Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Orientadores: Anete Pereira de Souza, Valéria Maia Merzel / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T03:05:12Z (GMT). No. of bitstreams: 1 Beloti_LilianLuzia_D.pdf: 11283696 bytes, checksum: dfedaea17b072c7d899caa1ccd4e9032 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The complete abstract is available with the full electronic document / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

Abstract Two regions of the genome of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H were characterized, representing 14 % of the phage genome. The first region of 2497 bp contained genes encoding phage structural proteins and the second region of 2498 bp genes involved in lytic functions. The nucleotide sequences of the major capsid protein gene g34, a putative capsid morphogenesis gene (ORF178A), the gene mur encoding phage cell wall hydrolase (lysin), the gene hol (ORF107) encoding the cell membrane permeabilizing phage holin, and six other genes with unknown function were found. Identification of these genes was performed by amino acid sequencing of their encoded proteins (genes g34 and mur), by their physiological effect on E. coli (genes hol and mur), by sequence comparison (genes mur, hol, ORF178A), and by biochemical analysis of their encoded purified protein (gene mur). A promoter for the capsid protein encoding gene cluster was determined by primer extension method. A purification method suitable for large scale processing (cation exchange chromatography by expanded bed adsorption method) was developed for the phage LL-H lysin protein Mur. Purified Mur was biochemically determined as a N-acetylmuramidase, which was effective on cell walls of Lb. delbrueckii, Lb. helveticus, Lb. acidophilus and Pediococcus damnosus. Some biotechnological applications for the lysis genes hol and mur or the purified protein Mur are suggested. Mur digests E. coli cell walls inefficiently, but could still be used for lysis of E. coli. Coexpression of the phage LL-H lysin and holin genes yielded to lysis of the E. coli host only at low culture densities. Therefore, some chemicals were tested for their ability to trigger lysis of E. coli cells overexpressing the phage LL-H gene mur. Thymol was found to mimic the physiological effects of the phage holin in a bacterial growth state independent manner. An efficient lysis method utilizing intracellular production of Mur and triggering the lysis with thymol was developed.

Polyether compounds are a subgroup of natural products with regular occurrence of multiple C-O-C motifs. The biosynthetic origin of polycylic polethers has been studied and the majority of them are derived from polyketide or terpene pathways. Normally, the polycyclic polyethers can be divided into two groups based on their structural features: the first group features multiple rings that are interconnected by carbon-carbon single bond, which are produced by a biosynthetic cascade of exo epoxide-opening reactions; the other group has multiple fused cyclic ethers and are formed by an cascade of endo epoxide-opening reactions. Karenia brevis (K. brevis) is known as principle harmful bloom (HAB) organism of the Gulf of Mexico which can cause red tides. Brevetoxins (PbTx) are a suit of cyclic polyether ladder compounds produced by K. brevis. Brevetoxins are neurotoxins that can bind to voltage-gated sodium channels in nerve and muscle cells, resulting in disruption of normal neurological processes causing the human illness which is clinically described as neurotoxic shellfish poisoning (NSP). Inspired by Cane-Celmer-Wesley’s proposal regarding monensin biosynthesis, Nakanishi and Shimizu proposed a biosynthetic pathway for brevetoxin which suggests that PKS-mediated synthesis of the polyene is followed by epoxidation to afford a polyepoxide which then undergoes an epoxide-opening cascade, catalyzed by an epoxide hydrolase (EH). To find evidence to support the hypothesis that an epoxide hydrolase from polyether ladder producing dinoflagellates will catalyze the construction of the polyether ladder framework from polyepoxide substrates, and to study the role of epoxide hydrolase in the biosynthesis of polyether ladder compounds, it is necessary to identify and produce one or more epoxide hydrolase from dinoflagellates. The methods to detect epoxide hydrolase activity in K. brevis and different techniques to obtain epoxide hydrolases from K. brevis are discussed. A microsomal EH identified from a K. brevis EST library was cloned and expressed. The characterization of this EH, including substrate selectivity and enantioselectivity as well as its potential to catalyze the critical ento-tet cyclization epoxy alcohol, is discussed.

8 Abstract Epoxide hydrolases (EHs) demonstrating high degree of enantioselectivity or enantioconvergence are useful biocatalysts for the production of optically active epoxides and vicinal diols, which can serve as chiral building blocks for syntheses of biologically active drugs. EHs can play an important role also in degradations of xenobiotics. Genes encoding EHs Kau2 and Kau8 were expressed in E. coli host strains TOP10 and RE3. Enantioselectivities and regioselectivities of Kau2 and Kau8 in supernatants of desintegrated cells were determined for four substrates: tert-butylglycidyl ether, para-chlorostyrene oxide, para-nitrostyrene oxide, α-methylstyrene oxide. The highest values of enantioselectivity and regioselectivity were achieved with Kau2 and para-nitrostyrene oxide as a substrate. The Kau2 was chosen for further experiments on the basis of these results. Kau2 was overexpressed in the recombinant strain RE3(pSEKau2). We performed two batch cultures and one fed-batch culture in stirred bioreactor. The highest volumetric activity of 4500 U/l was obtained in the case of fed-batch culture. Two phase system consisting of polyethylenglycole 6000 and sodium citrate (pH 7.7) was used for Kau2 purification from the supernatant of desintegrated cells. Purification factor 2.6 +/- 0.3 was achieved and...

