Aplicação de lipase e monoglicerídeo em pão de forma enriquecido com fibras

Neste trabalho, estudou-se a aplicação da enzima lipase e do emulsificante monoglicerídeo em pão de forma enriquecido com fibras, com o objetivo de verificar a possibilidade de substituição do emulsificante pela enzima. Inicialmente, foi realizada a caracterização das matérias-primas principais (farinha e farelo de trigo). Os pães de forma foram elaborados pelo método de massa direta. Foi utilizado um planejamento experimental do tipo composto central rotacional com duas variáveis independentes: i) dosagem de lipase; e ii) dosagem de monoglicerídeo e, paralelamente, realizou-se um teste controle (sem adição de lipase e monoglicerídeo) para comparação. As variáveis dependentes foram as características de qualidade dos pães: i) volume específico; ii) aceitação sensorial (aparência, textura, aroma e sabor); e iii) vida de prateleira avaliada pela umidade do miolo e firmeza dos pães após 1, 4 e 7 dias do forneamento. Dentro das faixas estudadas, foi possível verificar que somente a umidade dos pães no quarto e sétimo dia após o processamento foi influenciada pela variação das dosagens de lipase e monoglicerídeo. Na avaliação sensorial, verificou-se que as notas médias atribuídas aos pães do teste controle foram inferiores à menor nota média dos ensaios do planejamento experimental, com exceção do sabor e aroma. Como não foi possível obter modelos matemáticos para todas as respostas, foram selecionados os ensaios 5 (1% monoglicerídeo), 7 (25 ppm lipase) e 9 (25 ppm lipase e 1% monoglicerídeo) do planejamento experimental, e o teste controle, para a avaliação dos resultados por análise de variância. Nas condições em que os ensaios foram conduzidos e para as faixas de lipase (0 a 50 ppm) e monoglicerídeo (0 a 2%) estudadas, verificou-se a possibilidade de substituir o monoglicerídeo por lipase em formulação de pão de forma enriquecido com fibras.

Production and partial characterization of lipase from Penicillium verrucosum obtained by submerged fermentation of conventional and industrial media

The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. Current studies on lipase production by submerged fermentation involve the use of agro-industrial residues aiming at increasing economic attractiveness. Based on these aspects, the objective of this work was to investigate lipase production by Penicillium verrucosum in submerged fermentation using a conventional medium based on peptone, yeast extract, NaCl and olive oil, and an industrial medium based on corn steep liquor, Prodex Lac (yeast hydrolysate), NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained. Kinetics of lipase production was evaluated and the highest enzymatic activities, of 3.15 and 2.22 U.mL-1, were observed when conventional and industrial media were used, respectively. The enzymatic extract showed optimal activity in the range from 30 to 40 °C and at pH 7.0. Although the industrial medium presents economical advantages over the conventional medium, the presence of agro-industrial residues rich in nitrogen and other important nutrients seemed to contribute to a reduction in lipase activity.

Fatty acid ethyl esters production using a non-commercial lipase in pressurized propane medium

The objective of this work is to investigate the production of fatty acid ethyl esters from soybean oil in compressed propane using a non-commercial lipase from Yarrowia lipolytica and two commercial ones as catalysts, Amano PS and Amano AY30. The experiments were performed in the temperature range of 35-65 °C. at 50 bar, enzyme concentration of 5 wt%, oil to ethanol molar ratio of 1:6 and 1:9, and solvent to substrates mass ratio of 2:1 and 4:1. The results indicated that low reaction conversions were generally obtained with the use of commercial and non-commercial lipases in pressurized propane medium. On the other hand, the aspects of low solvent to substrates mass ratio and mild temperature and pressure operating conditions used to produce ethyl esters justify further investigations to improve reaction yields.

The role of organic solvent amount in the lipase-catalyzed biodiesel production

This research note addresses the role of organic solvent amount in the production of fatty acid ethyl esters from soybean oil. N-hexane was chosen as solvent and two commercial immobilized lipases as catalysts, Novozym 435 and Lipozyme IM. The reactions were conducted in 6 hours, varying the solvent to oil ratio from zero to 50 (v/wt) and adopting adopting for Novozym 435: 65 ºC, enzyme concentration (E, wt%) = 5, oil to ethanol molar ratio (R) = 1:10, water addition (H, wt%) = 0, and for Lipozyme IM: 35 ºC, E = 5 wt%, R = 1:3, H = 10 wt%. For Lipozyme IM, an increase in solvent amount is shown to lead to an enhancement of reaction conversion, while a negligible effect was found for Novozym 435. When using 30 mL of solvent the reaction conversions were 88% for Lipozyme IM and 15% for Novozym 435.

Avaliação da lipase extracelular de Pseudomonas cepacia para purificação em sistema bifásico aquoso

O presente trabalho teve como objetivo a produção de lipase a partir de Pseudomona cepacia por fermentação líquida em biorreator do tipo Bioflo III. As fermentações foram conduzidas a 150 rpm durante 96 horas a 30 °C. Analisou-se a atividade enzimática em diferentes condições de temperatura (20 a 50 °C) e pH (3,0 a 11,0), e obtiveram-se 37 °C e 8,0, as condições ótimas, respectivamente. Para avaliar a estabilidade térmica, a enzima foi incubada em temperaturas de 40, 50 e 60 °C durante 120 minutos. Em uma segunda etapa, foram realizados experimentos preliminares para verificar as condições adequadas de partição da enzima, bem como sua estabilidade e condições ótimas de hidrólise frente às modificações de temperatura e pH. Foram preparadas soluções de PEG 1500, 4000 e 6000 a 50% p/p e soluções tampão fosfato de pHs 6, 7 e 8. Foi feita a caracterização de um sistema bifásico aquoso (SBA) a partir da preparação de soluções estoques de PEG com massas molares de 1500, 4000 e 6000 (50% w/w) e tampão fosfato pH 6,7 e 8,0 (20% w/w de KH2PO4/K2HPO4). Esta caracterização do SBA posteriormente poderá ser utilizada para partição de lipases, bem como de biomoléculas que estejam dentro dessa faixa de pH.

Production and partial characterization of alkaline polygalacturonase secreted by thermophilic Bacillus sp. SMIA-2 under submerged culture using pectin and corn steep liquor

Polygalacturonase production by the thermophilic Bacillus sp. SMIA-2 cultivated in liquid cultures containing 0.5% (w/v) apple pectin and supplemented with 0.3% (w/v) corn steep liquor, reached its maximum after 36 hours with levels of 39 U.mL-1. The increase in apple pectin and corn steep liquor concentrations in the medium from 0.5 and 0.3%, respectively, to 0.65%, markedly affected the production of polygalacturonase, whose activity increased four times, reaching a maximum of 150.3 U.mL-1. Studies on polygalacturonase characterization revealed that the optimum temperature of this enzyme was between 60-70 °C. Thermostability profile indicated that the enzyme retained about 82 and 63% of its activity at 60 and 70 °C, respectively, after 2 hours of incubation. The optimum pH of the enzyme was found to be 10.0. After incubation of crude enzyme solution at room temperature for 2 hours at pH 8.0, a decrease of about 29% on its original activity was observed. At pH 10.0, the decrease was 25%.

Lipase and esterase: to what extent can this classification be applied accurately?

Enzyme technology is an ever-growing field of knowledge and, in recent years, this technology has raised renewed interest, due to the search for new paradigms in several productive processes. Lipases, esterases and cutinases are enzymes used in a wide range of processes involving synthesis and hydrolysis reactions. The objective of this work was to investigate and compare the specific lipase and esterase activities of five enzymes - four already classified as lipases and one classified as cutinase - in the presence of natural and synthetic substrates. All tested enzymes presented both esterase and lipase specific activities. The highest specific esterase activity was observed for Aspergillus 1068 lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate, while the highest specific lipase activity was observed for Geotrichum sp. lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate. These results display some interface-independent lipolytic activity for all lipases tested. This is in accordance with the rationale that a new and broader definition of lipases may be necessary.

The kinetics of lipase activity and hydrolytic rancidity of raw, parboiled, and extruded rice bran during storage

The main problem related to rice bran use is that it goes rancid right after its production. The objective of the present study was to apply a mathematical model to evaluate the kinetics of the lipase activity and hydrolytic rancidity of the raw rice bran (RRB), extruded rice bran (ERB), and parboiled rice bran (PRB) stored in low density polyethylene bags at room temperature for 180 days. Extrusion and parboiling were efficient in preventing free fatty acid formationin ERB and PRB.Extrusion reduced the velocity constant of lipase activity as compared to that of RRB while parboiling increased it, and both decreased the lipase activity after equilibrium from 150 days. The extrusion and parboiling treatments increased the velocity constants for the liberation of free fatty acids although the equilibrium was reached with reduced production of free fatty acids in relation to the production of raw rice bran after 150 days ofstorage. Extrusion proved the best treatment under the storage temperature conditions of rice bran from cultivar BRS Primavera.

Physical volcanology, sedimentology, stratigraphy and petrochemistry of the Berry Creek metavolcanics: an Archean calc-alkaline complex, Lake of the Woods, Ontario

The steeply dipping, isoclinally folded early Precambrian (Archean) Berry Creek Metavolcanic Complex comprises primary to resedimented pyroclastic, epiclastic and autoclastic deposits. Tephra erupted from central volcanic edifices was dumped by mass flow mechanisms into peripheral volcanosedimentary depressions. Sedimentation has been essentially contemporaneous with eruption and transport of tephra. The monolithic to heterolithic tuffaceous horizons are interpreted as subaerial to subaqueous pumice and ash flows, secondary debris flows, lahars, slump deposits and turbidites. Monolithic debris flows, derived from crumble breccia and dcme talus, formed during downslope collapse and subsequent gravity flowage. Heterolithic tuff, lahars and lava flow morphologies suggest at least temporary emergence of the edifice. Local collapse may have accompanied pyroclastic volcanism. The tephra, produced by hydromagmatic to magmatic eruptions, were rapidly transported, by primary and secondary mechanisms, to a shallow littoral to deep water subaqueous fan developed upon the subjacent mafic metavolcanic platform. Deposition resulted from traction, traction carpet, and suspension sedimentation from laminar to turbulent flows. Facies mapping revealed proximal (channel to overbank) to distal facies epiclastics (greywackes, argillite) intercalated with proximal vent to medial fan facies crystal rich ash flows, debris flows, bedded tuff and shallow water to deep water lava flows. Framework and matrix support debris flows exhibit a variety of subaqueous sedimentary structures, e.g., coarse tail grading, double grading, inverse to normal grading, graded stratified pebbly horizons, erosional channels. Pelitic to psammitic AE turbidites also contain primary stru~tures, e.g., flames, load casts, dewatering pipes. Despite low to intermediate pressure greenschist to amphibolite grade metamorphism and variably penetrative deformation, relicts of pumice fragments and shards were recognized as recrystallized quartzofeldspathic pseudomorphs. The mafic to felsic metavolcanics and metasediments contain blasts of hornblende, actinolite, garnet, pistacitic epidote, staurolite, albitic plagioclase, and rarely andalusite and cordierite. The mafic metavolcanics (Adams River Bay, Black River, Kenu Lake, Lobstick Bay, Snake Bay) display _holeiitic trends with komatiitic affinities. Chemical variations are consistent with high level fractionation of olivine, plagioclase, amphibole, and later magnetite from a parental komatiite. The intermediate to felsic (64-74% Si02) metavolcanics generally exhibit calc-alkaline trends. The compositional discontinuity, defined by major and trace element diversity, can be explained by a mechanism involving two different magma sources. Application of fractionation series models are inconsistent with the observed data. The tholeiitic basalts and basaltic andesites are probably derived by low pressure fractionation of a depleted (high degree of partial melting) mantle source. The depleted (low Y, Zr) calc-alkaline metavolcanics may be produced by partial melting of a geochemically evolved source, e.g., tonalitetrondhjemite, garnet amphibolite or hydrous basalt.

ORGANOSILICON BIOTECHNOLOGY: A BIO-INSPIRED APPROACH TO THE HYDROLYSIS OF ALKOXYSILANES and THE LIPASE-CATALYZED SYNTHESIS OF SILOXANE-CONTAINING POLYESTERS AND POLYAMIDES

The first part of this thesis studied the capacity of amino acids and enzymes to catalyze the hydrolysis and condensation of tetraethoxysilane and phenyltrimethoxysilane. Selected amino acids were shown to accelerate the hydrolysis and condensation of tetraethoxysilane under ambient temperature, pressure and at neutral pH (pH 7±0.02). The nature of the side chain of the amino acid was important in promoting hydrolysis and condensation. Several proteases were shown to have a capacity to hydrolyze tri- and tet-ra- alkoxysilanes under the same mild reaction conditions. The second part of this thesis employed an immobilized Candida antarctica lipase B (Novozym-435, N435) to produce siloxane-containing polyesters, polyamides, and polyester amides under solvent-free conditions. Enzymatic activity was shown to be temperature dependent, increasing until enzyme denaturation became the dominant pro-cess, which typically occurred between 120-130ᵒC. The residual activity of N435 was, on average, greater than 90%, when used in the synthesis of disiloxane-containing polyesters, regardless of the polymerization temperature except at the very highest temperatures, 140-150ᵒC. A study of the thermal tolerance of N435 determined that, over ten reaction cycles, there was a decrease in the initial rate of polymerization with each consecutive use of the catalyst. No change in the degree of monomer conversion after a 24 hour reaction cycle was found.

Lipase-Mediated Enzymatic Catalysis For The Synthesis of New Chiral Polymers

Immobilized lipase B from Candida antarctica (N435) was investigated as a potential biocatalyst to generate silicone-based chiral polymers from monomers derived from the enzymatic dihydroxylation of bromobenzene. Several conditions and parameters have been investigated for this purpose and lipase transesterification preference to each of the free secondary alcohols in the chiral monomers was documented. The N435 was challenged with a series of substrates where the free alcohol moieties were systematically protected in order to study the substrate preference(s) for the transesterification reactions.

Régulation de la lipoprotéine lipase macrophagique et de LOX-1 par des facteurs métaboliques. Implications dans l’athérosclérose associée au diabète de type 2.

Les maladies cardiovasculaires (MCV) sont la principale cause de décès dans les pays occidentaux et constituent la principale complication associée au diabète. La lipoprotéine lipase (LPL) est une enzyme clé du métabolisme des lipides et est responsable de l'hydrolyse des lipoprotéines riches en triglycérides (TG). Plusieurs études ont démontré que la LPL sécrétée par les macrophages dans la paroi artérielle est pro-athérogénique. La dysfonction endothéliale caractérise les stades précoces du processus athérosclérotique. Il a été observé qu’un récepteur nouvellement identifié des lipoprotéines de basse densité oxydées (LDLox), le récepteur de type lectine des LDLox (LOX-1), est fortement exprimé dans les lésions athérosclérotiques humaines et dans l’aorte de rats diabétiques, suggérant un rôle clé de LOX-1 dans la pathogénèse de l’athérosclérose diabétique. Au vu du rôle potentiel de la LPL macrophagique et du LOX-1 dans l’athérosclérose associée au diabète de type 2, nous avons évalué la régulation de ces deux molécules pro-athérogéniques par des facteurs métaboliques et inflammatoires augmentés dans le diabète, soit la leptine, l’acide linoléique (LA) et la protéine C-réactive (CRP). Nos résultats démontrent que : 1) Dans les cellules endothéliales aortiques humaines (HAECs), LA augmente l’expression protéique de LOX-1 de façon temps- et dose-dépendante; 2) La pré-incubation de HAECs avec des antioxydants et des inhibiteurs de la NADPH oxydase, de la protéine kinase C (PKC) et du facteur nucléaire-kappa B (NF-kB), inhibe l’effet stimulant de LA sur l’expression protéique de LOX-1; 3) Dans les HAECs traitées avec LA, on observe une augmentation d’expression des isoformes classiques de la PKC; 4) LA augmente de manière significative l’expression génique de LOX-1 ainsi que la liaison des protéines nucléaires extraites des HAECs à la séquence régulatrice NF-kB présente dans le promoteur du gène de LOX-1; 5) LA augmente, via LOX-1, la captation des LDLox par les cellules endothéliales. Pris dans leur ensemble, ces résultats démontrent que LA augmente l’expression endothéliale de LOX-1 in vitro et appuient le rôle clé de LA dans la dysfonction endothéliale associée au diabète. Au vu de nos études antérieures démontrant qu’une expression accrue de LPL macrophagique chez les patients diabétiques de type 2 et que l’augmentation de facteurs métaboliques dans cette maladie, soit l’homocystéine (Hcys), les acides gras et les produits terminaux de glycation (AGE), accroissent l’expression de la LPL macrophagique, nous avons par la suite déterminé l’effet, in vitro, de deux autres facteurs métaboliques et inflammatoires surexprimés dans le diabète, soit la leptine et la CRP, sur l’expression de la LPL macrophagique. Les concentrations plasmatiques de leptine sont élevées chez les patients diabétiques et sont associées à un accroissement des risques cardiovasculaires. Nous avons démontré que : 1) Dans les macrophages humains, la leptine augmente l’expression de la LPL, tant au niveau génique que protéique; 2) L’effet stimulant de la leptine sur la LPL est aboli par la pré-incubation avec un anticorps dirigé contre les récepteurs à la leptine (Ob-R), des inhibiteurs de la PKC et des antioxydants; 3) La leptine augmente l’expression membranaire des isoformes classiques de la PKC et la diminution de l’expression endogène de la PKC, abolit l’effet de la leptine sur l’expression de la LPL macrophagique; 4) Dans les macrophages murins, la leptine augmente le taux de synthèse de la LPL et augmente la liaison de protéines nucléaires à la séquence protéine activée-1 (AP-1) du promoteur du gène de la LPL. Ces observations supportent la possibilité que la leptine puisse représenter un facteur stimulant de la LPL macrophagique dans le diabète. Finalement, nous avons déterminé, in vitro, l’effet de la CRP sur l’expression de la LPL macrophagique. La CRP est une molécule inflammatoire et un puissant prédicteur d’événements cardiovasculaires. Des concentrations élevées de CRP sérique sont documentées chez les patients diabétiques de type 2. Nous avons démontré que : 1) Dans les macrophages humains, la CRP augmente l’expression de la LPL au niveau génique et protéique et la liaison de la CRP aux récepteurs CD32 est nécessaire pour médier ses effets; 2) La pré-incubation de macrophages humains avec des antioxydants, des inhibiteurs de la PKC et de la protéine kinase mitogénique activée (MAPK), prévient l’induction de la LPL par la CRP; 3) La CRP augmente l’activité de la LPL, la génération intracellulaire d’espèces radicalaires oxygénées (ROS), l’expression d’isoformes classiques de la PKC et la phosphorylation des kinases extracellulaires régulées 1/2 (ERK 1/2); 4) Les macrophages murins traités avec la CRP démontrent une augmentation de la liaison des protéines nucléaires à la séquence AP-1 du promoteur du gène de la LPL. Ces données suggèrent que la LPL puisse représenter un nouveau facteur médiant les effets délétères de la CRP dans la vasculopathie diabétique. Dans l’ensemble nos études démontrent le rôle clé de facteurs métaboliques et inflammatoires dans la régulation vasculaire de la LPL et du LOX-1 dans le diabète. Nos données suggèrent que la LPL et le LOX-1 puissent représenter des contributeurs clé de l’athérogénèse accélérée associée au diabète chez l’humain. Mots-clés : athérosclérose, maladies cardiovasculaires, diabète de type 2, macrophage, LPL, cellules endothéliales, LOX-1, stress oxydatif, leptine, LA, CRP.

Contribution de la déficience en lipoprotéine lipase (LPL) au profil cardiométabolique lié à l'adiponectine chez les femmes

La déficience partielle en lipoprotéine lipase (LPLD) est associée à une augmentation du risque cardiométabolique chez les hommes et les femmes. L’adiponectine, le syndrome métabolique et la ménopause sont des modulateurs importants de ce risque. L’objectif de cette étude était d’évaluer la contribution de l’adiponectine au profil de risque cardiométabolique de femmes porteuses de variants dans le gène LPL connus pour être associés avec la LPLD. L'échantillon étudié comprenait 568 femmes d'origine canadienne-française, dont 127 avec une LPLD et 441 non LPLD (contrôles). L'influence de l'adiponectine sur le risque associé à la LPLD a été évaluée en utilisant des analyses de régression multiples prenant en compte l’influence du statut ménopausique, des variables anthropométriques, du bilan lipidique, de la glycémie à jeun et du tabagisme. Les résultats montrent que les niveaux d'adiponectine étaient significativement plus faibles dans les groupes LPLD. La contribution des valeurs faibles d’adiponectine au profil de risque cardiométabolique des sujets LPLD était indépendante du statut ménopausique et de toutes les autres covariables étudiées. Cela suggère que l'adiponectine contribue au profil de risque cardiométabolique chez les femmes porteuses d’une mutation connue pour être associée avec la LPLD.

Age-Dependent Properties of Acid & Alkaline DNases in Chick Brain

Acid and alkaline DNase activities in partially purified preparations from young and old chick brain were measured. The specific activity of acid DNase from old brain was lower by about 50% than that of enzyme from young brain , whereas alkaline DNase exhibited only marginal difference in activity of the two preparations . Study of various properties, viz. heat-stability and effect of exogenous compounds like Mg=', Hgl', Zn=', PHM B , on these enzymes revealed that while acid DNase in old brain is more susceptible to heat and heavy metal ion inhibition , alkaline DNase is devoid of any age-dependent variation in its properties.

Alkaline protease production by marine fungus engyodontium BTMFS 10

The thesis entitled “Alkaline Protease Production by Marine Fungus Engyodontium BTMFS 10”.Proteases are the single class of enzymes, which occupy a pivotal position with respect to their application in both physiological and commercial filed. Protease in the industrial market is expected to increase further in the coming year. The current trend is to use microbial enzymes since they provide a greater diversity of catalytic activities and can be produced more economically. Main objective of theses studies are the optimization of various physicochemical factors in the solid state fermentation for the production of alkaline protease enzyme, characterization of the enzyme, evaluation of the enzyme for various industrial application. The result obtained the during the course of theses study indicate the scope for the utilization of this study Marine Fungus E. Album for extra cellular protease production employing solid state fermentation