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Cellulosic nanofibrils from cocoa shells were used in this study to produce biodegradable films from a residue naturally rich in bioactive compounds, while reserving these compounds. The nanofibrils were obtained by mechanical defibrillation without chemical or enzymatic pretreatment. The nanofibril gel was evaluated in biodegradable films, for the first time, with the addition of glycerol as a plasticizer in the proportions of 0, 1, 3, 5, and 7% (w/w). The morphological characteristics, thermal analysis, mechanical properties (rupture tensile, maximum rupture elongation, and Young’s modulus), water vapor permeability, and water solubility of the films were investigated. In addition, the residue was analyzed and compared to the films by the FTIR technique to prove the maintenance of functional groups characteristic of antioxidant activity. The use of 3% and 5% glycerol as a plasticizer showed the best mechanical characteristics—reduced water vapor permeation and increased degradation in water. The pure nanofibril film demonstrated greater thermal stability. It was possible to identify the maintenance of aromatic groups and phenols in the residue from all cocoa shell nanofibril films. This study contributes to the future application of cocoa shell nanofibers in the production of edible films, coatings, and packaging materials. It recommends their application in cocoa and chocolate products due to the compatibility of smell and color.

Adding chemicals that alter the microbial metabolism and reduce cell growth, without affecting organic matter removal, is an alternative technology to reduce sludge production. This study was intended to determine the best initial conditions to conduct a rapid test (4 h), which evaluates the potential of chemicals to reduce microbial growth and chemical oxygen demand removal. A commercial biosurfactant was used as model product and a central composite design (face centered) was performed using substrate/inoculum ratio and rhamnolipid/inoculum ratio as independent variables, and cellular yield coefficient, substrate consumption rate, and specific oxygen uptake rate for exogenous respiration as response variables. Lower values of substrate/inoculum ratio permitted larger reductions of cellular yield coefficient with lower rhamnolipid concentrations. The best condition was 1.06 g chemical oxygen demand/g total suspended solids and 25 mg rhamnolipid/L or 25 mg rhamnolipid/g total suspended solids, which achieved a reduction of 50-75% in cellular yield coefficient.

Acai residual biomass can be a potential source for a wide range of applications, especially biotechnological, such as solid-state fermentation processes and production of fermentable sugars (mannose), such as raw material for food, pharmaceutical, and other industries. Fiber and seed of acai fruit (Euterpe oleracea) were characterized, after pulp extraction, in samples collected at different maturation stages (seasonality). The seed contained a remarkable amount of mannose (75%) followed by glucose (6%) and galactose (2%), with mannan as the main polysaccharide. The fiber was a lignocellulosic biomass containing glucose (30%) and xylose (19%). Mannan and cellulose in the seed and fiber, respectively, were the polysaccharide responsible for the crystallinity of both materials, with the seed showing higher values. The period of fruit harvesting (winter or summer crops) influenced the maturity and chemical composition of the residues, which had higher crystallinity in the summer season. Hydrothermal treatment was very efficient for fiber but failed for seed, indicating a high recalcitrance of mannan in acai seed.

This work evaluates the reduction of scum accumulation on the top surface of upflow anaerobic sludge blanket (UASB) reactors by the addition of hydrolytic enzymes in their feed. For over 1 year, two UASB reactors of 1.4 L were maintained at 30 °C and continuously fed with synthetic domestic wastewater (containing 150 mg/L of soybean oil) under a hydraulic retention time of 10 h. The Control reactor was only fed with synthetic wastewater. Beginning at the 226th day of operation, low-cost hydrolytic enzymes (obtained by solid-state fermentation of Aspergillus terreus, a fungus isolated from a primary sewage sludge) were added into the feed of the other reactor (Test) for a lipase activity of 24 U/L, considerably reducing the formation of scum. In the Test reactor, the scum showed oil and grease (O&G) concentration between 0.8 and 1.3 g/L and an accumulation rate of 20 to 27 mg O&G/d. In the Control reactor, the scum had values twice as high (1.5–2.5 g/L and 34–51 mg O&G/d, respectively) and there were more operational problems. During the entire period of operation, both reactors presented high chemical oxygen demand removal (>80%), with no loss of effluent quality due to the addition of the enzymes.

