| Lignin peroxidase (Lip) is a monomeric haemoglycoprotein peroxidase secreted by Phanerochaete chrysosporium. However, lignin peroxidase production is hampered by several factors such as expression of these enzymes under nutrient limitation and unbalanced media, and the sensitivity of this basidiomycete fungus to high shear forces in the fermentor , besides the rapid inactivation of these enzymes even in the absence of mycelia.The expression of lignin peroxidase gene in Pichia methanolica was investigated. The cDNA encoding lignin peroxidase was cloned by RT-PCR using RNA of P.chrysosporium as template. The lignin peroxidase cDNA was inserted into plasmid pMET a A and transformed into Ade defective mutant Pichia methanolica PMAD16.Electroporation conditions for Pichia methanolica were investigated.By changing parameters,a method of high efficient electrotransformation for plasmid pMET a A-lipH8 to Pichia methanolica was established.The optimal electroporated stage to Pichia methanolica was at intermediate logarithmic phage .Under the condition of electric field intensity at 3kV, with 30ug/mL plasmid DNA,the transformation efficiency reach its maximum :57 transformants/ ug plasmid DNA.By the identification,all of transformants were positive clones.The experiments provided a modle for high efficient electrotransformation with Pichia methanolica as host cells.Transforments were obtained by screening on MD plates and PCR technique.The expression products of lipH8 gene were analyzed by SDS-PAGE and assay of lignin peroxidase. The results indicated that the lip gene from P.chrysosporium was successfully transformed and overexpressed in Pichia methanolica. Some factors such as media,buffer,pH,methanol concentration, media volume, inoculated volume were investigated to optimize the cultivation condition for expression of lip. Under optimized condition, The extracellular lignin peroxidase activity was up to 1997U/L.Fermentation coefficient was 27.69U/h.L.
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