The members of the genus Streptomyces are well-known for their capacity to synthesize a vast repertoire of secondary metabolites, including many useful antibiotics and proteins. Streptomyces clavuligerus is the producer of the medically important &amp / #946 / -lactam antibiotics such as cephamycin C and the potent &amp / #946 / -lactamase inhibitor clavulanic acid. The aspartate pathway of S. clavuligerus is an important primary metabolic pathway providing substrates for &amp / #946 / -lactam synthesis. This pathway uses L-aspartic acid as the precursor for the biosynthesis of the amino acids L-lysine, L-methionine, L-isoleucine, L-threonine and several important metabolic intermediates. L-&amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) required for &amp / #946 / -lactam synthesis is a catabolic product of L-lysine produced from the lysine branch of the aspartate pathway. The carbon flow through the L-lysine-specific branch of aspartate pathway is limiting for the formation of cephamycin C. Formation of L-homoserine from aspartate semialdehyde (ASA) is the first step of the other branch of the aspartate pathway leading to L-threonine, L-isoleucine and L-methionine synthesis and is catalyzed by homoserine dehydrogenase (HSD, EC 1.1.1.3). Regulation of the activity or biosynthesis of the HSD of S. clavuligerus determines the availability of ASA for the biosynthesis of L-lysine and &amp / #945 / -AAA. The gene encoding for homoserine dehydrogenase (hom) was previously cloned from S. clavuligerus NRRL 3585 and characterized in our laboratory. In this study, the hom gene was disrupted via insertion of a kanamycin resistance cassette into this gene which was subsequently transferred to S. clavuligerus cells using the Streptomyces plasmid vector pIJ486. A hom mutant of S. clavuligerus (AK39) was formed through integration into the chromosome by double crossing over and the effects of hom disruption on cephamycin C yields were investigated. Disruption of hom gene resulted in a 1.7 to 2.0 fold increase in specific cephamycin C production in chemically defined medium (CDM).

Streptomyces clavuligerus is a gram-positive, filamentous bacterium which has a great ability to produce secondary metabolites including isopenicillin N, cephamycin C and a beta-lactamase inhibitor clavulanic acid. Clavulanic acid (CA) which is a bicyclic beta-lactam, inhibits most of class A beta-lactamases by binding irreversibly to the serine hydroxyl group at the active center of beta-lactamases and resulting in the stable acyl-enzyme complexes. Clavaminate synthase (CAS) is one of the best characterized enzymes in the CA biosynthesis pathway regarding its mechanism, activity and structure. It exists as two isoenzymes, CAS1 and CAS2. cas1 gene is located in clavam biosynthetic gene cluster and its expression is nutritionally regulated. cas2 gene encodes for the rate limiting CAS2 isoenzyme which catalyzes the three distinct oxidative transformations. Conversion of deoxyguanidinoproclavaminic acid into guadinoproclavaminic acid takes part in early steps of CA biosynthesis and is catalysed by CAS2. Similarly, proclaviminic acid is converted into claviminic acid by a two-step reaction and also catalysed by CAS2. Integration of an extra copy of the cas2 gene into the chromosome of a clavulanic acid over-producer industrial strain of Streptomyces clavuligerus by means of an integration vector to improve CA production capacity of the industrial strain is the main goal of this study. Via comperative CA analysis based on HPLC, it was estimated that the recombinant strains produced higher amount of CA in comparison to the parental industrial S. clavuligerus strain. The highest CA production achieved by the recombinant strain, namely S. clavuligerus GV61 (1583.3 &mu / g/g) corresponded to more than 2 fold increase in the maximal CA titer of the parental strain.

