Modification of a xylanase cDNA isolated from an anaerobic fungus Neocallimastix patriciarum for high-level expression in Escherichia coli.
A Neocallimastix patriciarum xylanase cDNA with the core coding sequence essentially identical to xynA was isolated and modified for high-level expression in Escherichia coli. The xylanase cDNA was truncated into individual catalytic domains, which were modified at the N-terminus. These modified xylanases were synthesised as non-fusion proteins under the control of the tac promoter. High-level expression was obtained with the modified domain II construct, accounting for approx. 25% of total cellular protein. However, with the same vector and expression cassette, expression levels of constructs containing domain I or domains I and II fused in tandem were very low. RNA analysis revealed that the striking difference in expression levels of these three constructs was not due to transcription efficiency, but was mainly related to transcript stability. Further analysis of the domain II construct revealed that the high-level expression of the domain II xylanase was largely attributed to the presence of a favourable N-terminal coding sequence, as mutation at the N-terminus of the domain II dramatically reduced the expression level. The modified domain II xylanase produced in E. coli had a specific activity of 1229 U mg-1 protein at pH 7 and 50 degrees C without purification. The availability of a recombinant fungal xylanase with high specific activity and in high yield offers a potentially attractive source of xylanase for industrial applications.
Xue
G P
GP
CSIRO Division of Tropical Crops and Pastures, St. Lucia, Qld, Australia.
Denman
S E
SE
Glassop
D
D
Johnson
J S
JS
Dierens
L M
LM
Gobius
K S
KS
Aylward
J H
JH
D000818:Animals; D001483:Base Sequence; D008411:Chytridiomycota / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins; D012417:Rumen / Q000382:microbiology; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase