Triagem de suportes orgânicos e protocolos de ativação na imobilização e estabilização de lipase de Thermomyces lanuginosus

Lipase from Thermomyces lanuginosus was covalently immobilized on activated poly-hydroxybutyrate, sugarcane bagasse and the chemically modified hybrid hydrogel chitosan-alginate prepared by different strategies. Among the tested supports, chitosan-alginate chemically modified with 2,4,6-trinitrobenzenesulfonic acid rendered derivatives with the highest hydrolytic activity and thermal-stability, 45-fold more stable than soluble lipase and was then selected for further studies. The pH of maximum activity was similar for both immobilized and free lipase (pH 8.0) while optimum temperature was 5 - 10 ºC higher for the immobilized lipase. Higher yields in the butyl butyrate synthesis were found for the derivatives prepared by activation with glycidol and epichlorohydrin.

Imobilização e caracterização de lacase e seu uso na biodegradação de efluentes de indústrias papeleiras

Laccase from Aspergillus sp was immobilized on glutaraldehyde-activated chitosan beads. A comparative study between free and immobilized laccase was conducted and the potential of the resulting immobilized derivative in the biodegradation of pulp and paper mill effluent was evaluated. The immobilized laccase is more resistant to various denaturing conditions, which allows for the reduction of 65% of the phenols (total and low molecular weight) and loss of 60% of total color in the effluent. These results show the potential of the immobilized laccase in the biodegradation of phenols, the chemical agents responsible for the high toxicity of the effluent generated in cellulose pulp industries.

Imobilização de lacase de Aspergillus sp. em quitosana e sua aplicação na bioconversão de fenóis em reatores de leito fixo

The immobilization of laccase on chitosan by cross-linking and application of the immobilized laccase in the bioconversion of phenolic compounds in batch and fixed bed reactors were studied. The process for immobilization of enzyme was optimized using a rotational central composite design. The optimized conditions to generate immobilized laccase with maximal activity were determined to be a glutaraldehyde concentration of 1.0% (v/v), a pH of 6.0, an immobilization time of 5.0 hours and an enzyme concentration of 5.2 g L-1. In packed bed reactors, the activity of the immobilized enzyme is maintained for a longer time in the bioconversion of 2,6-dimethoxyphenol than in the bioconversion of syringaldazine.

Obtenção e caracterização de matriz apropriada para sistemas de liberação prolongada: estudos de liberação dos herbicidas atrazina e diuron

This study aimed to produce and characterize a novel material from fish scales and chitosan for use as a medium for the extended release of herbicides. The mechanism of release for the herbicides atrazine and diuron was influenced by diffusion and swelling according to the power law kinetic model. The atrazine release time was seven days, while that of diuron was four days. The results of this study will contribute to the development of environmental matrices for herbicide release systems.

Nanocompósito de polpa de mamão e nanopartículas de quitosana para aplicação em embalagens

Nanocomposite materials have been incorporated into biopolymers, (e.g. hydroxypropyl methylcellulose), to improve their physical and chemical properties and enable them to be applied in food packaging, especially for their biodegradable and renewable properties. With this addition, fruit puree has been incorporated into the films to confer nutritional properties besides color and flavor. Chitosan is of interest in the packaging field since it is a biodegradable, bioabsorbable, antimicrobial agent. Furthermore, chitosan nanoparticles have been widely explored for their interesting properties and potential applications in food packaging. This work was divided into two stages: (1) chitosan nanoparticle synthesis; (2) addition of nanoparticles into HPMC and papaya puree films. Addition of chitosan nanoparticles to HPMC and papaya puree films improved film properties: mechanical, thermal and water vapor barrier. We have developed a novel nanomaterial with great potential for application in packaging to prolong the shelf life of food.

