Bacterial Lipase Research Articles

Characterization of a novel lipase from Pseudomonas aeruginosa

Abstract Lipases cleaving oils into fatty acids and glycerol are of great interest for the use in increasing the efficiency of fuels. In this work, a novel lipase from Pseudomonas aeruginosa, P. aeruginosa A12, was isolated by ion-exchange and hydrophobic chromatography. The purity of lipase was shown by electrophoresis and its molecular weight was estimated to be ~ 31.6 kDa. The whole amino acid sequence was analyzed by an LC-MS/MS method. Temperature- and pH-dependent optimum of the enzyme compiled 30 °C and 7.5, respectively. The obtained enzyme exhibited 79 % similarity of amino acid sequence to a lipase isolated from the same strain of P. aeruginosa. Thus, the novel lipase was determined to belong to I.1 subfamily of bacterial true lipases. Three dimensional structure of the isolated lipase isoform was modeled based on obtained sequences. Amino acids forming the catalytic domain were shown in the model. Lid domain is suggested to be in the open conformation. These results provide a potential alternative for enzymatic digestion of fuel oils and serve for the development of fundamental knowledge of lipase activity.

An Overview of Bacterial Lipases and their Enormous Applications

An Overview of Bacterial Lipases and their Enormous Applications

Three Dimensional Structural Modelling of Lipase Encoding Gene from Soil Bacterium <i>Alcaligenes</i> sp. JG3 Using Automated Protein Homology Analysis

Bacterial lipases have significant potential to be used as the biocatalyst for many chemical reactions. In this study, a novel gene encoding lipase was isolated from an Alcaligenes sp. JG3. A pair of designed primer has successfully isolated 1 kb (LipJG3) that shares 98% identity towards lipase from Alcaligenes faecalis during sequence analysis. By using in silico tools, LipJG3 was related to the transporter protein sequences. Three highly conserved regions consisting of EASGSKT, VILLD, and LSGGQQQRVAIA were found. These regions were known as ATP-binding signature at Walker-A and Walker-B motifs and the S signature of ABC transporter family respectively. In addition, the 3-D structure of LipJG3 has been suggested but the role of the catalytic triad residues have been not fully understood.

N2 Gas-Flushing Prevents Bacteria-Promoted Lipolysis and Proteolysis and Alleviates Auto-Oxidation in Bovine Raw Milk During Cold-Storage

Worldwide, tremendous amounts of food, including milk and dairy products, are lost or wasted. Although refrigeration permits longer storage of raw milk, low temperatures still do not prevent the action of other abiotic and biotic factors that promote biochemical, chemical, microbial, sensorial and nutritional changes in raw milk, which also affect milk-derived dairy products. Treatment by N2 gas flushing showed great potential in preserving the microbiological quality of raw milk during storage at low and milder temperatures. Here, we examined the impact of cold storage (at 6°C up to 7 days), on the ascorbic acid content, extent of auto-oxidation, and on levels of lipolysis and proteolysis along with bacterial growth, in three raw milk samples in the presence or absence of N2. When N2 gas was applied, lower levels of lipolysis and proteolysis were found to coincide with the detection of lower numbers of bacterial lipase and protease producers in raw milk. Furthermore, lower auto-oxidation was detected in N2-treated samples than in only cold stored controls. By demonstrating that key components of milk were better preserved during cold storage, the present study further highlights the advantages of the N2 flushing treatment in terms of preserving the quality and safety of raw milk and its derived dairy products.

