofilobasidium macerans and Sporobolomyces shibatanus. Valorisation of animal fat can lead to production of unsaturated fatty acids, single cell oils, carotenoid pigments, sterols and enriched red yeast biomass.

Production of dry extract extracellular lipase from Aspergillus niger by solid state fermentation method to catalyze biodiesel synthesis

Lipase can be produced from Aspergillus niger using solid state fermentation method. This research aimed to produce lipase in powder form. The highest activity unit (38.67 U/g) was obtained from rice bran substrate at 5th day with inducer concentration of 2%. The best drying method to produce dry extract with activity unit of 566.67 U/g was freeze drying with maltodextrin additive. The best drying method to produce fine powder with activity unit of 333.33 U/g was spray drying with skim powder additive. Supernatant and powder lipase worked optimally at 30 °C with activity unit of 44.00 U/g and 355.56 U/g respectively.

Turning oil palm empty fruit bunch waste into substrate for optimal lipase secretion on solid state fermentation by Trichoderma strains

Abstract This study is aimed to improving extracellular lipase production by Trichoderma strains using oil palm empty fruit bunch (OPEFB) medium on solid state fermentation with the assistance of statistical optimization. Two selected Trichoderma strains (Trichoderma sp.1 and Hypocrea neorufa.1) with high lipase producing potential were employed in a stepwise optimization. Initially, the influence of nutritional (carbon, nitrogen sources, and inducers) and environmental (moisture and temperature) factors on lipase production was evaluated using One-Factor-At-A-time (OFAT) method. Next, three most influential factors (Glucose, Urea and Olive oil) resulting from OFAT were optimized for lipase production by Trichoderma sp.1 using the central composite design under response surface methodology. Experiments conducted under optimum conditions (10.38% (w/w) glucose, 0.86% (w/w) urea, and 7.38% (v/w) olive oil) were further improved the lipase production from 0.195 to 0.390 Ug−1, which is about 2 folds greater than un-optimized condition. This study demonstrated that OPEFB can be utilized as inexpensive substrate for improving the lipase secretion by Trichoderma sp.1.

Improvement of extracellular lipase production by a newly isolated Yarrowia lipolytica mutant and its application in the biosynthesis of L-ascorbyl palmitate

Yarrowia lipolytica Wt-11 producing an extracellular lipase was isolated and identified. To improve the lipase production, Y. lipolytica Wt-11 was subjected to low-energy ion implantation mutation breeding, and a best mutant, Y. lipolytica Mut-96, was obtained after screening. Under the optimal cultivation conditions, the scaled-up production of lipases were performed, and the lipase activity of Y. lipolytica Mut-96 was enhanced nearly 5.5-fold compared with that of Y. lipolytica Wt-11. After fermentation, the lipases were purified, and the characteristics of the purified lipases were studied. The optimum temperatures and pHs for lipases from Wt-11and Mut-96 were 30°C and 8.0, respectively. The purified lipases were stable between pH 7.0 and 8.5 and unstable at temperatures above 40°C. The lipase activities were enhanced by Ca2+, Ba2+, Mn2+, Fe2+ and SDS. The synthesis of L-ascorbyl palmitate via esterification with L-ascorbic acid and palmitic acid by immobilized lipases from Wt-11 and Mut-96 in organic media was investigated, and the L-ascorbyl palmitate can be respectively produced at levels of 14.8 and 27.5g/L.

Screening of Microorganisms and Raw Materials for Lipase Production by Solid-State Fermentation

The production of biodiesel from vegetable oils using eco-friendly processes is a hot topic actually. These processes are based on enzymatic biocatalysts, namely lipases, and present many advantages over classical processes i.e. they do not require the use of sodium hydroxide, nor huge quantities of water. Lipases are widespread in nature, being produced by many microorganisms. However, fungal lipases have benefits over bacterial lipases due to their low cost of production, thermal and pH stability, substrate specificity and activity in organic solvents. These low cost production processes rely, most of the time, on solid-state fermentation (SSF). The aim of this research was to select microorganisms for their ability to secrete lipolytic enzymes and to grow on a solid support compatible with SSF. Thirty-five yeast and mold strains were tested in term of growth rate and extracellular lipase production. Different solid support such as vermiculite, crushed wheat husk, cacao seed-husk and carbon sources such as soy oil, sunflower oil, olive oil or sucrose were also tested for their ability to support cell growth and lipase production.