Streptomyces clavuligerus produces industrially important secondary metabolites such as cephamycin C (a &beta / -lactam antibiotic) and clavulanic acid (a potent &beta / -lactamase inhibitor). Cephamycin C is active against penicillin-resistant bacteria due to presence of methoxyl group in C-7 position of cephalosporin nucleus. Clavulanic acid is prescribed in combination with &beta / -lactams for treatment of various bacterial infections. Cephamycin C and clavulanic acid gene clusters form &beta / -lactam supercluster in S. clavuligerus genome. CcaR (Cephamycin C-Clavulanic Acid Regulator), encoded by ccaR, located in cephamycin C gene cluster, is a positive regulator of &beta / -lactam supercluster. Previous studies on cephamycin C gene cluster have used different techniques, such as S1 nuclease (Paradkar et al., 1994), Northern blot (Perez-Llarena et al., 1997), and Western blot (Alexander and Jensen, 1998) to determine expression of cephamycin C genes at mRNA level and to identify their functions at protein level, and they have studied on different parts of the cluster. Hence, a comprehensive study is needed to understand molecular mechanisms of pathway-specific regulation of cephamycin C production by S. clavuligerus. In this study, time-dependent expression levels of cephamycin C gene cluster in a ccaR-disrupted mutant and ccaR-overexpressed recombinant strain of S. clavuligerus as compared to those in the wild strain were analysed by RT-PCR and qRT-PCR. In addition, DNA-binding sequences of CcaR on cephamycin C gene cluster were examined by EMSA. The effect of ccaR disruption and overexpression on cephamycin C and clavulanic acid yields were determined by bioassay and HPLC. Three polycistronic and two monocistronic transcripts were obtained by RT-PCR. CcaR regulation showed its effect on mostly ccaR, lat, cmcI, cefD, blp and cefF expression levels. qRT-PCR data was supported by EMSA showing CcaR binding to lat, cefD&ndash / cmcI and ccaR promoters. ccaR overexpression from multi-copy recombinant plasmid resulted in significant increase in cephamycin C and clavulanic acid yields, making the respective recombinant strain as an attractive industrial strain. qRT-PCR data presented herein constitute the first that reveal the effect of CcaR activator on the expression of cephamycin C genes in a time-dependent manner.

No description available.

In Streptomyces clavuligerus, the route to the biosynthesis of &amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) represents an important primary metabolic pathway providing carbon flux to the synthetases of antibiotic formation. This carbon flow comes through the lysine-specific branch of the aspartate pathway and is rate limiting in the formation of cephamycin C, a second generation cephalosporin produced by this organism. In this study, the lysA gene which encodes for an important key enzyme of aspartate pathway / meso-diaminopimelic acid (DAP) decarboxylase (E.C.4.1.1.20) catalyzing the conversion of diaminopimelate to lysine was cloned and characterized for the first time from S. clavuligerus NRRL 3585. The attempts to clone the gene by constructing libraries of S. clavuligerus genomic DNA and screening of the libraries either by homologous probing or complementation approach gave no positive results. Then, PCR-based cloning was taken as the approach and the gene was amplified with PCR using the primers derived from the conserved sequences of lysA genes in two fragments (620 and 983 bp) which had overlapping regions. Fragments were then cloned and nucleotide sequencing revealed a complete open reading frame (ORF) encoding a protein of 463 aa (Mr 49, 907). The GC content of the gene was identified as 70.98 %. The gene sequence showed 83 % identity to the sequence of S. coelicolor lysA gene and 81 % identity to S. avermitilis lysA gene. By comparing the amino acid sequence of this protein to those available in database, the sites of the enzyme important for catalysis were identified.

Streptomyces clavuligerus is a gram-positive filamentous bacterium well known for its ability to produce an array of &amp / #61538 / -lactam compounds (secondary metabolites) including cephamycin C, clavulanic acid and other structurally related clavams. Of these, cephamycin C is a second generation cephalosporin antibiotic having great medical significance. Biosynthesis of the &amp / #946 / -lactam nucleus begins with the non-ribosomal condensation of L-&amp / #945 / -aminoadipic acid (&amp / #945 / -AAA), L-cysteine and L-valine to form the tripeptide &amp / #945 / -aminoadipiyl-cysteinyl-valine (ACV). In Streptomyces clavuligerus, &amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) is a catabolic product of L-lysine produced from the lysine branch of the aspartate pathway and its biosynthesis represents a key secondary metabolic regulatory step in carbon flow to &amp / #946 / -lactam synthesis through this core pathway. The ask (aspartokinase)-asd (aspartate semialdehyde dehydrogenase) gene cluster which encodes for the first key enzymes of aspartate pathway has already been cloned from S. clavuligerus, characterized and heterologously expressed for the first time in our laboratory. Amplification of ask-asd cluster or ask gene alone in a multi-copy Streptomyces plasmid vector and determination of the effects of multiple copies on cephamycin C biosynthesis were the goals of the present study. For this purpose, three different strategies were employed. Of these, two strategies involving the use of vector pIJ702 did not work because of the instability of resulting recombinant plasmids. In the third and last strategy, we used another multicopy Streptomyces vector, pIJ486, which we showed in this study to be very stable for the same purpose. Meanwhile, an efficient protoplast transformation protocol was developed in our laboratory. Ask gene was cloned into this vector and S. clavuligerus protoplasts were efficiently transformed with the recombinant plasmid (pTB486) using the newly-developed protocol. After stable recombinants were obtained, the effects of the multiple copies of ask gene on cephamycin C biosynthesis were determined. There was a profound reduction in the rate and extent of growth of Ask overproducers, as experienced by testing two independent ask-multicopy recombinants. Although one such recombinant strain (designated S. clavuligerus TB 3585) had a 5.5 fold increased level of Ask activity as compared to the parental strain, it displayed only a 1.1 fold increase in specific production of cephamycin C.

Made available in DSpace on 2016-06-02T19:56:59Z (GMT). No. of bitstreams: 1 623.pdf: 1119771 bytes, checksum: 03ec886c5b7c1e6b64f80f767a197553 (MD5) Previous issue date: 2005-03-28 / Financiadora de Estudos e Projetos / The importance of the airlift bioreactor has grown in the biotechnology field by aspects as high oxygen transfer, with power input low. In this work were study the conditions heat and mass transfer, as well as the condenser efficiency in the water retention, of an airlift bench bioreactor of internal circulation of useful capacity 6 L. Such bioreactor is endowed with hollow draft tube working as a heat charger for temperature control and of aspersor type crosspiece for aspersion of air. Values of the heat transfer global coefficient (U) were certain in different operation conditions starting from applied energy balance in the system after the same to reach the steady state. Values of U varied in the range from 300 to 700 W.m-2.°C-1, showing that the device of thermal change proposed was efficient to heat removal. The results were correlated with the water flow inside the draft tube and with the feeding air flow in the bioreactor. After this stage, it was evaluated water drags of the bioreactor system plus condenser in different conditions of air flow and temperatures in the bioreactor and in the condenser. The condenser was shown efficient in the water retention, obtaining efficiency superior values to 80%, but it didn't solve the problem of water loss by water drag for the high air flows in that the airlift bioreactor is operated. The variable air flow was the variable that influenced more significantly water drags. The transfer oxygen was evaluated through the oxygen transfer volumetric coefficient (kLa). For comparison, kLa values were obtained in the airlift bioreactor operated in a range of specific feeding air flow from 1 to 5 vvm and in conventional bioreactor of 4 L, in different agitation conditions (600, 800 and 1000 rpm) and aeration (0,5 and 1,0 vvm). Two methods for kLa determination were employees, the sulfite method and the dynamic method considering the delay of the electrode answer. This last one that generated results between 2 and 6 times inferior, depending of bioreactor type, because it evaluates the kLa in coalescent media, similar to most of the media fermentation. Finally, to validate the use of the system as bioreactor, cultivations with Streptomyces clavuligerus seeking to the production of clavulanic acid, a potent inhibitor of β-lactamase, enzyme producted by patogenics microorganism that broke β-lactamic rings of antibiotics. Four cultivations were accomplished, two in airlift bioreactor and two in bioreactor conventional (Bioflo III) in two different conditions of oxygen transfer (kLa) and the results were compared in terms of clavulanic acid production. The maximum clavulanic acid concentrations obtained in the cultivations in airlift bioreactor were superior those obtained in conventional bioreactor, with minors consistence index (K), showing larger shear rate in the airlift bioreactors in the same conditions of transfer oxygen. In global terms, the obtained results suggest that bioreactor as the most appropriate for this cultivation type, justified for the appropriate heat and mass transfers formed an alliance with the smallest energy consumption. / A importância do biorreator airlift tem crescido no campo da biotecnologia por aspectos como a sua alta transferência de oxigênio, obtida através de consumo de potência. No presente trabalho foram avaliadas as condições de transferência de calor e massa, bem como a eficiência do condensador na retenção de água, de um biorreator airlift de circulação interna, onde o ar é injetado pelo centro do tubo interno, de bancada de 6 L de capacidade útil. Tal biorreator é dotado de tubo interno ( draft tube ) oco funcionando como um trocador de calor para controle de temperatura e de aspersor tipo cruzeta para aspersão de ar. Valores do coeficiente global de transferência de calor (U) foram determinados em diferentes condições de operação a partir de balanço de energia aplicado no sistema após o mesmo atingir o estado estacionário. Valores de U variaram na faixa de 300 a 700 W.m-2.