Purification And Characterization Of Feruloyl Esterases From Panus Giganteus,Russula Virescens And Lactarius Hatsudake

Feruloyl esterases (FAEs) are able to hydrolyse the ester bond between hydroxycinnamic acids and hemicellulosic sugars and releasing ferulic acid from the plant cell wall, which demonstrate a wide variety of applications in food, cosmetic, medical, feed industries and producing bioethanol. In this study, three FAEs were purified from the fruiting bodies of Panus giganteus, Russula virescens, and Lactarius hatsudake using purification procedures which involved in ion exchange chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, SP-Sepharose and gel filtration by FPLC.A novel FAE from Panus giganteus was found to be a monomeric protein with a molecular mass of 61 kDa as estimated by FPLC and SDS-PAGE. Maximal FAE activity was observed at a pH of 4.0 and at a temperature of 40 ?, respectively. The Km and Vmaxfor P. giganteus FAE on methyl ferulate (MFA)was 0.36 mM and 18.98 mM · min-1 · mg-1, respectively. The purified FAE exhibited activity towards three tested hydroxycinnamic acid methyl esters, and it exerted maximal activity on MFA. The P.giganteus FAE was sensitive to metal ions and all tested metal ions showed various degree of inhibitory effect. Ferulic acid can be released from de-starched corn bran and de-starched wheat bran under the action of the single purified P. giganteus FAE, and the release amount have risen to 6.4 and 2.9 times,respectively, by the synergistic action of xylanase.A monomeric FAE with a molecular mass of 62 kDa was acquired from Russula virescens. Two internal amino acid sequences showed some homology with the esterase of some fungi. Maximal activity was observed at pH 5.0 and at 50 0C. The Km and Vmax for this enzyme on MFA was 0.19 mM and 1.65 mM · min-1 · mg-1,respectively. The purified FAE prefers methyl ferulate over methyl caffeate,and was least active on methyl p-coumarate. The FAE activity was not significantly affected by the presence of metal ions except Al3+, Hg2+, Fe2+ and Pb2+ ions. Ferulic acid can be released from de-starched wheat bran under the action of the single purified R. virescens FAE, and the release amount had risen to 5 times by the synergistic action of xylanase. The R. virescens FAE had no hydrolytic activity towards de-starched corn bran.Lactarius hatsudake FAE was a monomeric protein with a molecular mass of 55 kDa. Three internal amino acid sequences exhibited 100% identity with the Laccaria bicolor carbohydrate esterase family 9 protein. The enzyme demonstrated high activity toward MFA at an optimum pH of 4.0 and an optimum temperature at 30 ?, and under these conditions, the Km and Vmax were 0.32 mM and 3.77 mM · min-1 · mg-1, respectively. The purified FAE exhibited preference of methyl caffeate over methyl p-coumarate, and was least active on methyl ferulate. L, hatsudake FAE was able to release ferulic acid from de-starched wheat bran both in the absence and presence of xylanase, and 89.3% (of the total amount of) FA was released by the synergistic action of xylanase.