1887

Abstract

Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. , a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of DNA, constructed in λZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in revealed an ORF of 1137 bp (), encoding a polypeptide with a deduced of 43413, defined as xylanase C (XylC). Probing a gene library of subsp. with identified a homologue (designated ) encoding XylG; XylG and were 67% and 63% identical to the corresponding sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.

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2000-08-01
2019-10-23
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