No description available.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. We identified FAAH in Physcomitrella patens and expressed heterologously in E. coli using Gateway cloning system. Radiolabeled NAE 16:0 and 20:4 were used as substrates to test amide hydrolase activity in vitro. In order to understand the role of PpFAAH in vivo, knock out (KO) and overexpressors (OE) were generated by homologous recombination. PpFAAH KO construct was generated by inserting 5‟- and 3‟-flanking regions into pMP1159 plasmid. Full length PpFAAH with stop codon was cloned into pTHUBlGATE vector in order to make OE construct. KO and OE constructs were then transformed into protoplasts of P. patens by using PEG-mediated transformation to generate mutant lines. To identify potential interacting proteins of PpFAAH, it was cloned into pDEST15 plasmid with N-terminus GST tag. Interaction between GST-tagged PpFAAH and proteins from 14-day old protonema will be visualized by SDS-PAGE and then subjected to LC-MS/MS analysis for identification. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in P. patens.

N-acylethanolamines (NAEs) are present in wide range of organisms and belong to family of functionally diverse signaling lipids. They consist of a fatty acid with varying chain lengths and ethanolamine linked via an amide bond. The level of NAEs is modulated by their hydrolysis to ethanolamine and fatty acid by an enzyme fatty acid amide hydrolase (FAAH). FAAH is an integral membrane protein that belongs to “amidase signature” superfamily of proteins, which is characterized by highly conserved region rich in serine, glycine and alanine. FAAH directly or indirectly plays a role in modulation of various physiological processes by regulating NAE levels. Although the role of NAEs and its key modulator FAAH has been studied in other plants, their role in tomato model is limited and unknown. More recently, SlFAAH1, an ortholog of AtFAAH1, was identified in tomato and cloned into bacterial expression system. However, putative SlFAAH1 function and distinct features are yet to be determined. It is hypothesized that the putative SlFAAH1 catalyzes the hydrolysis of NAEs and modulates the level of NAEs during the seedling development in tomato. To this extent, a putative SlFAAH1 (previously identified and cloned in pET-23a vector) will be biochemically characterized and also effect of NAEs on seedling development will be studied. Thus far, SlFAAH1 cloned in pET-23a vector was expressed in RIL cell line (prokaryotic expression system) followed by conformation of positive transformant by colony PCR. Currently, protein expression and confirmation of SlFAAH in the positive transformant is being done. The expressed protein will be characterized for its hydrolytic activity using radiolabelled substrate. The effect of exogenous NAEs during seedling development will be studied with regards to expression level of SlFAAH1 by qPCR and composition of NAE during the seedling development to determine the role of NAE during seedling development. Thus, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development. Long-term studies will identify the significance of highly conserved NAE pathway in eukaryotes.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. We identified FAAH in Physcomitrella patens and expressed heterologously in E. coli using Gateway cloning system. Radiolabeled NAE 16:0 and 20:4 were used as substrates to test amide hydrolase activity in vitro. In order to understand the role of PpFAAH in vivo, knock out (KO) and overexpressors (OE) were generated by homologous recombination. PpFAAH KO construct was generated by inserting 5'- and 3'-flanking regions into pMP1159 plasmid. Full length PpFAAH with stop codon was cloned into pTHUBlGATE vector in order to make OE construct. KO and OE constructs were then transformed into protoplasts of P. patens by using PEG-mediated transformation to generate mutant lines. To identify potential interacting proteins of PpFAAH, it was cloned into pDEST15 plasmid with Nterminus GST tag. Interaction between GST-tagged PpFAAH and proteins from 14-day old protonema will be visualized by SDS-PAGE and then subjected to LC-MS/MS analysis for identification. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in P. patens.

N-acylethanolamines (NAEs) are present in wide range of organisms and belong to family of functionally diverse signaling lipids. They consist of a fatty acid with varying chain lengths and ethanolamine linked via an amide bond. The level of NAEs is modulated by their hydrolysis to ethanolamine and fatty acid by an enzyme fatty acid amide hydrolase (FAAH). FAAH is an integral membrane protein that belongs to “amidase signature” superfamily of proteins, which is characterized by highly conserved region rich in serine, glycine and alanine. FAAH directly or indirectly plays a role in modulation of various physiological processes by regulating NAE levels. Although the role of NAEs and its key modulator FAAH has been studied in other plants, their role in tomato model is limited and unknown. More recently, SlFAAH1, an ortholog of AtFAAH1, was identified in tomato and cloned into bacterial expression system. However, putative SlFAAH1 function and distinct features are yet to be determined. It is hypothesized that the putative SlFAAH1 catalyzes the hydrolysis of NAEs and modulates the level of NAEs during the seedling development in tomato. To this extent, a putative SlFAAH1 (previously identified and cloned in pET-23a vector) will be biochemically characterized and also effect of NAEs on seedling development will be studied. Thus far, SlFAAH1 cloned in pET-23a vector was expressed in RIL cell line (prokaryotic expression system) followed by conformation of positive transformant by colony PCR. Currently, protein expression and confirmation of SlFAAH in the positive transformant is being done. The expressed protein will be characterized for its hydrolytic activity using radiolabelled substrate. The effect of exogenous NAEs during seedling development will be studied with regards to expression level of SlFAAH1 by qPCR and composition of NAE during the seedling development to determine the role of NAE during seedling development. Thus, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development. Longterm studies will identify the significance of highly conserved NAE pathway in eukaryotes.