°C-1, mostrando que o dispositivo de troca térmica proposto foi eficiente na remoção de calor.Os resultados foram correlacionados com a vazão de água no interior do tubo interno e com a vazão de alimentação de ar no biorreator. Após esta etapa, avaliou-se o arraste de água do sistema biorreator mais condensador em diferentes condições de vazão de ar e de temperaturas no biorreator e no condensador. O condensador mostrou-se eficiente na retenção de água, obtendo valores de eficiência superiores a 80%, mas não solucionou por completo o problema de perda de água do sistema por arraste pelos altos fluxos de ar em que se opera o biorreator airlift. A variável vazão de ar foi a variável que influenciou mais significativamente o arraste de água. A transferência de oxigênio foi avaliada através do coeficiente volumétrico de transferência de oxigênio (kLa). Para efeito de comparação, valores de kLa foram obtidos no biorreator airlift operado numa faixa de vazão específica de alimentação de ar de 1 a 5 vvm e em biorreator convencional de 4 L, em diferentes condições de agitação (600, 800 e 1000 rpm) e aeração (0,5 e 1,0 vvm). Dois métodos para determinação do kLa foram empregados, o método do sulfito e o método dinâmico considerando o atraso da resposta do eletrodo. Este último que gerou resultados entre 2 e 6 vezes inferiores, dependendo do tipo do biorreator, pois avalia o kLa em meios coalescentes, similares à maioria dos caldos de fermentação. Por fim, para validar a utilização do sistema como biorreator, foram realizados cultivos com Streptomyces clavuligerus visando à produção de ácido clavulânico, um potente inibidor de β-lactamases, enzimas produzidas por microrganismos patogênicos que clivam o anel dos antibióticos β-lactâmicos. Foram realizados quatro cultivos, dois em biorreator airlift e dois em biorreator convencional (Bioflo III) em duas diferentes condições de transferência de oxigênio (kLa) e os resultados foram comparados em termos de produção do ácido clavulânico. As concentrações máximas de ácido clavulânico obtidas nos cultivos em biorreator airlift foram superiores àquelas obtidas em biorreator tipo tanque agitado e aerado, com menores índices de consistência (K), mostrando maior cisalhamento nos biorreatores airlift nas mesmas condições de transferência de oxigênio. Em termos globais, os resultados obtidos sugerem esse biorreator como o mais adequado para este tipo de cultivo, justificado pelas adequadas transferências de calor e massa aliadas ao menor consumo de energia.

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2008Bitstream added on 2014-06-13T18:50:20Z : No. of bitstreams: 1 correa_t_me_araiq.pdf: 384400 bytes, checksum: 29862180365116d9b884951e094a9a20 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Muitos compostos beta-lactâmicos são sintetizados por bactérias do gênero Streptomyces. As cefalosporinas, entre elas a cefamicina C (CefC) são apenas alguns exemplos de antibióticos sintetizados por Streptomyces clavuligerus. Os antibióticos beta-lactâmicos são formados, em procariotos e eucariotos, a partir de três aminoácidos, o ácido L-alfaaminoadípico (AAA), a L-cisteína e a L-valina. Em procariotos, o AAA é formado por desaminação da L-lisina pela enzima lisina ε-aminotransferase (LAT). A L-lisina exerce um papel fundamental na síntese destes compostos atuando como indutora da LAT, a primeira enzima da rota biossintética de CefC. Desta forma, uma variação adequada na concentração de lisina no meio de produção deve proporcionar melhorias na produção da CefC. Além da lisina, as fontes de carbono e nitrogênio também exercem fundamental importância, uma vez que participam de vários mecanismos reguladores, tanto do metabolismo primário como do secundário, durante o processo fermentativo. O presente trabalho teve como objetivo investigar a influência da presença e concentração de fontes