Extracellular Lipase Activity Research Articles

Novel extracellular lipase gene Lip1728 influences nutrient-dependent performance bacterial quorum sensing of Burkholderia pyrrocinia WZ10-3

Quorum sensing (QS) is a cellular communication mechanism in which bacteria secrete and recognize signaling molecules to regulate group behavior. Lipases provide energy for bacterial cell growth but it is unknown whether they influence nutrient-dependent QS by hydrolyzing substrate. A high-yield lipase-producing strain, Burkholderia pyrrocinia WZ10–3, was previously identified in our laboratory, but the composition of its crude enzymes was not elucidated. Here, we identified a key extracellular lipase, Lip1728, in WZ10–3, which accounts for 99 % of the extracellular lipase activity. Lip1728 prefers to hydrolyze triglycerides at sn-1,3 positions, with pNP-C16 being its optimal substrate. Lip1728 exhibited activity at pH 5.0–10.0 and regardless of the presence of metal ions. It had strong resistance to sodium dodecyl sulfate and short-chain alcohols and was activated by phenylmethanesulfonylfluoride (PMSF). Lip1728 knockout significantly affected lipid metabolism and biofilm formation in the presence of olive oil. Finally, oleic acid, a hydrolysate of Lip1728, influenced the production of the signal molecule N-acyl homoserine lactone (AHL) and biofilm formation by downregulating the AHL synthetase gene pyrI. In conclusion, Lip1728, as a key extracellular lipase in B. pyrrocinia WZ10–3, exhibits superior properties that make it suitable for biodiesel production and plays a crucial role in QS.

Exploring the Interplay between Nutrients, Bacteriophages, and Bacterial Lipases in Host- and Bacteria-mediated Pathogenesis.

Pathogenic bacteria and host cells counteract or neutralize each other's effect in two fundamental ways: Direct invasion and secretion of various substances. Among these, lipases secreted by pathogenic bacteria and host cell lysozyme are key actors. Secreted lipases from pathogenic bacterial are suggested as a key player in the pathogen-host interaction. Among the gut microbial energy sources, glucose and fats have been referred to as one of the best inducers and substrates for bacterial lipases. Enrichment of bacterial growth medium with extra glucose or oil has been shown to induce lipase production in pathogenic bacteria. More recently, research has focused on the role of human gut phage alterations in the onset of dysbiosis because the bacteria-phage interactions can be dramatically affected by the nutrient milieu of the gut. However, the reciprocal role of bacterial lipases and phages in this context has not been well studied and there is no data available about how high glucose or fat availability might modulate the cellular milieu of the pathogenic bacteria-phageeukaryotic host cell interface. The purpose of this study was to evaluate the immunologic outcome of pathogenic bacteria-phage interaction under normal, high glucose, and high butter oil conditions to understand how nutrient availability affects lipase activity in pathogenic bacteria and, ultimately, the eukaryotic host cell responses to pathogenic bacteria-phage interaction. 10 groups of co-cultured T84 and HepG2 cells were treated with Pseudomonas aeruginosa strain PAO1 (P.a PAO1) in the presence and absence of its KPP22 phage and incubated in three different growth media (DMEM, DMEM + glucose and DMEM + butter oil). Structural and physiological (barrier function and cell viability), inflammatory (IL-6 and IL-8), metabolic (glucose and triglycerides), and enzymatic (lipases and lysozyme) parameters were determined. Excess glucose or butter oil enhanced additively extracellular lipase activity of P.a PAO1. Excess glucose or butter oil treatments also magnified P. a PAO1- induced secretion of inflammatory signal molecules (IL-1β, IL-6) from co-cultured cells, concomitant with the enhancement of intracellular triglycerides in co-cultured HepG2 cells, these effects being abolished by phage KPP22. The results of the present study imply that KPP22 phage influences the interplay between food substances, gut bacterial lipases, and the gut cellular milieu. This can be applied in two-way interaction: by affecting the microbial uptake of excess free simple sugars and fats from the gut milieu leading to decreased bacterial lipases and by modulating the immune system of the intestinal -liver axis cells. Further studies are needed to see if the biological consequences of these effects also occur in vivo.

Bioprospecting Antibacterial Properties of Fungi Isolated from Kakinada Sea Coast and Eucalyptus Foliage

Background: Fungi produce a wide range of secondary metabolites with bactericidal or bacteriostatic properties. In search of novel antibacterial compounds recently, many fungi of marine and plant origin have been studied for their antimicrobial properties. Aims: This work aimed to study bio-prospect marine and endophytic fungi for their antibacterial properties. objective: To determine the antibacterial properties of ethyl acetate extracts of fungal isolates. Methods: Intensive microbiological methods were followed for isolation, differential growth, and qualitative screening of enzyme production. The isolates were characterised and identified based on morpho-taxonomy, 18S rRNA gene sequencing, and phylogenetic analysis of the tar-get genes. The antimicrobial activity of fungal ethyl acetate extracts against S. aureus, B. sub-tilis, and E. coli was evaluated using a well-diffusion method, and MIC was determined by the microdilution method. Cell lysis was observed through Transmission Electron Microscopy. Results: A BLAST search of 18S rRNA gene sequences of the marine isolates GSBT S13 and GSBT S14 showed 99.3 % sequence similarity with A. glaucus for both isolates and that of endophyte GSBT E3 showed 99.7 % sequence similarity with B. pinkertoniae. Cellulase pro-duction was comparatively higher in GSBT E3 and lipase from GSBT S13 and GSBT S14. Ethyl acetate extracts of GSBT S14 and GSBT E3 showed a clear zone of inhibition by the well-diffusion method, further confirmed by electron microscopy. HR-TEM showed that the ethyl acetate extracts of the isolates appeared to damage the cell membrane, leading to cell shrinkage and death in E. coli and S. aureus. Conclusion: GSBT S13 and GSBT S14 exhibited extracellular amylase, cellulase, and lipase activities. Ethyl acetate extracts of both GSBT S14 and B. pinkertoniae GSBT E3 showed better antibacterial properties against S. aureus.

Phenotypic and genetic characterization of a lipolytic Ralstonia mannitolilytica isolate from petroleum-contaminated soil in Iraq.

Lipases play a crucial role in various industrial applications, and microbial lipases, particularly those from bacteria, possess significant properties. With increasing concerns about the environmental and health impacts of hydrocarbons from pipelines and refineries, there is a growing need to mitigate the risks associated with these compounds. In this study, 40 bacterial isolates were recovered from contaminated soil samples collected from multiple refineries across Iraq. Using the Vitek system, bacterial isolates were identified up to the species level, revealing that only 12 isolates exhibited lipase-producing capabilities. Among the lipase-producing isolates, Ralstonia mannitolilytica demonstrated the highest extracellular lipase activity, as determined by an olive oil plate assay supplemented with rhodamine B. Confirmation of the species identity was achieved through 16S rRNA gene sequencing, with the obtained sequence deposited under accession number LC772176.1. Further sequence analysis revealed single nucleotide polymorphisms (SNPs) in the genome of Ralstonia mannitolilytica strain H230303-10_N19_7x_R2 (CP011257.1, positions 1,311,102 and 1,311,457). Additionally, the presence of the lipase gene was confirmed through amplification and sequencing using a thermocycler PCR. Sequence analysis of the gene, aligned using Geneious Prime software, identified SNPs (CP010799, CP049132, AY364601, CP011257, and CP023537), and a phylogenetic tree was constructed based on genetic characterization. Our findings highlight the potential of Ralstonia mannitolilytica as a promising candidate for lipase production and contribute to our understanding of its genetic diversity and biotechnological applications in hydrocarbon degradation and industrial processes.

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.

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.

Purification and characterization of lipase produced from Bacillus cereus (PCSIR NL-37)

There is a growing trend to produce lipase from microorganisms owing to their commercial demand in various industries. Bacillus cereus has been shown to have extracellular lipase activity and high growth rates. This study explains the purification of microbial lipase to homogeneity by dialysis, precipitation and chromatography. The purified enzyme with 56kDa relative molecular mass exhibited the highest activity at 60°C (95.56U/ml) and pH 7 (124.50U/ml). The enzyme activity was highly promoted in the presence of K+ (136.17U/ml) and Zn++(133.07 U/ml), and SDS did not affect the enzyme activity, whereas in the company of triton X100 activity of lipase is maximum (23.90 U/ml). The enzyme activity was enhanced by using almond oil (120.00 U/ml) as a substrate. We deduce cheaper protocols for producing extracellular lipase via simple laboratory techniques, which could be a good insight for its production at the commercial level. Keywords: Lipase; Bacillus cereus; chromatography; enzyme activity; purification.

Homologous High-Level Lipase and Single-Cell Protein Production with Engineered Yarrowia lipolytica via Scale-Up Fermentation for Industrial Applications

Yarrowia lipolytica is a promising feed additives. Here, we aimed to produce extracellular lipases and single-cell proteins (SCPs) at high levels simultaneously through fed-batch fermentation of engineered Y. lipolytica. The parameters for 500 mL shake flask cultures were optimized with a single factorial design. The resultant activity of lipase reached 880.6 U/mL after 84 h of fermentation, and 32.0 g/L fermentation broth of dry SCP was obtained at 120 h. To attain high SCP and lipase productivity, the high-density fed-batch fermentation of Y. lipolytica was scaled up in 10 L, 30 L, and 100 L fermentors. Using glycerol as the sole carbon source, the lipase activity peaked to 8083.3 U/mL, and the final dry SCP weight was 183.1 g/L at 94.6 h in 10 L fermentors. The extracellular lipase activity and SCP weight reached 11,100.0 U/mL and 173.3 g of dry SCP/L at 136 h in 30 L fermentors, respectively. Following 136 h of fed-batch fermentation, the extracellular lipase activity and dry SCP weight reached 8532.0 U/mL and 170.3 g/L in 100 L fermentors, respectively. A balance between the lipase secretion and growth of Y. lipolytica recombinant strain was achieved, indicating that an efficient fermentation strategy could promote further scale-up for industrial SCP production from engineered Y. lipolytica.

Production of Enriched Biomass by Carotenogenic Yeasts Cultivated on by-Products of Poultry Processing—A Screening Study

Carotenogenic yeasts are a group of microorganisms producing valuable metabolites such as carotenoids, ergosterol, ubiquinone or fatty acids. Their exceptional adaptability allows them to grow in diverse conditions. Owing to their extracellular lipase activity, they are capable of processing many lipid-type waste substrates. This study discusses the processing of poultry waste, specifically fat and feathers by using carotenogenic yeasts. Poultry fat does not require any pre-treatment to be utilized by yeast, but hydrolytic pre-treatment is required for the utilization of the nitrogen contained in feathers. Glycerol was used as a supplementary substrate to support the culture in the early stages of growth. Seven yeast strains were used for the experiments, of which the strain Rhodotorula mucilaginosa CCY19-4-25 achieved exceptional results of biomass production: 29.5 g/L on poultry fat + 10% glycerol at C/N ratio 25 and 28.3 g/L on media containing poultry fat + 25% glycerol at C/N 50. The bioreactor cultivation of the Rhodosporidium toruloides strain in media containing glycerol and feather hydrolysate as a nitrogen substrate achieved a biomass yield of 34.92 g/L after 144 h of cultivation. The produced enriched yeast biomass can be used as a component for poultry feeding; thus, the study is performed under the biorefinery concept.

The potential of biodiesel production from grasses in Thailand through consolidated bioprocessing using a cellulolytic oleaginous yeast, Cyberlindnera rhodanensis CU-CV7

Biodiesel can be produced from lipids derived from the cellulolytic oleaginous yeast Cyberlindnera rhodanensis CU-CV7 when directly cultivated on undetoxified Thai-grass hydrolysates through consolidated bioprocessing (CBP). The lipid content of CU-CV7 grown on a glucose-based medium was 26.77 ± 0.56% of the cell dry weight (CDW). Strain CU-CV7 is a great CBP-enabling microbe because of its native capacity to generate lipid (22.19 ± 0.72% of CDW) while simultaneously expressing endoglucanase, exoglucanase, and β-glucosidase activities (0.33, 0.06, and 0.20 IU/mL, respectively) on carboxymethylcellulose. Strain CU-CV7 was demonstrated to be a lipase-producing strain and could potentially be employed as a biocatalyst to produce biodiesel with a maximum extracellular lipase activity of 1.47 IU/mL. The lipid production from 13 types of grass without the need for additional cellulase was evaluated. The highest lipid yield was obtained from Napier (Lampang ecotype; 1.01 ± 0.14 g/L) and was comprised of a high proportion of saturated fatty acids. The predicted biodiesel properties were found to be suitable with respect to the biodiesel international standards. Thus, lipid production from these grasses by CU-CV7 appears to open a new path for the renewable biodiesel industry and to satisfy the SDG 7 and SDG 13 goals.

Listeria monocytogenes is a solvent tolerant organism secreting a solvent stable lipase: potential biotechnological applications

PurposeThe emerging biobased economy will require robust, adaptable, organisms for the production and processing of biomaterials as well as for bioremediation. Recently, the search for solvent tolerant organisms and solvent tolerant enzymes has intensified. Resilient organisms secreting solvent stable lipases are of particular interest for biotechnological applications.MethodsScreening of soil samples for lipase-producing organisms was carried out on Rhodamine B plates. The most productive lipase-producing organisms were further screened for their resistance to solvents commonly used in biotechnological applications.ResultsIn the course of screening, one of the isolated organisms that exhibited extracellular lipase activity, was identified as the human pathogen Listeria monocytogenes through 16S rRNA sequencing. Further exploration revealed that this organism was resistant to solvents ranging from log P − 0.81 to 4.0. Moreover, in the presence of these solvents, L. monocytogenes secreted an extracellular, solvent tolerant, lipase activity. This lipase retained approximately 80% activity when incubated in 30% (v/v) methanol for 24 h.ConclusionThese findings identify L. monocytogenes as a potentially useful organism for biotechnological applications. However, the fact that Listeria is a pathogen is problematic and it will require the use of non-pathogenic or attenuated Listeria strains for practical applications. Nonetheless, the ability to adapt to rapidly changing environmental conditions, to grow at low temperatures, to resist solvents and to secrete an extracellular solvent tolerant lipase are unique and highly useful characteristics. The potential application of L. monocytogenes in wastewater bioremediation and plastics degradation is discussed.

Activated sludge microbial communities and hydrolytic potential in a full-scale SBR system treating landfill leachate

Landfill leachate, due to its recalcitrant nature and toxicity, poses a serious environmental threat, which requires the implementation of effective treatment processes. In this work, a full-scale treatment system consisting of two Sequencing Batch Reactors (SBRs) was used for the processing of landfill leachate of intermediate to mature age (BOD/COD ratio of 0.16). Biosystem operation resulted in BOD5, COD and TKN removal efficiencies of 81%, 39% and 76%, respectively, whereas the low residual NO3 --N concentration in the effluent (4.01 ± 0.10 mg/L) was indicative of the efficient denitrification process. Assessment of hydrolytic potential of activated sludge revealed high endocellular and extracellular lipase activities, which reached values up to 206 and 141 U/g protein respectively, possibly as the consequence of plastics degradation during maturation process. Implementation of Illumina sequencing indicated the predominance of Alphaproteobacteria, accompanied by members of Bacteroidetes, Betaproteobacteria and Chloroflexi. Paracoccus was the predominant genus identified, followed by representatives of the genera Bellilinea, Flavobacterium, Thauera and Truepera. Nitrosomonas was the major ammonia-oxidizing bacterium (AOB), while nitrite oxidation was mainly achieved by the uncultured nitrite-oxidizing bacterium (NOB) Candidatus Nitrotoga.

Fungal Extracellular Lipases from Coffee Plantation Environments for the Sustainable Management of Agro-Industrial Coffee Biomass

Coffee wastes have large amounts of by-products rich in phenolic compounds such as chlorogenic and caffeic acid, with potential applications for developing fine chemicals such as caffeic acid phenethyl ester (CAPE). A screening for microorganisms was undertaken in a coffee plantation environment to isolate native tropical species able to modify secondary metabolites present in this kind of biomass enzymatically. From the screening, 130 fungal strains could grow in lipase inducer media. Fungal strains were identified via ITS-based sequencing. Classification based on BLAST assigned 51 isolates to 12 different genera, including Absidia, Aspergillus, Cunninghamella, Fusarium, Metarhizium, Meyerozyma, Mucor, Neocosmospora, Papiliotrema, Penicillium, Rhizopus, and Trichoderma. DNA sequencing identified 14 putative extracellular lipases. According to the extracellular lipase activity, the most promising strain was identified as Fusarium sp. by DNA barcoding. Extracellular lipases from this strain exhibited maximal hydrolytic activity at a temperature of 45 °C, a pH of 7.00, and 200 ppm of NaCl, with an affinity towards substrates having carbon chain lengths of 8 or longer. Under these conditions, lipase instead of esterase activity is the main feature. The Km and Vmax values calculated using p-nitrophenyl palmitate (pNPP) as hydrolysis substrate were 0.003 mM and 299.8 μmol min−1 mg−1, respectively. Fusarium sp. lipases presented high stability during freeze–thawing, allowing the storage of enzyme solutions at −20 °C, but not as a lyophilized powder. According to our kinetic study, these lipases catalyzed CAPE hydrolysis, showing a progressive decrease in the concentration of the CAPE and a correspondent increase in the caffeic acid concentration as a product of this hydrolysis. Being able to carry out this type of reaction under mild conditions shows that Fusarium sp. lipases recognize CAPE as substrate and suggest CAPE synthesis (reverse reaction) and transformation can be engineered, using caffeic acid from coffee biomass, as a potential industrial application for these lipases.

Purification and Optimization of Extracellular Lipase from a Novel Strain Kocuria flava Y4.

The exogenous lipolytic activities of Kocuria sp. have been recognized earlier but the genus further contains many more unexplored strains. In this study, the extracellular lipase activity of Kocuria flava Y4 (GenBank accession no. MT773277), isolated from Dioscorea villosa during our previous study, was regulated by different physicochemical parameters, such as pH, temperature, shaking speed, and incubation time. For efficient immobilization of the extracellular lipase, 4% sodium alginate, 50 mL of 25 nM CaCl2.2H2O solution, and 15 min. Hardening time of gel beads in calcium chloride was used. For the first time, K. flava Y4 lipase was purified using ammonium sulphate precipitation followed by dialysis and DEAE-Sepharose anion exchange chromatography with Sepharose-6B gel filtration chromatography, yielding ∼15-fold purified lipase with a final yield of 96 U/mL. The SDS-PAGE of purified lipase displayed a single strong band, indicating a monomeric protein of 45 kDa. At a temperature of 35°C and pH 8, the purified lipase showed maximum hydrolytic activity. Using p-nitrophenyl acetate (p-NPA) as the hydrolysis substrate, the values of Km and Vmax derived from the Lineweaver–Burk plot were 4.625 mM and 125 mol/min−1mg−1, respectively.

Detection and diversity of the mannosylerythritol lipid (MEL) gene cluster and lipase A and B genes of Moesziomyces antarcticus isolated from terrestrial sites chronically contaminated with crude oil in Trinidad

