Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A biotecnologia microbiana tem evoluído muito nas últimas décadas, principalmente pela utilização de diferentes micro-organismos para produção de inúmeros bioprodutos utilizados nos diferentes setores industriais e ambientas. As enzimas desempenham um importante papel catalítico em inúmeras reações metabólicas existentes, e as de origem microbiana apresentam diversas vantagens em relação as de origem animal e vegetal. A reutilização de resíduos agroindustriais oriundos da indústria alimentícia na elaboração de meios de produção de produtos biotecnológicos, tem surgido como uma alternativa econômica viável, pois diversos nutrientes descartados, apresentam um elevado teor nutricional que pode ser reaproveitado pelos micro-organismos nos processos fermentativos. As proteases são enzimas que constituem um grande grupo de enzimas hidrolíticas que catalisam a hidrólise de proteínas e as degradam em pequenos peptídeos e aminoácidos. A relevância deste grupo de enzimas, ricas em diversidade e mecanismos de ação estrutural se reflete na importância de suas aplicações em processos industriais. Foram realizados ensaios de seleção de amostras de Aspergillus niger (SIS 18, SIS 19 e SIS 20) em diferentes temperaturas (28, 30, 40oC) e diferentes pH ( 6, 7, 8) para a obtenção da melhor amostra para a produção de protease. Após a seleção da melhor amostra, foram realizados ensaios de produção da enzima através de fermentação submersa utilizando 4 diferentes meios. Os ensaios ocorreram em câmara incubadora com agitação orbital, 150 rpm, 37oC, 96 horas, onde foram realizados a determinação do pH, da atividade enzimática e curva de crescimento. Em seguida, após a seleção do melhor meio de produção, foram realizados novos ensaios utilizando planejamento fatorial 22 completo com 4 repetições, utilizando os resíduos de soro de leite e de sorvete, nas mesmas condições cinéticas pré-estabelecidas. Os resultados obtidos indicaram que a amostra de A. niger (SIS 18) apresentou a formação do maior halo característico de 3,0 cm, com 96 h de crescimento. Os ensaios realizados com diferentes meios de produção em fermentação submersa, indicaram que o meio denominado 3, apresentou uma atividade proteolítica de 0,104 U/mL, após 96 horas de fermentação. No planejamento fatorial 22, utilizando meios contendo substratos agroindustriais, o melhor resultado obtido foi no ensaio 2, que apresentou uma atividade proteolítica de 0,118U/mL com o soro de leite e 0,093U/mL para o resíduo de sorvete.Esses resultados validam que a utilização de resíduos agroindustriais tem sido uma alternativa viável para produção de protease em fermentação submersa, e com isso pode reduzir os custos de produção e o descarte desses resíduos no meio ambiente. / Microbial biotechnology has evolved a lot in the last decades, mainly by the use of different microorganisms for the production of numerous bioproducts used in the different industrial and environmental sectors. Enzymes play an important catalytic role in numerous metabolic reactions, and those of microbial origin have several advantages over those of animal and plant origin. The reuse of agroindustrial residues from the food industry in the elaboration of means of production of biotechnological products has emerged as a viable economic alternative because several discarded nutrients present a high nutritional content that can be reused by the microorganisms in the fermentative processes. Proteases are enzymes that constitute a large group of hydrolytic enzymes that catalyze the hydrolysis of proteins and degrade them in small peptides and amino acids. The relevance of this group of enzymes, rich in diversity and mechanisms of structural action, is reflected in the importance of their applications in industrial processes. Sampling assays of Aspergillus niger (SIS 18, SIS 19 and SIS 20) were carried out at different temperatures (28, 30, 40C) and different pH (6, 7, 8) to obtain the best sample for the production of Protease. After the selection of the best sample, enzyme production assays were performed by submerged fermentation using 4 different media. The tests were carried out in an orbital shaker, 150 rpm, 37oC, 96 hours, where the pH, the enzymatic activity and growth curve were determined. Then, after the selection of the best production medium, new assays were performed using a 22 complete factorial design with four replicates using milk serum and ice cream residues, under the same pre-established kinetic conditions. The results indicated that the A. niger (SIS 18) sample showed the formation of the largest characteristic halo of 3.0 cm, with 96 h of growth. The assays performed with different production media in submerged fermentation indicated that the medium 3 was a proteolytic activity of 0.104 U/mL after 96 hours of fermentation. In factorial design 22, using media containing agroindustrial substrates, the best result obtained for test 2, which presented a proteolytic activity of 0.118 U/mL with serum of milk and 0.093 U/mL for ice cream residue.These results validate that the use of agroindustrial residues has been a viable alternative for the production of protease in submerged fermentation, and with this can reduce the costs of production and the discard of these residues in the environment.

The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.

Bibliography: pages 147-169. / Butyrivibrio fibrisolvens H17c is a gram-negative obligate anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to investigate the enzymes produced by B. fibrisolvens H17c involved in the degradation of cellulose, xylan, and protein. A library of chromosomal DNA fragments from B. fibrisolvens H17c was established in the plasmid pEcoR251, an Escherichia coli positive selection vector. The library was screened for genes expressing cellulase, xylanase, and protease activity. Two genes expressing endo-β-1,4-glucanase and cellodextrinase activity were cloned in E. coli as host. The gene expressing endo-β-1,4-glucanase activity (end1) was cloned on a recombinant plasmid pES400. The end1 gene was located on a 6.8 kb DNA fragment and expressed from its own promoter in the E. coli host. It was shown that 64% of the endoglucanase activity was located in the periplasm of the E. coli host. TnphoA mutagenesis indicated the presence of a functional E. coli-like signal peptide. The nucleotide sequence of end1 was determined and the amino acid sequence (547 amino acids) deduced. The catalytic domain of End1 showed very good similarity to the catalytic domain of the Clostridium thermoceiium EGE endoglucanase. End1 also has a non-catalytic domain similar to the binding domains of the CenA and Cex cellulases from Ceilulomonas fimi The gene expressing cellodextrinase activity (ced1) was cloned on a recombinant plasmid pES500. This gene was located on a 3.55 kb fragment and was also expressed from its own promoter in the E. coli host. The Ced1 enzyme was also exported to the periplasm of the E. coli host, but did not contain a functional E. coli-like signal peptide. The nucleotide sequence was determined and the deduced amino acid sequence (547 residues) showed high similarity to the catalytic domain of the C. thermocellum EGD endoglucanase. The proteins of End1 and Ced1 showed no similarity. The End1 and Ced1 enzymes were characterized using a range of different substrates. The End1 enzyme showed optimal activity at pH 5.6 and 45°C. Optimal activity for the Ced1 enzyme was obtained at pH 6.6 and 50°C. The proteolytic activity of B. fibrisolvens H17c was characterized using gelatin-SD5-PAGE. Ten bands of protease activity with apparent molecular weights ranging between 42 000 and 101 000 were detected at different stages during the growth cycle. The effect of protease inhibitors indicated that all ten protease bands were serine proteases. Optimal activity was observed between pH 6.0 to 7.5 and at a temperature of 50°C. The proteolytic activity of B. fibrisolvens H17c varied depending on the type of carbohydrate substrate in the medium, and was positively correlated with the growth rate.

Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.

Orientador: Silvio Roberto Andrietta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-09-11T21:08:51Z (GMT). No. of bitstreams: 1 Aquino_DeniseSilvade_M.pdf: 2817268 bytes, checksum: 75e30246006dba478e363bb0b37da638 (MD5) Previous issue date: 2006 / Resumo: Dextranas são polissacarídeos produzidos pela bactéria pertencente à família Lactobacileae constituídos de moléculas de glicose unidas por ligações a-(1-6) na cadeia principal e ligações a-(1-4), a-(1-3) e a-(1-2) nas ramificações. A enzima dextranasacarase, responsável por sua síntese, é extracelular e tem a sacarose como principal indutor. Este biopolímero possui aplicações nas indústrias farmacêuticas, químicas e de alimentos. Na indústria farmacêutica é que a dextrana tem a sua maior aplicação. A dextrana de baixa massa molecular, dextrana clínica, é utilizada como expansor volumétrico sanguíneo e como facilitador da fluidez do sangue. Este trabalho teve como objetivo otimizar o processo de produção de dextrana por três novas linhagens de bactérias denominadas 4-03, 4-30 e 4-26 isoladas da cana-de-açúcar, dentro deste objetivo principal estão incluídos a caracterização da estrutura de cadeia destes biopolímeros e a identificação das linhagens das bactérias isoladas. Para a otimização, tanto da produção de dextrana como da produção de dextrana-sacarase, realizaram-se ensaios em frascos agitados, analisando a influência das seguintes variáveis: concentração de tampão (controle do pH), concentração de substrato (sacarose) e concentração da fonte de nitrogênio (extrato de levedura). Conduziram-se os ensaios de otimização com base em planejamento experimental fatorial e análise de superfície de resposta. As linhagens 4-26 e 4-30 apresentaram modelos estatisticamente significativos, portanto, podem ser utilizados na previsão da composição do meio de fermentação para produção da dextrana-sacarase. Para a otimização da produção de dextrana, somente a linhagem 4-26 mostrou variação significativa estatisticamente das variáveis respostas frente ás variáveis estudadas na região avaliada. Realizaram-se ensaios para a obtenção da dextrana a qual teve sua estrutura de cadeia determinada utilizando o método da perioxidação e o método da degradação de Smith. A primeira metodologia consiste em determinar os tipos e as proporções das ligações a-(1-6), a-(1-4) e a-(1-2), e a-(1-3) por meio do consumo do oxidante e produção de ácido quando as dextranas são sujeitas a uma oxidação com periodato. A segunda metodologia citada, consiste na oxidação do polissacarídeo seguida de redução ao poliálcool correspondente e por fim realiza-se hidrólise ácida gerando fragmentos de Dgliceraldeído, característico de ligações a- (1,2), D-glicose, característico de ligações a-(1,3), eritritol, característico de ligações a- (1,4), glicerol e glicoaldeído, característicos de ligações a- (1,6) e terminal não-redutor. Quando compara-se os dois métodos de determinação da estrutura de cadeia da dextrana, a solubilidade em água da dextrana formada pela linhagem 4-03 é confirmada, pois a sua cadeia em ambas as análises apresentou uma maior porcentagem das ligações a-(1,6), característica contrária a goma produzida pela linhagem 4-30, que apresenta-se de forma insolúvel por ter uma cadeia ramificada. O resultado para o biopolímero formado pela linhagem 4-26 apresentou-se os mesmos valores para as duas metodologias, porém no método da degradação de Smith sua estrutura não aproximou-se da dextrana padrão / Abstract: Dextran is a polysaccharide produced by bacteria of the Lactobacillaceae family and it consists of D-glucose monomeric units linked at the position a-(1,6) in the linear chain and a-(1-4), a-(1-3) and a-(1-2) positions at the branching points. The enzyme dextransucrase, responsible for dextran synthesis, is extracellular and has sucrose as its main inductor. This biopolymer is used by the pharmaceutical, chemistry and the food industry. Dextran has its main application in the pharmaceutical industry. The low molecular weight dextran, the clinical dextran, is used as blood volume expander and blood flow improver. This work had as objective to optimize the dextran production by three new bacteria strains named 4-03, 4-26 and 4-30 isolated from sugar cane, into this main objective are including the characterization the structure of the chain of these bacteria biopolymers and to identification bacteria strains isolated. The influence of the following variable was analyzed for the dextransucrase and dextran production optimization: buffer (pH control), substrate (sucrose) and nitrogen source (yeast extract) concentrations. The experimental assays were performed on the basis of factorial design and response surface techniques. The strains 4-26 and 4-30 presented statically significant models, therefore, these models may be used for predict the optimum composition of the fermentation medium for dextransucrase production. Only strain 4-26 showed statically significant variation of the dependent variables in the evaluated region dextran production optimization. The dextran structure of the three strains was determined by the periodate oxidation and Smith degradation methods. The first methodology consist in to determine kinds and proportions of the a-(1,6), a-(1-2) e a-(1-4) e a-(1-3) linkages by the consumption of oxidant and the production of acid when the dextran is subject to periodate oxidation analysis. The second methodology consists in the oxidation of the polysaccharide followed by reduction and hydrolysis with dilute acid which produces characteristic fragments: D-glyceraldehydes, characteristic of the a-(1,2) linkages, D-glucose, characteristic of the a-(1,3) linkages, erythritol, characteristic of the a-(1,4) linkages and glycerol or glycolaldehyde, characteristic of the a-(1,6) linkages and no reducing terminal. The results obtained confirmed the high solubility of the dextran produced by strain 4-03 due to its high proportion of the a-(1,6) linkages in contrast with gum produced by strain 4- 30 that is highly insoluble due to its branched chain. The biopolymer produced by strain 4- 26 presented the same values for the both methodologies, however, the Smith degradation result showed that its structure is not close to the standard dextran / Mestrado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

With respect to a multienzyme complex of deoxyribonucleoside triphosphate (dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory has demonstrated the existence of a multienzyme complex in T4-infected E. coli, named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP synthesis to DNA replication and organization of the complex has not been well established. This study had two objectives. The first objective was to test the specific hypothesis that gp32, the single-stranded DNA binding protein encoded by gene 32, plays a role in recruiting enzymes of dNTP synthesis to the replisome and in organizing the dNTP synthetase complex. By use of two newly created gene 32 mutants along with several experimental approaches, DNA-cellulose chromatography, coimmunoprecipitation, and glutathione-S-transferase pull downs, interactions of gp32 with thymidylate synthase (gptd), ribonucleotide reductase (gpnrdA/B), and E. coli NDP kinase have been identified. These results support the hypothesis that gp32 helps to recruit enzymes of dNTP synthesis to DNA replication sites. As the second objective, I investigated contributions of two host proteins, E. coli nueleoside diphosphate kinase (NDP kinase) and adenylate kinase (Adk), to the organization of the complex. As an important step to understand roles of E. coli NDP kinase in the complex, I identified direct interactions of E. coli NDP kinase with gpnrdA/B, dCMP hydroxymethylase (gp42), and dihydrofolate reductase (gpfrd) by means of coimmunoprecipitation and glutathione-S-transferase pull-down experiments. Interestingly, these interactions were influenced by the presence of substrate nucleotides or an analog for E. coli NDP kinase, suggesting that metabolite flux may affect the preference of E. coli NDP kinase binding to enzymes in the complex in vivo. Meanwhile, Adk involvement in DNA precursor synthesis has been suggested, particularly in phage T4-infected E. coli, from observations of increased thermostability of temperature-sensitive Adk in situ. The involvement of E. coil Adk in the complex was demonstrated by identifying some proteins of the T4 dNTP synthetase complexgp42, dNMP kinase (gpl), gpfrd, and E. coli NDP kinasedirectly interacting with Adk, implying that E. coil Adk would be properly located in the complex to efficiently carry out the conversion of dNDPs to dNTPs. This implication was supported by measurements of T4 DNA synthesis. Taken together, this research strongly supports the idea of connection of dNTP synthesis to DNA replication and allows us to take a step toward understanding the organization of the complex at DNA replication sites. / Graduation date: 2005

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase is the enzyme responsible for catalyzing the first reaction in the biosynthesis of KDO. KDO is an essential component in the cell wall of Gram-negative bacteria and plants. This compound is not present in mammals; therefore the enzymes responsible for its biosynthesis are potential targets for the development of new antibiotic agents. KDO8P synthase catalyzes the condensation reaction between phosphoenol pyruvate (PEP) and D-arabinose 5-phosphate (A5P) to form KDO8P. Two types of KDO8P synthase have been identified; a metal-dependent type and a non metal-dependent type. KDO8P synthase from the organism Chlorobium tepidum (Cte) has been partially purified and partially characterized. In line with predictions based on sequence alone, the activity of this enzyme is dependent on the presence of a divalent metal ion and is sensitive to the presence of the metal chelating agent EDTA. Cte KDO8P synthase was found to have the highest activity in the presence of Mn2+ or Cd2+. KDO8P synthase from the organism Acidithiobacillus ferrooxidans (Afe) has also been cloned, purified and biochemically characterized. Afe KDO8P synthase was also found to be a metallo enzyme and the catalytic activity is highest in the presence of Mn2+ or Co2+. Afe KDO8P synthase was found to exist as a tetramer in solution and is most active within the pH range of 6.8 to 7.5 and within a temperature range of 35 ºC to 40 ºC. Sequence analysis suggests that this enzyme has characteristics conserved throughout the metallo and the non-metallo KDO8P synthases and is closely related to the metal-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthases. The role of several active-site residues of Afe KDO8P synthase has been investigated. A C21N mutant of Afe KDO8P synthase was found to retain 0.5% of wildtype activity and did not require a divalent metal ion for catalytic activity. This suggests that the metallo and non-metallo KDO8P synthases have similar catalytic mechanisms.

Thesis (Ph.D.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

Orientadores: Telma Teixeira Franco, Sueli Rodrigues / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-19T11:18:02Z (GMT). No. of bitstreams: 1 Honorato_TalitaLopes_D.pdf: 6860909 bytes, checksum: 13811fc8b567923905d4a11f6cfd1a1a (MD5) Previous issue date: 2011 / Resumo: Estudos sobre quitina e quitosana têm atraído interesse devido à sua possível conversão em oligossacarídeos, que são solúveis em água e apresentam atraentes atividades biológicas. Para este fim, métodos eficazes para a produção de amino-oligossacarídeos (AGO's) potencialmente bioativos são necessários. O trabalho objetivou estudar a viabilidade de Fermentação em Estado Sólido (FES) para a produção de um extrato enzimático adequado para a obtenção de AGO's. Inicialmente foi identificado um fungo adequado para a FES utilizando cascas de camarão como substrato, sendo uma cepa de Trichoderma polysporum selecionada. A otimização das condições da fermentação para obtenção de um extrato enzimático capaz de produzir AGO's com grau de polimerização em torno de 5, grau de polimerização considerado o mínimo adequado para bioatividades foi realizada e as enzimas produzidas por T. polysporum sob diferentes condições de fermentação (submersa e sólida) foram analisados por eletroforese, seguida pela atividade de coloração e por atividades enzimáticas utilizando substratos cromogênicos específicos. Atividades enzimáticas de ß-N-acetilglucosaminidase, quitobiosidase e uma pequena quantidade de exo-quitinase e endo-quitinase foram observadas. O extrato da FES foi utilizado para degradar parcialmente diferentes quitosanas com fração molar de N-acetilglicosamina (FA) de valor 0,27 ou 0,56, e uma quitosana cormecial com FA em torno de 0,15. Os AGO's produzidos foram caracterizados por cromatografia em camada delgada e espectrometria de massa. Uma metodologia para separação de padrões oligoméricos acetilados e desacetilados por cromatografia líquida com detecção amperométrica pulsada dos oligossacarídeos foi desenvolvida e otimizou-se a hidrólise enzimática para produção dos AGO's de quitosanas. Hetero-oligômeros bioativos com grau de polimerização entre 2 e 7 foram produzidos, apresentando atividade antimicrobiana em Pseudomonas syringae e potencializando a explosão oxidativa em células de arroz, utilizando quitosana como elicitor (ambas atividades foram encontradas em concentrações de 50 µg/mL de AGO's) / Abstract: Studies on chitin and chitosan have drawn interest because of their possible conversion into oligosaccharides, which are water-soluble and present attractive biological activities. To this end, cost effective methods for the production of potentially bio-active chito-oligosaccharides (COS) are required. In this work we decided to study the feasibility of using solid state fermentation (SSF) for the production of a suitable enzyme extract for the eventual obtention of COS. The first step was the identification of a suitable fungus for solid satet fermentation (SSF) using shrimp shells as a substrate, and a strain of Trichoderma polysporum was eventually selected. The second step involved the optimisation of growth conditions to obtain enzyme preparations capable of producing COS with degrees of polymerisation of at least 5, this typically being the minimum DP for reliable bio-activities. The SSF extract was used to partially degrade different well characterised chitosans with a molar fraction of N-acetylglucosamine (FA) value of 0.27 or 0.56, and a commercial chitosan obtained from Sigma (FA around 0.15). The chitosanolytic enzymes produced by T. polysporum under different fermentation conditions were analysed by electrophoresis followed by activity staining and the enzyme activities of ß-N-acetylglucosaminidase, chitobiosidase and a small amount of exo-chitinase and endo-chitinase were observed. After hydrolysis, the complex mixture was precipitated, and the supernatant containing the COS produced was analysed using thin layer chromatography (TLC) and quadrupole time-of-flight tandem mass spectrometry. We detected the presence of chitinase rather than chitosanase activity and the production of hetero-oligomers with degrees of polymerisation between 2 and 7. The bio-activity of these oligomer mixtures in rice plants cells and against the growth of Pseudomonas syringae was founded / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