de carbono, de L-lisina e de compostos relacionados à rota biossintética de antibióticos beta-lactâmicos em S. clavuligerus ATCC 27064, com ênfase no aumento da produção de CefC. Foram realizadas fermentações variando-se a natureza e a concentração de determinados compostos no meio de produção (fontes de carbono, de nitrogênio, etc) e, também, as condições de suplementação de L-lisina e outros compostos ao longo do processo. Com relação à fonte de carbono e energia (substrato limitante), os resultados demonstraram que o amido propiciou a maior produção de cefalosporinas totais, enquanto o glicerol foi mais adequado ao crescimento celular. Os resultados permitiram obter um meio padrão adequado para a... / Many beta-lactamic compounds are synthesized by bacteria from the Streptomyces ssp. The cephalosporins and among then the cephamycin C (CefC) are some examples of antibiotics synthesized by Streptomyces clavuligerus. The beta-lactam antibiotics are formed, both in eukaryote and prokaryote, by three amino acids, the L-alfa-aminoadipic acid (AAA), the L-cysteine and the L-valine. In prokaryote the AAA is formed by desamination of the Llysine by the lysine ε-aminotransferase (LAT) enzyme. The L-lysine plays a fundamental roll in the synthesis of these compounds acting as an inductor of the LAT, the first enzyme in the biosynthetic route of the CefC. Therefore, an adjusted variation on the lysine concentration in the production medium should provide improvement in the production of CefC. Besides lysine, the carbon and nitrogen sources also exert a crucial importance, once they are part of many regulating mechanisms from primary and secondary metabolism during fermentation. The objective in this paper is to investigate the influence of the presence and concentration of sources of carbon, L-lysine and composites related to the beta-lactam antibiotics biosynthetic route in S. clavuligerus ATCC 27064, with emphasis in the increase of the CefC production. Fermentation had been carried through varying the nature and concentration of certain composites in the production environment (sources of carbon, nitrogen, etc) and also the lysine and other compounds supplementation conditions along with the process. In relation to the sources of carbon and energy (limiting substrate), the results show that starch provides a higher production of total cephalosporins while glycerol has been more appropriate to cellular growth. The results allowed obtaining an appropriate standard medium to develop cephalosporins, ...(Complete abstract click electronic access below)

Made available in DSpace on 2016-06-02T19:55:22Z (GMT). No. of bitstreams: 1 1771.pdf: 1457129 bytes, checksum: 013319207cf51a783696430206a0ceed (MD5) Previous issue date: 2008-03-26 / Universidade Federal de Sao Carlos / The clavulanic acid (CA) is a powerful inhibitor of β-lactamases, enzymes that clives the β- lactam ring of penicillins and cephalosporins, inactivating these compounds. CA is traditionally produced by Streptomyces clavuligerus in batch and fed-batch cultivations. Considering that the products of the catabolism of easily assimilable carbon source repress the biosynthesis of secondary metabolites, including CA, its production in fed-batch mode is preferable because the control of nutrient level can be performed. In the present work the influence of glycerol and ornithine feeding on the clavulanic acid (CA) production by S. clavuligerus in batch and fed-batch cultivations was studied. Batch and fed-batch cultivations were performed at 800 rpm, 0.5 vvm and pH at 6.8. In batch experiments under different concentrations of ornithine, the maximum concentration of CA was within of a closed range between 0.46 and 0.56 g.L-1. Nevertheless, the maximum value of volumetric productivity in CA (Ppmax=13.7 mg.L-1.h-1) was obtained in the experiment with ornithine at concentration of 0.66 g.L-1, a value that is 40% higher than the Ppmax obtained in run control without ornithine. In fed-batch experiments in 5 L bioreactor, with different ornithine