Cloning and characterization of the first GH10 and GH11 xylanases from Rhizopus oryzae.
The only available genome sequence for Rhizopus oryzae strain 99-880 was annotated to not encode any β-1,4-endoxylanase encoding genes of the glycoside hydrolase (GH) family 10 or 11. Here, we report the identification and cloning of two such members in R. oryzae strain NRRL 29086. Strain 29086 was one of several selected fungi grown on wheat or triticale bran and screened for xylanase activity among other hydrolytic actions. Its high activity (138 U/ml) in the culture supernatant led to the identification of two activity-stained proteins, designated Xyn-1 and Xyn-2 of respective molecular masses 32,000 and 22,000. These proteins were purified to electrophoretic homogeneity and characterized. The specific activities of Xyn-1 and Xyn-2 towards birchwood xylan were 605 and 7,710 U/mg, respectively. Kinetic data showed that the lower molecular weight Xyn-2 had a higher affinity (K m=3.2 ± 0.2 g/l) towards birchwood xylan than Xyn-1 by about 4-fold. The melting temperature (T m) of the two proteins, estimated to be in the range of 49.5-53.7 °C indicated that they are rather thermostable proteins. N-terminal and internal peptide sequences were obtained by chemical digestion of the purified xylanases to facilitate cloning, expression in Escherichia coli, and sequencing of the respective gene. The cloned Rhizopus xylanases were used to demonstrate release of xylose from flax shives-derived hemicellulose as model feedstock. Overall, this study expands the catalytic toolbox of GH10 and 11 family proteins that have applications in various industrial and bioproducts settings.
Aquatic and Crop Resource Development, National Research Council Canada, Montreal, Quebec, Canada, H4P 2R2.
Zhizhuang
Z
Xiao
Stephan
S
Grosse
Hélène
H
Bergeron
Peter C K
PC
Lau
D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D005656:Fungal Proteins; D043364:Endo-1,4-beta Xylanases