Puuhydrolysaatin esikäsittely foulaantumisen vähentämiseksi ultrasuodatuksessa

Tässä kandidaatintyössä tutkittiin, voidaanko puuhydrolysaatin esikäsittelyllä vähentää sen ultrasuodatuksen aikana esiintyvää kalvojen foulaantumista. Kokeellinen osa rajattiin koskemaan hydrolysaatin käsittelyä lakkaasientsyymillä, flokkulanteilla, kitosaanilla sekä adsorbentin ja lakkaasientsyymin yhdistelmällä. Esikäsittelyjen tavoitteena oli poistaa tai hajottaa hydrolysaatin sisältämää ligniiniä. Käsittelyjen teho testattiin suodattamalla eri tavoin käsiteltyä hydrolysaattia laboratoriokokeissa. Kokeissa adsorbentin ja lakkaasientsyymin yhdistelmäkäsittely osoittautui parhaaksi. Suodatuskapasiteetti parani ja foulaantuminen väheni verrattuna esikäsittelemättömän hydrolysaatin suodatukseen. Ligniiniä poistui käsittelyssä, kuten tavoitteena oli. Suodatuksessa kuitenkin edelleen tapahtui foulaantumista ja esikäsittelyn tehoa tulisi siksi vielä parantaa. Myös esikäsittely lakkaasientsyymillä paransi suodatuskapasiteettia ja vähensi foulaantumista, mutta vähemmän kuin entsyymi-adsorbenttikäsittely.

Development of porous glass-fiber reinforced composite for bone implants. Evaluation of antimicrobial effect and implant fixation

Cranial bone reconstructions are necessary for correcting large skull bone defects due to trauma, tumors, infections and craniotomies. Traditional synthetic implant materials include solid or mesh titanium, various plastics and ceramics. Recently, biostable glass-fiber reinforced composites (FRC), which are based on bifunctional methacrylate resin, were introduced as novel implant solution. FRCs were originally developed and clinically used in dental applications. As a result of further in vitro and in vivo testing, these composites were also approved for clinical use in cranial surgery. To date, reconstructions of large bone defects were performed in 35 patients. This thesis is dedicated to the development of a novel FRC-based implant for cranial reconstructions. The proposed multi-component implant consists of three main parts: (i) porous FRC structure; (ii) bioactive glass granules embedded between FRC layers and (iii) a silver-polysaccharide nanocomposite coating. The porosity of the FRC structure should allow bone ingrowth. Bioactive glass as an osteopromotive material is expected to stimulate the formation of new bone. The polysaccharide coating is expected to prevent bacterial colonization of the implant. The FRC implants developed in this study are based on the porous network of randomly-oriented E-glass fibers bound together by non-resorbable photopolymerizable methacrylate resin. These structures had a total porosity of 10–70 volume %, of which > 70% were open pores. The pore sizes > 100 μm were in the biologically-relevant range (50-400 μm), which is essential for vascularization and bone ingrowth. Bone ingrowth into these structures was simulated by imbedding of porous FRC specimens in gypsum. Results of push-out tests indicated the increase in the shear strength and fracture toughness of the interface with the increase in the total porosity of FRC specimens. The osteopromotive effect of bioactive glass is based on its dissolution in the physiological environment. Here, calcium and phosphate ions, released from the glass, precipitated on the glass surface and its proximity (the FRC) and formed bone-like apatite. The biomineralization of the FRC structure, due to the bioactive glass reactions, was studied in Simulated Body Fluid (SBF) in static and dynamic conditions. An antimicrobial, non-cytotoxic polysaccharide coating, containing silver nanoparticles, was obtained through strong electrostatic interactions with the surface of FRC. In in vitro conditions the lactose-modified chitosan (chitlac) coating showed no signs of degradation within seven days of exposure to lysozyme or one day to hydrogen peroxide (H2O2). The antimicrobial efficacy of the coating was tested against Staphylococcus aureus and Pseudomonas aeruginosa. The contact-active coating had an excellent short time antimicrobial effect. The coating neither affected the initial adhesion of microorganisms to the implant surface nor the biofilm formation after 24 h and 72 h of incubation. Silver ions released to the aqueous environment led to a reduction of bacterial growth in the culture medium.

DNA-lipid systems: A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.

Experimental Studies on Non-Metallic Composite Bone Implants With a Special Reference to Staphylococcal Biofilm Infection

Non-metallic implants made of bioresorbable or biostable synthetic polymers are attractive options in many surgical procedures, ranging from bioresorbable suture anchors of arthroscopic surgery to reconstructive skull implants made of biostable fiber-reinforced composites. Among other benefits, non-metallic implants produce less interference in imaging. Bioresorbable polymer implants may be true multifunctional, serving as osteoconductive scaffolds and as matrices for simultaneous delivery of bone enhancement agents. As a major advantage for loading conditions, mechanical properties of biostable fiber-reinforced composites can be matched with those of the bone. Unsolved problems of these biomaterials are related to the risk of staphylococcal biofilm infections and to the low osteoconductivity of contemporary bioresorbable composite implants. This thesis was focused on the research and development of a multifunctional implant model with enhanced osteoconductivity and low susceptibility to infection. In addition, the experimental models for assessment, diagnostics and prophylaxis of biomaterial-related infections were established. The first experiment (Study I) established an in vitro method for simultaneous evaluation of calcium phosphate and biofilm formation on bisphenol-Aglycidyldimethacrylate and triethylenglycoldimethacrylate (BisGMA-TEGDMA) thermosets with different content of bioactive glass 45S5. The second experiment (Study II) showed no significant difference in osteointegration of nanostructured and microsized polylactide-co-glycolide/β-tricalcium phosphate (PLGA /β-TCP) composites in a minipig model. The third experiment (Study III) demonstrated that positron emission tomography (PET) imaging with the novel 68Ga labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) CD33 related sialic-acid immunoglobulin like lectins (Siglec-9) tracer was able to detect inflammatory response to S. epidermidis and S. aureus peri-implant infections in an intraosseous polytetrafluoroethylene catheter model. In the fourth experiment (Study IV), BisGMATEGDMA thermosets coated with lactose-modified chitosan (Chitlac) and silver nanoparticles exhibited antibacterial activity against S. aureus and P. aeruginosa strains in an in vitro biofilm model and showed in vivo biocompatibility in a minipig model. In the last experiment (Study V), a selective androgen modulator (SARM) released from a poly(lactide)-co-ε-caprolactone (PLCL) polymer matrix failed to produce a dose-dependent enhancement of peri-implant osteogenesis in a bone marrow ablation model.

Roll-to-roll atomic layer deposition process for flexible electronics applications

Atomic Layer Deposition (ALD) is the technology of choice where very thin and highquality films are required. Its advantage is its ability to deposit dense and pinhole-free coatings in a controllable manner. It has already shown promising results in a range of applications, e.g. diffusion barrier coatings for OLED displays, surface passivation layers for solar panels. Spatial Atomic Layer Deposition (SALD) is a concept that allows a dramatic increase in ALD throughput. During the SALD process, the substrate moves between spatially separated zones filled with the respective precursor gases and reagents in such a manner that the exposure sequence replicates the conventional ALD cycle. The present work describes the development of a high-throughput ALD process. Preliminary process studies were made using an SALD reactor designed especially for this purpose. The basic properties of the ALD process were demonstrated using the wellstudied Al2O3 trimethyl aluminium (TMA)+H2O process. It was shown that the SALD reactor is able to deposit uniform films in true ALD mode. The ALD nature of the process was proven by demonstrating self-limiting behaviour and linear film growth. The process behaviour and properties of synthesized films were in good agreement with previous ALD studies. Issues related to anomalous deposition at low temperatures were addressed as well. The quality of the coatings was demonstrated by applying 20 nm of the Al2O3 on to polymer substrate and measuring its moisture barrier properties. The results of tests confirmed the superior properties of the coatings and their suitability for flexible electronics encapsulation. Successful results led to the development of a pilot scale roll-to-roll coating system. It was demonstrated that the system is able to deposit superior quality films with a water transmission rate of 5x10-6 g/m2day at a web speed of 0.25 m/min. That is equivalent to a production rate of 180 m2/day and can be potentially increased by using wider webs. State-of-art film quality, high production rates and repeatable results make SALD the technology of choice for manufacturing ultra-high barrier coatings for flexible electronics.

