Molecular Clone Of CDNA Encoding Lignin Peroxidase And Manganese Peroxidase From Phanerochaete Chrysosporium And Expression In Pichia Methanolica

Lignin peroxidase (Lip) and manganese peroxidase is a monomeric haemoglycoprotein peroxidase (38-43kDa) secreted by Phanerochaete chrysosporium. However, lignin peroxidase and manganese 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.In this dissertation, molecular clone of cDNA encoding lignin peroxidase (LipH8) and manganese peroxidase(Mnp2) from Phanerochaete chrysosporium and expression in Pichia methanolica are studied. At the same time, Fermentation condition and medium for the expression of lignin Peroxidase in Pichia methanolica PMAD16 was investigated.(1) The cDNA encoding lignin peroxidase was cloned by RT-PCR using RNA of P.chrysosporium as template. The lignin peroxidase cDNA with the native signal sequence was inserted into plasmid pMETA and transformed into Pichia methanolica pMAD16. PCR results indicated that the lignin peroxidase gene from P.chrysosporium has been successfully cloned into Pichia methanolica. The transforments was screened to get expression strain by shake-flask culture. The extracellular lignin peroxidase activity in this construct was 932U/L.(2) The cDNA of lignin peroxidase without the native signal sequence(lipH8-ss) was cloned by PCR using pMETA / lipH8 as template. lipH8-ss gene was cloned into pMET α A and transformed into Pichia methanolica pMAD16 in the same manner as for pMETA, resulting in the vector pMET α A-lipH8-ss .PCR results indicated that the lignin peroxidase gene from P.chrysosporium has been successfully cloned into Pichia methanolica. The transforments was screened to get high expression strain by shake-flask culture. The extracellular lignin peroxidase activity in this construct was 1933U/L.(3) The transforments was screened to get high expression strain pMET a A-lipO8 by shake-flask culture. The optimum fermentation condition of lignin peroxidase was studied. In the YEPD with the lOg/L glucose, The lignin peroxidase reached the largest activity of 4888U/Lat pH3.0, 24°C, 12h culture, l.l%(v/v)methanol, 72h induction.(4) The effect of wort and inorganic salt on producing Lip was studied, The results indicated that malt extract induced higher OD and Lip activity, composed to the inorganic salt, moreover the malt extract as a natural medium is more nutritive and cheaper. In brief the malt extract is superior to producing Lip.(5) The cDNA encoding manganese peroxidase was cloned by RT-PCR using RNA ofP.chrysosporium as template, manganese peroxidase gene was cloned into pMET a A and transformed into Pichia methanolica pMAD16 . The transforments was screened to get expression strain by shake-flask culture. The extracellular lignin peroxidase activity in this construct was 686U/L.