The rumen bacteria Ruminococcus flavefaciens plays an important role in the degradation of plant cell wall polysaccharides. Pectin is a valuable component of feed of ruminants and although there are some studies on metabolism of pectin in mixed and pure cultures of rumen pectinolytic bacteria, there is little information on molecular properties of pectinase enzymes involved in the degradation process. We report here preliminary results on enzymes that act on the pectin polymer, in particular we have evidence of pectate lyases (PL) and pectin methyl esterases (PME). The bacteria was grown anaerobically on media containing cellobiose and 30% or 67% esterified citrus pectin. In all culture conditions examined substantial levels of extracellular PME activity were detected but in the presence of high-methylated pectins a ten fold increase of PME activity was observed. Furthermore, PME enzymes produced in the different cultures were examined by activity staining of pectin esterase (PE) following SDS-PAGE. Results revealed the presence of PE isoforms which are different between the substrates used for the growth. The most abundant PE enzyme produced, of about 35 kDa, in the cellobiose medium has been partially purified. As far as pectate lyase is concerned, the activity was detected by dot-spot experiments using the supernatant colture of the bacteria grown both on cellobiose and pectin. Moreover a genomic library was constructed in Escherichia coli and screened. One positive clone exhibiting pectate lyase activity was directly detected by an in situ plate assay. The plasmid isolated from the clone contained a 1.7 Kb genomic fragment from Ruminococcus whom sequence analysis is in progress. Further investigations on the enzyme characterisation and gene cloning are in progress in order to improve our knowledge on this system.
Pectinases in the rumen bacteria Ruminococcus flavefaciens
Palmieri G;Ferrara L;Aurilia V
2002
Abstract
The rumen bacteria Ruminococcus flavefaciens plays an important role in the degradation of plant cell wall polysaccharides. Pectin is a valuable component of feed of ruminants and although there are some studies on metabolism of pectin in mixed and pure cultures of rumen pectinolytic bacteria, there is little information on molecular properties of pectinase enzymes involved in the degradation process. We report here preliminary results on enzymes that act on the pectin polymer, in particular we have evidence of pectate lyases (PL) and pectin methyl esterases (PME). The bacteria was grown anaerobically on media containing cellobiose and 30% or 67% esterified citrus pectin. In all culture conditions examined substantial levels of extracellular PME activity were detected but in the presence of high-methylated pectins a ten fold increase of PME activity was observed. Furthermore, PME enzymes produced in the different cultures were examined by activity staining of pectin esterase (PE) following SDS-PAGE. Results revealed the presence of PE isoforms which are different between the substrates used for the growth. The most abundant PE enzyme produced, of about 35 kDa, in the cellobiose medium has been partially purified. As far as pectate lyase is concerned, the activity was detected by dot-spot experiments using the supernatant colture of the bacteria grown both on cellobiose and pectin. Moreover a genomic library was constructed in Escherichia coli and screened. One positive clone exhibiting pectate lyase activity was directly detected by an in situ plate assay. The plasmid isolated from the clone contained a 1.7 Kb genomic fragment from Ruminococcus whom sequence analysis is in progress. Further investigations on the enzyme characterisation and gene cloning are in progress in order to improve our knowledge on this system.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