Molecular adjuvant interleukin-33 enhances the antifertility effect of Lagurus lagurus zona pellucida 3 DNA vaccine administered by the mucosal route

It has been shown that cytokines can act as molecular adjuvant to enhance the immune response induced by DNA vaccines, but it is unknown whether interleukin 33 (IL-33) can enhance the immunocontraceptive effect induced by DNA vaccines. In the present study, we explored the effects of murine IL-33 on infertility induced by Lagurus lagurus zona pellucida 3 (Lzp3) contraceptive DNA vaccine administered by the mucosal route. Plasmid pcD-Lzp3 and plasmid pcD-mIL-33 were encapsulated with chitosan to generate the nanoparticle chi-(pcD-Lzp3+pcD-mIL-33) as the DNA vaccine. Sixty female ICR mice, divided into 5 groups (n=12/group), were intranasally immunized on days 0, 14, 28, and 42. After intranasal immunization, the anti-LZP3-specific IgG in serum and IgA in vaginal secretions and feces were determined by ELISA. The results showed that chi-(pcD-Lzp3+pcD-mIL-33) co-immunization induced the highest levels of serum IgG, secreted mucosal IgA, and T cell proliferation. Importantly, mice co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) had the lowest birth rate and mean litter size, which correlated with high levels of antibodies. Ovaries from infertile female mice co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) showed abnormal development of ovarian follicles, indicated by atretic follicles and loss of oocytes. Our results demonstrated that intranasal delivery of the molecular adjuvant mIL-33 with chi-pcD-Lzp3significantly increased infertility by enhancing both systemic and mucosal immune responses. Therefore, chi-(pcD-Lzp3+pcD-mIL-33) co-immunization could be a strategy for controlling the population of wild animal pests.

β-carotene biotransformation to obtain aroma compounds

Carotenoids are important constituents of food due to their color and because their degradation products generate important volatile compounds in foods. Aroma compounds derived from carotenoids are widely distributed in nature, and they are precursors of many important aromas in foods such as fruits and in flowers as well. They present high aromatic potential and are therefore of great interest to the industries of aromas and fragrances. In this study, more than 300 previously isolated microorganisms with potential for biotransformation of β-carotene present in the culture medium were selected using the plate method; about 80 strains presented capacity to produce aroma compounds and 7 strains were selected by an untrained panel of tasters to generate aroma compounds. The β-ionone was the main compound produced by CS1 (34.0 mg.L-1) and CF9 (42.4 mg.L-1) microorganisms at 72 and 24 hours of fermentation, cultured with and without pre-inoculation, respectively. The β-damascone and pseudoionone were found in low concentrations, 1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalen (TTN) was tentatively identified and other compounds such as apocarotenoids, apparently obtained from the cleavage of the central part of the carotenoid, were detected.

Functionalisation of spruce O-acetyl-galactoglucomannans for barrier and composite applications

