【作者】 王海宽；

【导师】 路福平；

【作者基本信息】 天津科技大学 ， 发酵工程， 2005， 博士

【摘要】 木质素过氧化物酶(Lignin peroxidase,Lip)和锰过氧化物酶(manganese peroxidase,Mnp)是由黄孢原毛平革菌(Phanerochaete chrysosporium)分泌的单血红素糖蛋白。利用黄孢原毛平革菌生产木质素过氧化物酶和锰过氧化物酶受到许多因素的限制。例如:只有在限氮或其它不均衡的培养基中才能分泌并且酶活较低,同时其菌丝在发酵罐中易受高剪切力的损伤。本论文重点研究木质素过氧化物酶和锰过氧化物酶基因的克隆及在甲醇毕赤酵母中的表达,并对工程菌发酵条件和培养基进行了研究。主要研究内容和结果如下: [1]包含信号肽的lipH8基因在甲醇毕赤酵母中的表达 以黄孢原毛平革菌RNA为模板,通过RT-PCR获得包含信号肽的lipH8基因片段,克隆进入pMETA载体得到在甲醇酵母表达的pMETA-lipH8重组质粒,将其线性化后用电穿孔法导入pichia methabolica pMAD16,筛选得到1株表达水平较高的酵母工程菌株,酶活可达932U/L。 [2]去自身信号肽的lipH8基因在甲醇毕赤酵母中的表达 克隆去自身信号肽的lipH8基因,克隆进入pMETαA载体得到在甲醇酵母表达的pMETαA-lipH8—ss重组质粒,将其线性化后用电穿孔法导入pichia methabolica pMAD16,筛选出表达水平高的酵母工程菌株,酶活可达1933U/L。 [3]pMETαA-lip08表达木质素过氧化物酶发酵条件的优化 确定了一步法发酵的最优葡萄糖浓度,优化其发酵培养条件,结果表明葡萄糖的添加量为10g/L,pH为3.0,诱导温度24℃,培养时间12h,甲醇添加量1.1%,诱导时间72h后发酵液中酶活可达4888U/L。 [4]pMETαA-lip08表达木质素过氧化物酶发酵培养基的研究 进行了麦芽汁培养条件的研究,用1∶6麦芽汁培养工程菌,然后用高温浸提液诱导产酶较好。进一步通过发酵罐培养来研究产酶情况,结果表明产酶稳定。而用无机盐培养酶活性较低且不稳定。 [5]去信号肽的mnp2基因在甲醇毕赤酵母中的表达 以提取自Phanerochaete chrysosporium的总RNA为模板,采用RT-PCR技术扩增得到了去除自身信号肽的mnp2基因;成功的将mnp2基因与含酿酒酵母α-因子信号肽的载体pMETαA相连,构建了重组表达载体pMETαA-mnp2;首次在Pichia methanolica pMAD16中实现了锰过氧化物酶基因的表达。更多还原

【Abstract】 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.更多还原