feeding conditions, the maximum concentration of CA varied within the limited range of 1.254 and 1.405 g.L-1. Therefore, under these experimental conditions, it can be concluded that the presence of ornithine increases the Ppmax in CA but not its maximum concentration, contradicting literature. The kinetic model considering non-associated production with CA degradation was fitted to the experimental results of the batch and fed-batch cultivations. The experimental results showed that the rate of production of CA and glycerol consumption, were directly related, condition not predicted by model, besides, its negative effect on the AC production after the feeding indicate a possible inhibition/repression effect of glycerol. With the model, it was possible to simulate conditions that the cultivations should be performed with the intention of obtaining larger production of AC. With respect to the influence of glycerol in fed-batch experiments, in the presence of ornithine at the concentration of 3.7 g.L−1 in feeding medium, the maximum CA concentration of 1.506 g.L-1 was obtained in the run BA7 showing a positive effect of the combination ornithine/glycerol in the biosynthesis of CA. In fed-batch experiments in 10-L bioreactor, with different ornithine feeding conditions, the maximum concentrations of CA were near of 1.6 g.L-1, validating the results obtained in 5-L bioreactor. / O ácido clavulânico (AC) é um potente inibidor de enzimas β-lactamases que clivam o anel β- lactâmico de antibióticos como penicilinas e cefalosporinas, tornando esses compostos inativos. O AC é tradicionalmente produzido por Streptomyces clavuligerus em cultivos operados em batelada (B) e em batelada alimentada (BA). Pelo fato dos produtos do catabolismo de fontes de carbono facilmente assimiláveis acabarem por reprimir as enzimas responsáveis pela biossíntese de metabólitos secundários, entre os quais se inclui o AC, a produção em BA é preferível, uma vez que torna possível controlar os níveis de nutrientes no caldo. No presente trabalho foi estudado a influência da alimentação de glicerol e ornitina na produção de ácido clavulânico (AC) por S. clavuligerus em cultivos em batelada e batelada alimentada operados a 800 rpm, 0,5 vvm e pH 6,8. Nos experimentos em batelada com diferentes concentrações de ornitina, as concentrações máximas de AC obtidas variaram na estreita faixa entre 0,46 e 0,56 g.L-1. Contudo, o valor máximo da produtividade volumétrica em AC (Ppmax=13,7 mg.L-1.h-1) obtida no experimento com concentração de ornitina de 0,66 g.L-1, foi 40% superior que o valor de Ppmax obtido no cultivo padrão sem adição de ornitina. Nos experimentos em batelada alimentada operados em biorreator de 5L, com diferentes concentrações de ornitina na alimentação, a concentração de AC variou entre 1,254 e 1,405 g.L-1. Portanto, sob as condições experimentais utilizadas, pode-se concluir que a presença de ornitina proporciona aumentos na Ppmax em CA, mas não sua produção máxima, contrariando trabalhos publicados recentemente na literatura. Um modelo cinético para produto não associado ao crescimento considerando degradação do AC foi ajustado aos resultados experimentais dos cultivos em batelada e batelada alimentada, a partir do qual foi possível simular condições que se deveria conduzir os cultivos em batelada alimentada com o intuito de se obter maior produção de AC. Com respeito à influência do glicerol na presença de ornitina no meio de alimentação numa concentração de 3,7 g.L−1, a concentração máxima de AC de 1,506 g.L-1 foi obtida no cultivo BA7 mostrando o efeito positivo da combinação ornitina/glicerol na biossíntese de AC. Nos experimentos em BA em biorreator de 10 L, com diferentes alimentações de ornitina, as produções máximas de AC foram próximas, em torno de 1,6 g.L-1, validando os resultados obtidos em escala de 5 L. Como principal conclusão, a presença de ornitina aumentou a produtividade volumétrica máxima em AC (Ppmax), mas não sua produção máxima, contrariando resultados anteriores publicados em literatura.

