Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei.
Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80 degrees C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80 degrees C.
Leskinen
S
S
Primalco Ltd. Biotec, Rajamäki, Finland.
Mäntylä
A
A
Fagerström
R
R
Vehmaanperä
J
J
Lantto
R
R
Paloheimo
M
M
Suominen
P
P
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases