Role of spacer length in interaction between novel gemini imidazolium surfactants and Rhizopus oryzae lipase

An insight into the effects of new ionic liquid-type gemini imidazolium cationic surfactants on the structure and function of the lipases is of prime importance for their potential application. Changes in the activity, stability and structure of Rhizopus ouzae lipase in the presence of novel gemini surfactants, C-16-3-C(16)im]Br-2 and C-16-12-C(16)im]Br-2 were probed in the present study. Surfactant with shorter spacer length, C-16-3-C(i6)im]Br-2 was found to be better in improving the hydrolytic activity and thermal stability of the lipase. For both the surfactants, activation was concentration dependent. CD spectroscopy results showed a decrease in a-helix and an increase in beta-sheet content in the presence of these surfactants. A higher structural change observed in presence of C-16-12-C(16)im]Br-2 correlated with lower enzyme activity. Isothermal titration calorimetric studies showed the binding to be spontaneous in nature based on sequential two site binding model. The forces involved in binding were found to differ for the two surfactants proving that the spacer length is an important factor which governs the interaction. These surfactants could be used as promising components both in enzyme modification and media engineering for attaining the desired goals in biocatalytic reactions. (C) 2015 Elsevier B.V. All rights reserved.

Effect of solvents on the enzyme mediated degradation of copolymers

The biodegradation of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA) and their copolymers, poly (lactide-co-glycolide) and poly (D, L-lactide-co-caprolactone) (PLCL) was investigated. The influence of different solvents on the degradation of these polymers at 37 degrees C in the presence of two different lipases namely Novozym 435 and the free lipase of porcine pancreas was investigated. The rate coefficients for the polymer degradation and enzyme deactivation were determined using continuous distribution kinetics. Among the homopolymers, the degradation of PGA was nearly an order of magnitude lower than that for PCL and PLA. The overall rate coefficients of the copolymers were higher than their respective homopolymers. Thus, PLCL degraded faster than either PCL or PLA. The degradation was highly dependent on the viscosity of the solvent used with the highest degradation observed in acetone. The degradation of the polymers in acetone was nearly twice that observed in dimethyl sulfoxide indicating that the degradation decreases with increase in the solvent viscosity. The degradation of the polymers in water-solvent mixtures indicated an optimal water content of 2.5 wt% of water.

Effect of solvents on the enzyme mediated degradation of copolymers

The biodegradation of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA) and their copolymers, poly (lactide-co-glycolide) and poly (D, L-lactide-co-caprolactone) (PLCL) was investigated. The influence of different solvents on the degradation of these polymers at 37 degrees C in the presence of two different lipases namely Novozym 435 and the free lipase of porcine pancreas was investigated. The rate coefficients for the polymer degradation and enzyme deactivation were determined using continuous distribution kinetics. Among the homopolymers, the degradation of PGA was nearly an order of magnitude lower than that for PCL and PLA. The overall rate coefficients of the copolymers were higher than their respective homopolymers. Thus, PLCL degraded faster than either PCL or PLA. The degradation was highly dependent on the viscosity of the solvent used with the highest degradation observed in acetone. The degradation of the polymers in acetone was nearly twice that observed in dimethyl sulfoxide indicating that the degradation decreases with increase in the solvent viscosity. The degradation of the polymers in water-solvent mixtures indicated an optimal water content of 2.5 wt% of water.

Food composition and digestive enzymes in the gut of the African electric catfish Malapterurus electricus (Gmelin 1789) (Malapteruridae)

Analyses of stomach contents of 330 Malapterurus electricus (standard length, 10.1-30.5 cm) in Mahin Lagoon (southwestern Nigeria) established it as a bottom feeder. There was a preponderance of insects accounting for >80% occurrence and >25% of total volume in stomachs of specimens, suggesting a stenophagous predatory habit. Qualitative and quantitative assays of digestive enzymes in the different regions of the gut (oesophagus, stomach, duodenum, ileum, rectum) were investigated. Carbohydrases (amylase, maltase), chitinase, proteases (pepsin, chymotrypsin, trypsin) and lipases were detected in different gut regions with different activity. The pattern of distribution and relative activity of the enzymes correlated with the predatory diet

Obtenção de biodiesel por transesterificação enzimática de óleo de soja com etanol empregando t-butanol como solvente

Neste estudo foi investigada a alcoólise enzimática do óleo de soja com etanol, utilizando t-butanol como solvente e enzimas imobilizadas Lipozyme TL IM, Lipozyme RM IM e Novozym 435 como catalisadores. As reações foram realizadas em um reator batelada fechado acoplado a um condensador e com constante agitação. Foram avaliadas a influência do t-butanol, do tipo de enzima utilizada, da razão molar álcool/óleo e da temperatura no rendimento em biodiesel. A etanólise do óleo de soja por sucessivas adições de álcool foi investigada e as melhores condições foram obtidas em presença de t-butanol, razão molar etanol/óleo igual a 3, temperatura de 50C e 5% (m/m) de Novozym 435. Nas reações conduzidas em presença de t-butanol não foram observadas diferenças significativas entre a adição direta e a escalonada do álcool. Os efeitos da adição de álcool só foram observados na ausência de t-butanol. O rendimento máximo em ésteres etílicos atingido foi cerca de 66% após 4h de reação com Novozym 435 na presença de solvente.

Produção de biodiesel a partir da alcoólise do óleo de palma

No presente trabalho foi investigada a produção de biodiesel a partir da alcoólise do óleo de palma catalisada por lipase imobilizada comercial. O efeito da razão molar de álcool:óleo, da forma de adição do álcool (única e escalonada), da temperatura de reação, da concentração de enzima, do tipo de álcool (metanol e etanol), do tipo de enzima e da reutilização da enzima no rendimento final de reação foi avaliado. As reações conduzidas com etanol apresentaram rendimentos superiores aos obtidos com o emprego de metanol devido à maior desativação da lipase pelo álcool de menor número de átomos de carbono. O maior rendimento em biodiesel (54%) foi obtido empregando razão molar de álcool:óleo de 3:1, com adição escalonada de etanol (0, 30 e 60 minutos), 9% (m/m) de Lipozyme TL IM a 50C. Não foi possível recuperar a lipase ao final das reações, pois a matriz de imobilização se solubilizou no meio. Além disso, para comparação, foi investigada a utilização das lipases comerciais imobilizadas Lipozyme RM IM e Novozym 435 e dos catalisadores químicos KOH, MgO e La2O3. O rendimento em biodiesel nas reações catalisadas pelas lipases foi maior do que os obtidos com catalisadores químicos. A menor eficiência dos catalisadores químicos pode ser justificada pelo alto índice de acidez do óleo de palma (6,26 mg KOH.g-1) que promove o consumo do catalisador (KOH), devido à neutralização dos ácidos graxos livres presentes no óleo, e o bloqueio dos sítios ativos dos catalisadores químicos sólidos devido à adsorção dos ácidos graxos nestes sítios

Produção de biodiesel a partir da transesterificação de óleo de soja ácido por via enzimática

Neste trabalho, foi estudada a transesterificação enzimática do óleo de soja com álcool catalisada por lipase comercial imobilizada. Inicialmente foram investigados os efeitos da temperatura, do tipo de enzima (Novozym 435, Lipozyme RM-IM e Lipozyme TL-IM) e do tipo de álcool (etanol ou butanol) na síntese de biodiesel utilizando óleo de soja refinado. A melhor temperatura observada para as reações empregando Lipozyme TL IM e Lipozyme RM IM foi de 50C, enquanto que para a Novozym 435, a temperatura ótima foi de 70C. O maior teor em biodiesel (~60%) foi obtido na etanólise do óleo de soja a 70C utilizando Novozym 435 a 5% m/m. Também foi avaliada a síntese de biodiesel por via enzimática a partir da etanólise de óleos ácidos com índice de acidez (IA) (de 8,5, 54,4 e 93,7). A conversão do ácido graxo livre foi superior a 90% nas reações conduzidas com Novozym 435 nas reações com os três óleos ácidos testados. O teor em biodiesel foi próximo a 50% na etanólise do óleo de soja com índice de acidez de 8,5 empregando Lipozyme TL IM, porém para índices de acidez maiores este rendimento diminuiu. Para as reações conduzidas com Lipozyme RM-IM, o teor em biodiesel manteve-se em torno de 30% para todos os óleos ácidos investigados. A etanólise do óleo de soja refinado empregando reator de leito fixo em modo contínuo, a 50C, foi investigada variando a velocidade espacial dos reagentes (0,255 e 0,508 h-1), o tipo de lipase (Novozym 435, Lipozyme RM-IM e Lipozyme TL-IM) e a possibilidade de reuso do biocatalisador após lavagem com butanol. Na reação com Novozym 435, o teor em biodiesel foi de aproximadamente 50% e se manteve estável durante 4,5 h para as duas velocidades espaciais testadas. A lipase Lipozyme TL-IM apresentou teor em biodiesel maior do que o obtido nas reações com os outros biocatalisadores (~80%), porém não apresentou a mesma estabilidade. Foi observada uma queda significativa na produção de biodiesel nas reações empregando o reuso do biocatalisador

Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough

The Southern Okinawa Trough is an area of focused sedimentation due to particulate matter export from the shelf of the East China Sea and the island of Taiwan. In order to understand the geomicrobiological characteristics of this unique sedimentary environment, bacterial cultivations were carried out for an 8.61 m CASQ core sediment sample. A total of 98 heterotrophic bacterial isolates were characterized based on 16S rRNA gene phylogenetic analysis. These isolates can be grouped into four bacterial divisions, including 13 genera and more than 20 species. Bacteria of the gamma-Proteobacteria lineage, especially those from the Halomonas ( 27 isolates) and Psychrobacter ( 20 isolates) groups, dominate in the culturable bacteria assemblage. They also have the broadest distribution along the depth of the sediment. More than 72.4% of the isolates showed extracellular hydrolytic enzyme activities, such as amylases, proteases, lipases and Dnases, and nearly 59.2% were cold-adapted exoenzyme-producers. Several Halomonas strains show almost all the tested hydrolases activities. The wide distribution of exoenzyme activities in the isolates may indicate their important ecological role of element biogeochemical cycling in the studied deep-sea sedimentary environment.

Interleukin 10 reduces the incidence of pancreatitis after therapeutic endoscopic retrograde cholangiopancreatography.

BACKGROUND & AIMS: Prophylactic administration of interleukin (IL)-10 decreases the severity of experimental pancreatitis. Prevention of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis in humans is a unique model to study the potential role of IL-10 in this setting. METHODS: In a single-center, double-blind, randomized, placebo-controlled study, the effect of a single injection of 4 microg/kg (group 1) or 20 microg/kg (group 2) IL-10 was compared with that of placebo (group 0), all administered 30 minutes before therapeutic ERCP. The primary endpoint was the effect of IL-10 on serum levels of amylases and lipases measured 4, 24, and 48 hours after ERCP. The secondary objective was to evaluate changes in plasma cytokines (IL-6, IL-8, tumor necrosis factor) at the same time points and the incidence of acute pancreatitis in the 3 groups. Subjects undergoing a first therapeutic ERCP were eligible for inclusion. RESULTS: A total of 144 patients were included. Seven were excluded based on intention to treat (n = 1) or per protocol (n = 6). Forty-five, 48, and 44 patients remained in groups 0, 1, and 2, respectively. The 3 groups were comparable for age, sex, underlying disease, indication for treatment, type of treatment, and plasma levels of C-reactive protein (CRP), cytokines, and hydrolases at baseline. No significant difference was observed in CRP, cytokine, and hydrolase plasma levels after ERCP. Forty-three patients developed hyperhydrolasemia (18 in group 0, 14 in group 1, and 11 in group 2; P = 0.297), and 19 patients developed acute clinical pancreatitis (11 in group 0, 5 in group 1, 3 in group 2; P = 0.038). Two severe cases were observed in the placebo group. No mortality related to ERCP was observed. Logistic regression identified 3 independent risk factors for post-therapeutic ERCP pancreatitis: IL-10 administration (odds ratio [OR], 0.46; 95% confidence interval [95% CI], 0.22-0.96; P = 0.039), pancreatic sphincterotomy (OR, 5.04; 95% CI, 1.53-16.61; P = 0.008), and acinarization (OR, 8.19; 95% CI, 1.83-36.57; P = 0.006). CONCLUSIONS: A single intravenous dose of IL-10, given 30 minutes before the start of the procedure, independently reduces the incidence of post-therapeutic ERCP pancreatitis.

Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa.

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.