A multipurpose fixed-bed bioreactor was evaluated, to simplify the process of producing lipase and its application in biocatalysis. Rhizomucor miehei lipase was produced by solid-state fermentation (SSF) in the fixed-bed bioreactor and generated a fermented solid with 30 U/g lipase activity and a synthesis capacity of 80% (conversion at 6 h of reaction in a standard stirred reactor). The fermented solid was then dried by forced aeration in the same column (1.0 L air/min, 35 °C) and the dried fermented solid (5 wt.% water) showed the same synthesis capacity as the freeze-dried standard, with high storage stability at 4 °C and 25 °C (14 months with no decrease in synthesis capacity). The reaction performed in the multipurpose bioreactor (one-pot system) showed relatively low conversion (30% of the conversion obtained in the stirred reactor at 6 h) when only babassu cake was employed as the fermentation substrate, due to the preferred pathways formed as a consequence of bed retraction during drying. Therefore, SSF was carried out with a mixture of 60:40 wt.% babassu cake and palm fiber (used to give structure to the fermented solid and prevent retraction during drying), and the reaction in the one-pot system reached a maximum conversion of 71%.

Cocoa meal, the residue from cocoa processing, when used in animal feed can negatively affect health and performance because of the high concentrations of methylxanthines (theobromine and caffeine) and high fiber content. Solid-state fermentation (SSF) is one method to increase the use and value of the residue and to remove undesirable compounds. The fungus Aspergillus awamori IOC-3914 used in SSF was able to grow in cocoa meal, reduce methylxanthines and produce xylanase. Enzyme production reached about 66.5 U/g in 48 h before and 72 U/g after optimization. The xylanase showed good thermal stability, with residual activity between 97% and 90% after 4 h of incubation between 30 and 40 °C. The highest residual activity was obtained at pHs between 5.0 and 6.0. Aspergillus awamori reduced the theobromine and caffeine contents by about 69% and 63%, respectively, after the optimization process. Addition of feather meal significantly increased the reduction of methylxanthines. SSF in cocoa meal using A. awamori can expand the options for waste use, by producing enzymes and reducing the contents of undesirable caffeine and theobromine.

Several value-added products can be produced from glycerol and among these products glycerol carbonate (GC) has received much attention in recent years because of its physical properties and wide application in the chemical, pharmaceutical and food industries. As a continuation of our efforts to produce GC under environmentally acceptable conditions, we proposed a consecutive approach where lipase B from Candida antarctica was immobilized on Accurel MP1000 (CalBAcc) under continuous-flow conditions followed by glycerol carbonate production by a cascade process of triacylglycerol hydrolysis, biodiesel synthesis and esterification of the remaining glycerol with dimethyl carbonate towards GC production. Results of the immobilization of lipase B from Candida antarctica on a continuous-flow protocol demonstrated that a short residence time of 25 minutes could lead to 83% of protein loading. Screening of glycerol carbonate production in a packed bed reactor revealed that CalBAcc and N435 showed the best results of conversion and selectivity, with more selectivity by CalBAcc, that could lead to quantitative yields and excellent selectivities of the desired glycerol carbonate at residence times of 176 minutes when working with soybean and palm oil. Comparatively, the results obtained with CalBAcc were better than the ones with Novozym 435 since no full conversion was observed.

