Isolation and characterization of xylanase from Fusarium graminearum.

摘要

Fusarium head blight (FHB), caused by Fusarium graminearum (teleomorph Gibberella zeae), results in severe yield losses and crop quality reductions in wheat and barley, and it is the predominant species of the FHB complex in North America. Cell wall degrading enzymes secreted by the pathogen have been suggested to be involved in pathogenesis. The objective of this project was to purify and characterize xylanases from F. graminearum. The in-vitro production of xylanases by F. graminearum was from cultures grown at 25°C for 5 days on wheat bran supplemented with a synthetic media. Two endo-xylanases were purified 52- and 40-fold by a combination of ion exchange, gel filtration, high performance liquid chromatography (HPLC) ion exchange and HPLC hydrophobic interaction chromatography. First endo-xylanases were separated by ion exchange chromatography and then were purified individually through subsequent steps. The two xylanases were identified as the products of genes FG03624 (62% coverage, 20 kDa) and FG06445 (87% coverage, 40 kDa) by LC/MS/MS and were termed HMW (high molecular weight xylanase) and LMW (low molecular weight xylanase), respectively. The LMW xylanase exhibited optimal activity at pH of 6.0 and 50°C. It had an isolectric point of 8.4 and a molecular mass of 20.9 kDa as measured by ESI-MS. The enzyme exhibited activity over a broad pH range (5.5 to 7.5) and was stable for 5 hr at 35°C. The HMW xylanase exhibited optimal activity at pH 6.0 and 45°C. It had an isoelectric point of 8.5 and a molecular mass of 41 kDa as estimated by SDS-PAGE. The HMW xylanase was also stable over a broad pH range (5.5 to 8.5) but lost up to 40% of activity following 5 hr at 35°C. Kinetic studies showed beechwood xylan to be the preferred substrate for both enzymes, when compared to arabinoxylan.