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Background: Fermented katsuobushi, a traditional Japanese seasoning, is produced from skipjack tuna through smoking, drying and fermentation by xerophilic Aspergillus molds, primarily Aspergillus chevalieri and Aspergillus pseudoglaucus. In this study, we characterized lipolytic enzymes (cLip_1 to cLip_5 and pLip_1 to pLip_3) to clarify their roles in lipid hydrolysis during katsuobushi production under low water activity., Results: The enzymes showed significant diversity in their activity, stability and substrate specificity, and in the hydrolysis profiles of their reactions with fish oil. Phylogenetic analyses revealed that cLip_5 showed a high identity with pLip_2 (94%) and these enzymes formed a phylogenetic cluster with filamentous fungal lipases. Purified recombinant enzymes (rcLip_1, rcLip_2, rcLip_4 and rcLip_5) and wild-type enzymes (cLip_3 and pLip_3) showed varying substrate preferences toward p-nitrophenyl esters. The addition of glycerol to reduce the water activity in the reaction mixture led to increased activities of rcLip_1 and rcLip_4, but it did not affect the activity of the other three enzymes. Among the tested six enzymes, cLip_5 showed the highest hydrolytic activity toward fish oil. The cLip_5 and pLip_2 gene transcript levels were moderately high in strains MK86 and MK88, respectively., Conclusion: cLip_5 and its homolog pLip_2 were identified as the most promising enzymes for katsuobushi fermentation, because of their high hydrolytic activities toward fish oil and adaptability to low water activity conditions. These findings support the selection of optimal Aspergillus strains as starter cultures to potentially shorten the fermentation time and improve the quality and shelf life of katsuobushi. © 2025 Society of Chemical Industry., (© 2025 Society of Chemical Industry.)

A lipolytic enzyme was isolated from seeds of Nigella sativa after pressing the oil to develop a waste-free technology. An enzyme with a maximum lipolytic activity of 150,000 U/g was obtained by selecting the optimum temperature (37°C) and pH (10.5). A comparative study of the lipolytic enzyme and sebelipase alfa in the Tween hyperlipidemia model in rats showed that the former decreased the concentration of total cholesterol by 19.6%, which indicated the expediency of using this enzyme to improve absorption processes in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

The present study investigated high-yield monoacylglycerol (MAG) synthesis by bacterial lipolytic enzymes in a solvent-free two-phase system. Esterification by monoacylglycerol lipase from Bacillus sp. H-257 (H257) required a high glycerol/fatty acid molar ratio for efficient MAG synthesis. Screening of H257 homologues revealed that carboxylesterase derived from Geobacillus thermodenitrificans , EstGtA2, exhibited a higher esterification rate than H257. Moreover, neutralizing the pH of the acidic reaction solution by adding potassium hydroxide (KOH) solution further increased the esterification rate. The esterification rate by EstGtA2 reached 75% under conditions of equivalent molar amounts of glycerol and fatty acid, and the MAG rate (MAG/total glyceride) was 97%. The neutralized pH of the reaction solution likely affected the thermal stability of EstGtA2 during the esterification reaction. Screening for thermal-tolerant variants revealed that the EstGtA2S26I variant was stable at 75 °C for 30 min, a condition under which wild-type EstGtA2 was completely inactivated. The esterification rate by the EstGtA2S26I variant reached 90%, and the MAG rate was 96%. The addition of alkali and the use of a thermal-tolerant enzyme were important for obtaining high-yield MAG in a solvent-free two-phase system utilizing EstGtA2. [ABSTRACT FROM AUTHOR]

