Seventeen species of facultative anaerobic bacteria belonging to three genera (Serratia, Cellulomonas, and Corynebacterium) were screened for the production of xylitol; a sugar alcohol used as a sweetener in the pharmaceutical and food industries. A chromogenic assay of both solid and liquid cultures showed that 10 of the 17 species screened could grow on D-xylose and produce detectable quantities of xylitol during 24-96 h of fermentation. The ten bacterial species were studied for the effect of environmental factors, such as temperature, concentration of D-xylose, and aeration, on xylitol production. Under most conditions, Corynebacterium sp. NRRL B 4247 produced the highest amount of xylitol. The xylitol produced by Corynebacterium sp. NRRL B 4247 was confirmed by mass spectrometry. Corynebacterium sp. NRRL B 4247 was studied for the effect of initial D-xylose concentration, glucose, glyceraldehyde, and gluconate, aeration, and growth medium. Corynebacterium sp. NRRL B 4247 produced xylitol only in the presence of xylose, and did not produce xylitol when gluconate or glucose was the substrate. The highest yield of xylitol produced in 24 h (0.57 g/g xylose) was using an initial D-xylose concentration of 75 g/l. Under aerobic conditions the highest xylitol yield was 0.55 g/g while under anaerobic conditions the highest yield was 0.2 g/g. Glyceraldehyde in concentrations greater than 1 g/l inhibited Corynebacterium sp. B 4247 growth and xylitol production. Corynebacterium sp. NRRL B 4247 culture grown in the presence of potassium gluconate (96 g/l) for 48 h and on addition of D-xylose to the media increased accumulation to 10.1 g/l of xylitol after 150 h. Corynebacterium sp. NRRL B 4247 exhibited both NADH and NADPH-dependent xylose reductase activity in cell-free extracts. The NADPH-dependent activity was substrate dependent. The activity was 2.2-fold higher when DL-glyceraldehyde was used as substrate than with D-xylose. In cell-free extracts the difference in xylose reductase and xylitol dehydrogenase activity was highest at 24 h, whereas for cell cultures that were grown in gluconate and xylose, the difference in the reductase and dehydrogenase activities was highest at 12 h after xylose addition. The NAD+ dependent xylitol dehydrogenase activity was low compared to the cells grown without gluconate. The molecular weight of NADPH-dependent xylose reductase protein obtained by gel filtration chromatography was 58 kDa. Initial purification was performed on a DE-52 anion exchange column. Purification using Red Sepharose affinity column resulted in a 58 kDa protein on the SDS PAGE gel and was further purified on a Mono-Q column. The activity stained band on the native gel yielded 58, 49, 39 and 30 kDa bands on the denaturing gel. The peptides of the 58 kDa protein of Corynebacterium sp. B 4247 sequenced by mass spectrometry, identified with E2 and E3 (Bacillus subtilis) components of multi-enzyme system consisting of pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex and oxo-acid dehydrogenase complex. A 75% match was shown by the peptide "QMSSLVTR" with E-value of 8e-04 to the Saccharomyces cerevisiae protein that was capable of reducing xylose to xylitol. The peptide "LLNDPQLILMEA" had conserved match "LL + DP" over several aldose reductases. The xylose reductase of the yeast Candida tropicalis ATCC 96745 was also purified. The molecular weight of the yeast NADPH-dependent xylose reductase was about 37 kDa on an SDS PAGE / Ph. D.

