Extracellular Lipase Production Research Articles

Waste cooking oil and molasses for the sustainable production of extracellular lipase by Saitozyma flava.

Organic waste valorization is one of the principal goals of the circular economy. Bioprocesses offer a promising approach to achieve this goal by employing microorganisms to convert organic feedstocks into high value products through their metabolic activities. In this study, a fermentation process for yeast cultivation and extracellular lipase production was developed by utilizing food waste. Lipases are versatile enzymes that can be applied in a wide range of industrial fields, from detergent, leather, and biodiesel production to food and beverage manufacturing. Among several oleaginous yeast species screened, Saitozyma flava was found to exhibit the highest secreted lipase activity on pNP-butyrate, pNP-caproate, and pNP-caprylate. The production medium was composed of molasses, a by-product of the sugar industry, which provided nutrients for yeast biomass formation. At the same time, waste cooking oil was employed to induce and enhance extracellular lipase production. After 48h of process, 20g/L of yeast biomass and 150mU/mgdw of lipase activity were achieved, with a productivity of 3mU/mgdw /h. The purified lipase from S. flava showed optimal performances at temperature 28°C and pH 8.0, exhibiting a specific activity of 62U/mg when using p-NPC as substrate.

Agricultural waste upcycling into improved production of triacyl glycerol acyl hydrolases

Abstract Agricultural waste upcycling is crucial in the context of climate change. Utilizing seven agricultural by-products as basal substrates, coconut meal (Cocos nucifera) emerged as the most efficient, supporting the highest extracellular lipase (triacyl glycerol acyl hydrolases) yield i.e., 5.27 ± 1.75a U mL−1. This study explored the production of extracellular lipases through solid-state fermentation by using co-cultures of Aspergillus niger, wild and mutant strains. Optimization experiments revealed that 30 g of coconut meal resulted is optimal for supporting highest lipases activity of 6.16 ± 0.16a U mL−1. Incubation at 30 °C, 1 mL inoculum size, and distilled water as a diluent further enhanced lipolytic activity. The study identified sucrose as the preferred carbon source, with 4 % concentration demonstrating the highest activity at 13.66 ± 0.33a U mL−1. Supplementary sources like 1 % olive oil and nitrogen sources such as ammonium chloride and peptone significantly increased lipases production. Magnesium sulfate (0.25 %) among metal ions exhibited the highest lipolytic potential i.e., 19.98 ± 0.01a U mL−1. The crude lipases displayed optimal activity at pH 5.0 and 30 °C, with positive effects observed for Fe2+, Ca2+, and Mg2+, while Co2+, Na+, and Hg2+ had negative impacts on lipases activity. This research not only contributes to understanding the factors influencing lipase production and activity, but also serves as an agricultural waste upcycling technique.

Isolation, Identification and Screening of Potential Marine Actinomycete for Lipase Production under Submerged Fermentation by using Tributyrin substrate

Ninety-six isolates were collected at various depths and sea shores of the Bay of Bengal near Visakhapatnam Coastal of Andhra Pradesh. These isolates were screened for the isolation of lipase activity by tributyrin agar clearing method and cultured under submerged fermentation and assayed for extra cellular lipase production by using tributyrin as substrate. Cultural characteristics of the selected strain was done by incorporating different media. Various parameters namely morphological, biochemical, physiological and molecular were used for the characterization and identification of actinomycetes isolates. Results indicated that all the isolates showed lipolytic activity after primary screening. The marine isolate T9 was the most active one thus the same was selected for production of lipase. The 16S rDNA amplification for phylogenetic study revealed that the isolate was highly related to Nocardiopsis dassonvillei. In conclusion, these results increased the scope of finding industrially important marine actinomycetes from Visakhapatnam Coastal of Andhra Pradesh.