Plastic films are widely used in current agricultural practices; however, most mulch films used are discarded and buried in the land after harvest, having adverse environmental impacts. To solve this environmental problem, the demand for biodegradable mulch has been increasing in recent years. Polybutylene succinate-co-adipate (PBSA) is a biodegradable polymer with good ductility and can be used for packaging and mulching. In this study, we isolated two elite fungal strains for PBSA degradation from farmlands, i.e., Aspergillus fumigatus L30 and Aspergillus terreus HC, and the latter showed better degradation ability than the former. It is noteworthy that biodegradation of PBSA by A. terreus is reported for the first time, which revealed unique characteristics. In the soil burial test, even the soil with relatively poor degradation ability could be improved by the addition of elite fungal mycelia. In substrate specificity analyses of soil samples, PBSA could induce the synthesis of lipolytic enzymes of indigenous microbes to degrade substrates with medium and long carbon chains in soil. Furthermore, PBSA residues or fungal mycelia supplementation in soils had no adverse effect on the seed germination rate, seedling growth, or mature plant weight of the test green leafy vegetable. Taken together, the results of this study not only advance our understanding of the biodegradation of PBSA films by filamentous fungi but also provide insight into improving the efficiency of biodegradation in soil environments. [ABSTRACT FROM AUTHOR]

Enzymes are pervasive substances created by living cells that transform a specific substrate into a product, either alone or in combination with a prosthetic group. Lipases are a class of enzymes with a wide range of uses in the food, chemical, and pharmaceutical industries, as well as a growing interest in the treatment of lipid-rich waste from the oil sector. The goal of this work was to isolate, describe, and select lipolytic bacteria from oil industry waste that generate lipase, as well as to investigate the enzymatic kinetics acquired using the shake flask fermentation method. The results shown that it is possible to identify lipase-producing bacteria, particularly Bacillus subtilis. With six factors such as pH, temperature, minimal mineral medium, soya bean oil, glucose, and sodium nitrate, mostly gram positive bacteria showed the highest enzymatic reaction. Temperature was the most positively influenced the enzymatic activity. The lipolytic enzyme extracted from oil industry waste was found to have potential for reducing environmental consequences. [ABSTRACT FROM AUTHOR]

A novel esterase, EstCS3, was isolated from a metagenomic library constructed from a compost. The EstCS3, which consists of 409 amino acids with an anticipated molecular mass of 44 kDa, showed high amino acid sequence identities to predicted esterases, serine hydrolases and β-lactamases from uncultured and cultured bacteria. Phylogenetic analysis suggested that EstCS3 belongs to family VIII of lipolytic enzymes. EstCS3 had catalytic Ser78 residue in the consensus tetrapeptide motif SXXK, which is characteristic of family VIII esterases. Two conserved YXX and W(H or K)XG motifs in an oxyanion hole of family VIII esterases were also present in EstCS3. EstCS3 demonstrated the highest activity toward p -nitrophenyl butyrate (C4) and was stable up to 70 °C with optimal activity at 55 °C. EstCS3 had optimal activity at pH 8 and maintained its stability within pH range of 7–10. EstCS3 had over 70% activity in the presence of 20% (v/v) methanol and DMSO and hydrolyzed sterically hindered tertiary alcohol esters of t -butyl acetate and linalyl acetate. EstCS3 hydrolyzed ampicillin, cephalothin and cefepime. The properties of EstCS3, including moderate thermostability, stability against organic solvents and activity toward esters of tertiary alcohols, indicated that it has the potential to be used in industrial applications. • A novel esterase was isolated from a compost metagenomic library. • The novel esterase belonged to the family VIII family of lipolytic enzyme. • The esterase was stable up to 70 °C with an optimal activity at 55 °C. • The esterase hydrolyzed two tertiary alcohol esters. • The esterase showed β-lactam hydrolyzing activity. [ABSTRACT FROM AUTHOR]