Identification of a general secretory pathway in a human isolate of Burkholderia vietnamiensis (formerly B. cepacia complex genomovar V) that is required for the secretion of hemolysin and phospholipase C activities

Members of the Burkholderia cepacia complex can secrete proteases, lipases, and hemolysins. We report in this study the identification of a general secretory pathway present in a B. vietnamiensis (formerly genomovar V) clinical isolate, which is required for the efficient secretion of phospholipase C and hemolysin activities. Southern blot hybridization experiments revealed that this general secretion pathway is highly conserved among the different genomovars of the B. cepacia complex and is homologous to a similar system described in B. pseudomallei. We also show that this pathway appears not to be necessary for intracellular survival of B. vietnamiensis within Acanthamoeba polyphaga.

Fatores de virulência em Aeromonas hydrophila causadora de septicémia em leitões

This work aims to contribute to determine the resistance profile to different antibiotics (ampicillin, gentamicin, penicillin G, oxytetracycline, lincomycin, neomycin, streptomycin, enrofloxacin, colistin sulfate, trimethoprim, sulfamide, tulathromycin, ceftiofur, amoxicillin/clavulanic acid), to assess genetic determinants associated to aminoglycoside antibiotics resistance, namely the presence of genes encoding acetyltransferases (AAC), phosphotransferases (APH) and nucletildiltranferases (ANT), determined by PCR studies, and to search for potentially pathogenic features as the production of extracellular lipases and proteases and the presence of genes encoding for putative virulence factors as aerolysin and related toxins, lipase proteins and type III secretion system component.

Production of lipase by extractive fermentation with ionic liquids

Novos processos fermentativos, designados por processos de Fermentação Extractiva, são caracterizados por apresentarem etapas de produção e extracção em simultâneo. A extracção líquido-líquido como técnica de separação é amplamente usado na indústria química pela sua simplicidade, baixo custo e facilidade de extrapolação de escala. No entanto o uso de solventes orgânicos nestes processos potencia os riscos ocupacionais e ambientais. Neste contexto, o uso de sistemas de duas fases aquosas baseados em líquidos iónicos, apresenta-se como uma técnica eficaz para a separação e purificação de produtos biológicos. Este trabalho apresenta um estudo integrado sobre o uso de líquidos iónicos não aromáticos foram determinados. A capacidade para a formação de sistemas de duas fases foi estudada para uma vasta gama de líquidos iónicos hidrofílicos com diferentes aniões, catiões e cadeias alqúilicas. A capacidade de separação e purificação de um largo conjunto de líquidos iónicos foi posteriormente investigada, recorrendo-se ao uso de várias biomoléculas modelo de diferentes graus de complexidade, um amino-acido (L-triptofano) e duas enzimas lipolíticas (enzima produzida pela bactéria Bacillus sp. e Candida antarctica lipase B – CaLB). Esta última foi ainda usada para um estudo de biocompatibilidade, tendo sido determinado o efeito de diferentes LIs hidrofílicos na sua actividade enzimática. Este trabalho mostra um estudo ecotoxicológico duma vasta gama de líquidos iónicos e espécies aquáticas, inseridas em diversos níveis tróficos. A bioacumulação foi investigada através do estudo dos coeficientes de distribuição 1-octanol-água (Dow).

Effect of photodynamic therapy on the virulence factors of Staphylococcus aureus

Staphylococcus aureus are Gram-positive bacteria who integrate the human microbiota. Nevertheless, these bacteria can be pathogenic to the humans. Due to the increasing occurrence of antibiotic-resistant S. aureus new approaches to control this pathogen are necessary. The antimicrobial photodynamic inactivation process (PDI) is based in the combined use of a light source, an oxidizing agent like oxygen and an intermediary agent (a photosensitizer). These three components interact to form cytotoxic reactive oxygen species that irreversibly damage vital constituents of the microbial cells and ultimately lead to cell death. In fact, PDI is being shown to be a promising alternative to the antibiotic approach in the inactivation of pathogenic microorganisms. However, information on effects of photosensitization on particular virulence factors is strikingly scarce. The objective of this work was to evaluate the effect of PDI on virulence factors of S. aureus. For this, as photosensitizer the 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) and six strains of S. aureus (one reference strain, one strain with 1 enterotoxin, two strains with 3 enterotoxins and two strains resistant to methicillin, MRSA – one with 5 enterotoxins and the other without enterotoxins) were used. The effect of photosensitization on catalase activity, beta hemolysis, lipases, thermonuclease, enterotoxins, coagulase production and resistance to methicillin was assessed. The results indicate that the expression of some virulence factors in the cells subjected to this therapy is affected. Additionally the susceptibility of the strains to PDI did not decrease upon successive treatments.