Characteristics of a cluster of xylanase genes in Fibrobacter succinogenes S85.
Xylanase genes xyn10D, xyn10E, and xyn10B, located sequentially on the Fibrobacter succinogenes S85 chromosome, were separately cloned and their properties characterized. Analysis of the sequences documented that xylanases Xyn10D, Xyn10E, and Xyn10B each consist of an N-terminal catalytic domain (glycosyl hydrolase family 10) and a C-terminal carbohydrate-binding module (CBM, family 6) connected by proline-rich linker sequences. The amino acid sequences exhibited similarities of between 53 and 60%. The xyn10D, xyn10E, and truncated xyn10deltaACBM were expressed in Escherichia coli and purified to homogeneity. The purified Xyn10D, Xyn10E, and Xyn10BdeltaCBM exhibited the same temperature optimum (40 degrees C) and pH optimum (6.5) and the highest specific activity against arabinoxylan, oat spelt xylan, and birchwood xylan, respectively. Xyn10D exhibited an affinity for cellulose and xylan with 47 and 33% binding, respectively, while the truncated Xyn10DdeltaCBM did not bind to the substrates. The main hydrolysis products of the three xylanases acting on oat spelt xylan and arabinoxylan were xylose and xylobiose. RT-PCR analysis showed that the three genes were co-transcribed as a single transcript. Western immunoblot analysis revealed that the three xylanases were expressed at a very low level by F. succinogenes grown on either glucose or cellulose as the source of carbohydrate.
Jun
Hyun S
HS
Department of Microbiology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Ha
Jong K
JK
Malburg
Laercio M
LM
Verrinder
Gibbins Ann M
GA
Forsberg
Cecil W
CW
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D014158:Transcription, Genetic; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000235:genetics*
D011994:Recombinant Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase