期刊
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
卷 40, 期 12, 页码 1433-1441出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s10295-013-1339-6
关键词
Cloning and expression; Feruloyl esterase; Aspergillus usamii; Ferulic acid; Pichia pastoris
资金
- National Nature Science Foundation of China [31101229]
- Fundamental Research Funds for the Central Universities of China [JUDCF11032]
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 A degrees C, and highly stable at pH 4.0-6.5 and 45 A degrees 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.
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