Lipase Biosynthesis Research Articles

Application of artificial neural networks for enhancing Aspergillus flavipes lipase synthesis for green biodiesel production

Biodiesel is a sustainable, and renewable alternative to fossil fuels that can be produced from various biological sources with the aid of lipases. This study developed a simple and novel fungal system for lipase biosynthesis to be used for catalyzing the oily residuals into biodiesel, employing the artificial neural network (ANN), and semi-solid-state fermentation (SSSF). Nigella sativa was selected among agro-industrial oily residuals as a substrate for lipase biosynthesis by Aspergillus flavipes MH47297. The effect of cultural humidity (X1), the surfactant; Brij 35 (X2), and inoculum density (X3) on lipase biosynthesis were researched based on the matrix of Box-Behnken design (BBD). The ANN together with a new fungal candidate and SSSF were then applied for the first time to model the biosynthesis process of lipase. The optimum predicted cultural conditions varied according to the model. The optimum predicted conditions were estimated separately by BBD (X1 = 5.8 ml water/g, X2 = 46.6 μl/g, and X3 = 62156610 spore/g) and ANN (X1 = 5.4 ml water/g, X2 = 54.2 μl/g, and X3 = 100000000 spore/g) models. Based on the modeling process, the response of lipase was calculated to be 214.95 (BBD) and 217.72 U (ANN), which revealed high consistency with the experimental lipase yield (209.13 ± 3.27 U for BBD, and 218 ± 2.01 U for ANN). Despite both models showing high accuracy, ANN was more accurate and surpassed the BBD model. Gas chromatography analysis showed that lipase successfully converted corn oil to biodiesel (29.5 mg/l).

Testing for adaptive changes linked to range expansion following a single introduction of the fall webworm.

Adaptive evolution following colonization can affect the impact of invasive species. The fall webworm (FWW) invaded China 40 years ago through a single introduction event involving a severe bottleneck and subsequently diverged into two genetic groups. The well-recorded invasion history of FWW, coupled with a clear pattern of genetic divergence, provides an opportunity to investigate whether there is any sign of adaptive evolution following the invasion. Based on genome-wide SNPs, we identified genetically separated western and eastern groups of FWW and correlated spatial variation in SNPs with geographical and climatic factors. Geographical factors explained a similar proportion of the genetic variation across all populations compared with climatic factors. However, when the two population groups were analysed separately, environmental factors explained more variation than geographical factors. SNP outliers in populations of the western group had relatively stronger response to precipitation than temperature-related variables. Functional annotation of SNP outliers identified genes associated with insect cuticle protein potentially related to desiccation adaptation in the western group and genes associated with lipase biosynthesis potentially related to temperature adaptation in the eastern group. Our study suggests that invasive species may maintain the evolutionary potential to adapt to heterogeneous environments despite a single invasion event. The molecular data suggest that quantitative trait comparisons across environments would be worthwhile.

Carposome productivity of Pleurotus ostreatus and Pleurotus eryngii growing on agro-industrial residues enriched with nitrogen, calcium salts and oils

The suitability of the abundant agro-industrial residues wheat straw (WS; control), barley and oats straw (BOS) and rice husk (RH), supplemented with various sources of oils (sunflower, corn oil), nitrogen (peptone, yeast extract) and calcium salts (CaSO4·2H2O, CaCl2), as novel substrates in solid-state fermentation of selected Pleurotus ostreatus and P. eryngii mushrooms was investigated. The possible effect of different additives on mycelial growth rate, biomass production and endoglucanase, laccase and lipase biosynthesis were evaluated. Moreover, their impact on essential cultivation aspects (earliness, total mushroom yield, biological efficiency) and carposome quality parameters (weight, morphological characteristics) was assessed. Both fungi showed their highest growth rates on BOS substrates and the most positive implementation was CaSO4·2H2O 6 % w/w (Kr = 9.58 mm/ day; P. ostreatus, Kr = 9.42 mm/ day; P. eryngii), while different additives led to enhancement of biomass production. Pleurotus species demonstrated minimal levels of endoglucanase activity, with values ranging from 0.01 to 0.42 U/g of dry weight, regardless of the substrate and the stage of colonization. On the contrary, the maximum values of laccase activity were observed at 50 % of colonization on BOS and RH, while supplementation with nitrogen and calcium sources positively affected its biosynthesis. P. ostreatus and P. eryngii cultivated on BOS supplemented with peptone at 2 and 5 % w/w, synthesized significant laccase amounts, i.e., 12,165.78 and 8,624.55 U/g d.w., respectively. Satisfactory amounts of lipase were produced, especially in substrates supplemented with sunflower 2 % w/w, in quantities up to 1.42 U/g d.w., whereas the highest lipase activity was achieved by P. eryngii on WS supplemented with corn oil at 2 % w/w, with a value of 4.25 U/g d.w. being recorded. Regarding fermentation of Pleurotus species in polypropylene bags, WS and BOS supported faster colonization and shorter earliness period than RH substrates, whereas supplementation did not seem to affect these culture parameters. Furthermore, oils supplementation had a positive effect on BE of both species, with values up to 100 % for P. ostreatus and 80 % for P. eryngii on WS and BOS, whereas on RH the lowest BE values were detected. Morphological characteristics were not significantly affected by the additives. Results indicate the positive impact that certain additives have on mushroom productivity and production of enzymes with great financial and environmental importance.

Изменение липазной активности Acinetobacter radioresistens при культивировании на пшеничных отрубях

The aim of the work was to study the lipase activity of the bacterial culture Acinetobacter radioresistens, a part of the microbiota of wheat bran which is secondary raw materials of wheat processing. Secondary raw materials of food and agricultural production are of scientific and practical interest as sources of microorganisms – producers of hydrolytic enzymes. In this work, the effect of the composition of wheat bran for food and feed purposes on the lipase activity of the A. radioresistens isolate was studied. The studied bacterial culture was isolated by VNIIPD employees from a batch of dietary wheat bran. As a result of deep fermentation of bran in rocking flasks under conditions of a Multitron shaker incubator with A. radioresistens isolate, it was found that feed bran is more preferable as a source of carbohydrate, protein, and lipid substrates for the vital activity of bacterial culture and the manifestation of lipase activity. The amount of biomass was 17 ± 2 g/l, the achieved level of lipase activity was 45 ± 2 units/ml. The regularity of the effect of fermentation duration on the change in lipase activity has been established and it has been shown that substrates from feed bran induce the synthesis of lipolytic enzymes to a greater extent. Productive biosynthesis of lipase with an activity in the range of 10–47 units/ml, depending on the time of cultivation of A. radioresistens isolate on wheat bran for both food and feed purposes, is possible, moreover, with one-stage (sterilization of wheat bran hydromodule) preparation of raw materials for the biotechnological process. The achieved values of lipase activity are at the level of some well-known enzyme preparations for feed purposes. When studying the fatty acid composition of the obtained culture liquid by gas chromatography with mass spectrometric detection, fatty acids with an alkyl chain length from C12 to C22 carbon atoms, including Omega-9, Omega-6, and Omega-3 acids, were identified. The results obtained can be used to develop technologies for new food and feed ingredients, biologically active additives.

Biosynthesis of Lipases from an Alkaliphillic Fungal Isolate and its Potential Application in Detergent Formulation

Biosynthesis of Lipases from an Alkaliphillic Fungal Isolate and its Potential Application in Detergent Formulation

Aeration and Stirring in Yarrowia lipolytica Lipase Biosynthesis during Batch Cultures with Waste Fish Oil as a Carbon Source

