RESUMO
Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg-1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency (kcat/Km) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v/v ethanol and 70% v/v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.
Assuntos
Estabilidade Enzimática , Geobacillus , Lipase , Proteínas Recombinantes , Solventes , Geobacillus/enzimologia , Geobacillus/genética , Lipase/genética , Lipase/química , Lipase/metabolismo , Lipase/isolamento & purificação , Solventes/química , Regiões Antárticas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Concentração de Íons de Hidrogênio , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cinética , Especificidade por Substrato , Temperatura , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
BACKGROUND: Lipases are extensively exploited in lots of industrial fields; cold-adapted lipases with alkali-resistance are especially desired in detergent industry. Penicillium cyclopium lipase I (PCL) might be suitable for applications of detergent industry due to its high catalytic efficiency at low temperature and relatively good alkali stability. In this study, to better meet the requirements, the alkali stability of PCL was further improved via directed evolution with error-prone PCR. RESULTS: The mutant PCL (N157F) with an improved alkali stability was selected based on a high-throughput activity assay. After incubating at pH 11.0 for 120 min, N157F retained 70% of its initial activity, which was 23% higher than that of wild type PCL. Combined with the three-dimensional structure analysis, N157F exhibited an improved alkali stability under the high pH condition due to the interactions of hydrophilicity and ß-strand propensity. Conclusions: This work provided the theoretical foundation and preliminary data for improving alkali stability of PCL to meet the industrial requirements, which is also beneficial to improving alkali-tolerance ability of other industrial enzymes via molecular modification.
Assuntos
Penicillium/enzimologia , Estabilidade Enzimática , Indústria de Detergentes , Lipase/metabolismo , Penicillium/isolamento & purificação , Penicillium/genética , Reação em Cadeia da Polimerase/métodos , Temperatura Baixa , Álcalis , Biocatálise , Interações Hidrofóbicas e Hidrofílicas , Concentração de Íons de Hidrogênio , Lipase/isolamento & purificação , Lipase/genética , MutaçãoRESUMO
Knowledge regarding the enzymatic machinery of fungi is decisive to understand their ecological role. The species of the genus Geastrum are known to grow extremely slowly in pure culture, which makes it difficult to evaluate physiological parameters such as enzyme activity. Qualitative assays were performed on isolates of four species of this genus, showing evidence of laccase, cellulase, pectinase, amylase and lipase activity and suggesting that a wide range of carbon sources can be exploited by these species. For the first time in this genus, quantitative assays verified manganese peroxidase activity (up to 0.6 mU/g) in 30-day old cultures, as well as laccase, β-glycosidase and β-xylosidase activities.
El conocimiento de la maquinaria enzimática de un hongo es decisivo para entender su rol ecológico. Las especies del género Geastrum son conocidas por su crecimiento extremadamente lento en cultivos puros, lo que hace difícil la evaluación de parámetros fisiológicos como las actividades enzimáticas. Se realizaron ensayos cualitativos sobre aislamientos de 4 especies de este género, mostrando evidencias de actividades lacasa, celulasa, pectinasa, amilasa y lipasa, mostrando el amplio rango de fuentes de carbono que pueden ser explotadas por estas especies. Ensayos cuantitativos verificaron por primera vez en este género la actividad manganeso peroxidasa (hasta 0,6 mU/g) en cultivos de 30 días, así como también β-glucosidasa y β-xilosidasa.
Assuntos
Fungos/enzimologia , Xilosidases/isolamento & purificação , Biotransformação/fisiologia , Celulase/isolamento & purificação , Lacase/isolamento & purificação , Fungos/fisiologia , Lipase/isolamento & purificaçãoRESUMO
Abstract Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.
Assuntos
Aspergillus/enzimologia , Lipase/metabolismo , Cátions Bivalentes/metabolismo , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Concentração de Íons de Hidrogênio , Lipase/química , Lipase/isolamento & purificação , Peso Molecular , Mercaptoetanol/metabolismo , Metais/metabolismo , TemperaturaRESUMO
ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.
Assuntos
Compostos Orgânicos , Solventes , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Acinetobacter/enzimologia , Lipase/isolamento & purificação , Lipase/biossíntese , Compostos Orgânicos/química , Solventes/química , Especificidade por Substrato , Temperatura , Proteínas de Bactérias/química , Estabilidade Enzimática , Cinética , Cromatografia por Troca Iônica , Ativação Enzimática , Espaço Extracelular/enzimologia , Concentração de Íons de Hidrogênio , Íons , Lipase/química , Lipólise , Metais , Peso MolecularRESUMO
Background Oil and grease laden wastewaters pose hindrance to the treatment units and further threaten the receiving water bodies. Lipase-producing microbial strains are increasingly being exploited for the remediation of such effluents. Results When bacterial strains isolated from oil mill effluent were screened for their lipolytic activity, two isolates, COM-4A and COM-6B showed significant extracellular lipase activity. They were identified to be Staphylococcus pasteuri and Bacillus subtilis, respectively. S. pasteuri COM-4A was cultivated in nutrient media based on coconut oil mill waste (CMW), in which it showed good growth at concentrations up to 20 g/L. While growing in such media, it was capable of producing lipase and other important extracellular hydrolytic enzymes. Furthermore, the isolate was able to effectively biodegrade the CMW supplemented in the medium. Applying the Box Behnken Design of Response Surface Methodology, lead to a 1.4-fold increase in both lipase production and oil removal by the isolate. The lipase was purified 9.02-fold and the molecular weight of the monomeric enzyme was deduced to be around 56 kDa. Characterization of the enzyme revealed it to be alkaliphilic and moderately thermophilic in nature, with pH and temperature optima of 9.0 and 50°C, respectively. The enzyme was also quite stable in the presence of water-miscible organic solvents. Conclusion Hence, the COM-4A lipase could be considered to be suitable for a variety of industrial applications such as in detergent formulations and in biodiesel production as well, apart from the possibility of applying it for bioremediation of fat and oil contaminants.
Assuntos
Staphylococcus/enzimologia , Óleo de Palmeira/metabolismo , Lipase/isolamento & purificação , Lipase/biossíntese , Temperatura , Bacillus subtilis/enzimologia , Biodegradação Ambiental , Cromatografia por Troca Iônica , Biomassa , Detergentes , Biocombustíveis , Águas Residuárias , Concentração de Íons de HidrogênioRESUMO
The practice of refrigerating raw milk at the farm has provided a selective advantage for psychrotrophic bacteria that produce heat-stable proteases and lipases causing severe quality problems to the dairy industry. In this work, a protease (AprX) and a lipase (LipM) produced by Pseudomonas fluorescens 041, a highly proteolytic and lipolytic strain isolated from raw milk obtained from a Brazilian farm, have been purified and characterized. Both enzymes were purified as recombinant proteins from Escherichia coli. The AprX metalloprotease exhibited activity in a broad temperature range, including refrigeration, with a maximum activity at 37 °C. It was active in a pH range of 4.0 to 9.0. This protease had maximum activity with the substrates casein and gelatin in the presence of Ca+2. The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from 7.0 to 10. It exhibited the highest activity, in the presence of Ca+2, on substrates with long-chain fatty acid residues. These results confirm the spoilage potential of strain 041 in milk due to, at least in part, these two enzymes. The work highlights the importance of studies of this kind with strains isolated in Brazil, which has a recent history on the implementation of the cold chain at the dairy farm.
Assuntos
Animais , Lipase/metabolismo , Leite/microbiologia , Peptídeo Hidrolases/metabolismo , Pseudomonas fluorescens/isolamento & purificação , Brasil , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Lipase/química , Lipase/genética , Lipase/isolamento & purificação , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificação , Pseudomonas fluorescens/genética , Refrigeração , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
Lipases produced by a newly isolated Sporidiobolus pararoseus strain have potential catalytic ability for esterification reactions. After production, the enzymatic extracts (conventional crude and precipitated, 'CC' and 'CP', and industrial crude and precipitated, 'IC' e 'IP') were partially characterized. The enzymes presented, in general, higher specificity for short chain alcohols and fatty acids. The precipitated extract showed a good thermal stability, higher than that for crude enzymatic extracts. The 'CC' and 'CP' enzymes presented high activities after exposure to pH 6.5 and 40 ºC. On the other hand, the 'IC' and 'IP' extracts kept their activities in a wide range of pH memory but presented preference for higher reaction temperatures. Preliminary studies of application of the crude lipase extract in the enzymatic production of geranyl propionate using geraniol and propionic acid as substrates in solvent-free system led to a reaction conversion of 42 ± 1.5%.
