Cloning, expression of a feruloyl esterase from Aspergillus usamii E001 and its applicability in generating ferulic acid from wheat bran.
A cDNA gene (AufaeA), which encodes a mature polypeptide of the type-A feruloyl esterase from Aspergillus usamii E001 (abbreviated to AuFaeA), was cloned and heterologously expressed in Pichia pastoris GS115. One transformant, labeled as P. pastoris GSFaeA4-8, expressing the highest recombinant AuFaeA (reAuFaeA) activity of 10.76 U/ml was selected by the flask expression test. The expressed reAuFaeA was purified to homogeneity with an apparent molecular weight of 36.0 kDa by SDS-PAGE analysis, and characterized using the model substrate of methyl ferulate (MFA). The purified reAuFaeA was optimally active at pH 5.0 and 45 °C, and highly stable at pH 4.0-6.5 and 45 °C or below. Its activity was not significantly affected by metal ions tested and EDTA. The K m and V max of reAuFaeA towards MFA were 4.64 mM and 115.5 U/mg, respectively. High-performance liquid chromatography analysis showed that only 9.7 % of total alkali-extractable ferulic acid (FA) was released from destarched wheat bran by reAuFaeA alone. The released FA increased to 36.5 % when reAuFaeA was used together with a recombinant Aspergillus usamii GH family 11 xylanase A, indicating a synergistic interaction between them.
School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People's Republic of China.
Yan-Yan
YY
Gong
Xin
X
Yin
Hui-Min
HM
Zhang
Min-Chen
MC
Wu
Cun-Duo
CD
Tang
Jun-Qing
JQ
Wang
Qing-Feng
QF
Pang
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D019295:Computational Biology; D003373:Coumaric Acids / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004043:Dietary Fiber / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003373:Coumaric Acids; D018076:DNA, Complementary; D004043:Dietary Fiber; D005656:Fungal Proteins; D011994:Recombinant Proteins; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D043364:Endo-1,4-beta Xylanases