Gene structure and comparative study of two different plastic-degrading esterases from Roseateles depolymerans strain TB-87

The structures of two genes from Roseateles depolymerans strain TB-87 encoding the esterases Est-H and Est-L, which can degrade aliphatic-aromatic copolyesters, were annotated. Two open reading frames (ORFs) consisting of 1083 bp and 870 bp nucleotides, corresponding to est-H and est-L, encoding enzymes of 290 and 289 amino acids, respectively, were predicted. In addition, another ORF consisting of 735 bp encoding a chaperone-like protein (Est-Ch) of 244 amino acids was identified in the intergenic region of est-H and est-L. The presence of a promoter region upstream of est-H and the absence of a terminator region downstream of the ORF and vice versa for est-Ch, suggests that est-H and est-Ch are polycistronically expressed. A homology search for Est-H and Est-L revealed homology with plastic degrading enzymes, such as esterases and cutinases, while Est-Ch showed homology with a bacterial lipase chaperone. As consensus lipase sequences (-Gly-His-Ser-Met-Gly-) were observed in these enzymes, Est-H and Est-L were hypothesized to be hydrolases with serine (Ser) in their active center. Three-dimensional structures of Est-H and Est-L without their putative signal sequences were constructed using Est119 from Thermobifida alba strain AHK119 as the template; the structures and positions of the catalytic triad (Ser, Asp, His) active centers were similar to those of Est119. A mutant strain in which the annotated esterase-encoding genes were disrupted using a homologous recombination method lost the ability to form a clear zone on poly(butylene succinate-co-adipate (PBSA) emulsion-overlaid nutrient agar plates. Characterization of the esterases from strain TB-87 could contribute to the development of novel biodegradable plastics with unique properties such as recyclable monomers.

Identification of new members of alkaliphilic lipases in archaea and metagenome database using reconstruction of ancestral sequences

The application of bioinformatics in lipase research has the potential to discover robust members from different genomic/metagenomic databses. In this study, we explored the diversity and distribution of alkaliphilic lipases in archaea domain and metagenome data sets through phylogenetic survey. Reconstructed ancestral sequence of alkaphilic lipase was used to search the homologous alkaliphilic lipases among the archaea and metagenome public databases. Our investigation revealed a total 21 unique sequences of new alkaliphilic lipases in the archaeal and environmental metagenomic protein databases that shared significant sequence similarity to the bacterial alkaliphilic lipases. Most of the identified new members of alkaliphilic lipases belong to class Haloarchaea. The searched list of homologs also comprised of one characterized lipase from alkalohyperthermophilic Archaeoglobus fulgidus. All the newly identified alkaliphilic lipase members showed conserved pentapeptide [X-His-Ser-X-Gly] motif, a key feature of lipase family. Furthermore, detailed analysis of all these new sequences showed homology either with thermostable or alkalophilic lipases. The reconstructed ancestral sequence-based searches increased the sensitivity and efficacies to detect remotely homologous sequences. We hypothesize that this study can enrich our current knowledge on lipases in designing more potential thermo-alkaliphilic lipases for industrial applications.

Application of Bacterial Thermostable Lipolytic Enzymes in the Modern Biotechnological Processes: A Review

Review focuses on the modern applications of bacterial lipolytic enzymes in biotechnology and covers the scope of their properties including their activity and functional stability at different temperatures, pH, substrate specificity, and activity in the presence of different chemicals. The recent data on the production of genetically engineered strains producing the bacterial lipolytic enzymes and approaches to improving their productivity are presented. The applications of bacterial lipases in biotechnological processes used in the production of biofuel, chemicals and detergents, in the food industry, and in wastewater treatment are considered.

Shiunko and Chuoko, topical Kampo medicines, inhibit the expression of gehA encoding the extracellular lipase in Cutibacterium acnes.

Antimicrobial agents have been used for eradication of Cutibacterium (formerly Propionibacterium) acnes that is an exacerbation factor of the skin disease acne vulgaris. However, the use of antibiotics is associated with an increased risk of promoting the emergence of resistant bacteria and leading to skin dysbiosis. Traditional Japanese Kampo medicines, such as Keigairengyoto, are used to treat acne. However, there is incomplete understanding regarding their functional mechanism in treatment of acne. In this study, we examined the antimicrobial and anti-lipase activity of the Kampo medicines used empirically for acne treatment. Three oral medicines, Keigairengyoto, Seijoboufuto and Jumihaidokuto, were found to inhibit the growth of C.acnes and decrease the lipase activity. Especially, Keigairengyoto caused remarkable decrease of bacterial lipase activity. Furthermore, topical medicines such as Shiunko and Chuoko significantly decreased the lipase activity in a dose-dependent manner, without inhibiting C.acnes growth. The topical medicines were found to inhibit the expression of gehA, which codes for extracellular lipase. Our results indicate that Shiunko and Chuoko have potential as effective acne therapeutic agents, especially because they do not promote the emergence of antimicrobial-resistant bacteria and skin dysbiosis.

Bacterial lipolysis of immune-activating ligands promotes evasion of innate defenses

Commensal and pathogenic bacteria hydrolyze host lipid substrates with secreted lipases and phospholipases for nutrient acquisition, colonization, and infection. Bacterial lipase activity on mammalian lipids and phospholipids can promote release of free fatty acids from lipid stores, detoxify antimicrobial lipids, and facilitate membrane dissolution. The gram-positive bacterium Staphylococcus aureus secretes at least two lipases, Sal1 and glycerol ester hydrolase (Geh), with specificities for short- and long-chain fatty acids, respectively, each with roles in the hydrolysis of environmental lipids. In a recent study from our group, we made the unexpected observation that Geh released by S. aureus inhibits activation of innate immune cells. Herein, we investigated the possibility that S. aureus lipases interface with the host immune system to blunt innate immune recognition of the microbe. We found that the Geh lipase, but not other S. aureus lipases, prevents activation of innate cells in culture. Mutation of geh leads to enhancement of proinflammatory cytokine production during infection, increased innate immune activity, and improved clearance of the bacterium in infected tissue. These in vitro and in vivo effects on innate immunity were not due to direct functions of the lipase on mammalian cells, but rather a result of inactivation of S. aureus lipoproteins, a major pathogen-associated molecular pattern (PAMP) of extracellular gram-positive bacteria, via ester hydrolysis. Altogether, these studies provide insight into an adaptive trait that masks microbial recognition by innate immune cells through targeted inactivation of a broadly conserved PAMP.

Shift in Bacillus sp. JR3 esterase LipJ activity profile after addition of essential residues from family I.5 thermophilic lipases

We recently described LipJ, a mesophilic esterase clustering in a specific clade of bacterial lipase family I.5, bearing the same structure but several distinct sequence motifs. Two mutagenesis approaches were used here to modify LipJ by addition of common features of family 1.5 lipases, searching for changes on the activity profile. Iterative saturation mutagenesis of a residue possibly involved in Zn2+ coordination produced a LipJ variant –H110N– displaying a remarkable shift in substrate specificity, possibly boosted by changes in number and distance of relevant polar interactions for Zn2+ coordination. Also, variants E27 A, G136 A and M139Q were obtained by site directed mutagenesis as single, double or triple mutants, showing a general shift in specificity towards higher temperature and longer chain-length substrates, all of them displaying higher activity than LipJ on C7 substrates. The results obtained suggest that the N-terminal region of LipJ may indeed contribute to substrate accommodation, whereas mutations at the pentapeptide displayed maximum activity at 50 °C, with the triple mutant showing activity even at 80 °C, greatly enhanced by Mn2+. 3D modelling and molecular docking analysis of selected mutants provided clues for a better accommodation of medium chain-length substrates, and for a modified Zn2+ environment, in agreement with the substrate and temperature profile shifts observed.

Synergistic antibacterial effects of curcumin modified silver nanoparticles through ROS-mediated pathways

Due to their remarkable antibacterial properties, silver nanoparticles (Ag NPs) and curcumin (CCM) have been widely used in the antimicrobial field. In our study, we have fabricated the uniform and stable silver/curcumin composite nanoparticles by a facile ultrasound treatment process and the synergistic antibacterial activity were evaluated. The curcumin not only played a role of reducing agent but also acted as a capping agent. The antibacterial effects of silver/curcumin (cAgNPs) were studied by measuring the growth curve and surface plate assay based on the E. coli and B. subtilis, which showed concentration dependent bacteriostatic and bactericidal effects of cAgNPs. The presence of CCM enhance the binding of Ag to bacterial membrane and Ag+ release in comparison to that without CCM, so that creating a temporary and local high Ag+ concentration near the surface of the bacterium, meanwhile, generation of more reactive oxygen species, lead to membrane damage, bacterial lipases and induce leakage of intracellular contents followed by bacterial death that lead to growth inhibition of the bacteria. The antibacterial effects were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the effect which were further found to decrease by introducing antioxidant N-acetyl-l-cysteine (NAC) act as a reactive oxygen species (ROS) scavenging agent. These initial data suggest that cAgNPs have a highly antibacterial efficient and might have potential to be developed as an effective antimicrobial nanomaterial.

Characterization and mutation anaylsis of a cold-active bacterial hormone-sensitive lipase from Salinisphaera sp. P7-4

In mammals, hormone sensitive lipase (EC 3.1.1.79, HSL) catalyzes the hydrolysis of triacylglycerols as well as the modifications of a broad range of hydrophobic substrates containing ester linkages. HSLs are composed of an N-terminal ligand-binding domain and a C-terminal catalytic domain. Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the C-terminal domain of mammalian HSLs, have a catalytic triad composed of Ser, His, and Asp. Here, a novel cold-active hormone-sensitive lipase (SaHSL) from Salinisphaera sp. P7-4 was identified, functionally characterized, and subjected to site-directed mutations. The enzymatic properties of SaHSL were investigated using several biochemical and biophysical methods. Interestingly, SaHSL exhibited the ability to act on a broad range of substrates including glyceryl tributyrate and glucose pentaacetate. Homology modeling and site-directed mutagenesis indicated that hydrophobic residues (Leu156, Phe164, and Val204) around the substrate-binding pocket were involved in substrate recognition. In addition, highly conserved amino acids (Glu201, Arg207, Leu208, and Asp227) in the regulatory regions were found to be responsible for substrate specificity, thermostability, and enantioselectivity. In summary, this work provides new insights into the understanding of the C-terminal domain of HSL family and evidence that SaHSL can be used in a wide range of industrial applications.

Parallel Computations in the Development of Thermostable Lipase Mutants

The advanced high performance computing methods are used to study the stability and conformational dynamics of the bacterial enzyme lipase LipA, its mutants, and the close homologous enzyme CLE whose substrate is polylactic acid-based plastics. From the analysis of the GPU molecular dynamics of native lipases and their mutants the amino acid residues whose point substitutions can markedly improve the thermostability of the enzymes under study without deteriorating their activity are determined.

Purification, Characterization of Alkaline Cold Active Lipase from Acinetobacter radioresistens PR8 and Development of a New Zymography Method for Lipase Detection.

Bacterial lipases find so many industrial applications. Therefore, new source of lipase suitable for industrial conditions is always required. Lipase zymography methods use costly chromogenic substrates and indicator dyes and are few in numbers. The objectives of this work include lipase purification and its characterization from Acinetobacter radioresiens PR8 and development of new zymography method for lipase detection. The lipase was purified using conventional method and cation exchange chromatography and it was characterized biochemically and analytically. Based on these characterization new in-gel lipase zymography method was developed. In this present work, an alkalophilic lipase producing bacterium was isolated from soil; screened for extracellular lipase activity and identified to be Acinetobacter radioresistens PR8 (Genbank accession ID: MF073322). Enzyme production kinetics showed maximum production (4.16 U/ml at pH 9) of enzyme after 72 h. The lipase activity was found to be highest in olive oil (1% v/v; 8.1 U/ml). Low molecular weight (27 kDa) alkaline (pH 9) cold active (20 °C) lipase was purified from Acinetobacter radioresistens PR8. Lipase was characterized using PMF, FT-IR and its high conformational stability (Transition temperature: 122.3 °C) was attributed from its DSC spectrum. The importance of magnesium and sodium ions for enhancing lipase activity was obtained from flux balance analysis. Based on the lipase activating role of Mn2+ and Na+ ions, optimum temperature, pH with no chromogenic substrates and indicator dyes, a new in gel zymography method for lipase detection was developed.