Enzymatic hydrolysis and anaerobic treatment of effluent similar to that generated in the fish processing industry were evaluated at 30 °C and 50 °C. Hydrolysis used lipase produced by fungus Penicillium simplicissimum in solid state fermentation with babassu cake as substrate, which has optimal activity at 50 °C. Hydrolysis kinetics was conducted with mixtures of effluent (containing 1500 mg oils and greases/L) and different lipase activities (0–0.67 U/ml of effluent), verifying that with 0.16 U/ml of effluent, 9.69 μmol/ml of free acids were produced after 4 h at 50 °C. Anaerobic biodegradation assays were conducted with effluent submitted to three different treatments: thermophilic (hydrolysis and anaerobic treatment at 50 °C), mesophilic (hydrolysis and anaerobic treatment at 30 °C) and hybrid (hydrolysis at 50 °C and anaerobic treatment at 30 °C). The best results (97.5% of chemical oxygen demand [COD] removal and 105.4 ml CH 4 /g COD removed ) were obtained with the hybrid treatment in only 68 h. The thermophilic hydrolysis not only reduced the amount of enzyme and the hydrolysis time but also reduced the time and the cost of mesophilic anaerobic treatment, favoring the application of this treatment on an industrial scale.

During cocoa (Theobroma cacao L.) processing, the accumulated cocoa shell can be used for bioconversion to obtain valuable compounds. Here, we evaluate the effect of solid-state fermentation of cacao flour with Penicillium roqueforti on secondary metabolite composition, phenol, carotenoid, anthocyanin, flavonol, and fatty acids contents, and antioxidant activity. We found that the total concentrations of anthocyanins and flavonols did not change significantly after fermentation and the phenolic compound and total carotenoid concentrations were higher. The fermentation process produced an increase in saponin concentration and antioxidant activity, as well as significant changes in the levels of oleic, linoleic, gamma-linolenic, and saturated fatty acids. Based on our findings, we propose that the reuse of food residues through solid state fermentation is viable and useful.

Enantiomerically pure product synthesis plays a key role in production of pharmaceuticals. The application of biocatalytic approaches represents a convenient and sustainable way for this purpose. The enormous potential of enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enantioselectivity is on increasing awareness. In particular, lipases show a high versatility to recognize non-natural substrates being successfully applied in regio- and stereoselective biotransformations. The immobilization technology on lipases is not only a requisite for the industrial implementation but it has been a great methodology to alter their catalytic properties to obtain highly enantio- and regiose-lective biocatalysts in the enantioselective synthesis of chiral drugs or drug intermediates. This review focuses on showing some of the latest advances on the use of immobilized lipases on pharmaceutical biotransformations.

The growth and magnetosome production of the marine magnetotactic vibrio Magnetovibrio blakemorei strain MV-1 were optimized through a statistics-based experimental factorial design. In the optimized growth medium, maximum magnetite yields of 64.3 mg/liter in batch cultures and 26 mg/liter in a bioreactor were obtained.

The need to find more stable catalysts has encouraged the study of naturally resilient enzymes, such as those found in extremophile organisms. In the present work, the influence of rare codons on the expression in Escherichia coli of the lipase Pf2001Δ60 from Pyrococcus furiosus was evaluated. Expression was carried out in two E. coli strains, BL21(DE3)pLysS and the rare tRNA supplemented Rosetta(DE3)pLysS. 32 factorial design was used to appraise the influence of temperature and inducer concentration on enzyme expression every hour for the 4-h expression period. Four response surfaces were constructed for each time, and the statistical parameters were evaluated. Lipase production was twice as high for Rosetta(DE3)pLysS than for BL21(DE3)pLysS. The factorial design indicated that optimal expression occurred at 30°C after 4h, with lipase production of 240U/L. The analysis of statistical parameters during the expression time showed that the velocity at which the enzyme was produced affected cell growth and maximum activity, with a higher speed leading to lower expression and cell growth. The presence of rare tRNAs prevented bottlenecks in lipase expression, and the experimental design was shown to be important for maximizing the production strategies and minimizing the metabolic load to which the host is subjected.

Enzyme immobilization has achieved important goals during the recent years trying to overcome the drawbacks of free enzyme catalysis. Up to now many strategies can be used in order to immobilize enzymes, some methods can be very simple such as adsorption or more complex as covalent immobilization. This chapter will cover some aspects of enzyme immobilization and its use on organic synthesis transformations.