Made available in DSpace on 2016-06-02T19:56:28Z (GMT). No. of bitstreams: 1 DissAC.pdf: 1100634 bytes, checksum: a59508afe52240474931f49ecbfe180d (MD5) Previous issue date: 2007-03-12 / Universidade Federal de Sao Carlos / In biochemical processes, shear rate ( γ&) in the system is an important variable on design and operation of bioreactors. Shear can result in damage to the cells and the microorganisms, taking to the viability loss and even to the cellular breaking and the microbial death. In the present work was proposed a methodology to quantify average shear rate ( av γ & ) in stirred bioreactor with a 4 L working volume, through volumetric oxygen transfer coefficient (kLa), varying the operating conditions, as the impeller rotational speed (N: 600, 700, 800, 900 and 1000 rpm) and the specific air flow rate (Φar: 0.50 and 1.00 vvm). Five different glycerol solutions and distilled water were utilized as Newtonian fluids and six different xantham gum solutions were utilized as non-Newtonian fluids. Dynamic viscosity (µ) of the glycerol solutions and rheological parameters (K, n) for the xantham gum solutions, were determined at 28ºC from rheograms using a digital concentric-cylinders rheometer. Based on the obtained results, a correlation for average shear rate ( av γ & ) as function of the impeller rotational speed (N), rheological parameters (K, n) and volumetric oxygen transfer coefficient (kLa) was proposed. The correlation obtained for the average shear rate ( av γ & ) showed appropriate to estimate av γ & , because the estimated values approximated of others values described in literature for another correlations. In a second stage, four cultivations of Streptomyces clavuligerus ATCC 27064 was realized in a stirred bioreactor, aerated 0.50 vvm and agitated in 700, 800, 900 and 1000 rpm. Process variable values, as consistency index (K), flow index (n), cellular concentration (Cx), glicerol concentration (CGlic) and 2 CO n& produced ag of the cultivations were evaluated. It was observed that the consistency index (K), flow index (n) and cellular concentration (Cx) varied due to shear conditions imposed to the cultivations. Finally, based on experimental literature results, average shear rate ( av γ & ) was determined in function of the time for cultivation of S. clavuligerus in stirred bioreactor for 600, 800 and 1000 rpm and specific air flow rate (Φar) of 0.50 vvm, using the proposed methodology, the obtained values were more consistent, because the experimental values of kLa were obtained by gaseous balance. / Em processos bioquímicos, a velocidade de cisalhamento ( γ &) no meio reacional é uma importante variável no projeto e operação de biorreatores. O cisalhamento pode resultar em dano às células e aos microrganismos, levando à perda de viabilidade e até mesmo ao rompimento celular e à morte microbiana. No presente trabalho propôs-se uma metodologia para quantificar a velocidade de cisalhamento média ( γ &m) em biorreator convencional tipo tanque agitado e aerado com 4 litros de volume útil, através do coeficiente volumétrico de transferência de oxigênio (kLa), variando-se as condições de operação, no caso freqüência de rotação do impelidor (N: 600, 700, 800, 900 e 1000 rpm) e vazão específica de alimentação de ar (Φar: 0,50 e 1,00 vvm). Utilizaramse cinco diferentes soluções de glicerol e água destilada como fluidos Newtonianos, e seis diferentes soluções de goma xantana como fluidos não-Newtonianos. As viscosidades dinâmicas (µ) para as soluções de glicerol e as constantes reológicas (K e n) para as soluções de goma xantana foram obtidas a 28ºC em reômetro de cilindros concêntricos a partir de reogramas. Com base nos resultados obtidos, propôs-se uma correlação para a velocidade de cisalhamento média ( γ &m) em função da freqüência de rotação do impelidor (N), das propriedades reológicas dos fluidos (K e n) e do coeficiente volumétrico de transferência de oxigênio (kLa). A correlação obtida para a velocidade de cisalhamento média ( γ &m) mostrou-se adequada para estimativas de γ &m, pois os valores estimados aproximaram-se de outros estimados por diferentes correlações propostas na literatura. Numa segunda etapa, realizaram-se quatro cultivos com Streptomyces clavuligerus ATCC 27064 em biorreator convencional tipo tanque agitado e aerado a 0,50 vvm e agitado em 700, 800, 900 e 1000 rpm. Valores de variáveis do processo como o índice de consistência (K), índice de comportamento de escoamento (n), concentração celular (Cx), concentração de glicerol (CGlic) e quantidade de dióxido de carbono produzido ( 2 CO n& ) foram obtidos ao longo dos cultivos. Observou-se que o índice de consistência (K), índice de comportamento de escoamento (n) e a concentração celular (Cx) sofreram variações decorrentes das condições de cisalhamento impostas aos cultivos. Por fim, com base em resultados experimentais de literatura, determinou-se a velocidade de cisalhamento média ( γ &m) em função do tempo para cultivos de S. clavuligerus em biorreator convencional, a 600, 800 e 1000 rpm e vazão específica de alimentação de ar de 0,50 vvm, empregando-se a metodologia proposta, os resultados obtidos foram mais consistentes, pois se utilizou valores experimentais de kLa obtidos pelo balanço gasoso.