Evaluation of Anti-Foodborne Bacterial Activity, Digestive Enzyme Secretion, and Antimicrobial Resistant Genes as Probiotic Strains Selection for Industrial Interest

Beneficial microbes, such as probiotic bacteria, are increasingly in demand in the food and feed industry. Lactic acid bacteria and bifidobacteria are commonly used as commercial probiotics, only a few species have been isolated from Southeast Asia areas. This study employed criteria including antimicrobial activity, the release of digestive enzymes, and the absence of antibiotic-resistant (AMR) genes to screen potential local isolates. The results revealed that 4 out of 16 isolates met these criteria, displaying anti-foodborne bacterial activities and a lack of fifty-one tested AMR genes. Furthermore, the four selected isolates demonstrated the production of extracellular digestive enzymes, including amylase, lipase, protease, β-glucanase, and cellulase, with enzyme indices ranging from 1.09–1.31. Among these isolates, two potential probiotics were identified as Bifidobacterium animalis subsp. lactis (strain H9-01) and Lactobacillus reuteri (strain P4-S03). Importantly, both species are approved for use as food and feed supplements in accordance with Thai regulations. This research outlines an approach for screening potential probiotics for industrial-scale applications.

Upcycling of the industrial waste as a sustainable source of axenic fungal strain (Aspergillus oryzae) for scale up enzymatic production with kinetic analysis and Box–Behnken design application

Abstract The utilization of industrial waste for valuable product synthesis is essential for mitigating the environmental impact of climate change and managing waste disposal challenges. Industrial waste is always a big challenge for global warming. In this study, a novel fungus strain was isolated, and identified as Aspergillus oryzae (AKMS), from the oily surface of industrial effluent from an oil industry. Submerged fermentation was exploited to achieve maximum lipases production. A sequence of batch experiments was performed, altering five primary components: sucrose (for carbon), molasses (constituting nutrient mix), yeast extract (as nitrogen supply), sunflower oil (used as an activating agent), and Tween-80 (functioning as an emulsifying agent). These elements are recognized for their substantial impact on the proliferation of microorganisms and, consequently, the synthesis of lipases. The maximum lipase activity for each ingredient was determined, with sucrose showing 2.4 ± 0.539 U/mL at 0.15 g/L, molasses at 1.499 ± 0.291 U/mL at 0.25 g/L, Tween 80 % at 3.33 ± 0.484 U/mL at 0.25 g/L, sunflower oil at 7.49 ± 0.282 U/mL at 1 % v/v, and yeast extract at 4.165 ± 0.841 U/mL at 0.075 % v/v. The optimization of the lipase production and activity was done by using RSM (Box–Behnken design) with the 41st run yielding the maximum activity of 9 U/mL, while the 25th run showed the minimum activity of 4 U/mL, enzyme estimation (U/mL and U/g), glucose (g/L), and dry cell mass estimation (g/L) were monitored up to 120 h of fermentation in a custom-made fermenter with a working volume of 7 L. The highest production of extracellular and intracellular lipases (27.4 ± 0.378 U/mL and 25.4 ± 0.208 U/g, respectively) was observed at the 40 h interval, with the highest value of dry cell mass at 20 g/L. Glucose concentration initially peaked and subsequently decreased over time. A. oryzae exhibited the capability to produce triacyl glycerol acyl hydrolases, demonstrating its potential significance in biotechnological applications. These novel findings shed light on the efficient utilization of oil industry waste soil for isolation of microorganism for sustainable lipase production and highlight the biotechnological promise of the novel isolated fungal strain, which was reported first time in this study.

PHYSICOCHEMICAL PROPRIETIES OF LIPASE FROM NEWLY STREPTOMYCES SP. OLIVE POMACE ISOLATE

Screening of new producing strains is required to meet the constant industrial demand for useful enzymes. In this context, an actinobacteria was isolated from the olive pomace, tested for extracellular lipase production, and identified by partial 16S rDNA sequencing. Then, the physicochemical characteristics of the enzyme are determined. Strain is a member of Streptomyces genus. Lipase was partially purified at 62.5 times, with a yield of 14.83% and a specific activity of 337.5 U/mg. Optimum activity was achieved at pH 7.0 and 60°C; it was completely preserved at pH 6.0 to 9.0, and more than 70% at 40 to 70°C, after 1 hour. The metal ions: K+, Na+, Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, and Zn2+ had no significant impact. Lipase activity was stable with ethylene diamine tetra acetic acid (EDTA), sodium dodecyl sulfate (SDS), H2O2, Triton x100, and Tween 80. Moreover, it was enhanced in butanol, chloroform, ethyl acetate, isopropanol, isobutyl acetate, hexane, petrol ether, and toluene. This enzyme can be suitable for various industrial applications.

Extracellular lipase production by Yarrowia lipolytica under magnetic fields.

This study aimed at estimating cultivation conditions to enable Yarrowia lipolytica NNRLY-1095 to produce extracellular lipase and at evaluating the influence of magnetic fields (MF) on the lipase production and on its catalytic conditions. Culture conditions of carbon sources and surfactant defined to produce extracellular lipase were 10 g L-1 glucose, 15 g L-1 olive oil and 2 g L-1 Triton X-100. The highest lipase activity (34.8 U mL-1) was reached after 144 h when MFs were applied from 72 to 144 h of culture. It corresponds to an increase of 287.5% by comparison with the highest lipase activity in the control culture. MF application from 72 to 144 h did not change the optimal temperature of lipase, which was 37°C, by comparison with the control. However, the optimal pH of the control was 7.0 while the one of lipase produced with MF was 8.0. Findings highlighted that the presence of MFs led to increase in synthesis of lipase by Y. lipolytica, with changes in the catalytic profile. This is one of the first studies of MF application to Y. lipolytica NRRL Y-1095 cultures to produce lipase.

A Study on the Isolation and Characterization of Bacillus subtilis Strains with Extracellular Lipase Production from Specific Environments

A Study on the Isolation and Characterization of Bacillus subtilis Strains with Extracellular Lipase Production from Specific Environments

Study of Hydrolytic Enzymes Activity and Stability of the Isolated Yeast Close to Zygoascus hellenicus

Background: Lipase is one of the most well-known and essential biocatalysts in the de-tergent, food, and pharmaceutical industries. Microbial lipase sources such as yeasts are applicable due to their stability in harsh conditions. Objective: In this study, the effect of temperature, initial pH, and incubation time were investigated to improve the extracellular lipase production by yeast, named Zygoascus hellenicus strain MZ_574439 T. Methods: Strain MZ_574439 T has already been isolated and registered from Iran. In the current project, strain MZ_574439 T with 99% similarity to Z. hellenicus was isolated from water samples. Results: Our findings showed that the isolated strain has a remarkable difference from its close phy-logenetic species in the production of lipase and can produce extracellular lipase up to 7.2 U/ml while the Z. hellenicus has no ability of lipase production. The isolated strain was not able to pro-duce other hydrolytic enzymes. The enzyme activity results showed that the best activity for the iso-lated lipase is pH= 7 and 37°C. The best stability condition for the enzyme occurs at 50°C and pH =7. Conclusion: From the current study, it can be concluded that Z. hellenicus produces lipase. The li-pase enzyme production was optimized with different physiological conditions. Yeast extract could be a better source for maximum lipase production.

Optimization of fermentation medium components by response surface methodology (RSM) and artificial neural network hybrid with genetic algorithm (ANN-GA) for lipase production by Burkholderia cenocepacia ST8 using used automotive engine oil as substrate

Bioconversion of used automotive engine oil (UEO) into lipase was conducted via submerged fermentation by Burkholderia cenocepacia ST8, as a strategy for value-added product generation and waste management. Response surface methodology (RSM) and artificial neural network hybrid with genetic algorithm (ANN-GA) were employed to optimize the fermentation medium for enhancing extracellular lipase production by B. cenocepacia ST8. Employing a four-factor-five-level central composite rotatable experimental design (CCRD), a reduced quartic polynomial RSM model and ANN model (4-4-1) trained using Bayesian Regularization were developed to attain the optimized fermentation medium for maximum level of lipase production. The RSM model predicted the following as the optimum media constituents: 2.28% v/v of Tween 80, 2.26% v/v of UEO, 0.79% w/v of nutrient broth, and 1.33% w/v of gum arabic, with an actual lipase yield of 216 U/mL. While, ANN-GA predicted the optimum media constituents to be 3% v/v of Tween 80, 3% v/v of UEO, 0.72% w/v of nutrient broth, and 3.38% w/v of gum arabic, with actual lipase yield of 225 U/mL. In comparison to the unoptimized medium, optimized RSM and ANN-GA systems both demonstrated a 1.6-fold increment in lipase production. Tween 80 and nutrient broth concentrations were the most important variables influencing the lipase production. The findings of this study indicated that the ANN-GA and RSM could be useful for effective optimization of the fermentation medium for enzyme production.

A comprehensive proteomics analysis of the response of Pseudomonas aeruginosa to nanoceria cytotoxicity

The increased commercial use and spread of nanoceria raises concerns about the risks associated with its effects on living organisms. Although Pseudomonas aeruginosa may be ubiquitous in nature, it is largely found in locations closely linked with human activity. P. aeruginosa san ai was used as a model organism for a deeper understanding of the interaction between biomolecules of the bacteria with this intriguing nanomaterial. A comprehensive proteomics approach along with analysis of altered respiration and production of targeted/specific secondary metabolites was conducted to study the response of P. aeruginosa san ai to nanoceria. Quantitative proteomics found that proteins associated with redox homeostasis, biosynthesis of amino acids, and lipid catabolism were upregulated. Proteins from outer cellular structures were downregulated, including transporters responsible for peptides, sugars, amino acids and polyamines, and the crucial TolB protein of the Tol-Pal system, required for the structural formation of the outer membrane layer. In accordance with the altered redox homeostasis proteins, an increased amount of pyocyanin, a key redox shuttle, and the upregulation of the siderophore, pyoverdine, responsible for iron homeostasis, were found. Production of extracellular molecules, e.g. pyocyanin, pyoverdine, exopolysaccharides, lipase, and alkaline protease, was significantly increased in P. aeruginosa san ai exposed to nanoceria. Overall, nanoceria at sublethal concentrations induces profound metabolic changes in P. aeruginosa san ai and provokes increased secretion of extracellular virulence factors, revealing the powerful influence this nanomaterial has on the vital functions of the microorganism.

Production of extracellular lipase by Enterococcus faecium E68 with olive oil waste as substrate

With green technologies, the damages caused by agro-technological wastes to the environment are eliminated. In our study, it was aimed both to prevent the harm of olive oil waste to the environment and to produce lipase enzyme, which is an important biotechnological product.. E. faecium E68 obtained from milk and dairy products was used in the production of lipase enzyme. E. faecium E68 was developed in lipase production medium containing 10% olive waste, pH6.5, at 37oC with 120 rpm agitation for 48 hours. The effect of temperature, pH metal ion, surfactant and NaCl was also determined.
 The molecular weight of the partially purified extracellular lipase enzyme was estimated to be around 19-20 kDa by SDS-PAGE. The optimum temperature was 45°C, while the enzyme exhibited appreciable thermostability retaining of activity at 55°C for 48h. The lipase was most active in the pH range of 3-11 with an optimum activity at pH 10. 1mM, Ca 2+, Mn2+, Cu2+, Ni2+, Zn2+, Mg2+ and K+ ions modulated the activity of the enzyme but inhibited by Hg2+, SDS and Triton X-100. The enzyme is halophilic and 25% NaCl salt increased the activity.
 Olive oil waste may be the preferred substrate for lipase production.

Statistical optimization of media components for production of extracellular lipase from edible mushroom Cantharellus cibarius.

Cantharellus cibarius is a wild edible mushrooms and considered as a plethora of compounds with potential biotechnological applications. This study highlighted the utilization of C. cibarius mushroom in the production of extracellular lipase under submerged fermentation, representing the first report on lipase production by this mushroom. Various physicochemical factors were optimized via one-factor-at-a time (OFAT) method. Maximum enzyme production was recorded when the mushroom mycelium was grown at 30°C on pH 6.0 for 96h in the medium supplemented with 1% [(v/v)] olive oil. Productivity of enzyme was affected by variation in the nitrogen sources, carbon sources, metal ions and NaCl salt. Glucose and peptone significantly enhanced enzyme production as carbon and nitrogen sources, respectively. Stimulatory and inhibitory effects were found by Ca2+ and Zn2+ ions, respectively. Furthermore, Box-Behnken Design (BBD) of Response Surface Methodology (RSM) was employed to optimize the interactive effects of specific media components like glucose, olive oil and CaCl2. The regression model was significant with a coefficient of determination (R2) value of 0.9483. Statistically optimized design (RSM) resulted approximately two-fold increase (23.5-42.283UmL-1) of lipase production than classical optimization method (OFAT), confirmed the validation of model. The kinetic parameters for p-nitrophenyl palmitate hydrolysis, Kmand Vmax were 5.24mM and 0.768mmol/min/mg respectively, established a high affinity for the substrate.

Production of Extracellular Lipase by Bacillus halotolerans from Oil-Contaminated Soil in a Pilot-Scale Submerged Bioreactor

Microbial lipases are the biocatalyst of choice for the present and future because of their characteristics, including their ability to remain active as an enzyme throughout a broad pH, temperature, and substrate range. The goal of the current investigation was to find novel sources of substrates and isolates from soil contaminated by oil for the synthesis of lipase. On tributyrin media, 10 lipolytic bacterial strains that were isolated from oil-contaminated soil were grown. Using the zone of clearance, it was possible to identify the isolates with the highest activity. Following phylogenetic tree analysis, molecular characterization of the 16S rRNA sequence of the bacterial isolates revealed that it was Bacillus halotolerans (VSH 09). The enzyme was purified to near homogeneity. The enzyme activity was found to be optimum at a pH of 7.0 and a temperature of 35 °C. While Ni2+ and Cu2+ had no effect, the presence of Mg2+ and Ca2+ exhibited the highest levels of enzyme activity. At 1%, tributyrin as a substrate exhibited its highest level of activity. The molecular weight, as determined by SDS-PAGE, was found to be 38 kDa. The kinetics of the enzyme were found to be 41.66 and 9.37 mg/mL for Vmax and Km, respectively. The high yield of lipase produced by this method suggests that it holds potential for production on a large scale and could be used for various biotechnological applications.

Towards the Physiological Understanding of Yarrowia lipolytica Growth and Lipase Production Using Waste Cooking Oils

The yeast Yarrowia lipolytica is an industrially relevant microorganism, which is able to convert low-value wastes into different high-value, bio-based products, such as enzymes, lipids, and other important metabolites. Waste cooking oil (WCO) represents one of the main streams generated in the food supply chain, especially from the domestic sector. The need to avoid its incorrect disposal makes this waste a resource for developing bioprocesses in the perspective of a circular bioeconomy. To this end, the strain Y. lipolytica W29 was used as a platform for the simultaneous production of intracellular lipids and extracellular lipases. Three different minimal media conditions with different pH controls were utilized in a small-scale (50 mL final volume) screening strategy, and the best condition was tested for an up-scaling procedure in higher volumes (800 mL) by selecting the best-performing possibility. The tested media were constituted by YNB media with high nitrogen restriction (1 g L−1 (NH4)2SO4) and different carbon sources (3% w v−1 glucose and 10% v v−1 WCO) with different levels of pH controls. Lipase production and SCO content were analyzed. A direct correlation was found between decreasing FFA availability in the media and increasing SCO levels and lipase activity. The simultaneous production of extracellular lipase (1.164 ± 0.025 U mL−1) and intracellular single-cell oil accumulation by Y. lipolytica W29 growing on WCO demonstrates the potential and the industrial relevance of this biorefinery model.

Extracellular lipase production by local isolate of Penicillium citrinum

Extracellular lipase production by local isolate of Penicillium citrinum

Statistical optimization of lipase production from oil mill effluent by Acinetobacter sp. KSPE71

The present study investigated the valorisation of oil-rich residues of coconut oil mill effluent (COME) as a potential growth medium for the microbial production of extracellular lipase. The bacterial species isolated from oil mill effluent, Acinetobacter sp. KSPE71 was tested for its efficiency to grow and produce lipase in undiluted COME and 0.2 % yeast extract and 0.2 % NH4Cl supplemented COME. In this connection, the process parameters such as pH, temperature, agitation speed, and inoculum size were optimized to maximize the production using a central composite design in the Response surface methodology. At the optimized state of pH 7.5, 35?C, 150 rpm with 0.6 % inoculum size, a maximum of 3.95 U mL-1 activity was obtained, four-fold higher than the basal condition. At this stage, 73 % of the lipid content was degraded. The present work results imply that the oil mill effluent can be used as a cheaper production medium for lipase and the new isolate Acinetobacter sp. KSPE71 as a potential lipase producer. The degradation of oil waste along with the production of the valuable product has multiple advantages of cost reduction of lipase and environmental concern.

Production of new lipase from Preussia africana and partial characterization

The present work aims to study the production of a new extracellular lipase from the endophytic fungus Preussia africana isolated from red lapacho tree (Handroanthus impetiginosus). Tests were carried out in order to evaluate the influence of different inducing oils (sunflower, cotton, corn, palm kernel, canola and linseed) and carbon/nitrogen ratio (C/N of 11.73, 8.63 and 7.05) on submerged fermentation aiming at optimal production of a single extracellular lipase. Results show that an optimal lipase production of 14.5 kDa has been attained after 48 h of fermentation when sunflower oil was used in the C/N ratio of 8.63 in the fermentation medium. The produced lipase showed greater activity for oils that had higher percentage of unsaturated fatty acids in their composition. Characterization was performed using a two-factor central composite rotatable design (CCRD), and the pH 6 was found to be optimal (around 28 U/mL). The temperature range studied (from 20 to 54 °C) showed no difference for the lipase catalytic activity. This is an advantage, especially when aiming at its application in reactions of industrial interest.

Production of extracellular lipase from psychrotrophic bacterium Oceanisphaera sp. RSAP17 isolated from arctic soil.

Cold-active extracellular lipases produced by different psychrotrophs are important for various industrial applications. We have isolated a Gram-negative, rod-shaped, aerobe, non-pigment producing psychrotrophic bacterial strain RSAP17 (MTCC 12991, MCC 4275) from the unexplored Arctic soil sample of NyAlesund, Svalbard, Norway (78° 55″ N, 11° 54″ E). The detailed morphological, biochemical, and molecular characteristics were investigated to characterize the isolate RSAP17. Analyses of the 16S rDNA sequence of strain RSAP17 (Accession no. MK391379) shows the closest match with Oceanisphaera marina YM319T (99.45%) and Oceanisphaera sediminis TW92 JCM 17329T (97.40%). The isolate is capable of producing extracellular lipase but not amylase, cellulase or urease. The optimal parameters for lipase production have been found in tributyrin based (10mL/L) agar media supplemented with 3% (w/v) NaCl after 2-3days of incubation at 20-22°C temperature and pH 9 at shaking condition. We have purified the extracellular lipase from the RSAP17 grown culture supernatant through 75% ammonium sulfate precipitation followed by dialysis and DEAE cellulose column chromatography. The invitro lipolytic activity of the purified lipase enzymes has been done through zymogram analysis. The molecular weight found for the lipase is 103.8 kD. The optimal activity of the purified lipase has been found at 25°C and pH 9. MALDI-TOF-MS study of the purified lipase showed the highest match with the sequence of prolipoprotein diacylglyceryl transferase with 44% sequence coverage. Further study on large-scale production, substrate utilization and enzymatic kinetics of this lipase could unravel its possibility in future biotechnological applications.

Tepki Yüzey Metodu ile Aeromonas caviae LipT51'den Ekstrasellüler, Termo-Alkali Lipaz Üretiminin İstatistiksel Optimizasyonu

Extracellular thermo-alkaline lipase production from Aeromonas caviae LipT51 was statistically optimized by response surface methodology (RSM). First, the one factor at a time approach was implemented to screen the sources of carbon (olive oil, tributyrin, sunflower oil, waste frying oil, glycerol, Tween 80, Tween 20, palm oil, and Triton X100) and nitrogen (peptone, yeast extract, tryptone, whey, urea, NaNO2, NH4NO3) for the highest lipase production. Then, optimum values for waste frying oil selected as carbon source, tryptone selected as nitrogen source and initial pH of the medium were determined by RSM using Box-Behnken design (BBD). The quadratic model of BBD for lipase production was statistically significant and reliable (p < 0.0001, R2 = 0.9881). The validated optimal conditions for maximum lipase production (1.6 U mL-1) were determined as 1.13% waste frying oil, 1.5% tryptone and pH 7.9. For the first time in this study, optimization of lipase production from an A. caviae strain was carried out and under optimized culture conditions using cheap waste material. The production efficiency of lipase enzyme, which is known to be valuable with its detergent activity, increased 2.7 times compared to non-optimized conditions.