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The role of manganese peroxidase in biomass conversion technologies .

机译：锰过氧化物酶在生物质转化技术中的作用。

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摘要

Currently, the technologies used to separate lignocellulosic biomass into its component parts (cellulose, hemicellulose, and lignin) and enzymatically hydrolyze the cellulose to glucose for conversion to ethanol could be improved economically and in terms of efficiency. A major impediment to utilizing the biomass is the presence of residual lignin. The residual lignin "poisons" the cellulase enzymes and as a result, a lower glucose yield is obtained. A higher titer of cellulases could be used to increase the yield of glucose, but cellulases are expensive. A possibility to reduce the cost of cellulase enzymes and improve the efficiency, economics, extent and/or rate of hydrolysis is to use lignin-degrading fungal heme peroxidase such as manganese peroxidases.;An efficient and economical glucose assay was developed to monitor the rate and extent of enzymatic hydrolysis of cellulose. The glucose assay is based on the glucose oxidase horseradish peroxidase enzymatic method, but uses bulk enzymes making it more economical than commercial kits. The glucose assay also has a higher throughput rate and is more economical than HPLC.;In order to evaluate possible synergistic effects between cellulases and manganese peroxidase (MnP), rMnP was produced in high cell density fed-batch cultivations by the genetically engineered yeast, P. pastoris. In addition, a mathematic model was developed to describe the temperature dependent growth of P. pastoris and consumption of glucose and the production and temperature dependent degradation of rMnP in the bioreactor broth. The model successfully predicted the cell growth, substrate consumption, and rMnP production for the base case and also for cultivations with varying fed-batch air flow rates (kLa) and temperatures.;The production of rMnP requires cultures amended with exogenous heme and there are several sources of heme. Through shake flask experiments and bioreactor cultivations it was determined that 0.1 g/L of heme was necessary for producing high titers of rMnP (4,500 U/L). It was also determined that not all types of heme will yield the same rMnP titer.;The water soluble fraction post pretreatment of lignocellulosic biomass contains inhibitors to fermentation such as 5-hydroxymethyl furfural (HMF) and furfural. rMnP was shown to degrade HMF (1 g/L) and furfural (1 g/L) and detoxify medium containing these inhibitors. The rMnP reduced furfural and HMF, measured by absorbance at 276 and 286 nm respectively and the degree of absorbance decrease was dependent on rMnP concentration. Furfural was more readily degraded by rMnP than HMF. Growth assays using S. cerevisiae indicated rMnP treatment detoxified medium containing furfural and HMF.;The optimal conditions (temperature, pH, and buffer) for enzyme activity were determined for both Accellerase(TM) 1000 (commercially available combination of cellulases) and rMnP using filter paper as a substrate. Woody biomass and corn stover were pretreated and then exposed to simultaneous or sequential treatment with rMnP and Accellerase(TM) 1000. The results for the sequential treatment of Ponderosa pine and corn stover with rMnP and Accellerase(TM) 1000 was inconclusive as to whether or not rMnP effected the production of glucose due to the high variability between replicates.;Finally, part of my doctoral program was significant mentoring and facilitation of undergraduate research. A significant portion of my time was dedicated toward senior laboratory teaching assistance, serving as a mentor for high school students, and participating in research mentoring with 24 undergraduate students over five years, 19 of whom were women.
机译：目前，用于将木质纤维素生物质分离成其组成部分（纤维素，半纤维素和木质素）并酶促水解纤维素以转化为乙醇的技术可以在经济和效率方面得到改进。利用生物质的主要障碍是残留木质素的存在。残留的木质素“毒化”纤维素酶，结果，葡萄糖的产量降低。较高滴度的纤维素酶可用于增加葡萄糖的产量，但是纤维素酶是昂贵的。降低纤维素酶成本并提高水解效率，经济性，程度和/或水解速率的一种可能性是使用木质素降解真菌血红素过氧化物酶，例如锰过氧化物酶。开发了一种高效且经济的葡萄糖测定法来监测速率纤维素酶解的程度和程度。葡萄糖测定法是基于葡萄糖氧化酶辣根过氧化物酶的酶促方法，但使用大量酶使其比市售试剂盒更经济。葡萄糖测定法也比HPLC法具有更高的通量且更经济。为了评估纤维素酶和锰过氧化物酶（MnP）之间可能的协同作用，转基因酵母是在高细胞密度补料分批培养中产生的rMnP，巴斯德体育。另外，建立了数学模型来描述巴斯德毕赤酵母的温度依赖性生长和葡萄糖消耗以及生物反应器肉汤中rMnP的产生和温度依赖性降解。该模型成功地预测了基本情况以及不同补料分批空气流速（kLa）和温度的培养的细胞生长，底物消耗和rMnP的产生; rMnP的产生需要用外源血红素修饰的培养物，并且血红素的几种来源。通过摇瓶实验和生物反应器培养，可以确定0.1 g / L的血红素对于产生高滴度的rMnP（4,500 U / L）是必需的。还确定并非所有类型的血红素都能产生相同的rMnP滴度。木质纤维素生物质预处理后的水溶性级分含有发酵抑制剂，例如5-羟甲基糠醛（HMF）和糠醛。 rMnP被证明可降解HMF（1 g / L）和糠醛（1 g / L），并能解毒含有这些抑制剂的培养基。 rMnP还原糠醛和HMF，分别通过在276和286 nm处的吸光度测量，吸光度降低的程度取决于rMnP浓度。 rMnP比HMF更容易使糠醛降解。使用酿酒酵母的生长试验表明，含有糠醛和HMF的rMnP处理的解毒培养基。确定了Accellerase？1000（纤维素酶的市售组合）和rMnP的酶活性的最佳条件（温度，pH和缓冲液），滤纸作为基材。预处理木质生物质和玉米秸秆，然后将其暴露于rMnP和Accellerase™1000的同时处理或相继处理。使用rMnP和Accellerase™1000依次处理美国松和玉米秸秆的结果尚不确定由于重复之间的高度差异，rMnP不会影响葡萄糖的产生。最后，我的博士课程的一部分是对本科生研究的重要指导和促进。我的大部分时间都用于高级实验室教学协助，为高中生提供指导，并与24位5年以上的本科生一起参加研究指导，其中19位是女性。

著录项

  • 作者

    Yee, Kelsey L.;

  • 作者单位

    Oregon State University.;

  • 授予单位 Oregon State University.;
  • 学科 Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 270 p.
  • 总页数 270
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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