BackgroundMannosylerythritol lipids (MELs) belong to the class of glycolipid biosurfactants and are produced by members of the Ustilago and Moesziomyces genera. Production of MELs is regulated by a biosynthetic gene cluster (MEL BGC). Extracellular lipase activity is also associated with MEL production. Most microbial glycolipid-producers are isolated from oil-contaminated environments. MEL-producing yeast that are capable of metabolizing crude oil are understudied, and there is very limited data on indigenous strains from tropical climates. Analysis of the MEL BGC and lipase genes in Trinidad M. antarcticus strains, using a gene-targeted approach, revealed a correlation between their intrinsic capability to degrade crude oil and their adaptation to survive in a chronically polluted terrestrial environment.ResultsM. antarcticus was isolated from naturally-occurring crude oil seeps and an asphaltic mud volcano in Trinidad; these are habitats that have not been previously reported for this species. Genus identification was confirmed by the large-subunit (LSU) and the small-subunit (SSU) sequence comparisons and species identification was confirmed by ITS sequence comparisons and phylogenetic inference. The essential genes (Emt1, Mac1, Mac2, Mmf1) of the MEL BGC were detected with gene-specific primers. Emt1p, Mac1p and Mmf1p sequence analyses confirmed that the Trinidad strains harboured novel synonymous amino acid (aa) substitutions and structural comparisons revealed different regions of disorder, specifically for the Emt1p sequence. Functionality of each protein sequence was confirmed through motif mining and mutation prediction. Phylogenetic relatedness was inferred for Emt1p, Mac1p and Mmf1p sequences. The Trinidad strains clustered with other M. antarcticus sequences, however, the representative Trinidad M. antarcticus sequences consistently formed a separate, highly supported branch for each protein. Similar phylogenetic placement was indicated for LipA and LipB nucleotide and protein sequences. The Trinidad strains also demonstrated lipolytic activity in culture, with an ability to utilize different carbon sources. Comparative evolution of MEL BGC and LipA gene suggested early and late duplication events, depending on the gene, followed by a number of speciation events within Ustilaginaceae. M. antarcticus and M. aphidis were separated from all other members of Ustilaginaceae and two gene homologues were detected, one for each species.ConclusionsSequence analyses was based on a novel gene-targeted approach to analyze the essential genes of the MEL BGC and LipA and LipB genes of M. antarcticus strains from Trinidad. The findings indicated that these strains accumulated nucleotide mutations to a threshold level that did not affect the function of specific proteins encoded by the MEL BGC and LipA and LipB genes. The biosurfactant and lipase enzymes secreted by these Trinidad M. antarcticus strains facilitated their survival in oil-contaminated terrestrial environments. These findings suggest that the Trinidad strains should be explored as promising candidates for the commercial production of MEL biosurfactants and lipase enzymes.

Global industrial enzymes market projected to reach worth $9.2 bn by 2027

A novel lipase gene (McLipB) was cloned from a thermophilic fungus Malbranchea cinnamomea and expressed in Pichia pastoris. The deduced amino acid sequence of the lipase (McLipB) shared the highest identity of 46% with the Candida rugosa lipase LIP4. The extracellular lipase activity of 4304 U/mL with protein concentration of 7.7 mg/mL was achieved in a 5-L fermentor. The optimal pH and temperature of McLipB were 7.5 and 40 °C, respectively. The lipase showed high specificity towards triglycerides with short and medium chain fatty acids, and had non-position specificity. McLipB hydrolyzed butter to produce mainly butyric acid, hexanoic acid and a small amount of octanoic acid and decanoic acid. Furthermore, it degraded more than 90% dipropyl phthalate, dibutyl phthalate and dihexyl phthalate to their corresponding monoalkyl phthalates. The properties of McLipB indicate that it has great application potential for production of lipolyzed milkfat flavor and biodegradation of phthalate esters.

A Novel Approach towards Removal of Lipid Barrier from Wool Fibers’ Surface Using Thermophilic Lipase

ABSTRACT The hydrophobic nature of wool caused by the surface lipid barrier impedes any wet processes of wool fibers. The conventional methods that utilize alkaline solutions for removing this lipid layer are deteriorative for the fiber. The use of enzymes in textile industry is one of the most rapidly growing fields in industrial enzymology because of eco-friendly and nontoxic characteristics. Therefore, this work is aimed at isolation of thermophilic strains from a soil sample in a hot region. The bacterial strain Bacillus aerius strain 24 K showed the maximum extracellular lipase activity (150 ± 1.03 U/ml) among the seven investigated strains. The locally produced thermophilic lipase enzyme was utilized in scouring as well as one-bath scouring and dyeing of wool. The effect of enzyme concentration, treatment temperature, and time on the dyeability of wool fibers with acid, reactive, and basic dyes was investigated. The results of this investigation proved that the extracted thermophilic lipase enzyme is an appropriate candidate for enhancing the dyeability of wool prior to the dyeing process. One-bath scouring and dyeing of wool were successfully conducted to save energy and water. Unlike the conventionally scoured wool, bio-scoured wool exhibits similar chemical composition and solubility parameters to those of untreated wool.

