| Hohenbuehelia serotina, as wood rot fungi, secretes lignocellulose degrading enzymes that degrade compost to provide necessary nutrients during the cultivation process. A preliminary inquiry about lignocellulosic enzyme activity and structural changes of lignocellulose is helpful to learn more about the relationship between utilization of lignocellulose and the growth of H. serotina.In this study, four periods during cultivation process ( raw materials, mycelium growth stage,primordium stage and stage of fruiting body ) have been chosen to detect activities about lignocellulose degrading enzymes. The activity of amylase is high in the initial stage of cultivation. There are high activities of laccase and manganese peroxidase during the entire cultivation, and the highest value are both in the primordium stage. The activity of xylanase and CMCase increase rapidly after the formation of primordia, and peaked in fruiting body period.The structural characterization of lignocellulosic components of four periods have studied by means of molecular weight determination, sugar analysis, fourier transform infrared spectroscopy(FT-IR) and scanning electron microscopy analysis (SEM).Starch, as the instant carbon source, has been used in early stage of mycelium growth. Laccase,manganese peroxidase and lignin peroxidase begin to degrade lignin when starch runs out. Structure of lignin has been destroyed. Hyphae get into the interior of the lignocellulosic structure and secrets enzymes such as xylanase and CMCase to degrade the crosslinked cellulose and hemicellulose.Dry fruiting bodies of H. serotina, which have been identified by ITS and phylogenetic relationship,are used as experimental materials. Several degrading enzymes (xylanase, laccase and RNase) have been purified by the integrated use of ion exchange chromatography, affinity chromatography, gel filtration chromatography. The physicochemical properties and related applications of these enzymes have also been analyzed.Xylanase is a protein with double subunits. Its molecular weight is 29.6 kDa, and the optimal pH is 4.2, the optimal reaction temperature is 50 ?, Ca2+ can enhance the activity of H. serotina xylanase,while Hg2+ and Cu2+ having a high inhibitory activity on it. The optimal substrate of H. serotina xylanase is birchwood xylan, and without cellulase activity. The degradation products of xylan and xylooligosaccharide have been analyzed by HPAEC-PAD and MALDI-TOF-MS. And it has been proved that the xylanase is a multifunctional enzyme with ?-1,4-exo-xylanase, P-xylosidase and polymerase activity.Laccase is a protein with 57.8 kDa molecular weight, the optimum pH was 6.8, the optimum reaction temperature is 30 ?, the optimal substrate was ABTS. Five internal peptides of the laccase were determined by LC-LTQ including two peptide sequences that lie just in the highly conserved Cupredoxin super family. Fe2+ has a strong inhibitory effect on laccase activity, Al3+, Mg2+ and Ca2+followed, Mn2+ and K+ have little effect on it; while Cu2+ and Zn2+ can enhance the activity of H.serotina laccase in varying degrees; inhibition of DMSO, EDTA, ?-ME, SDS, and L-cysteine concludes laccase is a copper bonding protein having disulfide bonds and molecular hydrogen. Decolouration effect of 11 kinds of common industrial dyes of H. serotina laccase is different. Laccase have different degrees of degradation on RBBR, CBB, M-GR, R-RV, K-BR, A-OR, RES and RW, and the decolorization descending order is as follows: M-GR > RBBR > CBB > R-RV > A-OR > RES >K-BR > RW.Ribonuclease is a single subunit protein with molecular weight of 27 kDa, the optimum temperature is 80 ?, the optimum pH is 5.0. It has high degrading activity in poly (C). N-terminal sequence is TVGGSLAEKGN, having lower homology with ribonuclease of other fungal. H. serotina RNase can inhibit murine leukemia cell lines L1210, the proliferation of hepatoma cells MBL2 as well as HIV-1 reverse transcriptase activity. IC50 was 40.3 ?M, 20.8 ?M and 49.9 ?M, respectively. |
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