Ricin is a toxic protein present in castor bean seeds ( Ricinus communis ). A toxic residue named castor bean waste is generated during biodiesel production process, such as that developed by PETROBRAS (the national petroleum company of Brazil). Solid-state fermentation (SSF) was used to detoxify castor bean waste through the Penicillium simplicissimum growth. After 24 h of fungal growth, the ricin was no longer identified by Sephadex G-50 gel chromatography. In order to verify the biological activity of ricin after several treatment stages, an in vitro assay using Vero cell line was carried out. Through this methodology, it was verified that after 24 and 48 h of treatment, the cell culture showed slightly growth inhibition. The waste was completely detoxified only after 72 h of fungal growth. This fact shows that an in vitro assay is important to verify the real efficiency of detoxification. Moreover, a relationship between the fungal protease production and the waste detoxification was observed.

Tailor-made oligosaccharides and polymers were investigated for a specific surface glycosylation of Candida antarctica lipase (fraction B) (CAL-B) already immobilized on octyl-Sepharose by interfacial activation. The chemical modification was performed in the N-terminal amino acid enzyme residue by using low oxidized aldehyde–dextran polymers through a reductive amination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. Circular dichroism experiments revealed interesting conformational changes in secondary and tertiary structures of the protein after modification. The formed immobilized glycosylated lipase biocatalysts were more stable, active, and selective toward different substrates than unmodified CAL-B. These immobilized conjugates were compared with a genetically glycosylated version of CAL-B expressed in Pichia pastoris immobilized in the same way. Enzyme thermostability was improved after chemical modification with Dextran-1500 and also by the genetic glycosylation, retaining 90–96 % activity after 24 h at 55 °C. The catalytic activity of CAL-B was improved by the incorporation of dextran polymers (Mw=1500 or 6000) more than twofold in the hydrolysis of p-nitrophenylbutyrate and more than threefold in the hydrolysis of methyl mandelate at pH 7. However, the activity of the genetically glycosylated CAL-B was threefold lower in the hydrolysis of both substrates. The enantioselectivity of CAL-B increased for all formed bioconjugates, with the Dextran-1500–CAL-B conjugate being the most selective in the hydrolysis of racemic methyl mandelate (up to 88.1 % ee at pH 5). This glycosylated CAL-B also demonstrated the highest synthetic activity in the transesterification of methyl butyrate with glycerol, with 80 % yield of monoglyceryl ester at 100 % conversion compared to 57 % yield obtained with unmodified CAL-B or other polymer–lipase conjugates.

In countries with a strong agricultural base, such as Brazil, the generation of solid residues is very high. In some cases, these wastes present no utility due to their toxic and allergenic compounds, and so are an environmental concern. The castor bean (Ricinus communis) is a promising candidate for biodiesel production. From the biodiesel production process developed in the Petrobras Research Center using castor bean seeds, a toxic and alkaline waste is produced. The use of agroindustrial wastes in solid-state fermentation (SSF) is a very interesting alternative for obtaining enzymes at low cost. Therefore, in this work, castor bean waste was used, without any treatment, as a culture medium for fungal growth and lipase production. The fungus Penicillium simplicissimum was able to grow and produce an enzyme in this waste. In order to maximize the enzyme production, two sequential designs-Plackett-Burman (variable screening) followed by central composite rotatable design (CCRD)-were carried out, attaining a considerable increase in lipase production, reaching an activity of 155.0 U/g after 96 h of fermentation. The use of experimental design strategy was efficient, leading to an increase of 340% in the lipase production. Zymography showed the presence of different lipases in the crude extract. The partial characterization of such extract showed the occurrence of two lipase pools with distinct characteristics of pH and temperature of action: one group with optimal action at pH 6.5 and 45°C and another one at pH 9.0 and 25°C. These results demonstrate how to add value to a toxic and worthless residue through the production of lipases with distinct characteristics. This pool of enzymes, produced through a low cost methodology, can be applied in different areas of biotechnology.

