Résumé
ABSTRACT Rumen fungi inhabit the gastro-intestinal tract of ruminants and the most non-ruminant herbivores. Rumen fungi produce highly active plant cell wall degrading enzymes, therefore they have gained scientific interest. In this study, genes encoding xylanase (xynA-7) and cellulase (celA-5) were amplified from Neocallimastix sp. GMLF7 and Orpinomyces sp. GMLF5, respectively, and expressed in Escherichia coli. XynA-7 was found to be active only on xylan, however CelA-5 had activity both on carboxymethyl cellulose and lichenan. Lichenase activity of CelA-5 was found to be higher than carboxymethyl cellulase activity. The optimal conditions were at pH 6.0 and 40 °C for CelA-5 and at pH 6.5 and 50 °C for XynA-7. A coexpression vector was constructed to coproduce the XynA-7 and CelA-5 and then transformed into E. coli. The ability of the transformed E. coli strain to produce CMCase, xylanase and lichenase was evaluated. The transformed E. coli strain acquired the capacity to degrade CMC, xylan and lichenan.
Résumé
An alkaliphilic and highly thermostable alpha-amylase producing Bacillus sp. was isolated from Van soda lake. Enzyme synthesis occurred at temperatures between 25ºC and 40ºC. Analysis of the enzyme by SDS-PAGE revealed a single band which was estimated to be 66 kDa. The enzyme was active in a broad temperature range, between 20ºC and 90ºC, with an optimum at 50ºC; and maximum activity was at pH 10.5. The enzyme was almost completely stable up to 80ºC with a remaining activity over 90 percent after 30 min pre-incubation. Thermostability was not increased in the presence of Ca2+. An average of 75 percent and 60ºC of remaining activity was observed when the enzyme was incubated between pH 5 and 9 for 1 h and for 2 h, respectively. The activity of the enzyme was inhibited by SDS and EDTA by 38 percent and 34 percent, respectively.
Bacillus sp AB68 alcalif¨ªlico produtor de alfa-amilase alcalina termoest¨¢vel foi isolado do lago Van soda. A s¨ªntese da enzima ocorreu entre 25ºC e 40ºC. A an¨¢lise da enzima por SDS-PAGE revelou uma ¨²nica banda estimada em 66 kDa. A enzima foi ativa em uma ampla faixa de temperatura, entre 20ºC e 90ºC, com um ¨®timo a 50ºC. A atividade m¨¢xima foi em pH 10,5. A enzima foi est¨¢vel at¨¦ 80ºC, mantendo 90 por cento de atividade ap¨®s 30 min de pr¨¦-incubação. A termoestabilidade não aumentou na presença de Ca2+. Quando incubada em pH entre 5 e 9 por 1h e por 2h, a enzima manteve 75 por cento e 60 por cento de atividade, respectivamente. SDS e EDTA causaram redução de 38 por cento e 34 por cento na atividade da enzima, respectivamente.