Assuntos
Basidiomycota/enzimologia , Lipase/isolamento & purificação , Lipase/metabolismo , Álcoois/metabolismo , Basidiomycota/crescimento & desenvolvimento , Estabilidade Enzimática , Ácidos Graxos/metabolismo , Concentração de Íons de Hidrogênio , Lipase/química , Especificidade por Substrato , TemperaturaRESUMO
The global epidemiology of fungal infections is changing. While overall, Candida albicans remains the most common pathogen; several institutions in Europe, Asia and South America have reported the rapid emergence to predominance of Candida parapsilosis. This mini-review examines the impact of gene deletions achieved in C. parapsilosis that have been published to date. The molecular approaches to gene disruption in C. parapsilosis and the molecularly characterized genes to date are reviewed. Similar to C. albicans, factors influencing virulence in C. parapsilosis include adherence, biofilm formation, lipid metabolism, and secretion of hydrolytic enzymes such as lipases, phospholipases and secreted aspartyl proteinases. Development of a targeted gene deletion method has enabled the identification of several unique aspects of C. parapsilosis genes that play a role in host-pathogen interactions - CpLIP1, CpLIP2, SAPP1a, SAPP1b, BCR1, RBT1, CpFAS2, OLE1, FIT-2. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012 (AU)
La epidemiología mundial de las infecciones fúngicas está cambiando. Aunque Candida albicans sigue siendo el patógeno más común, varios centros en Europa, Asia y Sudamérica han descrito la rápida emergencia de Candida parapsilosis, que ha terminado por predominar. La presente revisión examina la influencia de las deleciones genéticas producidas en C. parapsilosis que se han publicado hasta la fecha. Se revisan las estrategias moleculares de la alteración de genes de C. parapsilosis y los genes caracterizados molecularmente hasta la fecha. Al igual que en C. albicans, los factores que influyen en la virulencia de C. parapsilosis incluyen la adherencia, formación de biopelículas, el metabolismo de lípidos y la secreción de enzimas hidrolíticas, como lipasas, fosfolipasas y aspartilproteinasas. El desarrollo de un método de deleción génica dirigido ha permitido la identificación de varios aspectos exclusivos de los genes de C. parapsilosis que participan en las interacciones huésped-patógeno-CpLIP1, CpLIP2, SAPP1a, SAPP1b, BCR1, RBT1, CpFAS2, OLE1, FIT-2.Este artículo forma parte de una serie de estudios presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)
Assuntos
Humanos , Masculino , Feminino , Virulência/genética , Virulência/fisiologia , Candida/genética , Candida/isolamento & purificação , Lipase/genética , Lipase/isolamento & purificação , Lisofosfolipase/isolamento & purificação , Lisofosfolipase/fisiologia , Ácidos Graxos/biossíntese , Virulência , Fungemia/genética , Fungemia/microbiologia , BiofilmesRESUMO
A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca2+, Mg2+ and K+, while heavy metals (Fe3+ and Zn2+) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes.