The increasing use of energy, food, and materials by the growing population in the world is leading to the situation where alternative solutions from renewable carbon resources are sought after. The growing use of plastics depends on the raw-oil production while oil refining are politically governed and required for the polymer manufacturing is not sustainable in terms of carbon footprint. The amount of packaging is also increasing. Packaging is not only utilising cardboard and paper, but also plastics. The synthetic petroleum-derived plastics and inner-coatings in food packaging can be substituted with polymeric material from the renewable resources. The trees in Finnish forests constitute a huge resource, which ought to be utilised more effectively than it is today. One underutilised component of the forests is the wood-derived hemicelluloses, although Spruce Oacetyl-galactoglucomannans (GGMs) have previously shown high potential for material applications and can be recovered in large scale. Hemicelluloses are hydrophilic in their native state, which restrains the use of them for food packaging as non-dry item. To cope with this challenge, we intended to make GGMs more hydrophobic or amphiphilic by chemical grafting and consequently with the focus of using them for barrier applications. Methods of esterification with anhydrides and cationic etherification with a trimethyl ammonium moiety were established. A method of controlled synthesis to obtain the desired properties by the means of altering temperature, reaction time, the quantity of the reagent, and even the solvent for purification of the products was developed. Numerous analytical tools, such as NMR, FTIR, SEC-MALLS/RI, MALDI-TOF-MS, RP-HPLC and polyelectrolyte titration were used to evaluate the products from different perspectives and to acquire parallel proofs of their chemical structure. Modified GGMs with different degree of substitution and the correlating level of hydrophobicity was applied as coatings on cartonboard and on nanofibrillated cellulose-GGM films to exhibit barrier functionality. The water dispersibility in processing was maintained with GGM esters with low DS. The use of chemically functionalised GGM was evaluated for the use as barriers against water, oxygen and grease for the food packaging purposes. The results show undoubtedly that GGM derivatives exhibit high potential to function as a barrier material in food packaging.

The effect of age on the structure and compostition of the cell walls of Choanephora cucurbitarum

The effect of age on the structure and composition of isolated and purified cell walls from cultures of Choanephora cucurbitarum was investigated by microchemical analyses, visible and infrared spectrophotometry, x-ray diffractometry and electron microscopy. Qualitative evaluation revealed the presence of lipids, proteins, neutral sugars, strong alkali soluble sugars, chitin, chitosan and uronic acids in the cell walls of both the 1 and 7 day old cultures. As the mycelium aged, there was a slight but statistically significant increase in the protein content, and a pronounced rise in the chitin and neutral sugar constituents of the cell walls. Conversely, the decrease in the chitosan content during this period had the net effect of altering the chitin: chitosan ratio from near unity in the younger cultures, to a 2:1 ratio in the 7 day old cell wall samples. Glutaraldehyde-osmium fixed thin sections of the 1 day old vegetative hyphae of £. curbitarum revealed the presence of a monolayered cell wall, which upon aging became bilayered. Replicas of acid hydrolysed cell walls demonstrated that both the 1 and 7 day old samples possessed an outer layer which was composed of finely granular amorphous material and randomly distributed microfibrils. The deposition of an inner secondary layer composed of parallel oriented microfibrils in the older hypha was correlated with an increase in the chitin content in the cell wall. The significance of these results with respect to the intimate relationship between composition and structure is discussed.

Investigations on the host-parasite interface of a mycoparasite system /

The cell wall composition of Choanephora cucur - bitarum and the host-parasite interface, after infection with Piptocephalis virginiana , were examined in detail. The cell walls of C_. cucurbitarum were determined to be composed of chitin (17%), chitosan (28.4%), neutral sugars (7.2%),uronic acid (2.4%), proteins (8.2%) and lipids (13.8%). The structure of hyphal walls investigated by electron microscopy of shadowed replicas before and after alkali-acid hydrolysis, showed two distinct regions: microfibrillar and amorphous. The microfibrils which were composed of mainly chitin, were organized into two distinct layers: an outer, thicker layer of randomly orientated microfibrils and an inner, thin layer of parallel microfibrils.Electronmicrographs of the host-parasite interface of C_. cucurbitarum and the mycoparasite , P_. virginiana , 30 h following inoculation, showed that the sheath zone has a similar electron density to that of the host cell wall. The sheath was not present around the young (18 h old) haustorium. High-resolution autoradiographs of infected host hyphae showed that radioactive N-acetyl-D-glucosamine , a precursor of chitin, was incorporated preferentially in the host cell wall and sheath zone. Cell fractionation of label fed hyphae showed that 84% of the label was present in the cell wall and specifically in the chitin portion of the wall. The antifungal antibiotic, Polyoxin D, a specific inhibitor of the enzyme, chitin synthetase, suppressed the incorporation of the label in the cell wall and sheath zone and resulted in a decrease in electron density of the developing sheath. The significance of these results is discussed in the light of host resistance.