A residue from the primary treatment of a Wastewater Treatment Plant (WWTP) was used to isolate filamentous fungi with lipase production potential. Two of the 27 isolated fungi presented high hydrolysis index and were selected for lipase production by solid-state fermentation (SSF). The fermentations were conducted at 30 °C for 48 h, with moist air circulation, using 20% (w/w) of the residue mixture with a basal medium (agroindustrial residue—babassu cake), obtaining a solid enzymatic preparation (SEP) with lipase activity of 19 U/g with the fungus identified as Aspergillus terreus. Scum, collected in an anaerobic reactor operating in a WWTP, was hydrolyzed with SEP and subjected to anaerobic biodegradability tests at 30 °C. Different dilutions of crude (Control) or hydrolyzed scum in raw sewage were evaluated. The dilution of 5% (v/v) of hydrolyzed scum in raw sewage proved the most adequate, as it resulted in higher methane yield compared to the raw sewage (196 and 133 mL CH4/g CODadded, respectively), without increasing the chemical oxygen demand (COD) of the treated sewage (138 and 134 mg/L). The enzymatic hydrolysis of the scum, followed by dilution in the influent sewage, is technically feasible and increases methane production in anaerobic reactors.

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

A comparative study of Penicillium simplicissimum morphology and lipase production was performed using solid-state (SSF) and submerged (SmF) fermentation. SSF was carried out on babassu cake as culture medium and SmF on a semi-synthetic medium and a medium based on suspended babassu cake grains. Yield of product on biomass, specific activity and conidia production were 3.3-, 1.3- and 2-fold higher in SSF. In SmF, the type of fungus growth differed according to the medium. Using the semi-synthetic medium, the fungus formed densely interwoven mycelial masses without conidia production, whereas using the babassu-based medium the fungus formed free mycelia and adhered to the surfaces of the grains, producing conidia. The results show that babassu cake induces conidiation in SmF. In SSF, the fungus not only grew on the surface of the grains, producing conidia abundantly, but also effectively colonized and penetrated the babassu particles. The high conidia production and lipase productivity in SSF may be related to the low availability of nutrients or to other stimuli associated with this type of fermentation. Thus, the high production of the thermostable P. simplicissimum lipase, using a non-supplemented, low-cost agro-industrial residue as the culture medium, demonstrates the biotechnological potential of SSF for the production of industrial enzymes.

The castor bean (Ricinus communis) represents a potential candidate for biodiesel production. The Petrobras Research Center is developing a biodiesel production process from castor bean seeds, in which an unwanted byproduct named castor bean waste is produced. This extremely alkaline waste is toxic and allergenic and, as such, poses a significant environmental problem. Solid-state fermentation (SSF) of castor bean waste was carried out to achieve ricin detoxification, reduce allergenic potential and stimulate lipase production. The fungus, Penicillium simplicissimum, an excellent lipase producer, was able to grow and produce lipase enzyme. After an optimization process, the maximum lipase activity achieved was 44.8 U/g. Moreover, the fungus P. simplicissimum was able to reduce the ricin content to non-detectable levels in addition to diminishing castor bean waste allergenic potential by approximately 16%. In this way, SSF of castor bean waste by P. simplicissimum may increase the utility of the waste by promoting enzyme production and eliminating the principal toxic element, ricin.

Two alternative inoculation strategies for lipase production by the fungus Penicillium simplicissimum were tested in solid-state fermentation using a residue from the babassu oil industry (babassu cake). Conventional spore inoculation was compared with fungal pellets grown in liquid medium and with inocula consisting of fermented cake. Fungal pellets delayed lipase production whereas fermented cake accelerated enzyme synthesis, yielding a productivity of 0.45 U g−1 h−1, which is equivalent to the highest values obtained with conventional inocula. Therefore, a 22 factorial design was used to determine the best conditions for lipase production with fermented cake as inoculum strategy, varying the inoculum propagation time and inoculum concentration. Lipase activity and productivity reached 30 U g−1 and 0.63 U g−1 h−1, respectively, with 10% inoculum and 36 h. Thus, fermented cake inocula increased production 1.5-fold with 10 times fewer spores than in conventional inoculation, indicating that fermented solids are an interesting alternative for inoculum development in solid-state fermentation, mainly for large-scale processes. Copyright © 2007 Society of Chemical Industry

The effectiveness of cell-free rhamnolipid biosurfactant, derived from the culture medium at the end of fermentation was investigated for the removal of two different kinds of oil from contaminated sandy soils. The crude cultivation medium, containing 13.2 g L−1 of rhamnolipids, had a surface tension, interfacial tension and critical micellar concentration of 30 mN m−1, 2 mN m−1 and 60 mg L−1, respectively. The evaluation of biosurfactant in the culture medium (BM) and oil concentrations in the removal of oil from different contaminated sandy soil was performed using a statistical experimental design tool. Oil in sandy soil, containing predominantly aromatic or paraffinic hydrocarbons (5 to 10% w/w), was removed by as much as 91 and 78%, respectively, in the presence of reduced amounts of BM (6.3 to 7.9 g L−1). The progress of oil removal was monitored for 101 days and results indicated that removal efficiency in sandy soil with aromatic characteristics was relatively stable over the entire period. Based on these studies, it is concluded that use of a BM was effective in reducing oil concentrations in contaminated sandy soil. Copyright © 2007 Society of Chemical Industry

In the present work, packed bed bioreactors were employed with the aim of increasing productivity and scaling up of lipase production using Penicillium simplicissimum in solid-state fermentation. The influence of temperature and air flow rate on enzyme production was evaluated employing statistical experimental design, and an empirical model was adjusted to the experimental data. It was shown that higher lipase activities could be achieved at lower temperatures and higher air flow rates. The maximum lipase activity (26.4 U/g) was obtained at the temperature of 27°C and air flow rate of 0.8 L/min.O fungo Penicillium simplicissimum se mostrou, em trabalhos anteriores, um ótimo produtor de lipase por fermentação no estado sólido, quando cultivado em biorreatores do tipo bandeja, utilizando a torta de babaçu como meio de cultura. Com o objetivo de aumentar a produtividade e possibilitar uma ampliação de escala, foi investigado, no presente trabalho, o emprego de biorreatores de leito fixo com aeração forçada. Os biorreatores utilizados tinham 4 cm de diâmetro interno e 14 cm de altura útil. Empregando-se planejamento estatístico de experimentos como ferramenta, foram avaliadas as influências da temperatura e da vazão de ar sobre a produção de lipase nestes biorreatores. Os resultados obtidos permitiram ajustar um modelo empírico, o qual indicou que maiores atividades lipásicas são alcançadas para temperaturas mais baixas e vazões de ar mais altas. A atividade lipásica máxima (26,4 U/g) foi obtida para temperatura de 27°C e vazão de ar de 0,8 L/min.

The SSF lipase production from Penicillium simplicissimum using an agro industrial residue and the biosurfactant from Pseudomonas aeruginosa were employed for a combined pre-treatment of wastewater from poultry processing industry. During the hydrolysis step of the wastewater fat better results were obtained at 34°C with biosurfactant 0.4% (v/v) and liquid enzyme preparation 6.2% (v/v) or at 46°C with biosurfactant 0.1% (v/v) and liquid enzyme preparation 3.8% (v/v). The pre-hydrolysis in two different conditions of enzyme and biosurfactant concentrations at 34°C and posterior anaerobic treatment allowed the COD removal and methane production, while the no hydrolyzed wastewater besides the COD removal, did not show methane production. Those results suggest the importance and efficiency of the pre-treatment with home-made lipases and biosurfactant during the high fat content wastewater pre-treatment.

Enzyme extraction from solid matrix is as important step in solid-state fermentation to obtain soluble enzymes for further immobilization and application in biocatalysis. A method for the recovery of a pool of lipases from Penicillium simplicissimum produced by solid-state fermentation was developed. For lipase recovery different extraction solution was used and phosphate buffer containing Tween 80 and NaCl showed the best results, yielding lipase activity of 85.7 U/g and 65.7 U/g, respectively. The parameters with great impacts on enzyme extraction detected by the Plackett-Burman analysis were studied by Central Composite Rotatable experimental designs where a quadratic model was built showing maximum predicted lipase activity (160 U/g) at 25°C, Tween 80 0.5% (w/v), pH 8.0 and extraction solution 7 mL/g, maintaining constant buffer molarity of 0.1 M and 200 rpm. After the optimization process a 2.5 fold increase in lipase activity in the crude extract was obtained, comparing the intial value (64 U/g) with the experimental design (160 U/g), thus improving the overall productivity of the process.