Made available in DSpace on 2017-06-01T18:20:27Z (GMT). No. of bitstreams: 1 dissertacao_ubirajara_samuel.pdf: 732291 bytes, checksum: ac5aa588af8d07c0d025bd3c436c0f36 (MD5) Previous issue date: 2009-12-29 / The sewage sludge is a resultant residue of the system of residuary biological water treatment proceeding, presents in its chemical composition one high concentration of organic components and inorganic, that after treatments, can be used in agriculture, however its application to the ground modifies many times the physical parameters, chemical and biological of the ground. The ground is a complex system that a variety of microhabitats with different physical and chemical gradients understands and discontinues ambient conditions. The metal presence weighed in the sewer sludge, limits its use in the processes of fertilization of the ground, had to the high degree of toxicity through the absorption for the plants, that will feed the herbivorous, the metals can enter in the alimentary chain, arriving at the consumers first-class and the man, and the high index of pathogenicity proceeding from pathogenic microorganisms gifts. The action of microbiota present in not barren ground and the plants, is one of the main factors of removal of pathogenic microorganisms that arrive with the sewer at the ground, contaminating this environment many times of irreversible form. The process of mineralization of the organic substance is catalyzed by different enzymes, produced in its majority for microorganisms gifts in the ground. Assays of isolation, identification and enzymatic detention in mesophilic bacteria had been carried through gifts in the silt of sewer collected in the Station Hose in different temperatures (28 and 37oC) and in the presence and/or absence of NaCl. They had been detected in both tested temperatures, pathogenic bacteria (Escherichia coli, Pseudomonas sp, Alcaligenes sp). In screening enzymatic carried through, in the resulted detention of amylase the best ones had been gotten in the samples of the 28 ºC isolated mesophilic bacteria and in the resulted detention of urease the best ones had been gotten in the 37 ºC samples / O lodo de esgoto é um sub-produto residuário das empresas de tratamento de águas, apresenta, é um dos principais fatores de remoção de microrganismos patogênicos que chegam com o esgoto ao solo, contaminando esse ambiente muitas vezes de forma irreversível. O processo de mineralização da matéria orgânica é catalisado por diferentes enzimas, em sua composição química uma alta concentração de componentes orgânicos e inorgânicos, que após tratamentos, pode ser utilizado na agricultura, porém a sua aplicação ao solo altera muitas vezes os parâmetros físicos, químicos e biológicos do solo. O solo é um sistema complexo que compreende uma variedade de microhabitats com diferentes gradientes físicos e químicos e condições ambientais descontínuas. A presença de metais pesados no lodo de esgoto, limita a sua utilização nos processos de fertilização do solo, devido ao alto grau de toxicidade através da absorção pelas plantas, que alimentarão os herbívoros, os metais podem entrar na cadeia alimentar, chegando aos consumidores de primeira ordem e ao homem, e o alto índice de patogenicidade proveniente de microrganismos patogênicos presentes. A ação da microbiota presente nos solos não estéreis e nas plantas produzidas na sua maioria por microrganismos presentes no solo. Foram realizados ensaios de isolamento, identificação e detecção enzimática em bactérias mesofílicas presentes no lodo de esgoto coletado na Estação Mangueira em diferentes temperaturas (28 e 37oC) e na presença e/ou ausência de NaCl. Foram detectados em ambas temperaturas testadas, bactérias patogênicas (Escherichia coli, Pseudomonas sp, Alcaligenes sp). No screening enzimático realizado, na detecção da amilase os melhores resultados foram obtidos nas amostras de bactérias mesofílias isoladas a 28 ºC e na detecção de urease os melhores resultados foram obtidos nas amostras a 37 ºC