Two polyurethanases PueA and PueB are major extracellular lipases partly secreted by the mediation of their cognate ABC exporter AprDEF in Pseudomonas protegens Pf‐5

Two polyurethanases PueA and PueB from Pseudomonas protegens Pf-5 have been reported to have hydrolytic activity against synthetic p-nitrophenyl palmitate of lipase substrate, and PueA may play a more effective role in this activity. However, it is still unknown whether PueA and PueB play similar parts in the lipase activity against natural acylglycerols and achieve the extracellular secretion via their cognate ABC exporter AprDEF. In this study, we investigated these questions through the construction of four markerless deletion mutants in Pf5139 (Δupp derivative of Pf-5), two heterologous co-expression strains and their three control strains in lipase-free Escherichia coli BL21(DE3), and detected their lipase activities by the tributyrin plate assay and the liquid culture assay. The results showed that PueA and PueB, classified as subfamily I.3 lipases, are major extracellular lipases involved in the uptake of oil in Pf-5, and PueA plays a leading role in extracellular lipase activity. In addition, the extracellular secretion of PueA and PueB can be partly mediated via AprDEF in Pf-5 and BL21(DE3). Finally, PueA and PueB are also able to achieve the extracellular secretion without the assistance of AprDEF in Pf-5 and BL21(DE3).

Heterologous expression and biochemical characterization of a cold-active lipase from Rhizopus microsporus suitable for oleate synthesis and bread making.

Cold-active lipases which show high specific activity at low temperatures are attractive in industrial applications in terms of product stability and energy saving. We aimed to identify novel cold-active lipase suitable for oleates synthesis and bread making. A novel lipase gene (RmLipA) from Rhizopus microsporus was cloned and heterologously expressed in Pichia pastoris. The encoding sequence displayed 75% identity to the lipase from R. niveus. The highest extracellular lipase activity of 7931 U/mL was achieved in a 5-L fermentation. The recombinant enzyme (RmLipA) was optimally active at pH 8.0 and 20-25°C, respectively, and stable over a wide pH range of 2.0-11.0. The enzyme was a cold-active lipase, exhibiting > 80% of its maximal activity at 0°C. RmLipA was a sn-1,3 regioselective lipase, and preferred to hydrolyze pNP esters and triglycerides with relatively long chain fatty acids. RmLipA synthesized various oleates using oleic acid and different alcohols as substrates (> 95%). Moreover, it significantly improved the quality of bread by increasing its specific volume (21.7%) and decreasing its crumb firmness (28.6%). A novel cold-active lipase gene from R. microsporus was identified, and its application potentials were evaluated. RmLipA should be a potential candidate in oleates synthesis and bread making industries.

Use of different kinds of wastes for lipase production: Inductive effect of waste cooking oil on activity

This study aimed to evaluate the effects of by-product molasses and waste orange peels (WOP) as the nutrient medium on lipase production with Rhizopus arrhizus NRRL 2286 and investigate the inductive effect of waste cooking oil (WCO) on activity. The results showed that the highest specific growth rate and the maximum biomass concentration were obtained 0.424 h-1 and 3.83g dry weight/L for 70g/L WOP, respectively. Additionally, the highest intracellular and extracellular lipase activities were attained in 50g/L WOP as 400 U/g and 13.2 U/mL, correspondingly. Next, the inductive effects of WCO and olive oil were tested in lipase production. Intracellular lipase activity enhanced and reached a maximum of 520 U/g in the presence of 10g/L WCO with an increase of 30% compared to the one without WCO. However, both inducers caused a diminish in extracellular lipase hydrolysis activity. Overall, this study reveals that the lipase production of R.arrhizus can be accomplished using WOP and WCO with the optimum conditions.