High-level production of recombinant Aspergillus niger cinnamoyl esterase (FAEA) in the methylotrophic yeast Pichia pastoris.
The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was isolated by reverse transcriptase-polymerase chain reaction, sequenced, and expressed in Pichia pastoris. Secretion yields up to 300 mg l(-1) were obtained in buffered medium. The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence. Specific activity, pH and temperature optimum, and kinetic parameters were also found similar to the native esterase. FAEA is thus the first fungal esterase efficiently produced using a heterologous system.
Juge
N
N
Nutrition, Health and Consumer Science Division, Institute of Food Research, Norwich Research Park, Colney, UK.
Williamson
G
G
Puigserver
A
A
Cummings
N J
NJ
Connerton
I F
IF
Faulds
C B
CB
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D007700:Kinetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012331:RNA, Fungal / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D003373:Coumaric Acids; D018076:DNA, Complementary; D012331:RNA, Fungal; D011994:Recombinant Proteins; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase