| Plant biomass, a renewable resource targeted for bioenergy production, is mainly composed of cellulose and hemicellulose. To completely hydrolyze plant cell wall polysaccharides into simple sugars, a suite of cellulases and hemicellulases are required to operate in synergy. For the hydrolyzation of mannans, it requires several glycoside hydrolase enzymes cooperatively acting together, such as endo-1, 4-mannanase, β-1,4-mannosidases, and a-galactosidases. The accepted model for enzymatic depolymerization of cellulose is centered on hydrolytic cellulase enzymes classified as exoglucanases (or cellobiohydrolases) and endoglucanases. However, it has been challenged by several reports of the occurrence and functional importance of a novel class of copper-dependent polysaccharide monooxygenases (PMOs) recently, which depolymerize cellulose through an oxidative mechanism. LPMOs are of increased biotechnological interest due to their implication in lignocellulosic biomass decomposition for the production of biofuels and high-value chemicals, and its main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation.To obtain new cellulase and hemicellulases with favorable properties and build cheap, efficient composite enzyme system of polysaccharide degradation, we herein cloned, expressed and purified the first P-mannosidase CbMan2A from C. bescii and the first lytic polysaccharide monooxygenases MiLPMO1417from Humicola insolens, the main work and results are as follows:1. CbMan2A was thermophilic, with an optimal temperature of 80℃. CbMan2A hydrolyzes mannooligosaccharides with degrees of polymerization from 2 to 6 mainly into mannose and shows strong synergy with CbMan5A, an endo-mannanase from the same bacterium, in releasing mannose from β-1,4-mannan. Thus CbMan2A formed the missing link in enzymatic conversion of mannan into the ready-to-use mannose by C. bescii. Based on these observations, a model illustrating how CbMan2A may assist C. bescii in mannan utilization was presented. In addition, CbMan2A appeared to bind to insoluble galactomannan in a pH-dependent fashion. Although the relation of this feature to mannan utilization remains elusive, CbMan2A representedr an excellent model for investigation of the binding of GH2 β-mannosidases to galactomannan.2.HiLPMO1417 was cloned and expressed in P. pastoris GS115 under control of the Neurospora crassa NCU02916 signal peptide and methanol inducible AOX1 promoter. C-terminal tags for purification were omitted. The production and purification of HiLPMO1417 was specifically followed by a newly developed, fast assay based on a side reaction of LPMO:the production of H2O2 in the presence of ascorbate which works as electron donor. We showed that HilPMO1417 was an enzyme that mainly catalyzes cleavage of glycosidic bonds in crystalline cellulose, include phosphoric acid swollen cellulose, Avicel and filter paper. We also evidenced using high performance anion exchange chromatography (HPAEC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that MiLPMO1417 behaves as oxidizing enzyme and produces oxidized chain end.HiLPMO1417 showed a strong synergistic effect with endoglucanase HiCelxw with high degree of synergy value (above 1.3), that will facilitate their application for the degradation and modification of nonrecalcitrant plant cell wall polysaccharides and the development of future biorefmeries. At last, we found that soluble low molecular weight lignin can also functions as a reservoir of electrons facilitating LPMO activity, such as vanillin,4-Hydroxybenzaldehyde, O-vanillin, vanillic acid and so on, the complex electron transfer system generates important novel insights into fungal biomass degradation and may eventually lead to more efficient industrial processing of biomass. |
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