Application of Thermostable Lipolytic Bacterial Enzymes for Modern Biotechnological Processes: Review

In the review paper, the modern investigations on the application of thermostable lipolytic bacterial enzymes for biotechnology are discussed, the properties of these enzymes discussed including their activity and functional stability at various temperatures, pH in organic solvents, as well as the substrate specificity and activity in the presence of various chemical compounds. The paper contains data on the development of recombinant producers of lipolytic bacterial enzymes and on approaches to improving their productivity. The application of the bacterial lipases for biotechnological processes of synthesis of biofuel, various chemicals and detergents, for food industry and wastewater treatment is considered.

Understanding domain movements and interactions of Pseudomonas aeruginosa lipase with lipid molecule tristearoyl glycerol: A molecular dynamics approach

Lipases are biocatalysts which exhibit optimal activity at the aqueous-lipid interface. Molecular Dynamics (MD) Simulation studies on lipases have revealed the structural changes occurring in the enzyme, at the loop-helix-loop, often designated as the “lid”, which is responsible for its interfacial activation. In recent years, MD simulation of lipases at molecular level have been studied in detail, whereas very few studies are carried over on its interaction with lipid molecules. Hence, in the current study we have investigated molecular interaction of bacterial lipase (Pseudomonas aeruginosa lipase, PAL) with a lipid molecule (tristearoyl glycerol, TGL). This provides an insight into the interfacial activation of the enzyme. The lipid molecule was placed near the lids of the enzyme and MD simulations were performed for 100 ns to understand the nature and site of the interaction. The results clearly indicate that, the presence of a lipid molecule near the lids affects the motion of the enzyme through changes in conformation. Lipid molecule near the lids reduces the movements of both lids, and the TGL molecule was observed moving towards the active site. The movement of the lids, surface accessibility and the domain movements of PAL are discussed and the results provide valuable insight in to the role played by the two lids in the interfacial activation of PAL with TGL.

Nanocarriers with conjugated antimicrobials to eradicate pathogenic biofilms evaluated in murine in vivo and human ex vivo infection models

Conventional antimicrobials are becoming increasingly ineffective for treating bacterial infection due to the emergence of multi-drug resistant (MDR) pathogens. In addition, the biofilm-mode-of-growth of infecting bacteria impedes antimicrobial penetration in biofilms. Here, we report on poly(ethylene)glycol-poly(β-amino esters) (PEG-PAE) micelles with conjugated antimicrobials, that can uniquely penetrate biofilms, target themselves to bacterial cell surfaces once inside the low-pH environment of a biofilm and release conjugated antimicrobials through degradation of their ester-linkage with PAE by bacterial lipases. In vitro, PEG-PAE micelles with conjugated Triclosan (PEG-PAE-Triclosan) yielded no inadvertent leakage of their antimicrobial cargo and better killing of MDR Staphylococcus aureus, Escherichia coli and oral streptococcal biofilms than Triclosan in solution. In mice, PEG-PAE-Triclosan micelles with conjugated Triclosan yielded better eradication efficacy towards a MDR S. aureus-infection compared with Triclosan in solution and Triclosan-loaded micelles at equal Triclosan-equivalent concentrations. Ex vivo exposure of multi-species oral biofilms collected from orthodontic patients to PEG-PAE-Triclosan micelles, demonstrated effective bacterial killing at 30–40 fold lower Triclosan-equivalent concentrations than achieved by Triclosan in solution. Importantly, Streptococcus mutans, the main causative organism of dental caries, was preferentially killed by PEG-PAE-Triclosan micelles. Thus PEG-PAE-Triclosan micelles present a promising addendum to the decreasing armamentarium available to combat infection in diverse sites of the body. Statement of SignificancepH-adaptive polymeric micelles with conjugated antimicrobials can efficiently eradicate infectious biofilms from diverse body sites in mice and men. An antimicrobial was conjugated through an ester-linkage to a poly(ethylene glycol) (PEG)/poly(β-amino ester) block copolymer to create micellar nanocarriers. Stable micelle structures were formed by the hydrophobic poly(β-amino ester) inner core and a hydrophilic PEG outer shell. Thus formed PEG-PAE-Triclosan micelles do not lose their antimicrobial cargo underway to an infection site through the blood circulation, but penetrate and accumulate in biofilms to release antimicrobials once inside a biofilm through degradation of its ester-linkage by bacterial lipases, to kill biofilm-embedded bacteria at lower antimicrobial concentrations than when applied in solution. PEG-PAE-Triclosan micelles effectively eradicate biofilms of multi-drug-resistant pathogens and oral bacteria, most notably highly cariogenic Streptococcus mutans, in mice and men respectively, and possess excellent clinical translation possibilities.

Phytochemical Profile and Anti-lipase Activity of Balkan Endemic Jurinea tzar-ferdinandii

Phytochemical study of the Balkan endemic J. tzar-ferdinandii Davidov allowed the identification of twenty-two components. β-Amyrin, lupeol, taraxasterol and Ψ-taraxasterol and their 3- O-acetates and 3- O-palmitates were identified by GC/MS. Six closely related sesquiterpene lactones and four flavones were isolated from the chloroform extract using column chromatography, and their structural identification was performed by spectral analyses. All isolated compounds are described for the first time in Jurinea species. The inhibitory potential of the total chloroform extract, fractions containing triterpenes, flavonoids and sesquiterpene lactones as well as individual compounds against a bacterial lipase from Candida rugosa (CRL) and a lipase from porcine pancrease (PPL) was evaluated. The half maximal inhibitory constants (IC50) for the two enzymes were in the range of 28–150 μg/mL. The strongest inhibitory activity was found for the total chloroform extract (29±1 μg/mL and 39±1 μg/mL for CRL and PPL, respectively.). Among the individual compounds, onopordopicrin was found to be the most potent inhibitor for CRL and PPL with IC50 values of 32±1 μg/mL and 36±1 μg/mL, respectively.

Statistical optimization for lipase production from solid waste of vegetable oil industry

ABSTRACTThe production of biofuel using thermostable bacterial lipase from hot spring bacteria out of low-cost agricultural residue olive oil cake is reported in the present paper. Using a lipase enzyme from Bacillus licheniformis, a 66.5% yield of methyl esters was obtained. Optimum parameters were determined, with maximum production of lipase at a pH of 8.2, temperature 50.8°C, moisture content of 55.7%, and biosurfactant content of 1.693 mg. The contour plots and 3D surface responses depict the significant interaction of pH and moisture content with biosurfactant during lipase production. Chromatographic analysis of the lipase transesterification product was methyl esters, from kitchen waste oil under optimized conditions, generated methyl palmitate, methyl stearate, methyl oleate, and methyl linoleate.

A new cold-adapted, alkali-stable and highly salt-tolerant esterase from Bacillus licheniformis

Bacterial esterases and lipases, especially extremozymes attract increasing attention due to various advantages both in good properties and wide applications. In the present study, a cold-adapted, alkali-stable and highly salt-tolerant esterase (Est700) was cloned from Bacillus licheniformis, expressed and purified with a molecular mass of 25 kDa. The optimal temperature of Est700 was 30 °C, with 35% maximal activity retaining at 0 °C. Its optimal pH was 8.0 and showed high stability at pH 5.0–11.0. Noticeably, Est700 was highly activated by 3.5 M NaCl and the extent of this activation is much stronger than that of currently reported halophilic ones. It was also stable in 5 M NaCl with no activity loss. High salt concentrations changed the secondary structure and folding properties of Est700 with formation of more α-helix and less β-sheet domains. With salt incubation, its melting temperature was estimated to be 57.2 °C, which is 12.8 °C higher than that of native one. Interestingly, Est700 lacks the acidic surface that is considered essential for enzyme stability at high salinity. However, it has a mainly positive surface electrostatic potential, which is probably different from most reported halotolerant esterases. These multiple properties make Est700 a valuable candidate in both basic research and industrial applications.