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Investigating the enzymatic hydrolysis of crystalline cellulose using fluorescence based assays---Implications of synergism, binding and product inhibition.

机译：使用基于荧光的测定法研究结晶纤维素的酶促水解---协同作用，结合和产物抑制的含义。

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摘要

The hydrolysis kinetics of bacterial microcrystalline cellulose (BMCC) by the cellulases of Thermobifida fusca was studied using fluorescence based assays in three ways. First, the binding of fluorescence-labeled Cel5A, Cel6B and Cel9A in ternary synergistic mixtures was assessed. A rapid high-throughput binding assay using microwell plates was developed to measure the bound fractions of the three cellulases at varying mole ratios of Cel6B and Cel9A, with Cel5A fixed at 10% of the total cellulase loading. This study revealed that the bound fractions of cellulases in ternary mixtures were additive, unlike the hydrolytic activity which was synergistic. Second, an experimental system was developed for the application of high resolution fluorescence microscopy to examine the binding of individual Cel5A, Cel6B and Cel9A to immobilized cellulose with varying morphologies. The immobilization technique allowed deposition of cellulose morphologies ranging from nanoscale cellulose fibers, to microscale cellulose fibril mats to sub-millimeter scale cellulose particles. Fluorescently labeled Cel5A, Cel6B and Cel9A were successfully integrated with fluorescently labeled cellulose to obtain a miniaturized reaction system which retained the intrinsic binding and hydrolytic capabilities of cellulases. Direct visualization of the binding behavior of individual cellulases on cellulose with different morphologies was achieved using this system which showed that the binding behavior depended strongly on the morphology and complexity of cellulose aggregates. Third, the significance of product inhibition by cellobiose as a rate-retarding factor in the hydrolysis of BMCC by Cel9A and Cel9A-68, its construct lacking the family 2 cellulose binding module, was investigated. Fluorescently labeled BMCC was used as the substrate for an analysis of initial rates in the presence of exogenous cellobiose. Increasing cellobiose concentrations ranging from 1--5mM were found to decrease the initial rate by 10--30% but increasing cellobiose concentrations from 5 to 60 mM did not cause a further decline in initial rates, clearly ruling out classical competitive inhibition as a possible mechanism. No definitive correlation was observed between binding and cellobiose concentrations for both enzymes indicating that the presence of cellobiose does not lead to significant enhancement or inhibition of binding.
机译：使用基于荧光的测定法以三种方式研究了嗜热栖热菌纤维素酶对细菌微晶纤维素（BMCC）的水解动力学。首先，评估了荧光标记的Cel5A，Cel6B和Cel9A在三元协同混合物中的结合。开发了使用微孔板的快速高通量结合测定法，以测量三种纤维素酶在Cel6B和Cel9A摩尔比变化时的结合级分，其中Cel5A固定为总纤维素酶负载的10％。这项研究表明，纤维素酶在三元混合物中的结合级分是累加的，这与协同的水解活性不同。其次，开发了一种用于高分辨率荧光显微镜检查的实验系统，以检查单个Cel5A，Cel6B和Cel9A与具有不同形态的固定化纤维素的结合。固定技术可以沉积从纳米级纤维素纤维到微米级纤维素原纤维毡到亚毫米级纤维素颗粒的纤维素形态。荧光标记的Cel5A，Cel6B和Cel9A与荧光标记的纤维素成功整合在一起，获得了一个微型化的反应系统，该系统保留了纤维素酶的固有结合和水解能力。使用该系统可以直接观察单个纤维素酶在具有不同形态的纤维素上的结合行为，这表明结合行为在很大程度上取决于纤维素聚集体的形态和复杂性。第三，研究了纤维二糖抑制产物作为Cel9A和Cel9A-68水解BMCC的速率减速因子的重要性，该构建体缺乏家族2纤维素结合模块。荧光标记的BMCC用作底物，用于分析外源纤维二糖存在时的初始速率。发现纤维二糖浓度范围从1--5mM降低了初始速率10--30％，但纤维二糖浓度范围从5至60mM并未引起初始速率的进一步降低，显然排除了经典竞争性抑制的可能性机制。在两种酶的结合和纤维二糖浓度之间未观察到明确的相关性，表明纤维二糖的存在不会导致结合的显着增强或抑制。

著录项

  • 作者

    Santhanam, Navaneetha.;

  • 作者单位

    Cornell University.;

  • 授予单位 Cornell University.;
  • 学科 Chemistry Biochemistry.;Engineering Biomedical.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 218 p.
  • 总页数 218
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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