Gene Cloning,Expression And Characterization Of Novel Polysaccharide Monooxygenases From Thermomyces Lanuginosus

Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels,chemicals,and materials.Lignocellulosic biomass is the major structural component of all plants and the largest source of renewable energy.It is covers more than 50%of the botanic body and the only renewable source of carbon.Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries.Its enzymatic degradation to the constituent monosaccharaides has attracted considerable attention for biofuel production.The high cost of enzymes for saccharification of lignocellulosic biomass is a major barrier to the production of second generation biofuels.Thermomyces lanuginosus is a soil-borne thermophilic fungus that grows well at45-50°C.As the first filamentous fungi with finished genome, it is a widely lanuginosus fungus and it has been used to identify novel cellulolytic activity, including exo-1,4-?-glucanases(EC 3.2.1.91;endo-1,4-?-glucanases(EC 3.1.1.4),EC 3.2.1.74)and?-glucosidases(EC 3.2.1.21).In this research,M.lanuginosus was cultured on a medium only with Avicel glucose as carbon source, and total RNA was isolated from the mycelia to sequencing using High-throughput sequencing.Four unknown functional genes were screened, termed PMO000810,PMO003424,PMO002033,PMO000154,respectively.The obtained nucleotide sequences were deposited in GEO DateSets under Accession No.GSE69763 for NCBI,respectively.The encoded proteins(PMO000810,PMO003424,PMO002033,PMO000154)comprised 816;822;1107;981 amino acid residues,and the predicted the molecular weight was 28.9kDa, 29.2kDa, 40.8kDa and 34.9 kDa, respectively.Blast showed that PMOs belonged to AA9 family.Each of the four proteins has a predicted signal peptide, it is suggested that all the proteins were the cell extracellular secreted proteins.Using NetNGlyc1.0 Server and online NetOGlyc 4.0Server to analysis the N-linked glycosylation sites and O-linked glycosylation sites,the results suggested that the four genes might be glycosylated.Four recombinant plasmids were constructed and electroporated into P.pastoris cells,and heterologous expression of the four genes under transcriptional control of the AOX1 promoter.Hundreds of transformants were obtained by electroporation on YPDs plates containzeocin.Multicopy transformants were detected by PCR and screened out on YPDs plate with the highest concentration of Zeocin.The clones with the highest yield of four proteins designated, were chosen for characterization respectively and proteins purification.After methanol induction, four proteins were secreted into the culture medium.The Oxidative cleavage activity of recombinant proteins in the culture supernatant reached highest after induced expressionfor 168 h.After purification using HisTrapTM FF crude,each of the four recombinant proteins displayed a single band in the electrophoresis gel image,corresponding to a molecular mass of 40 kDa,45kDa,50 kDa and 39kDa?Characteristics of those proteins showed that the four proteins could efficiently Oxidative cleavage cellulose the product is a fiber oligosaccharides.Thin-layer chromatography( TLC) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry showed the hydrolysis products were cello-oligosaccharides meaning the four proteins conformed to the characteristics of lytic polysaccharide.Recombinant PMOs lacked measurable hydrolytic activity by themselves,but as the presence of divalent metal ions and ascorbic acid significantly enhance traditional cellulase activity.In the absence of a substrate presence, The production and purification of all four enzymes was specifically followed by a newly developed of PMO:the production of H2O2 in the presence of reductants.While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH.Most characterized LPMOs,including all reported fungi LPMOs,form aldonic acids,i.e.,products oxidized in the C1 position of the terminal sugar.xidation at other positions has been observed,and there has been some debate concerning the nature of this position(C4 or C6).By flight mass spectrometry, thin cotton Thermomyces lanuginosus AA9 family polysaccharide monooxygenase that is having a C1 oxide, also having C4 oxidation.Novel lignocellulose oxidase found that not only increased people's awareness oxidase family, while the conversion of biomass into biofuels provides an advantageous resources,to lay the foundation for further industrial production.