Assuntos
Enterobacter/enzimologia , Lipase/isolamento & purificação , Lipase/metabolismo , Sequência de Aminoácidos , Técnicas de Tipagem Bacteriana , Sequência de Bases , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Estabilidade Enzimática , Enterobacter/classificação , Enterobacter/genética , Enterobacter/isolamento & purificação , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Irã (Geográfico) , Lipase/química , Lipase/genética , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Microbiologia do Solo , TemperaturaRESUMO
Extracellular lipases from the endophytic yeast Candida guilliermondii isolated from castor leaves (Ricinus communis L.) were produced using low-cost raw materials such as agro-industrial residues and applying them in the esterification of oleic acid for evaluating their potential use in biodiesel production. After partial purification using ammonium sulfate, the enzyme was characterized and presented higher activity (26.8 ± 1.5 U mL-1) in the presence of 5 mmol L-1 NaCl at 30 ºC and pH 6.5. The production through submerged fermentation was formerly performed in 150 mL erlenmeyer flasks and, once the enzyme production was verified, assays in a 14 L bioreactor were conducted, obtaining 18 ± 1.4 U mL-1. The produced enzyme was applied in the oleic acid esterification under different solvents: hexane, cyclohexane or cyclohexanone) and different acid:alcohol molar ratios. Higher ester conversion rate (81%) was obtained using hexane and the molar ratio of 1:9 was the best conditions using methanol. The results suggest the potential for development of endophytic yeast in the production of biocatalyst through submerged fermentation using agroindustrial residues as culture medium.
Assuntos
Candida/enzimologia , Candida/metabolismo , Lipase/isolamento & purificação , Lipase/metabolismo , Candida/crescimento & desenvolvimento , Candida/isolamento & purificação , Meios de Cultura/química , Esterificação , Endófitos/enzimologia , Endófitos/crescimento & desenvolvimento , Endófitos/isolamento & purificação , Endófitos/metabolismo , Concentração de Íons de Hidrogênio , Ácido Oleico/metabolismo , Folhas de Planta/microbiologia , Ricinus/microbiologia , Cloreto de Sódio/metabolismo , TemperaturaRESUMO
Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L-1; starch, 15.0 g.L-1; triton-X-100, 0.93 mL.L-1; incubation temperature, 34.12 ºC and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL-1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R²) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.
Assuntos
Alcalinização , Enzimas , Fermentação , Lipase/análise , Lipase/isolamento & purificação , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Ativação Enzimática , Metodologia como Assunto , Padrões de ReferênciaRESUMO
In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.
Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.
Assuntos
Esterases/isolamento & purificação , Lipase/isolamento & purificação , Metagenômica , Sequência de Aminoácidos , Bacillaceae/enzimologia , Proteínas de Bactérias/química , Butiratos/metabolismo , Sequência Conservada , DNA , Esterases/classificação , Alemanha , Concentração de Íons de Hidrogênio , Hidrólise , Lipólise , Lipase/classificação , Dados de Sequência Molecular , Concentração Osmolar , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Microbiologia do Solo , Especificidade por Substrato , Sais/farmacologia , Solventes/farmacologia , Temperatura , Árvores , Triglicerídeos/metabolismoRESUMO
An extracellular alkaline lipase from Pseudomonas aeruginosa mutant has been purified to homogeneity using acetone precipitation followed by anion exchange and gel filtration chromatography and resulted in 27-fold purification with 19.6% final recovery. SDS-PAGE study suggested that the purified lipase has an apparent molecular mass of 67 kDa. The optimum temperature and pH for the purified lipase were 45°C and 8.0, respectively. The enzyme showed considerable stability in pH range of 7.0-11.0 and temperature range 35-55 °C. The metal ions Ca2+, Mg2+ and Na+ tend to increase the enzyme activity, whereas, Fe2+ and Mn2+ ions resulted in discreet decrease in the activity. Divalent cations Ca+2 and Mg+2 seemed to protect the enzyme against thermal denaturation at high temperatures and in presence of Ca+2 (5 mM) the optimum temperature shifted from 45°C to 55°C. The purified lipase displayed significant stability in the presence of several hydrophilic and hydrophobic organic solvents (25%, v/v) up to 168 h. The pure enzyme preparation exhibited significant stability and compatibility with oxidizing agents and commercial detergents as it retained 40-70% of its original activities. The values of Km and Vmax for p-nitrophenyl palmitate (p-NPP) under optimal conditions were determined to be 2.0 mg.mL-1 and 5000 μg.mL-1.min-1, respectively.
Assuntos
Lipase/metabolismo , Pseudomonas aeruginosa/enzimologia , Precipitação Química , Cromatografia em Gel , Cromatografia por Troca Iônica , Cátions/metabolismo , Ativadores de Enzimas , Estabilidade Enzimática , Inibidores Enzimáticos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lipase/química , Lipase/isolamento & purificação , Metais/metabolismo , Oxidantes/metabolismo , Pseudomonas aeruginosa/genética , Solventes/metabolismo , TemperaturaRESUMO
A novel alkaline lipase-producing strain 1-7 identified as Acinetobacter calcoaceticus was isolated from soil samples collected from Bohai Bay, China, using an olive oil alkaline plate, which contained olive oil as the sole carbon source. The lipase from strain 1-7 showed the maximum activity at pH 9.0 under 40ºC. One interesting feature of this enzyme is that it exhibits lipase activity over a broad range of temperatures and good stability. It is also stable at a broad range of pHs from 4.0 to 10.0 for 24 h. Its catalytic activity was highly enhanced in the presence of Ca2+, Mg2+ and K+, but partially inhibited by Cu2+, Al3+, Fe3+ , Ba2+and Zn2+. The fact that it displays marked stability and activity in the presence of TritonX-100, Tween-20, Tween-80, SDS, Hydrogen peroxide, Sodium perborate, Sodium hypochlorite, Sodium citrate, Sodium taurocholate, Glycerine and NaCl suggests that this lipase is suitable as an additive in detergent formulations.
Assuntos
Acinetobacter calcoaceticus/enzimologia , Acinetobacter calcoaceticus/isolamento & purificação , Carbono/análise , Lipase/análise , Lipase/isolamento & purificação , Alcalinidade do Solo , Ativação Enzimática , Microbiologia do Solo , Gorduras VegetaisRESUMO
The lipase produced by a newly isolate Sporidiobolus pararoseus strain has potential catalysis ability for esterification reactions. In order to improve its synthetic activity, this work aimed at optimizing 'synthetic lipase' production by submerged fermentation of a conventional media based on peptone, yeast extract, NaCl and olive oil using experimental design technique. According to the results obtained in the first experimental design (2(4-1)), yeast extract and NaCl concentrations were tested to further optimization by response surface methodology. The maximum 'synthetic lipase' activity obtained was 26.9 U/mL in the optimized media (5.0, 6.8, 7.0 and 1.0% (wt/v) of peptone, yeast extract, NaCl and olive oil, respectively), representing a 6.36-fold increase compared to the initial medium. The time course of 'synthetic lipase' production in the optimized condition was evaluated in terms of synthetic activity, protease activity, biomass and total carbon and the maximum synthetic activity was observed during the stationary phase of growth.
Assuntos
Sequência de Bases , Blastomyces/isolamento & purificação , Fermentação , Fungos Mitospóricos/genética , Fungos Mitospóricos/isolamento & purificação , Leveduras/isolamento & purificação , Lipase/análise , Lipase/isolamento & purificação , Ativação Enzimática , Microbiologia Industrial , Metodologia como AssuntoRESUMO
Studies on lipase production were carried out with a bacterial strain (Bacillus sp LBN 2) isolated from soil sample of hotspring of Arunachal Pradesh, India. The cells were cultivated in a mineral medium with maximum production at 1% groundnut oil. The optimum temperature and initial medium pH for lipase production by the organism were 50ºC and 9.0 respectively. The molecular mass was found to be 33KDa by SDS PAGE. The optimal pH and temperature for activity were 10 and 60ºC respectively. The enzyme was found to be stable in the pH range of 8-11 with 90% retention of activity at pH 11. The enzyme retained 90% activity at 60ºC and 70% of activity at 70ºC for 1h. The lipase was found to be stable in acetone followed by ethanol. The present findings suggested the enzyme to be thermophilic alkaline lipase.
Assuntos
Bacillus/enzimologia , Bacillus/isolamento & purificação , Lipase/análise , Lipase/isolamento & purificação , Óleos/análise , Estruturas Vegetais , Microbiologia do Solo , Arachis , Amostras de Alimentos , Concentração de Íons de Hidrogênio , Métodos , SoloRESUMO
Kinetics of a lipase isolated from Bacillus sp. was studied. The enzyme showed maximum activity at pH 9 and temperature 60ºC. The Michaelis constant (K M 0.31 µM) obtained from three different plots i.e., Lineweaver-Burk, Hanes-Wolf and Hofstee, was found to be lower than already reported lipases that confirmed higher affinity of the enzyme for its substrate p-NPL (p-nitrophenyl laurate). Vmax of the enzyme was found to be 7.6 µM/mL/min. Energy of activation calculated from Arrhenius plot was found to be 20.607 kJmol-1. Activation enthalpy (ΔH*) had negative trend and the value for the hydrolysis of p-NPL by the enzyme at optimum temperature was -2.748 kJmol-1 . Activation entropy (ΔS*) and free energy of activation (ΔG*) of the enzyme were found to be 1.468 Jmol-1K-1 and -3.237 kJmol-1, respectively at optimum temperature. Low value of Q10 (0.04788) shows high catalytic activity of the enzyme. Mn2+, Fe2+ and Mg2+ enhanced the lipase activity whereas Cu2+, Na+ and Co2+ inhibited the enzyme activity. However, the enzyme activity was not affected significantly by K+ ions. EDTA and SDS also significantly inhibited the lipase activity. Activity of the enzyme was increased in n-hexane while decreased with increase in concentration of acetone, chloroform, ethanol and isopropanol.
Assuntos
Acetona/análise , Bacillus/enzimologia , Bacillus/isolamento & purificação , Catalase/análise , Microbiologia Ambiental , Reativadores Enzimáticos , Curtume , Lipase/isolamento & purificação , Compostos Orgânicos , Solventes , Ativação Enzimática , Cinética , Métodos , Métodos , ResíduosRESUMO
In this study, we investigate the antimicrobial effects of a mixture of a biosurfactant from Bacillus subtilis and an alkaline lipase from Fusarium oxysporum (AL/BS mix) on several types of microorganisms, as well as their abilities to remove Listeria innocua ATCC 33093 biofilm from stainless steel coupons. The AL/BS mix had a surface tension of around 30 mN.m-1, indicating that the presence of alkaline lipase did not interfere in the surface activity properties of the tensoactive component. The antimicrobial activity of the AL/BS mix was determined by minimum inhibitory concentration (MIC) micro-assays. Among all the tested organisms, the presence of the mixture only affected the growth of B. subtilis CCT 2576, B. cereus ATCC 10876 and L. innocua. The most sensitive microorganism was B. cereus (MIC 0.013 mg.mL-1). In addition, the effect of the sanitizer against L. innocua attached to stainless steel coupons was determined by plate count after vortexing. The results showed that the presence of the AL/BS mix improved the removal of adhered cells relative to treatment done without the sanitizer, reducing the count of viable cells by 1.72 log CFU.cm-2. However, there was no significant difference between the sanitizers tested and an SDS detergent standard (p<0.05).
Assuntos
Antibacterianos , Biofilmes , Bacillus subtilis/enzimologia , Bacillus subtilis/isolamento & purificação , Ativação Enzimática , Fusarium/isolamento & purificação , Lipase/análise , Lipase/isolamento & purificação , Listeria/isolamento & purificação , Métodos , Testes de Sensibilidade MicrobianaRESUMO
This study was aimed at producing protease and lipase simultaneously on a common medium by Bacillus licheniformis VSG1, which was isolated from a tannery effluent. The effect of media composition with respect to protein source, lipid source and emulsifier on the production of protease and lipase was analysed. Both those enzymes were produced under optimized conditions like pH, temperature and incubation time. The enzyme mixture comprising of both protease and lipase was purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography to obtain 20-fold pure enzymes. The purified enzyme mixture was characterized to determine the optimum pH and temperature of protease and lipase, the response of the enzymes to inhibitors, additives and solvents. The molecular weight of both the enzymes was determined as 40 kDa on SDS-PAGE. The concomitant production of protease and lipase and the purification of both the enzymes in a single mixture have industrial significance, as many industrial processes use both protease and lipase together.