Yarrowia lipolytica is one of the most studied non-conventional forms of yeast, exhibiting a high secretory capacity and producing many industrially important and valuable metabolites. The yeast conceals a great biotechnological potential to synthesize organic acids, sweeteners, microbial oil, or fragrances. The vast majority of bioprocesses are carried out in bioreactors, where suitable culture conditions are provided. In the current study, the effect of agitation speed (200–600 rpm) and air flow rate (0.0375–2.0 dm3/(dm3 × min)) on the biomass yield and lipase activity of Y. lipolytica KKP 379 is analyzed in a growth medium containing waste fish oil. The increase of aeration intensity limited the period of oxygen deficit in the medium. Simultaneously, an increase in lipolytic activity was observed from 2.09 U/cm3 to 14.21 U/cm3; however, an excessive agitation speed likely caused oxidative or shear stresses, and a reduction in lipolytic activity was observed. Moreover, it is confirmed that the synthesis of lipases is related to oxygen consumption, pH, and the yeast growth phase, and appropriate process selection may provide two advantages, namely, the maximum use of the waste carbon source and the production of lipolytic enzymes that are valuable in many industries.

Biosynthesis of lipase by Burkholderia cenocepacia ST8 using waste cooking oil as feedstock

Biosynthesis of lipase by Burkholderia cenocepacia ST8 using waste cooking oil as feedstock

Enhancing Lipase Biosynthesis by Aspergillus Melleus and its Biocatalytic Potential for Degradation of Polyester Vylon-200

Herein, response surface methodology (RSM) was employed to optimize the bioprocess parameters for improved production of extracellular lipase by Aspergillus melleus and evaluated its biocatalytic potential for degradation of polyester vylon-200. Our previous report showed that pH, incubation time, temperature, and additional nitrogen source had significant effects on lipase biosynthesis. The variance analysis revealed that the established RSM model based on a central composite design for lipase production was significant (p < 0.0001, R2 = 0.9925). Under the optimized bioprocess conditions of pH 5.68, incubation time 96 h, temperature 30 °C, and diammonium tartrate as a nitrogen source, maximum lipase titer of 1346.87 U/gds was achieved, 1.92-fold higher than lipase yield in basal medium. The optimally synthesized cell-free lipase extract was partially purified by ammonium sulfate fractionation and dialysis and used to degrade polyester vylon 200. The degradation profile revealed that the lipolytic enzyme demonstrated excellent hydrolytic potential resulting in a 76% weight of polyester vylon-200. Differential scanning calorimetry revealed a noticeable decrease in the glass transition temperature of PV-200 (from 72.6 oC to 63.9 oC). Scanning electron microscopy envisaged various micron-scale cracks and holes on the surface of film after enzymatic treatment. Likewise, significant weight loss of the PV-200 films was also corroborated by FTIR analysis. This study's findings illustrate lipase's potential as a green and ecofriendly biocatalyst for robust polyester degradation and depolymerization.

Biosynthesis lipase from the fungusPenicillium hordeiisolated from waste fat

The article provides a method of isolating a lipase producer and identification methods by classical methods in microbiology of an isolated micromycete producing lipase. The species affiliation ofPenicillium hordeihas been determined. The temperature of micromycete cultivation was studied, as well as the dynamics ofPenicillium hordeilipase biosynthesis. Screening plans were also carried out (Plackett-Berman Plan), then optimization according to the steep climb plan and an experiment based on the plan of a full-factor experiment (three-level CFE) was also conducted to determine the optimal concentrations that are significant for lipase biosynthesis of components. A mathematical model is constructed that describes the relationship between the influence of two significant factors on the volumetric activity ofP. Hordeiculture fluid. The most optimal composition for the culture fluid for lipase biosynthesis,Penicillium hordeimicromycete, was compiled.

Cellular and metabolic engineering of oleaginous yeast Yarrowia lipolytica for bioconversion of hydrophobic substrates into high-value products.

The non-conventional oleaginous yeast Yarrowia lipolytica is able to utilize both hydrophilic and hydrophobic carbon sources as substrates and convert them into value-added bioproducts such as organic acids, extracellular proteins, wax esters, long-chain diacids, fatty acid ethyl esters, carotenoids and omega-3 fatty acids. Metabolic pathway analysis and previous research results show that hydrophobic substrates are potentially more preferred by Y. lipolytica than hydrophilic substrates to make high-value products at higher productivity, titer, rate, and yield. Hence, Y. lipolytica is becoming an efficient and promising biomanufacturing platform due to its capabilities in biosynthesis of extracellular lipases and directly converting the extracellular triacylglycerol oils and fats into high-value products. It is believed that the cell size and morphology of the Y. lipolytica is related to the cell growth, nutrient uptake, and product formation. Dimorphic Y. lipolytica demonstrates the yeast-to-hypha transition in response to the extracellular environments and genetic background. Yeast-to-hyphal transition regulating genes, such as YlBEM1, YlMHY1 and YlZNC1 and so forth, have been identified to involve as major transcriptional factors that control morphology transition in Y. lipolytica. The connection of the cell polarization including cell cycle and the dimorphic transition with the cell size and morphology in Y. lipolytica adapting to new growth are reviewed and discussed. This review also summarizes the general and advanced genetic tools that are used to build a Y. lipolytica biomanufacturing platform.

Optimization of Lipase Production in Solid-State Fermentation by Rhizopus Arrhizus in Nutrient Medium Containing Agroindustrial Wastes

Background: Rhizopus arrhizus is a potential microorganism for lipase production. Solid-state fermentation is used for microbial biosynthesis of enzymes, due to advantages, such as high productivity, utilization of abundant and low-cost raw materials, and production of enzymes with different catalytic properties. Objective: The objective of the research is optimization of the conditions for lipase production in solid-state fermentation by Rhizopus arrhizus in a nutrient medium, containing agroindustrial wastes. Method: Biosynthesis of lipase in solid-state fermentation by Rhizopus arrhizus was investigated. The effect of different solid substrates, additional carbon and nitrogen source, particles size and moisture content of the medium on enzyme production was studied. Response surface methodology was applied for determination of the optimal values of moisture content and tryptone concentration. A procedure for efficient lipase extraction from the fermented solids was developed. Results: Highest lipase activity was achieved when wheat bran was used as a solid substrate. The addition of 1% (w/w) glucose and 5% (w/w) tryptone to the solid medium significantly increased lipase activity. The structure of the solid medium including particles size and moisture content significantly influenced lipase production. A mathematical model for the effect of moisture content and tryptone concentration on lipase activity was developed. Highest enzyme activity was achieved at 66% moisture and 5% (w/w) tryptone. The addition of the non-ionic surfactant Disponyl NP 3070 in the eluent for enzyme extraction from the fermented solids increased lipase activity about three folds. Conclusion: After optimization of the solid-state fermentation the achieved 1021.80 U/g lipase activity from Rhizopus arrhizus was higher and comparable with the activity of lipases, produced by other fungal strains. The optimization of the conditions and the use of low cost components in solid-state fermentation makes the process economicaly effective for production of lipase from the investigated strain Rhizopus arrhizus.

Biosynthesis of lipases through solid substrate fermentation technique using Penicillium sp. isolated from pickle (MBL) 40

Biosynthesis of lipases through solid substrate fermentation technique using Penicillium sp. isolated from pickle (MBL) 40

New sources of halophilic lipases: Isolation of bacteria from Spanish and Turkish saltworks

High-salinity biotopes in Turkey and Spain have been demonstrated to be a suitable source of lipolytic enzyme-producing halophilic microorganisms. Three strains turned out to grow at high NaCl concentration (greater than 15%) and their ability to synthesize lipolytic enzymes was demonstrated on tributyrin and olive oil-containing plates by the formation of transparent or orangish halos around the colonies. The three isolates were cultivated in liquid medium and one of them was cherry-picked in terms of the enzyme production levels. It was subsequently genetically identified as Halomonas sp. LM1C. Response Surface Methodology (RSM) based on central composite design was used to optimize the culture conditions for lipase production, concluding that the maximum levels of lipase biosynthesis were obtained at 21.6°C and pH 6.9. The impact of NaCl on the lipolytic activity of the obtained halophilic crude enzyme revealed an improved resistance against deactivation, when compared with a commercial lipase from Candida rugosa. The optimum reaction conditions (obtained by RSM) for the crude enzyme-biocatalyzed hydrolysis of p-nitrophenyl laurate turned out to be neutral pH and 29°C. The process at optimum conditions was kinetically characterized by means of logistic equations, obtaining maximum activities near to 250U/L and the use of a Luedeking & Piret type model allowed elucidating the metabolic characteristics of the synthesized lipolytic enzymes. The present investigation lays the foundation for implementing a biotechnological process to produce highly resistant lipolytic enzymes able to perform its biocatalytic function under extreme salt conditions.

Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10.

Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

Production of lipases by Alternaria sp. (mbl 2810) through optimization of environmental conditions using submerged fermentation technique

The present investigation was conducted for the biosynthesis of extracellular and intracellular lipases by Alternaria sp. (MBL 2810) through submerged fermentation technique. To increase the lipases production various parameters were optimized such as nitrogen, carbon sources and additional additives besides the basic cultural conditions e.g., rate of fermentation, volume of fermentation medium, inoculum size and type and initial pH of fermentation medium. The maximum extracellular and intracellular lipases production was achieved, when fermentation was optimized at room temperature for 24 h at the agitation speed of 100 rpm. Other cultural conditions such as inoculum type and inoculum size (4.0 mL) was also optimized, glycine as an additional carbon source and urea as an additional nitrogen source gave the maximum extracellular lipases activity. Maximum production of extracellular lipases (9.04 ± 0.05 a U/mL) was achieved when volume of fermentation was optimized at 25mL and maximized intracellular lipases (10.04 ± 0.13a U/mL) production was achieved when the medium pH was adjusted at 5.

Enhancement of Lipase Biosynthesis by Aspergillus Niger Using Gamma Radiation

Objectives: The present study describes the isolation, identification and screening of fugal isolates for the biosynthesis of extracellular lipase enzyme. Methodology:Thirteen fungal cultures were isolated from cotton seeds, waste cake of cotton seed oil and spent bleaching earth, samples by serial dilution method. All isolates were initially selected qualitatively on tributyrin agar plates and were shifted to the slants of PDA for maintenance and storage at 4 o C. Quantitative screening for extracellular lipase biosynthesis by isolated fungi was carried out in shake flasks and the most potent fungal isolate which producing 25 U ml-1 of enzyme was selected. The isolate was then identified on the basis of standard morphological measurements and was assigned the code Aspergillus niger ADM 110.Results:Enhanced lipase biosynthesis was observed at 25 °C, pH 7, 1.0 ml (4.60 × 10 7 ) of spore inoculum and after 72 h of incubation. Olive oil 5 % was observed as the most effective carbon source and yeast extract 5.0 % as the most effective nitrogen source for lipase biosynthesis. The optimum shaking value was 200 rpm. Aspergillus niger ADM 110 was subjected to Gamma radiation in order to improve its lipolytic potential. Conclusion:Using the optimized conditions, maximum lipase biosynthesis has been obtained by using 0.14 kGy of gamma radiation with enzyme activity 53 U/ml as compared to the parent strain (unirradiated) with enzyme activity 30 U/ml.

Purification and Characterization of a Novel Cold-Active Lipase from the Yeast Candida zeylanoides

Cold-active lipases have attracted attention in recent years due to their potential applications in reactions requiring lower temperatures. Both bacterial and fungal lipases have been investigated, each having distinct advantages for particular applications. Among yeasts, cold-active lipases from the genera Candida, Yarrowia, Rhodotorula, and Pichia have been reported. In this paper, biosynthesis and properties of a novel cold-active lipase from Candida zeylanoides isolated from refrigerated poultry meat are described. Heat-sterilized olive oil was found to be the best lipase biosynthesis inducer, while nonionic detergents were not effective. The enzyme was purified to homogeneity using hydrophobic chromatography and its enzymatic properties were tested. Pure enzyme activity at 7°C was about 60% of the maximal activity at 27°C. The enzyme had rather good activity at higher temperatures, as well. Optimal pH of pure lipase was between 7.3 and 8.2, while the enzyme from the crude extract had an optimum pH of about 9.0. The enzyme was sensitive to high ionic strength and lost most of its activity at high salt concentrations. Due to the described properties, cold-active C. zeylanoides lipase has comparative advantages to most similar enzymes with technological applications and may have potential to become an industrially important enzyme.

Lipase biosynthesis by Aspergillus carbonarius in a nutrient medium containing products and byproducts from the oleochemical industry

Abstract The process of lipase biosynthesis by Aspergillus carbonarius in a submerged cultivation was studied. It was established that inclusion of a lipid carbon source in the nutrient medium significantly increased enzyme production. Highest lipase activity was achieved when unrefined rapeseed oil (195.61 U/L), soybean oil (155.00 U/L), and soap stock from sunflower oil (149.00 U/L) were used as a carbon source. These three raw materials were characterized by a high acid value, indicating high content of free fatty acids. It was found that the high concentration of free fatty acids in the nutrient medium determined achievement of high lipase activity. Significant increase in lipase biosynthesis was observed when surfactants were included in the nutrient medium. In the presence of 20 g/L Tween 80 lipase activity was more than 2-fold higher in comparison to a control sample. Lipase production was considerably influenced by the presence of organic nitrogen source in the medium. Meat extract was found to stimulate enzyme biosynthesis.

Investigation of mutual interactions of physicochemical parameters on simultaneous Zn(II) bioaccumulation and lipase production of R. delemar

In this study, while Rhizopus delemar removes Zn(II) ions in the wastewater with the help of bioaccumulation mechanisms, the effects of increasing concentrations of Zn(II) ions both on micro-organism growth and lipase enzyme production which biodegrades lipids were investigated. The parameters which affect micro-organism growth, lipid biosynthesis, and Zn(II) bioaccumulation were determined as pH, temperature, and initial Zn(II) ion concentration. In single factor experiments, pH and temperature were studied in the ranges of 5.0–8.0 and 25–35°C, respectively, while initial Zn(II) ion concentration was changed between 5 and 200 mg/L. At pH 5.0, and temperature 35°C, maximum Zn(II) removal was obtained as 26.31 mg/L in the bioaccumulation medium containing 30 mg/L Zn(II) initially. The lipase activities and micro-organism concentrations obtained in Zn(II)-free and the preceding media were 656 and 537 U/L, 1.16 and 0.82 g/L, respectively. When the biomass concentration was kept constant at a required level, in order to maximize the bioaccumulation of Zn(II) and lipase biosynthesis, a statistical experimental design was applied. Maximum Zn(II) bioaccumulation was obtained as 32.38 mg/L at pH 6.8, 33.8°C and 41 mg/L initial Zn(II) concentration. At this optimum point, it was determined that lipase activity is 586 U/L and micro-organism concentration is 0.62 g/L.

Unraveling the Lipolytic Activity of Thermophilic Bacteria Isolated from a Volcanic Environment

In a bioprospecting effort towards novel thermostable lipases, we assessed the lipolytic profile of 101 bacterial strains isolated from the volcanic area of Santorini, Aegean Sea, Greece. Screening of lipase activity was performed both in agar plates and liquid cultures using olive oil as carbon source. Significant differences were observed between the two screening methods with no clear correlation between them. While the percentage of lipase producing strains identified in agar plates was only 17%, lipolytic activity in liquid culture supernatants was detected for 74% of them. Nine strains exhibiting elevated extracellular lipase activities were selected for lipase production and biochemical characterization. The majority of lipase producers revealed high phylogenetic similarity with Geobacillus species and related genera, whilst one of them was identified as Aneurinibacillus sp. Lipase biosynthesis strongly depended on the carbon source that supplemented the culture medium. Olive oil induced lipase production in all strains, but maximum enzyme yields for some of the strains were also obtained with Tween-80, mineral oil, and glycerol. Partially purified lipases revealed optimal activity at 70–80°C and pH 8-9. Extensive thermal stability studies revealed marked thermostability for the majority of the lipases as well as a two-step thermal deactivation pattern.