BACKGROUND To initiate insect infection, entomopathogenic fungi produce diverse cuticle‐degrading enzymes. Of those, lipolytic enzymes participate in epicuticular lipid hydrolysis and thus facilitate fungal penetration through the outermost cuticular barrier of the insect host. The Far/CTF1‐type zinc finger transcription factors play an important role in the regulation of lipolytic activity and fungal pathogenicity in plant pathogens but remain functionally unknown in fungal insect pathogens. RESULTS: Two Far/CTF1‐type transcription factor Bbctf1α and Bbctf1β, which are essential for differential expression of genes involved in the fungal lipid degradation, were identified and functionally characterized in a fungal entomopathogen Beauveria bassiana. Disruption of each gene led to drastic losses of extracellular lipolytic activities under lipidic substrate‐inducing conditions, followed by remarkable phenotypic defects associated with the fungal biocontrol potential. These defects mainly included severe impairments of mycelial growth and conidium formation, and drastic losses of tolerance to the stresses of oxidation and cell wall perturbation during colony growth under either normal or induction conditions. Bioassays showed that the virulence of each disruption mutant on the greater wax moth was remarkably attenuated in topical immersion. However, there was no significant difference in intrahemolymph injection when the cuticle penetration process was bypassed. CONCLUSIONS: Bbctf1α and Bbctf1β are multifunctional transcription factors that play vital roles in the regulation of fungal lipid utilization and contribute to the vegetative growth, sporulation capacity, environmental fitness and pest control potential in B. bassiana. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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A novel esterase, EstCS1, was isolated from a compost metagenomics library. The EstCS1 protein, which consists of 309 amino acid residues with an anticipated molecular mass of 34 kDa, showed high amino acid sequence identities to predicted esterases and alpha/beta hydrolases (59%) from some cultured bacteria and to predicted lipases/esterases from uncultured bacteria. The phylogenetic analysis suggested that the EstCS1 belongs to the hormone-sensitive lipase family of lipolytic enzyme classification and contains a catalytic triad including Ser155–Asp255–His285. The Ser155 residue of the catalytic triad in the EstCS1 was located in the consensus active-site motif, GXSXG. Besides, a conserved HGGG motif placed in an oxyanion hole of the hormone-sensitive lipase family was discovered, too. The EstCS1 demonstrated the highest activity toward p-nitrophenyl propionate (C3) and caproate (C6) and was normally stable up to 60°C with optimal activity at 50°C. In addition, an optimal activity was observed at pH 8, and the EstCS1 possessed its stability within the pH range between 5 and 10. Interestingly, EstCS1 had an outstanding stability in up to 30% (v/v) organic solvents and activity over 50% in the presence of 50% (v/v) acetone, ethanol, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide. The EstCS1 hydrolyzed sterically hindered tertiary alcohol esters of t-butyl acetate and linalyl acetate. Considering the properties, such as the moderate thermostability, stability against organic solvents, and activity toward esters of tertiary alcohols, the EstCS1 will be worthwhile to be used for organic synthesis and related industrial applications.

A novel esterase, EstCS1, was isolated from a compost metagenomics library. The EstCS1 protein, which consists of 309 amino acid residues with an anticipated molecular mass of 34 kDa, showed high amino acid sequence identities to predicted esterases and alpha/beta hydrolases (59%) from some cultured bacteria and to predicted lipases/esterases from uncultured bacteria. The phylogenetic analysis suggested that the EstCS1 belongs to the hormone-sensitive lipase family of lipolytic enzyme classification and contains a catalytic triad including Ser155–Asp255–His285. The Ser155 residue of the catalytic triad in the EstCS1 was located in the consensus active-site motif, GXSXG. Besides, a conserved HGGG motif placed in an oxyanion hole of the hormone-sensitive lipase family was discovered, too. The EstCS1 demonstrated the highest activity toward p -nitrophenyl propionate (C3) and caproate (C6) and was normally stable up to 60°C with optimal activity at 50°C. In addition, an optimal activity was observed at pH 8, and the EstCS1 possessed its stability within the pH range between 5 and 10. Interestingly, EstCS1 had an outstanding stability in up to 30% (v/v) organic solvents and activity over 50% in the presence of 50% (v/v) acetone, ethanol, dimethyl sulfoxide (DMSO), and N , N -dimethylformamide. The EstCS1 hydrolyzed sterically hindered tertiary alcohol esters of t -butyl acetate and linalyl acetate. Considering the properties, such as the moderate thermostability, stability against organic solvents, and activity toward esters of tertiary alcohols, the EstCS1 will be worthwhile to be used for organic synthesis and related industrial applications. [ABSTRACT FROM AUTHOR]

Rv0518, a hypothetical protein of Mycobacterium tuberculosis , was designated as possible exported protein. In-silico analysis suggested that the protein belonged to the family of GDSL lipases. In this study, rv0518 gene was cloned and expressed in E. coli followed by purification and characterization. It possessed lipolytic activity, preferably hydrolyzed p NP-decanoate at pH 9.0 and 40 °C. The enzyme was stable till 50 °C and wider pH range (5.0–11.0). The predicted active site residues, Ser-46, Asp-205, His-208, Gly-87, Asn-120 were confirmed by site directed mutagenesis. rv0518 gene expression in M. tuberculosis H37Ra was up-regulated under nutrient starvation and the protein was detected in membrane fraction. The expression of rv0518 in M. smegmatis altered the colony morphology/growth kinetics, provided resistance to SDS, lysozyme, anti-TB drugs and enhanced in vitro survival of M. smegmatis under nutritive stress. The total lipid content and trehalose dimycolate was elevated in M. smegmatis expressing rv0518 gene. The presence of Rv0518 enhanced infection ability and intracellular survival capability of M. smegmatis. Hence, Rv0518 is a cell wall associated GDSL lipase might helped bacteria to utilize glycerol/lipids for its growth as well as provided resistance to various intracellular stresses by cell wall modulation resulting in its enhanced intracellular survival. • Rv0518 belonged to GDSL superfamily of lipolytic enzymes that possessed lipase activity. • rv0518 gene expression up-regulated under nutrient starvation and localized in membrane fraction of M. tuberculosis H37Ra. • The expression of rv0518 in M. smegmatis provided resistance to SDS, lysozyme, anti-TB drugs. • M. smegmatis expressing rv0518 gene demonstrated to have more lipid content and TDM. • The presence of Rv0518 enhanced infection ability and intracellular survival capability of M. smegmatis. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis LipY protein, a prototype of the proline‐glutamic acid (PE) family, exhibits a triacylglycerol (TAG) hydrolase activity that contributes to host cell lipid degradation and persistence of the bacilli. LipY is found either as a full‐length intracytosolic form or as a mature extracellular form lacking the N‐terminal PE domain. Even though the contribution of the extracellular form in TAG consumption has been partly elucidated, very little information is available regarding the potential interactions of either full‐length LipY with the cytoplasmic membrane, or mature form LipY with the outer membrane. Herein, several LipY variants truncated in their N‐terminal domain were produced and biochemically characterized in lipid–protein interaction assays, using the monomolecular film technique and FTIR. Comparison of the catalytic activities of these recombinant proteins showed that LipY∆149, corresponding to the extracellular form of LipY lacking the PE domain, is more active than the full‐length protein. This confirms previous studies reporting that the PE domain negatively modulates the TAG hydrolase activity of LipY. Lipid–protein interaction studies indicate that the PE domain anchors LipY onto membrane lipids. Consistent with these findings, we show that LipY∆149 is loosely associated with the mycobacterial cell wall, and that this interaction is mediated by the sole lipase domain. Overall, our results bring new information regarding the molecular mechanisms by which LipY either binds and hydrolyses host cell lipids or degrades TAG, the major source of lipids within mycobacterial intracytosolic lipid inclusions. [ABSTRACT FROM AUTHOR]

