Gene Cloning, Expression And Application Of A Feruloyl Esterase A

Feruloyl esterases (FAEs, EC3.1.1.73), one of the key enzymes in the completehydrolysis of hemicellulose, can catalyze the hydrolysis of ester linkages between ferulic acid(FA) and polysaccharides. FA exhibits great commercial values as a natural antioxidant, foodpreservative and antimicrobial agent. In addition, FAEs deserve particular attention due totheir wide-ranging applications in the pharmaceutical, pulp, food, feed, and bio-fuel industries.However, few FAEs have been applied in industries owing to their low expression quantitiesand catalytic activities. This study intended to efficiently obtain excellent FAEs by geneticengineering and molecular biology technology.Using the A. usamii total RNA as the template, the AuFaeA-encoding cDNA gene(AufaeA) was amplified by means of RT-PCR and nested PCR techniques with primers FAE-Fand FAE-R. The sequence of AufaeA is exactly783bp in length, encoding a maturepolypeptide of260amino acids. Construction of the recombinant expression vectorpPIC9K-AufaeA, then linearized with SacⅠ, and transformed into P. pastoris GS115byelectroporation. One transformant expressing the highest recombinant AuFaeA (reAuFaeA)activity of7.05U/mL towards methyl ferulate (MFA) was selected by G418and flaskexpression test after induction by1%methanol for72h.The purification of reAuFaeA was performed by a combination of (NH4)2SO4precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, ultrafiltration andSephadex G-75gel filtration. SDS-PAGE analysis of the purified reAuFaeA displayed onesingle protein band with an apparent molecular weight of36.0kDa. The purified reAuFaeAwas optimally active at45℃and pH5.0, and highly stable at pH4.0-6.5and45℃or below.Its activity was not significantly affected by metal ions tested and EDTA. The Kmand VmaxofreAuFaeA towards MFA were4.64mM and115.5U/mg, respectively.Only1.9%,7.4%and5.6%of total alkali-extractable FA were released from straw,wheat bran and corncob by70U reAuFaeA alone. When reAuFaeA incubated with120UreAuXyn10A or reAuXyn11A, the released FA from wheat bran increased obviously, with thelevels of1.69and2.85-fold, respectively. The results indicated a good synergistic interactionbetween reAuFaeA and the two xylanases. On the basis of single factor experiments,orthogonal experiments were performed to obtain the conditions of efficient release of FAfrom wheat bran. The optimal amount of reAuFaeA and reAuXyn11A were56U and600U,and the hydrolysis time and temperature were12h and42℃, respectively. Under theseconditions, the release of FA was up to43.7%. The enzymatic hydrolysis product of FA waspreliminarily purified with macroporous resin, and its in vitro antioxidant activity was tested.The results indicated that FA is an efficient antioxidant.