The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.

Micro-isolation of PCR-ready from fresh and dried legumes seeds and food products containing legumes (hummus) was tested. For optimization process magnetic microparticles PGMAox was used. Optimum weight plants and food material was 200 mg. For isolation of DNA from fresh legumes, mixture containing 500 L of CTAB extraction buffer, 1 L -mercaptoethanol and 500 L chloroform-octanol was used. For isolation of DNA from dried legumes seeds and food products volume of CTAB extraction buffer was increased to 1 000 L. To achieve higher purity of DNA some prepared homogenates was precipitate by isopropylalcohol. The optimized process of DNA isolation was used to prepare a homogenate from food products which DNA was isolated by different types of magnetic carriers. For comparison, non-porous magnetic carriers poly(glycidyl methacrylate) PGMAox, poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) P(HEMA-co-GMA)ox covered by carboxyl groups and porous fully crosslinked microparticles poly(styrene-co-divinylbenzene) HPS-B-M-22-NH2, magnetic porous glass MPG and nanoparticles of iron oxides covered by poly(L-lysine) PLL were used. In average the highest concentration and the best purity of DNA was isolated by magnetic carriers P(HEMA-co-GMA)ox a PGMAox.

The aim of the work was the selective isolation of bacterial DNA with help of magnetic carriers covered by streptavidine (PGMA-NH2-STV, MPG® Streptavidin). Conditions of functionalisation of carriers using two biotinylated probes were optimized: the amount of carrier, the amount of probe, binding of biotinyled probe to streptavidine. Purified DNA Lactobacillus was used for hybridization. DNA binding to the probe (DNA/DNA hybridization) and nospecific adsorption of DNA to the carrier were tested. Target DNA eluted from the carrier was identified using PCR with primers R16-1 and LbLMA1-rev and with primers P_eub and F_eub. The amount of probe bound to the carrier was estimated using UV spectrophotometry. It was estimated that biotinyled probe can be used for functionalisation in concentration 5 pmol/µl added to the carrier in the ration carrier : probe 1:1. It was shown that nonspecific DNA adsorption to the MPG® Streptavidin is significantly lower than to the carrier PGMA-NH2-STV.Using DNA/DNA hybridization and the MPG® Streptavidin, DNA from pure culture Lactobacillus was isolated. Procedure was applicated for DNA isolation from milk products.

Polymerase chain reaction (PCR) is molecular diagnostic method which allows the identification of lactic acid bacteria used in food industry. In this work species-specific PCR primers (targeted on highly conserved 16S rDNA region) were used for identification of bacteria of species Streptoccocus thermophilus in 10 randomly commercially accessible fermented milk products and for identification of species Streptococcus thermophilus in 25 lyophilisates collected in Culture Collection of Dairy Microorganisms Laktoflora (CCDM, Tábor, Czech Republic). The PCR products (968 bp) were detected using electrophoresis in 1,2 % agarose gel. Bacterial DNA was isolated from crude cell lysates by magnetic carriers P(HEMA co GMA) containing carboxyl groups. DNA was reversibly bind on their surface in the presence of high concentrations of poly(ethylene glycol) (PEG 6000) and sodium chloride. Phenol extraction of DNA was used as control. Streptococcus thermophilus strains were identificated using PCR in all analysed samples.

The theoretical part of the diploma thesis was focused on core-shell type magnetic carriers, used mainly in medical, molecular-biological and biochemical applications. Encapsulation of the core is essential for these applications due to the decrease od non-specific protein adsorbtion, increase of biocompatibility and the possible functionalization of magnetic carriers. In the experimental part, the DNA (E. coli) was amplified by real-time PCR in the presence of poly(hydroxymethacrylate-co-glycidylmethacrylate) (P(HEMA-co-GMA)) magnetic carriers with/without carboxyl groups. The inhibitory effect of different concentrations of magnetic carriers in the PCR mixture was evaluated from the calibration curve parameter values obtained by regression analysis. The presence of a specific PCR product was verified by agarose gel electrophoresis. Most of magnetic carriers without carboxyl groups extinguished the fluorescence in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, without inhibition of DNA amplification - the carriers were biocompatible. Magnetic carriers with carboxyl groups extinguished the fluorescence in the lower concentration range (0,4 – 4,0 g.l-1 in the PCR mixture). Their inhibition of amplification was in the concentration range of 2,0 – 4,0 g.l-1 in the PCR mixture, from the concentration 0,8 g.l-1 in the PCR mixture, the inhibition did not occur and the carriers were biocompatible. The results do not depend on the characteristic properties of the magnetic carriers but on the presence of the carboxyl groups on the surface of the carrier and the degree of coverage of the magnetic core by the polymer. Real-time PCR has become an effective tool for studying magnetic core encapsulation and the influence of functional groups on the surface of the polymeric layer.

Made available in DSpace on 2016-06-02T19:02:43Z (GMT). No. of bitstreams: 1 5706.pdf: 7869131 bytes, checksum: 3a35e736b3418ca357ef4fc2e657c0af (MD5) Previous issue date: 2013-10-16 / Universidade Federal de Minas Gerais / Enzymes are potent catalysts, but operationally fragile, expensive and soluble. Industrial applications of enzymes, often, are possible only using immobilized enzyme. Nowadays, various studies have been performed aiming to immobilize enzymes onto magnetic carriers, which allow the selective recovery of the derivative by applying an external magnetic field even in complex reaction media containing other suspended solids. There are many studies using magnetic carriers in enzymes immobilization procedures, however there are no commercially available enzymes immobilized onto magnetic materials. In these studies usually are used carriers with not ideal characteristics for applications in industrial processes. The present study aimed to develop new magnetic carriers and methods for immobilization of enzymes in these carriers, penicillin G acylase (PGA) and cellulases have been used as model enzymes. The thesis was divided into five parts, in the first part (Chapter 1) the state-of-art is presented. The second part (Chapter 2) describes the synthesis of magnetic carriers robust, cheap and with good characteristics for applications in bioprocesses. For this purpose were tested the synthesis of silica magnetic microparticles (SMMps) in water-in-oil micro-emulsion using sodium silicate as silica source and superparamagnetic iron oxide nanoparticles as magnetic core. Materials with good magnetic properties, high surface area and mesoporous structure were obtained. SMMps structure was characterized, it was possible to control the final structure of the material according to the synthesis conditions. In the third part of this study (Chapter 3) was evaluated a new concept in enzymes immobilization using magnetic materials. Magnetic tags were co-aggregated with PGA and cross-linked with glutaraldehyde, producing magnetic cross-linked enzymes aggregates (M-CLEAs). Several reaction conditions were tested producing M-CLEAs with different characteristics and strong response to external magnetic fields. Derivatives with good recovered activity and increased thermal and methanol 50% (v/v) stabilities were obtained. M-CLEAs presented superior performance, in comparison with the free enzyme, in penicillin G hydrolysis experiments, being reused for three reaction cycles without loss of activity. In the fourth part of this study (Chapter 4) the immobilization of the Trichoderma reesei cellulolytic complex onto 17 carriers using 60 different immobilization conditions was evaluated. Covalent methods to cellulases immobilization resulted in total loss of the enzymatic activity. The immobilization by adsorption allowed preserving a portion of the enzymatic activity, however, the enzyme was desorbed from the carrier with the increase in the ionic strength. The best results were achieved for adsorption in MANAE-agarose followed by cross-linking with glutaraldehyde. Hydrolysis experiments using insoluble substrates showed that it is possible to hydrolyze such substrates even using immobilized enzyme onto porous carriers. The derivative was reused for ten reaction cycles (hydrolysis of filter paper) saving more than 90% of its activity. Finally, in Chapter 5, the T. reesei cellulolytic complex was immobilized by adsorption onto SMMp activated with amino groups followed by glutaraldehyde cross-linking achieving good results in terms of recovered activity. / Enzimas são potentes catalisadores, porém frágeis operacionalmente, caras e solúveis. Aplicações industriais desses catalisadores, muitas vezes, são possíveis apenas com o uso de enzima imobilizada. Estudos indicam que o uso de suportes magnéticos para imobilizar enzimas pode permitir a recuperação seletiva do derivado através da aplicação de um campo magnético externo mesmo em meios complexos contendo outros sólidos em suspensão. Apesar de existirem muitos estudos empregando suportes magnéticos para imobilização de enzimas, não existem enzimas imobilizadas em materiais magnéticos disponíveis comercialmente. Nestes estudos geralmente são utilizados suportes magnéticos com características não ideais para aplicações em bioprocessos. O presente estudo teve como principal objetivo o desenvolvimento de novos suportes magnéticos e métodos para imobilização de enzimas nestes suportes, a enzima penicilina G acilase (PGA) e celulases foram utilizadas como modelo. O estudo foi dividido em cinco partes, no Capítulo 1 é apresentada uma introdução indicando o estado da arte. O Capítulo 2 apresenta o preparo de novos suportes magnéticos robustos, baratos e com características ótimas para aplicações em bioprocessos. Nesta etapa foi testada a síntese de micro-partículas magnéticas de sílica (SMMps) em micro-emulsão água-em-óleo, empregando silicato de sódio como fonte de sílica e nanopartículas superparamagnéticas de óxido de ferro como núcleo magnético. Os materiais obtidos apresentaram excelentes propriedades magnéticas, alta área de superfície e estrutura mesoporosa. A partir da caracterização físico-química e morfológica das SMMps foi possível controlar a estrutura final do material de acordo com as condições de síntese. No Capítulo 3 foi avaliado um novo conceito em imobilização de enzimas empregando materiais magnéticos. Neste estudo etiquetas magnéticas foram co-agregadas com PGA e entrecruzadas com glutaraldeído, gerando agregados enzimáticos entrecruzados com propriedades magnéticas (M-CLEAs). Várias condições reacionais foram testadas rendendo M-CLEAs com diferentes características e com resposta robusta a campos magnéticos externos. Derivados imobilizados com boa atividade recuperada e incremento na estabilidade térmica e frente a metanol 50% (v/v) foram obtidos. M-CLEAs apresentaram desempenho superior ao observado para a enzima livre em experimentos de hidrólise de penicilina G, sendo reutilizados por três ciclos reacionais sem perda de atividade. No Capítulo 4 foi avaliada a imobilização do complexo celulolítico de Trichoderma reesei em 17 suportes, empregando 60 diferentes condições de imobilização. Os experimentos de imobilização realizados empregando técnicas de imobilização por união covalente ocasionaram perda total de atividade enquanto métodos de imobilização por adsorção permitiram conservar boa atividade enzimática, porém a enzima dessorveu do suporte com o aumento na força iônica do meio. Os melhores resultados foram alcançados para adsorção em MANAE-agarose seguido de entrecruzamento com glutaraldeído. Experimentos de hidrólise de substratos insolúveis mostraram que é possível hidrolisar este tipo de substrato mesmo com enzima imobilizada em suportes porosos. O derivado foi reutilizado por dez ciclos (hidrólise de papel filtro) conservando mais de 90% de sua atividade. Por fim, no Capítulo 5, o complexo celulolítico de T. reesei foi imobilizado por adsorção em SMMp ativado com grupos amino seguido de entrecruzamento com glutaraldeído apresentando bons resultados em termos de atividade recuperada.