Background: Bacillus subtilis lipase (BSL), Burkholderia sp. lipase (BCL) and Ophiostoma piceae sterol esterase (OPE) represent three typical structural features of lipolytic enzyme active sites. In this research, enzymatic characterization, especially polyester degradation activity, of these lipolytic enzymes was conducted, which might give clues to enzymatic degradation of polyester plastics in the future. Results: The optimum temperatures of BSL, BCL and OPE were 40, 40 and 35 °C, respectively. The optimum pH of BSL, BCL and OPE were 8.0, 8.0 and 7.5, respectively. All three lipolytic enzymes displayed a preference for esters with medium acyl chain length (C8–C10). The initiation reaction rates towards poly(ϵ‐caprolactone) hydrolysis catalyzed by BSL and BCL were 57 and 80 μg min−1 U−1, respectively. However, OPE showed very low activity towards poly(ϵ‐caprolactone) degradation. In contrast, OPE displayed high activity towards poly(vinyl acetate) degradation. Conclusions: Poly(ϵ‐caprolactone) could be enzymatically hydrolyzed using BSL and BCL, while poly(vinyl acetate) could only be enzymatically hydrolyzed using OPE. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Description of the subject. Lipolytic enzymes are widely distributed and fulfil important physiological functions in the microorganisms inhabiting diverse environments. Soils are rich, diversified environments containing microbial communities that remain largely unknown. Objectives. This work aimed to discover new lipolytic enzymes. Method. New enzymes were found by functional screening of two seasonal metagenomic libraries (a winter and a spring library) constructed from an agricultural soil. Screens were performed on 2xYT medium supplemented with 3% lipase reagent. Results. Nineteen positive clones were isolated. Analysis of the corresponding inserts led to identifying 23 putative lipolytic enzymes (13 for the winter library and 10 for the spring library) displaying between 31% and 62% identity to known enzymes and belonging to seven different families. Conclusions. As enzymes show low identity to known enzymes, the encoded enzymes may display novel biochemical features.

A novel lipolytic enzyme-producing endophytic strain PC2 was successfully isolated from the seeds of an ideal bioenergy plant Pistacia chinensis Bunge. Based on the analysis of morphology and 16S rRNA sequence, bacterial strain PC2 was identified as a subspecies of Pseudomonas putida, therefore named as P. putida PC2. Whole-genome sequencing showed PC2 contained a 1224-nucleotide lipase gene (named lip-PC2) predicted to encode a 407-amino-acid protein. Purified lipases from both the original PC2 strain and heterologously expressed Escherichia coli were nearly 50 kD with specific activity of 9.48 U/mL. LIP-PC2 displayed the maximal activity at 50°C or pH 8.0, and maintained above 80% relative activity in the range of from 40 to 60°C or pH in the range of from 6.0 to 8.0, indicating thermostable and alkaline properties. Enzyme activity was enhanced by Mg, Na and Mn, but strongly inhibited by Cu, Zn Co, EDTA as well as organic solvents and surfactants. Additionally, the analysis of amino acid sequence and structure indicated that LIP-PC2 was a novel member belonging to family I.3 of bacterial lipolytic enzymes and its catalytic triad was consisted of Ser-200, Asp-342 and His-374. [ABSTRACT FROM AUTHOR]

Insect morphogen decapentaplegic (Dpp) functions as one of the key extracellular ligands of the Bone Morphogenetic Protein (BMP) signaling pathway. Previous studies in insects mainly focused on the roles of Dpp during embryonic development and the formation of adult wings. In this study, we demonstrate a new role for Dpp in retarding lipolysis during metamorphosis in both Bombyx mori and Drosophila melanogaster. CRISPR/Cas9-mediated mutation of Bombyx dpp causes pupal lethality, induces an excessive and premature breakdown of lipids in the fat body, and upregulates the expressions of several lipolytic enzyme genes, including brummer (bmm), lipase 3 (lip3), and hormone-sensitive lipase (hsl), and lipid storage droplet 1 (lsd1), a lipid droplets (LD)-associated protein gene. Further investigation in Drosophila reveals that salivary gland-specific knockdown of the dpp gene and fat body-specific knockdown of Mad involved in Dpp signaling phenocopy the effects of Bombyx dpp mutation on pupal development and lipolysis. Taken together, our data indicate that the Dpp-mediated BMP signaling in the fat body maintains lipid homeostasis by retarding lipolysis, which is necessary for pupa-adult transition during insect metamorphosis. [Display omitted] • The loss of Bombyx Dpp function disrupts the pupa-adult transition. • Dpp retards lipolysis during Bombyx metamorphosis. • Dpp regulation of lipolysis is conserved in Drosophila. [ABSTRACT FROM AUTHOR]

Milk fat curdle is difficult to remove from sewage. In an attempt to identify an appropriate agent for bio-remediation of milk fat curdle, Mrakia strains were collected from the Skarvsnes ice-free area of Antarctica. A total of 27 strains were isolated and tested for their ability to decompose milk fat at temperatures ranging from 4 °C to 15 °C. All strains could decompose milk fat at 4 °C and 10 °C. Phylogenetic analysis and comparison of the decomposition ability of milk fat (DAMF) revealed that the DAMF may be useful for predicting the outcome of phylogenetic analysis based on ITS sequences. [ABSTRACT FROM AUTHOR]

This paper proposes a high-throughput, function-based screening approach of a metagenomic library for isolating novel microbial enzymes by droplet-based microfluidics. We used gel microdroplets (GMDs) dispersed in oil as picoliter-volume reaction vessels for lipolytic enzyme by encapsulating cells in individual GMDs. Using this approach, we monitored the growth of individual cells encapsulated in GMDs and assessed the enzyme reaction activities at the level of an individual GMD. We then applied this method to screen lipolytic enzyme genes from the metagenomic library constructed from soil collected from a quercus serrate forest of Mount Tsukuba, Ibaraki, Japan. In the workflow presented in this study, metagenomic library clones were encapsulated in 100-pL GMDs with a fluorogenic reporter substrate. A total of 67,000 metagenomic library clones can be screened in only 24 h with reduced consumption of reagents (i.e., <10 μL). As a result, we identified a novel lipolytic enzyme, EstT1, belonging to the EstD2 family of esterases and containing a putative signal peptide, which facilitates enzyme export and catalyzation of substrates in the periplasm. Our study demonstrates the potential of microfluidic GMDs as an efficient tool for metagenomic library screening of industrially relevant enzymes with the potential of significantly reducing the cost and time factors involved in successful practical application of microbial enzymes. [ABSTRACT FROM AUTHOR]

In this study, we report the characterization of a protein from Aspergillus oryzae, exhibiting sequence identity with paraben esterase from the genus Aspergillus. The coding region of 1,586 bp, including a 77-bp intron, encoded a protein of 502 amino acids. The gene without the signal peptide of 19 amino acids was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0-8.0 and 30 °C, respectively, and was stable at the pH range of 7.0-10.0 and up to 40 °C. The optimal substrate for hydrolysis by the purified recombinant protein, among a panel of α-naphthyl esters (C2-C16), was α-naphthyl butyrate (C4), with activity of 0.16 units/mg protein. The considerable hydrolytic activity of the purified recombinant enzyme toward tributyrin was determined. However, no paraben esterase activity was detected toward the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid. In addition, no activity was detected toward the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids that would indicate feruloyl esterase activity. [ABSTRACT FROM AUTHOR]

Pathogenic microbes secrete various enzymes with lipolytic activities to facilitate their survival within the host. Lipolytic enzymes include extracellular lipases and phospholipases, and several lines of evidence have suggested that these enzymes contribute to the virulence of pathogenic fungi. Candida albicans and Cryptococcus neoformans are the most commonly isolated human fungal pathogens, and several biochemical and molecular approaches have identified their extracellular lipolytic enzymes. The role of lipases and phospholipases in the virulence of C. albicans has been extensively studied, and these enzymes have been shown to contribute to C. albicans morphological transition, colonization, cytotoxicity, and penetration to the host. While not much is known about the lipases in C. neoformans, the roles of phospholipases in the dissemination of fungal cells in the host and in signaling pathways have been described. Lipolytic enzymes may also influence the survival of the lipophilic cutaneous pathogenic yeast Malassezia species within the host, and an unusually high number of lipase-coding genes may complement the lipid dependency of this fungus. This review briefly describes the current understanding of the lipolytic enzymes in major human fungal pathogens, namely C. albicans, C. neoformaris, and Malassezia spp. [ABSTRACT FROM AUTHOR]

Staphylococcus women M exhibited extracellular lipase activity. By zymogram analysis of extracellular proteins, multiple bands were detected and the profiles changed depending on the bacterial growth phase. N-terminal amino acid sequences of three bands (N1-N3) were determined. From the genome library of S. women M whole DNA, the gene-directing lipase activity (named gehCWM) was cloned and characterized. The gehCWM gene encoded a protein (GehCWM), whose calculated molecular mass was 83.4 kDa, and the sequence was similar to the other staphylococcal lipases. Though two lipases have been known from S. worneri 863, GehCWM differs from both of them, indicating that this enzyme is the third extracellular lipase of the S. women strain. The N-terminal sequences of the N1-N3 polypeptides completely coincided with the deduced amino acid sequences in GehCWM. GehCWM was predicted to be a prepro-protein. In vitro processing and protein sequencing suggested that pro-GehCWM is possibly processed by extracellular glutamyl endopeptidase, PROM. Inductively coupled plasma-atomic emission spectrometer analysis showed that purified his- tagged mature GehCWM possessed zinc ion. Age/iCWM knock- out mutant was constructed by insertion of an erythromycin resistance gene into the gehCWM Zymogram and immunoblot analyses of the gehCWM mutant indicated that GehCWM was a major extracellular lipase of S. women M. [ABSTRACT FROM AUTHOR]

An extracellular esterase, EstK, was purified from the psychrotrophic bacterium Pseudomonas mandelii grown at 25°C. Prior to harvest, cells were treated with 0.2 M MgCl to precipitate lipopolysaccharides in the outer membranes, which otherwise form aggregates with the secreted enzymes. EstK was purified to homogeneity using standard procedures. It had substrate specificity towards esters of short-chain fatty acids, particularly, p-nitrophenyl acetate. Optimum activity of EstK was at 40°C; at 4°C the activity was ~50% of its maximum. EstK has a unique substrate preference for p-nitrophenyl acetate and remains active at low temperatures. [ABSTRACT FROM AUTHOR]

Abstract: FarA is a Zn(II)2Cys6 transcription factor which upregulates genes required for growth on fatty acids in filamentous fungi like Aspergillus nidulans. FarA is also highly similar to the cutinase transcription factor CTF1α of Fusarium solani which binds to the cutinase gene promoter in this plant pathogen. This study determines whether FarA transcriptional factor also works in the regulation of genes responsible for the production of cutinase for the degradation of a biodegradable plastic, poly-(butylene succinate-co-adipate) (PBSA), in Aspergillus oryzae. The wild-type and the farA gene disruption strains were grown in minimal agar medium with emulsified PBSA, and the wild-type showed clear zone around the colonies while the disruptants did not. Western blot analysis revealed that the cutinase protein CutL1 and a hydrophobic surface binding protein such as HsbA were produced by the wild-type but not by the disruptants. In addition, the expressions of cutL1, triacylglycerol lipase (tglA), and mono- and di-acylglycerol lipase (mdlB) genes as well as the hsbA gene were significantly lower in the disruptants compared to the wild-type. These results indicated that the FarA transcriptional factor would be implicated in the expression of cutL1 and hsbA genes that are required for the degradation of PBSA as well as lipolytic genes such as mdlB and tglA for lipid hydrolysis. [Copyright &y& Elsevier]

Abstract: This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54kDa by SDS–PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution. [Copyright &y& Elsevier]

Microalgae constitute an interesting novel study area for characterizing new esterases, and so we decided to isolate a complete cDNA encoding a new putative microalgal esterase from the haptophyte Isochrysis galbana Parke. Rapid amplifications of both the 5′ and 3′ cDNA ends (RACE) were performed with specific primers, designed using an incomplete candidate gene from the I. galbana expressed sequence tag (EST) database. The full-length cDNA obtained was designated IgEst1. The coding sequence was 828 bp long, and the deduced amino acid sequence revealed a polypeptide of 275 amino acids with a predicted signal peptide of 23 residues in the N-terminal region. The following 252 amino acids formed, after in silico analysis, a mature protein with a molecular mass of ∼26.92 kDa and had a theoretical p I of 5.87. Alignment analyses revealed slight but significant identity and similarity with carboxylesterases, phospholipases, and lysophospholipases from various organisms including fungi, plants, and animals. The new sequence IgEst1 enclosed the catalytic triad Ser/Asp/His and the consensus pentapeptide Gly-X-Ser-X-Gly, two highly conserved patterns found in serine hydrolases. Phylogenetic analyses established a close relationship with putative esterases identified in microalgae genomes. [ABSTRACT FROM AUTHOR]