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Purification and characterization of Thermobifida fusca xylanase 10B

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Abstract:

Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 °C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl α-D-arabinofuranoside, p-nitrophenyl--D-xylobioside, p-nitrophenyl--D-xyloside, and p-nitrophenyl--D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.Key words: xylanase, enzyme purification, Thermobifida fusca, family 10 hydrolase.

Thermobifida fusca pousse bien sur de la cellulose et du xylane et produit une quantité de cellulases et de xylanases. Nous avons surexprimé xyl10B chez E. coli, un gène codant une endoxylanase qui n'avait pas été caractérisée par le passé, et avons purifié et caractérisé la protéine. La Xyl10B mature est une glycohydrolase de 43 kDa ayant un court domaine basique en terminus C. Elle démontre une thermostabilité modérée, conservant 50 % de son activité après une incubation de 16 heures à 62 °C et est la plus active entre les pH 5-8. Xyl10B est produite suite à une croissance de T. fusca sur du xylane ou du Solka Floc mais non sur de la cellulose pure. Une analyse par spectrométrie de masse a démontré que Xyl10B produisait majoritairement du xylobiose à partir de xylane de bouleau et d'épautre d'avoine et que ses produits d'hydrolyse étaient différents de ceux de la Xyl11A de T. fusca. Xyl10B hydrolyse divers sucres pNP incluant le p-nitrophényl α-D-arabinofuranoside, p-nitrophényl--D-xylobioside, p-nitrophényl--D-xyloside, and p-nitrophényl--D-cellobioside. Xyl11A démontre une activité supérieure sur des substrats de xylane, mais Xyl10B a produit davantage de sucres réducteurs à partir de fibres de maïs que Xyl11A.Mots clés : xylanases, purification d'enzymes, Thermobifida fusca, hydrolases de la famille 10.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: October 1, 2004

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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