ACTIVIDAD LIPOLITICA DE MICROORGANISMOS AISLADOS DE AGUAS RESIDUALES CONTAMINADAS CON GRASAS

Lipases are one of the groups of enzymes with the highest number of industrial and biotechnological applications, processing of dairy foods and oils, detergents, cosmetics, leather, pharmaceuticals, paper, production of surfactants, biodiesel and recently as a promising alternative for the treatment of wastewater contaminated with lipids. In this study, microorganisms were isolated from wastewater samples containing fats and extracellular lipase production was determined using p-nitrophenylpalmitate. From the 149 isolates obtained, 37 showed lipolytic activity and strain CCEI-1, identified as Serratia marcescens, had the highest activity at alkaline pH and at 30°C. Also degraded lipids and produced pigmentation on solid medium at pH 8,0 and 50°C. The culture supernatants of S. marcescens used to determine thermal stability at 50°C indicate that the enzyme is stable at this temperature and at alkaline pH. The enzyme was partially purified and electrophoresed (SDS-PAGE) where bands between 40 and 116 kDa were detected. The results obtained in this study indicate that the strain CCEI-1 of S. marcescens can be a possible source of thermostable lipases with industrial and biotechnological applications.

English

In this study, a wild (LPF-5) and a mutant (HN1) strain of A. niger were compared for lipase production. Several physical parameters (carbon source, nitrogen source, pH, temperature and incubation period) were optimized for maximization of lipase production. Lipase activity between wild type and mutant strain were compared. Among all carbon sources, mixture of glucose (1%, w/v) and olive oil (1%, v/v) exhibited maximum increase in the production of lipases by both the wild (94.91 &plusmn; 0.60 U mL-1 min-1) and mutant (118.23 &plusmn; 0.73 U mL-1 min-1) strain. Addition of glucose into the production medium (containing olive oil) increased the production of lipase up to 20% in case of both the strains. The production of lipase by both the strains was higher in the medium of pH 7.0 containing peptone (1%, w/v) as nitrogen source after 3 days of incubation at 28&deg;C. The activity of lipase from HN1 strain in optimized medium was 40% higher (147.65 &plusmn; 1.14 U mL-1 min-1) than in un-optimized medium (105.19 &plusmn; 0.91 U mL-1 min-1), while it was 38% higher for LPF-5 strain in optimized medium. Therefore, the mutant strain (A. niger HN1) is prospective for the development of industrial biotechnology for production of extracellular lipase. Lipase enzyme was partially purified by ammonium sulfate precipitation and 70% precipitate showed highest specific activity of 66.12 U mg-1 for mutant strain as compared to specific activity of 29.88 U mg-1 in crude lysate. &nbsp; Key words: Wild strain, mutant strain, Aspergillus niger, lipase activity, specific activity, ammonium sulfate.

Bioprospecting of lipolytic microorganisms obtained from industrial effluents.

The lipases have ability to catalyze diverse reactions and are important in different biotechnological applications. The aim of this work was to isolate and characterize microorganisms that produce lipases, from different food industry effluents localized in Pelotas, RS/Brazil. Bacteria were identified using Gram stain and biochemical tests (Vitek 2(r)). Fungi were identified according to macro and micromorphology characteristics. The extracellular lipase production was evaluated using the Rhodamine B test and the enzymatic activity by titration. Twenty-one bacteria were isolated and identified as Klebsiella pneumoniae ssp. pneumoniae, Serratia marcescens, Enterobacter aerogenes, Raoultella ornithinolytica and Raoultella planticola. Were characterized isolated filamentous fungi by the following genera: Alternaria sp., Fusarium sp., Geotrichum sp., Gliocladium sp., Mucor sp., Paecilomyces sp. and Trichoderma sp. Extracellular lipase production was observed in 71.43% of the bacteria and 57.14% of the fungi. The bacterium that presented better promising enzymatic activity was E. aerogenes (1.54 U/ml) however between fungi there was not significant difference between the four isolates. This study indicated that microorganisms lipase producers are present in the industrial effluents, as well as these enzymes have potential of biodegradation of lipid compounds.

Enhancement of Extracellular Lipase Production by Strain Improvement of Fungus Aspergillus niger LPF-5

Twelve fungal isolates belonging to different genera were quantitatively screened for extracellular lipase production in SmF (Submerged fermentation). Isolate LPF-5 demonstrated higher lipase activity and it was identified as Aspergillus niger based on morphology of its Petri plate culture, microscopic study of its slide culture and with the help of relevant literatures. Further an effort was made to increase the production of extracellular lipase by subjecting the most potent lipolytic fungal strain A. niger LPF-5 to the strain improvement by induced mutagenesis using UV radiations and nitrous acid. Among the all tested mutagens, nitrous acid was found to be the best mutagen (incubation time of 15 minutes) for inducing the favorable mutation in fungal strain A. niger LPF-5 which enhanced the lipase activity up to 30% (105.19 ± 0.91 U mL -1 min -1 ) when compared to lipase activity (81.00 ± 0.30 U mL -1 min -1 ) of wild strain while the lipase activity of the best UV mutant (A. niger UV3), obtained after an incubation of 6 minutes was 94.30 ± 0.54 U mL -1 min -1 , which was 16.41% higher as compared to wild strain of A. niger. These results indicate that UV light and nitrous acid both were effective mutagens but nitrous acid was more potent mutagen than UV light.