Extracellular Lipase Research Articles

Presence of pharmaceutical contaminants of emerging concerns in two rivers of western Cuba and their relationship with the extracellular enzymatic activity of microbial communities

In recent years, the concern derived from the presence of emerging contaminants in the environment and the possible effects on the One Health trilogy has increased. This study determined the concentration of pharmaceutical contaminants of emerging concern and their relationship with the extracellular enzymatic activity of microbial communities from two rivers in western Cuba. Two sampling stations were analyzed; one in the Almendares River (urban) and the other in the San Juan River (rural), taking into account the pollution sources that arrive at these stations and previous physicochemical characterizations. Extracellular protease, acid phosphatase, alkaline phosphatase, lipase, and catalase activities in water and sediments were determined and correlated with contaminants of emerging concern determined by liquid chromatography with mass spectrometry. This study evidenced the presence of different pharmaceutical contaminants found in the categories of antihypertensives, stimulants, anti-inflammatories, and antibiotics in both rivers. Concentrations of contaminants of emerging concern were greater in the Almendares River compared to the San Juan River. In addition, through the canonical redundancy analysis, the influence of these contaminants on the extracellular enzymatic activities of microbial communities was documented, where in most cases they inhibit protease, phosphatase, and lipase activities and enhance catalase activity in response to oxidative stress. The present investigation constitutes the first report in Cuba of the presence of pharmaceutical contaminants of emerging concern and one of the few works that exist in the Latin American region.

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.

Fever-like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo

BackgroundStaphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments.ResultsIn general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsβ genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host–pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39οC showed a decrease in cellular invasion and an increase in IL-8—but not in IL-6—production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39 °C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37 and 39 °C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39 °C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39 °C comparing with 37 °C.ConclusionsOur results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host–pathogen interactions and coevolution.

Optimization, Purification and Characterization of Extracellular Lipase Produced by Serratia marcescens EGHK-19

Optimization, Purification and Characterization of Extracellular Lipase Produced by Serratia marcescens EGHK-19

Evaluation of the Structure-Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach.

Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10-His216 and His216-Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

Actinomycetes isolated from rhizosphere of wild Coffea arabica L. showed strong biocontrol activities against coffee wilt disease.

Coffee, the second most traded commodity globally after petroleum and is the most exported cash crop of Ethiopia. However, coffee cultivation faces challenges due to fungal diseases, resulting in significant yield losses. The primary fungal diseases affecting coffee production include coffee berry disease, wilt disease (caused by Gibberella xylarioides), and coffee leaf rust. In this study, we aimed to isolate potentially antagonistic actinomycetes from the root rhizosphere of wild Coffea arabica plants in the Yayo coffee forest biosphere in southwestern Ethiopia. Soil samples were collected from the rhizosphere, and actinomycetes were selectively isolated and identified to the genus level by morphological, physiological, and biochemical characterization. These pure isolates were screened for their antagonistic activity against Gibberella xylarioides in vitro using a dual culturing method. Promising isolates demonstrating strong inhibition of fungal mycelial growth were further investigated through in vivo experiments using coffee seedlings. A total of 82 rhizobacteria were isolated. These isolates' inhibition of fungal mycelial growth varied from 0% to 83.3%. Among them, four isolates MUA26, MUA13, MUA52, and MUA14 demonstrated the highest percentage inhibition of fungal mycelial growth: 83.3%, 80%, 76.67%, and 73.3%, respectively. Seedlings inoculated with MUA13, MUA14, and MUA26 during the challenge inoculations (Rhizobacteria + Gibberella xylarioides) exhibited the lowest disease incidence compared to the infected fungi (P < 0.05). Notably, the seedlings inoculated with MUA26 demonstrated the highest disease control efficiency, reaching 83% (P < 0.05). MUA26 was found to produce extracellular enzymes, including chitinase, protease, and lipase, which acted as inhibitors. In summary, this study highlights that MUA26, among the actinomycete isolates, exhibited significant antagonistic activity against Gibberella xylarioides f.sp. coffea. Its efficacy in controlling coffee wilt disease, both in vitro and in vivo, positions it as a potential bioinoculant for managing coffee wilt disease.

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.

Effect of protatranes on the biosynthesis of extracellular enzymes of &lt;i&gt;Candida ethanolica&lt;/i&gt; BKM Y-2300 T

The study aims to explore the possibility of using synthetic biologically active compounds (protatranes) to increase the lipolytic and proteolytic activity of Candida ethanolica. Protatrane 1 (tris(2-hydroxyethyl) ammonium-4chlorophenyl-sulfanyl acetate) and protatrane 2 (tris(2-hydroxyethyl) ammonium-4-chlorophenyl-sulfonyl acetate) were added to the growth medium at trace concentrations of 1×10−6–1×10−8wt% separately and together. It was established that with the introduction to the growth medium, protatranes had a multidirectional effect on the biosynthesis of enzymes by Candida ethanolica yeast. This effect was dependent on the concentrations of these compounds, as well as on their separate or combined introduction. All the studied concentrations were found to be effective for lipase synthesis, as they improved enzyme synthesis by 1.7–8.6 times. The combined use of protatranes increased enzyme synthesis by 3.4–11.7 times. For protease formation, the combined introduction of the studied protatranes at a concentration of 10−6wt% was found to be the most effective, with enzyme synthesis reaching 184.8±7.02 U/mL in the culture broth. When co-introduced, the studied protatranes tris(2-hydroxyethyl) ammonium-4-chlorophenylsulfanyl acetate and tris(2-hydroxyethyl) ammonium-4-chlorophenyl-sulfonyl acetate can be used to increase the production efficiency of extracellular lipases and proteases by Candida ethanolica. The specific effect of protatranes was revealed to be the uneven dynamics of extracellular enzyme accumulation, manifested in the presence of sharp peaks during yeast cultivation. The reasons for this unevenness require further research.

Exploring the use of hexadecane by Yarrowia lipolytica: Effect of dissolved oxygen and medium supplementation

This work aimed to evaluate the effect of medium composition and volumetric oxygen transfer coefficient (kLa) on Y. lipolytica growth and production of microbial lipids and enzymes from hexadecane. In the stirred tank bioreactor, increasing kLa from 11 h−1 to 132 h−1 improved the hexadecane assimilation rate, biomass concentration, and lipids synthesis (0.90 g·L−1). A cost-effective hexadecane-based medium supplemented with corn steep liquor and a low amount of ammonium sulfate boosted lipids production up to 2.1 g·L−1, composed of palmitic, palmitoleic, oleic, and linoleic acids. The unsaturated/saturated fraction was dependent on the C/N ratio. Lipids of Y. lipolytica CBS 2075 are promising feedstock for animal feed, food additives, or the biodiesel industry. Simultaneous synthesis of extracellular lipase and protease from hexadecane was observed, which is a new feature that was not previously reported. The highest enzyme activity was obtained at the highest C/N ratio conditions. These results open new perspectives on the application of Y. lipolytica-based cultures for the biotransformation of hexadecane-polluted streams into valuable compounds, fulfilling an interesting strategy towards the circular economy concept.

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.

Isolation of Lactiplantibacillus plantarum producing Extracellular Lipase from Dairy Products and Optimization of the Enzyme Production

Recently, microbial lipases isolated from probiotic bacteria have attracted the attention of many scientists because they have significantly beneficial properties for human health. As a highly nutritious substrate, dairy products possess great potential for probiotic bacteria isolation. The main purpose of this study was to isolate probiotic bacteria in order to search for lipase producing bacteria. The bacteria were isolated from dairy products and purified by repeated sub-culturing on MRS medium at 37°C. The isolates were subjected to primary and secondary screening via fluorometric and colorimetric methods respectively. The isolates showing orange fluorescence under UV light at 350 nm around their colonies were selected for the colorimetric method. In this method, lipase activity was investigated by p-nitrophenyl palmitate. Probiotic characterizations of lipase-producing isolates were performed by acid resistance test, cell surface hydrophobicity assay, auto-aggregation and co-aggregation. The optimization of lipase production was performed by Central Composite Design (CCD). In the present study, 92 isolates were recovered from 71 samples. The results showed that 41 isolates were positive for fluorometric assay. According to the amount of enzyme production, seven strains were selected for probiotic characterizations. Finally, a potential probiotic strain of genus Lactobacillus showed the highest enzyme activity with 3.4 U.ml-1 compared to other isolates. This strain showed 100% similarity to Lactiplantibacillus plantarum. This research indicated that the lipase of Lactiplantibacillus plantarum can be used as a promising enzyme in the food and medicine industries.

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.

Analysis of the virulence, infection process, and extracellular enzyme activities of Aspergillus nomius against the Asian corn borer, Ostrinia furnacalis guenée (Lepidoptera: Crambidae)

ABSTRACT The control of Ostrinia furnacalis, a major pest of maize in Xinjiang, is challenging owing to the occurrence of resistant individuals. Entomopathogenic fungi (EPF) are natural insect regulators used as substitutes for synthetic chemical insecticides. The fungus Aspergillus nomius is highly pathogenic to O. furnacalis; however, its virulence characteristics have not been identified. This study aimed to analyse the lethal efficacy, mode of infection on the cuticle, and extracellular enzyme activity of A. nomius against O. furnacalis. We found that the mortality and mycosis of O. furnacalis were dose-dependent when exposed to A. nomius and varied at different life stages. The egg-hatching and adult emergence rates decreased with an increase in conidial suspension. The highest mortality (83.33%, 7 d post-infection [DPI]) and mycosis (74.33%, 7 DPI) and the lowest mortality response (8.52 × 103 conidia mL−1) and median lethal time (4.91 d) occurred in the 3rd instar larvae of O. furnacalis. Scanning electron microscopy indicated that numerous conidia germination and infection structure formation may have contributed to the high pathogenicity of A. nomius against O. furnacalis. There were significant correlations between O. furnacalis mortality and the activities of extracellular protease, lipase, and chitinase of A. nomius. This study revealed the infection process of the highly pathogenic A. nomius against O. furnacalis, providing a theoretical basis and reference for strain improvement and field application of EPF.

Exploration of phyto-beneficial traits of halophilic rhizobacteria isolated from the mangrove halophyte Avicennia marina

The salinity of the soil exerts an adverse effect on plant development and yield. However, amid saline stress, certain halophilic bacteria enhance the growth of plants. In this investigation, the mangrove halophyte Avicennia marina’s (A. marina) rhizosphere soil was used as the source for the isolation of halophilic bacteria. The isolates’ growth capacity at different pH, salinity, temperature, and polyethylene glycol (PEG) was assessed, and it was found that 56% showed growth in media supplemented with 2–7.5% NaCl; 50% showed efficient growth at pH 6–8, 62% grow optimally at temperature 25–35 °C, and about 88% showed growth in media with 5% PEG. The isolates possess growth-promoting traits evidenced by nitrogen fixation (18%), ammonia production (31%), phosphate solubilization (88%), zinc solubilization (44%), and indole acetic acid (IAA) (88%) production. The isolates can produce extracellular enzymes, viz., protease (88%), amylase (62%), cellulase (56%), and lipase (31%). The bacterial isolate S1B1 that displayed maximum plant growth-promoting traits was selected as the potent and subjected to plant growth promotion studies. The potent isolate S1B1 enhances Solanum lycopersicum’s germination under different salinity compared to untreated seeds with germination of 64% at the maximum concentration of 2% NaCl. The potential halophilic plant growth-promoting rhizobacteria (PGPR) isolate was determined as Rossellomorea aquimaris (R. aquimaris) by 16s rRNA analysis. The results demonstrated that R. aquimaris recovered from the rhizosphere of halophyte A. marina found possible applications for enhancing plant development and productivity in salinity-affected soils.

Preparation, purification, and biochemical of fat-degrading bacterial enzymes from pig carcass compost and its application

BackgroundA lot of kitchen waste oil is produced every day worldwide, leading to serious environmental pollution. As one of the environmental protection methods, microorganisms are widely used treating of various wastes. Lipase, as one of the cleaning agents can effectively degrade kitchen waste oil. The composting process of pig carcasses produces many lipase producing microorganisms, rendering compost products an excellent source for isolating lipase producing microorganisms. To our knowledge, there are no reports isolating of lipase producing strains from the high temperature phase of pig carcass compost.MethodologyLipase producing strains were isolated using a triglyceride medium and identified by 16S rRNA gene sequencing. The optimal fermentation conditions for maximum lipase yield were gradually optimized by single-factor tests. The extracellular lipase was purified by ammonium sulfate precipitation and Sephadex G-75 gel isolation chromatography. Amino acid sequence analysis, structure prediction, and molecular docking of the purified protein were performed. The pure lipase's enzymatic properties and application potential were evaluated by characterizing its biochemical properties.ResultsIn this study, a lipase producing strain of Bacillus sp. ZF2 was isolated from pig carcass compost products, the optimal fermentation conditions of lipase: sucrose 3 g/L, ammonium sulfate 7 g/L, Mn2+ 1.0 mmol/L, initial pH 6, inoculum 5%, temperature 25 ℃, and fermentation time 48 h. After purification, the specific activity of the purified lipase reached 317.59 U/mg, a 9.78-fold improvement. Lipase had the highest similarity to the GH family 46 chitosanase and molecular docking showed that lipase binds to fat via two hydrogen bonds at Gln146 (A) and Glu203 (A). Under different conditions (temperature, metal ions, organic solvents, and surfactants), lipase can maintain enzymatic activity. Under different types of kitchen oils, lipase has low activity only for ‘chicken oil’, in treating other substrates, the enzyme activity can exceed 50%.ConclusionsThis study reveals the potential of lipase for waste oil removal, and future research will be devoted to the application of lipase.