Made available in DSpace on 2016-12-08T16:24:10Z (GMT). No. of bitstreams: 1 PGCA12MA087.pdf: 1147805 bytes, checksum: 749e6e38b1399033aef2801d2ef1b25f (MD5) Previous issue date: 2012-03-09 / The bovine mastitis is a disease that causes more losses in dairy farming, significantly reducing the production and milk quality. A causative agent of mastitis is the Corynebacterium sp. , especially the specie Corynebacterium bovis, but their role in intramammary infection is not yet defined, since some studies consider this agent, a minor pathogen of the mammary gland, causing no change in production and composition milk, though others, realize the large capacity of Corynebacterium sp. in causing mastitis, placing it as a risk factor for the development of the activity. Meet the microbiological profile of milk samples, the specific action of the agents in the mammary gland and its effect on milk quality, are of great importance in mastitis control programs. This study aimed to determine the prevalence of Corynebacterium sp. n the milk of cows from dairy herds located in five Brazilian states and to correlate the presence of this agent with the management of the milking herd and the somatic cell count of bulk tank milk and milk of individual cows. We conducted a longitudinal study from January 2010 to July 2011, in dairy herds located in the states of SC, PR, RS, MG and PE, which were performed in four colletions (at six months intervals) of milk samples in each herd. A questionnaire was applied in each farm to obtain data and information on the practices of milking herds, such as teat disinfection and teat drying, which may influence the occurrence of mastitis by Corynebacterium sp. Of the 4949 milk samples analyzed, submitted to microbiological culture, 666 (13,46%) had isolation of Corynebacterium sp. Minas Gerais was the state with the highest pravelence off this agent (21,07%) compared to others states. Risk factors associated with the management of milking, such as teat disinfection before and after milking and dry cow treatment showed no statistically significant influence on the prevalence of Corynebacterium sp. in milk samples analyzed. The isolation of Corynebacterium sp. in milk samples did not affect the SCC of bulk tank milk of the herds, but in the CMT, the presence of this agent caused variations in the number of positive mammary quarters and the degree of score in their test. Therefore Corynebacterium sp. is a causative agent of mastitis of great relevance not only by the frequency of isolation observed in this study, but also by the effect of CMT in individual cows and consequently in the herd SCC / A mastite bovina é a enfermidade que mais causa prejuízos na pecuária leiteira, reduzindo, significativamente, a produção e qualidade do leite. Um dos agentes causadores da mastite é o Corynebacterium sp., principalmente a espécie Corynebacterium bovis, porém seu papel na infecção intramamária ainda não é definido, pois alguns estudos consideram este agente, um patógeno menor da glândula mamária, sem causar alteração da produção e composição do leite, entretanto outros, constatam a grande capacidade do Corynebacterium sp. em causar mastite, colocando-o como um fator restritivo para o desenvolvimento da atividade leiteira. Conhecer o perfil microbiológico de amostras de leite, a ação específica dos agentes na glândula mamária e seu efeito na qualidade do leite, são de grande relevância em programas de controle da mastite. Este estudo teve como objetivos determinar a prevalência de Corynebacterium sp. no leite de vacas de rebanhos leiteiros localizados em cinco estados brasileiros e, correlacionar a presença deste agente com o manejo de ordenha dos rebanhos e com a contagem de células somáticas do leite do tanque e do leite individual das vacas. Foi realizado um estudo longitudinal, de janeiro de 2010 a julho de 2011, em rebanhos leiteiros localizados nos estados de SC, PR, RS, MG e PE, nos quais foram realizadas quatro coletas (semestrais) de amostras de leite em cada rebanho. Foi aplicado um questionário, em cada propriedade, para obter dados e informações sobre o manejo de ordenha dos rebanhos, como a desinfecção pré e pós-ordenha, secagem dos tetos, que possam influenciar na ocorrência de mastite por Corynebacterium sp. Do total de 4949 amostras de leite analisadas, submetidas a cultura microbiológica, 666 (13,46%) apresentaram isolamento de Corynebacterium sp. Minas Gerais foi o estado que apresentou a maior prevalência deste agente (21,07%) comparado aos demais estados. Os fatores de risco associados ao manejo de ordenha das propriedades, como a desinfecção dos tetos pré e pós-ordenha e o tratamento de vaca seca não apresentaram influência estatística significativa na prevalência de Corynebacterium sp. nas amostras de leite analisadas. O isolamento de Corynebacterium sp nas amostras de leite não afetou a CCS do leite do tanque dos rebanhos, porém no CMT, a presença deste agente causou variações no número de quartos positivos e no grau de escore no respectivo teste. Portanto, Corynebacterium sp. é um agente causador de mastite de grande relevância, não só pela freqüência de isolamento observada no presente estudo, mas também pelo efeito no CMT individual das vacas e consequentemente na CCS do rebanho

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