We studied the immobilization of a recombinant thermostable lipase (Pf2001) from the hyperthermophilic archaeon Pyrococcus furiosus on supports with different degrees of hydrophobicity: butyl Sepabeads and octadecyl Sepabeads. The enzyme was strongly adsorbed in both supports. When it was adsorbed on these supports, the enzyme showed 140 and 237% hyperactivation, respectively. The assessment of storage stability showed that the octadecyl Sepabeads immobilized enzyme showed 100% of residual activity after 30 days of storage. However, the greatest stability at was obtained in butyl Sepabeads immobilized enzyme, which retained 77% activity after 1 hour incubation. The maximum activity of the immobilized preparations was obtained with the pH between 6 and 7, at . Thus, this study achieved a new extremophilic biocatalyst with greater stability, for use in several biotechnological processes.

Polyethyleneimine (PEI) modification on immobilized lipases greatly enhanced their enantioselectivity in the kinetic resolution of (±)-2-hydroxy-phenylacetic acid methyl ester. The enantiomeric ratio (E) of CNBr-agarose-CRL rose from 8 (without coating) to 20 (ee = 90%) after PEI coating in the hydrolysis at pH 5. In the case of CAL-B, the coating highly improved the enantioselectivity of the immobilized lipase from E = 1.5 (without coating) to E > 100 (ee > 99%). Moreover, this coating strategy improved the stability of the biocatalyst at high temperatures and in the presence of high co-solvent concentrations.

Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work’s aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions’ hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology.

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30 degrees C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28 degrees C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30°C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28°C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.

The immobilization of Candida antarctica (fraction B) lipase expressed in Pichia pastoris, a selective glycosylated protein at Asn 74, on a new heterofunctional support consisted of phenylboronic acid and aldehyde groups (Borald) has been performed. This method occurs via a two step mechanism: first orientation by organoborane interaction at neutral pH and a consecutive multi-point covalent attachment by aldehyde reaction at alkaline pH. The enzyme was specifically immobilized on this support in 70% yield at pH 7, oriented by the reaction of the hydroxyl groups on the sugar moiety with boronic acid on the support, whereas commercial CAL-B from Novozymes (non-glycosylated) was hardly immobilized at this pH ( 99% final immobilization yield. The Borald-CAL-B preparation was a very stable biocatalyst in the presence of high amount of solvent or high temperature (e.g. more than 10 fold in the presence of 60% (v/v) acetonitrile). An improvement of the specific activity up to 5 fold for example in the hydrolysis of methyl phenyl acetate was obtained compared with a one-point covalent preparation. An ee of 89% towards R isomer was achieved with this new immobilized biocatalyst in the enantioselective hydrolysis of methyl mandelate.

Neste estudo nós avaliamos o efeito da prostaglandina A1 (PGA1) na replicação do vírus Síndbis em células de macaco e em células de mosquito. Nas células tratadas com PGA1 nós observamos uma redução significativa na produção de partículas virais infecciosas. Em ambas as linhagens celulares tratadas com concentrações não-tóxicas de PGA1 (1-10 µg/ml) foi observado uma redução da replicação viral, de forma dose dependente, chegando a 90% na maior dose utilizada (10 µg/ml). A análise das proteínas virais e celulares marcadas com [35S]-metionina em SDS-PAGE mostrou que as proteínas virais (E1, E2 e C) foram normalmente sintetizas em células Vero tratadas com PGA1. Além disso, observamos também a indução das proteínas de estresse (HSP70 e HSP90) nas células Vero infectadas ou não infectadas. Em células Vero foi observado uma diminuição na incorporação de [3H] glicosamina nas glicoproteínas virais.