Immobilization microbial cellulase and lipase as bio-catalyst agent using maltodextrin for biofuel synthesis

The alternative method of using enzyme technology and modification methods will reduce the cost of biofuel production. New solution for biofuel production by improving the advantages from potential microbial to produce biocatalyst such as cellulase and lipase as an alternative catalyst for replacing the chemical catalyst for transesterification process. This study was developing new method of enzyme immobilization for feasibility biofuel production which is more efficient and effective process in industries. The result obtained in this work allowed concluding that the highest value for the total cellulolytic activity was obtain using carboxymethyl cellulose as substrate reaching value 4.225 U/ml for 24 h with optimization of pH 7 and temperature 50 °C. Instead of cellulase, the potential Aspergillus niger InaCC F538 can produce extracellular lipase and the value found for lipase activity in olive oil substrate is 3.710 U/ml for 24 h incubation time with optimization of pH 8 and temperature 50 °C. The immobilization process of cellulase and lipase using maltodextrin was enhancing the activity to 19.615 U/ml and 24.123 U/ml. The biofuel production based on microbial will be widely used for continuously used for renewable raw material from waste agriculture biomasses for biofuel production. New enzymes combination for improving enzyme stability for industrial application based on fermentation through the economically feasible.

Purification and characterization of extracellular lipase from a thermotolerant strain: Bacillus subtilis TTP-06.

In current study, lipase from a thermotolerant Bacillus subtilis TTP-06 was purified in a stepwise manner by using ammonium sulfate precipitation and column chromatography. Thenceforth, it was subjected to sodium dodecyl sulfate- and native-polyacrylamide gel electrophoresis to check the homogeneity of the purified enzyme. The ideal substrate concentration, pH, temperature, reaction duration and lipase specificity were identified. With a yield of 11.02%, purified lipase displayed activity of 8.51 U/mg. Thenceforward, the homogeneously purified enzyme was considered to be a homo-dimer of 30kDa subunits. Enzyme had Km and Vmax value of 9.498mM and 19.92molmg-1min-1, respectively. Additionally, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method was used to investigate the purified lipase and estimate its 3-D structure, which revealed a catalytic triad of serine, aspartate and histidine.

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.

Community diversity of psychrophilic bacteria in dairy farm raw milk and its characteristic enzyme production at different temperature

Psychrophilic bacteria and their heat-resistant enzymes are important risk factors affecting the quality of shelf life of UHT milk, and it is important for the development of the industry to uncover their action pattern. In these study, 40 raw milk samples were taken from dairy farms in Hebei (northern China) and Guangdong (southern China) in four seasons. The structure and composition of psychrophilic bacteria by PCR targeting 16S rRNA, as well as its extracellular protease, lipase activity and heat resistance was analyzed. The results showed that there was a significant temperature dependence but not geographical dependence in the distribution of the psychrophilic bacteria community. Secondly, P. fluorescens, P. azotoformans and P. paralactis were not only the dominant bacterium, but also had higher proteolytic and lipolytic activities. Lastly, Analysis of the enzyme-producing heat resistance of a typical psychrophilic bacteria, P. azotoformans B15-1. At 135 °C, the average residual rate of protease only 3.08%, however, lipase was more heat resistant and the average residual rate was still 19.16%. The present study investigated the psychrophilic bacteria and its hydrolytic enzymes activity in raw milk from different regions and seasons, in order to assess the baseline of psychrophilic bacteria contamination in the dairy farm.