Characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate-binding module.
We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 alpha-L-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (M(r)) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.
Koseki
Takuya
T
Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 9978555, Japan. tkoseki@tds1.tr.yamagata-u.ac.jp
Mochizuki
Keiji
K
Kisara
Hiroe
H
Miyanaga
Akimasa
A
Fushinobu
Shinya
S
Murayama
Tetsuya
T
Shiono
Yoshihito
Y
D001089:Arabinose / Q000031:analogs & derivatives / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D050260:Carbohydrate Metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
D003373:Coumaric Acids; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D014990:Xylans; C431714:arabinofuranose; C085118:arabinoxylan; C004999:ferulic acid; D001089:Arabinose; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase