【作者】 杨晓宽；

【导师】 路福平；

【作者基本信息】 天津科技大学 ， 发酵工程， 2004， 硕士

【摘要】 白腐担子菌（White-rot Basidiomycetes）分泌的胞外锰过氧化物酶（manganese peroxidase，MnP）是胞外降解木素体系的一种主要成分。来自白腐担子菌的典型种黄孢原毛平革菌（Phanerochaete Chrysosporium）的锰过氧化物酶是一种需要H₂O₂的血红素糖蛋白，其相对分子质量范围为4.5×10⁴-4.7×10⁴Dalton。本论文旨在确定黄孢原毛平革菌产锰过氧化物酶的最优培养基和最优发酵条件，并通过基因工程手段得到锰过氧化物酶（MnP2）基因的克隆，为进一步将其转入适宜的真核细胞表达系统进行蛋白异源表达奠定基础。其主要内容包括以下两个方面。 1．黄孢原毛平革菌产锰过氧化物酶最适条件的确定 在静置培养条件下，通过对产酶培养基的优化，得到最优组成为(L^-1)： 葡萄糖10g，酒石酸铵2mmol，吐温80 1g，醋酸缓冲液10mmol（pH4.0），V_B1 1mg，，Mn²⁺9.9×10^-6g。采用该发酵限氮培养基，于34℃静置培养5d，产MnP活力达1200 U／L，比未优化提高近17倍。 进一步对产酶最适的培养条件进行优化，最终在250mL三角瓶中装液量30mL：发酵限氮培养基pH4.5；接种后，孢子终浓度为6.7×10⁵个孢子／mL；温度37℃；静置培养5d；产锰过氧化物酶活力达1452U／L，比未优化条件MnP活力提高20％。 2锰过氧化物酶（MnP2）基因的克隆 以最适条件培养的菌丝为材料，采用Trizal法提取黄孢原毛平革菌5.776总RNA并以之为模板进行RT-PCR，成功扩增出预期大小约为1.3kb的特异性条带，将扩增产物提纯后与pUC19载体体外重组，转化E．coli JM109，经筛选及酶切鉴定后，获得锰过氧化物酶（MnP2）基因的克隆。通过SDS-PAGE分析表明重组质粒pUC19-mnp2／JM109比空质粒pUC19／JM109在相对分子质量4.5×10⁴Dalton处多一条蛋白带，其大小与黄孢原毛平革菌锰过氧化物酶单体的大小相同，证明MnP2克隆成功。更多还原

【Abstract】 Extracellular manganese peroxidase which is secret by white-rot Basidiomycetes is a major component of the extracellular lignin-degrading systems. Manganese peroxidase from a model strain of the white-rot Basidiomycetes-Phanerochaete Chrysosporium is a H2O2-requiring heme glycoprotein of Mr=4.5 ×104-4.7×104Dalton.The main purpose of this dissertation lies in establishing the optimum culture components and the optimum fermentation conditions that Phanerochaete Chrysosporium produce manganese peroxidase,and lies in that clone of manganese peroxidase was acquired by gene engineering means.the groundwork that manganese peroxidase albumen hetero-source expression in fitting fungal expression system was established. The mainly researching contents of the dissertation include two aspects as blow:1 .The most agreeable conditions that Phanerochaete Chrysosporium produce manganese peroxidase was established.Under static culture condition, the optimum culture components by optimizing culture which produce manganese peroxidase showed as blow(L-1):Glucose 10g, ammonium tartrate 2mmol, Tween 80 1g, acetate buffer (pH4.0) lOmmol, VB1 1mg , Mn2+ 9.9×10~6 g. The culture temperature was 34℃.Under above conditions,the strain produced MnP activity up to 1200 U/L during 5 days stationary culture ,which increased almost 17 times than that of initially fermentation culture.The most agreeable culture condition which produce manganese peroxidase was optimized farther. The volume of liquid culture is 30mL(250 mL Erlenmeyer flasks),the pH of the fementable limiting nitrogen culture is 4.5,the inoculation volume is 6.7×105 entries spores per milliliter,the culture temperature was 37 ℃ .Under above conditions,the strain produced MnP activity up to 1452U/L during 5 days stationary culture, which increased almost 20% than that of initially fermentation condition.2. Molecular clone of cDNA encoding manganese peroxidase (MnP2). total RNA of 5.776 was withdrawed from the fungi silk which is educated in the most agreeable conditions and RT-PCR was proceeded regarding it as template, cDNA encoding specialized fragment whose size is about 1.3kb was amplified successfully just as anticipation.Amplified produut was extracellularly recombined with pUC19 vector after purification,and was transformed into E. coli JM109,then,the MnP2 gene cloning was obtained after screening and enzyme-cut identify.The analysis through SDS-PAGE showed that recombination plasmidpUC19-mnp2/JM109 have a more albumen fragment than that of volume plasmid pUC19/JM109 and its size is about 4.5 X 104Dalton,the same as that of manganese peroxidase(MnP2) monomer come from Phanerochaete Chrysosporium which proved clone is success.更多还原