纤维材料酶解机理分析与应用策略探讨
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摘要
木质纤维素是最具有利用潜力的生物质资源，其生物降解产物可以用来生产多种生物产品以及渴望替代化石能源。对纤维素利用的工艺流程中，最主要的限制性因素存在于纤维素转化为可发酵的还原糖这一步。降低单位还原糖的生产成本的策略大体有两类途径：(1)提高纤维素酶生产的经济性，这主要涉及纤维素酶合成机理的研究，提高其合成效率以降低单位纤维素酶的生产成本(为本实验室另一研究组主要研究方向，不属本文主要研究内容)；(2)提高纤维素酶的比活力或者利用效率，主要涉及纤维素酶解纤维材料时催化作用机理的研究，以降低单位可发酵还原糖的生产成本，此为本文研究的主要内容。
与蛋白酶类、果胶酶类以及淀粉酶类相比较，纤维素酶(系)活力的准确测定十分困难，一直是理论和应用研究中的难题。由于纤维素的不溶性与结构异质性，以及纤维素酶系统的复杂性，建立以其为底物的实用且可靠的纤维素酶活力测定方法十分困难，而建立高效的活力测定或者筛选方法，以选择具有人们期望的特性的酶或者菌株，是酶工程学研究的基础。
阐明纤维素酶的催化机理与催化环境的影响是提高其转化率的基础。酶与底物结合是催化过程的首要阶段，目前对酶与底物的结合的研究仍然沿用可逆平衡的假说进行分析，但实际上该理论并不适用于如纤维素酶催化体系等多组分反应体系。另外，温度是影响酶催化效率的重要影响因素，在热力学、动力学与生物工程学中都有关于温度对酶催化影响的研究，但无论是思路还是方法都没有形成统一的认识，彼此之间存在很多矛盾。
山东大学微生物技术国家重点实验室从微生物学、生物化学、分子生物学、结构生物学等多学科出发对于解决上述问题进行了深入的研究。本文是从反应机理的角度，从反应动力学机理出发，探讨解决实际应用中的问题。本论文以纤维分解真菌Trichoderma pseudonkoningii S-38分泌的胞外纤维素酶以及从中分离到的外切纤维素酶1，4-β-D-glucan cellobiohydrolase I(CBHI)为材料，主要研究内容为：
●建立能够准确评价纤维素酶制剂对水解纤维材料的长时间总糖化能力，以及不同性质纤维材料酶解敏感性的测定方法；
●以反应过程的动态光谱变化为基础，建立CBHI酶活力的测定方法；
●深入的分析CBHI与其小分子底物对硝基酚纤维二糖苷(p-nitrophenyl-D-cellobioside,PNPC)的结合(Binding)过程的构象构型变化，以阐明结合在酶催化过程中的作用机理；
●研究了，温度和时间过程对CBHI催化PNPC反应的复合影响，以阐明动力学和热力学过程对酶催化的控制机理；
●对生物质资源的利用策略进行了探索和讨论。
研究工作取得具有一定创新性结果如下：
1．提出了一个可准确评价对纤维材料酶解糖化潜力的纤维素酶(系)催化活力的新方法，为应用研究中纤维素酶的准确定量测定这一难题，提供了解决方法。
2．建立了一个可准确测定外切纤维素酶(CBHI)活力的动态光谱法，为纤维素酶作用机理研究提供了技术支持。
3．证实OBHI结合底物对硝基苯纤维二糖(PNPO)时，OBHI的构象(Conformation)变化可导致PNPC构型(Configutation)的改变；证实了纤维二糖抑制纤酶活力的机理在于其能与OBHI活性中心附近的色氨酸残基结合发生位阻效应，是对酶催化机理的“诱导—契合”假说的进一步发展。
4．从反应动力学和热力学机理以及数学分析相结合的角度，指出传统的研究中温度影响酶催化能力(催化活力和热稳定性测试)以及Arrhenius活化能等方法的局限性。并指出以时间—温度叠加法则测定了温度对酶催化过程的作用。分别选择复合函数表征瞬时速率和以偏导数之和表征产物瞬时增量的方法，以分析温度和时间对催化的综合影响。
5．根据化学亲和力(Chemical affinity)并以净反应速率为工具，建立了一个可反映酶催化过程中自由能变化规律的方法。综合上述结果提出水解过程中，酶构象反复发生构型的变化，对以可逆平衡为基础等假说存在的问题进行分析的基础上，进一步提出酶是把环境中的热能转化为功的分子热能转换器，催化是一做功的过程，酶本身是参与反应的等关于酶反应机理的新假说。
最后，对木质纤维材料生物转化利用的新策略做了一些探索性的实验。
1．构建了能够准确评价纤维素酶制剂水解纤维材料的新方法，该方法可以用于评价不同性质纤维材料的酶解感受性。解决了目前应用CMC-Na方法和滤纸酶活法难以准确评价纤维材料被酶解糖化潜力的难题。
由于纤维素的不溶性与结构异质性，以及纤维素酶系统的复杂性，并且因为传统的酶活力测定方法由于没有考虑到酶、底物浓度以及反应时间的影响，限于经验性的结果，拟合而缺少反应动力学分析的支持。特别是酶解过程中，底物结构变化对结果的影响，使测定结果之间难以比较，且不能反应纤维素酶制剂的真实糖化能力和纤维底物可被糖化的潜力。
纤维素比底物转化率(Specific substrate conversion%,SSC)与纤维素酶的糖化能力密切相关，以其表征酶催化活力的变化，既能够反应纤维素酶制剂对底物的水解情况，又避免了酶与底物浓度对计算结果的影响，(Fig.1-A)。SSC的瞬时速率可以表征纤维素酶对底物的转化能力的变化，为了表征纤维素酶制剂对纤维素内禀的糖化能力，本文采用计算瞬时速率的曲线下总面积(The total area under the curve,AUC)的方法，该方法可以反映整个水解过程的信息(Fig.1-B)。在较大的纤维素酶与底物转化率范围之内，AUC-纤维素酶体积曲线在Y轴的截距与加入的酶浓度呈线性相关。
Fig.1 (A) Time courses of SSC, during hydrolysis for filter paper by different concentrations of crude cellulase; (B) The relationship between cellulase concentration and its AUC calculated as d[SSC]/dt.
本方法考虑到了纤维素酶浓度、纤维素底物浓度以及水解时间三个因素的共同影响，微分—积分方法，拟合数据合正确应用。所以能够准确测定纤维素酶粗酶对固体纤维素的糖化能力。经验证，本方法也适用于纤维素酶制剂对棉纤维、微晶纤维素PH101以及磷酸膨化纤维素等不同酶水解敏感度的纤维素底物水解活力的测定。经比较证实，本方法较国际纯粹和应用化学学会推荐的FPA(滤纸活力)法，能准确反映纤维材料被酶解糖化的潜力。
对纤维素Ⅰ比纤维素Ⅱ形态与结构测定以及两者对稀酸水解和酶水解的感受性的比较结果表明，必须结合纤维素的酶水解过程才能正确评价不同纤维素底物酶解感受性的变化。应用本文提出的测定方法同样可以评价不同纤维素底物对特定纤维素酶的水解感受性，并在实际应用中评价各种纤维素改性方法对酶水解效率的促进作用。本文选取具有不同的结晶度、聚合度，以及无定形区含量的棉纤维、微晶纤维素PH101以及磷酸膨化纤维素等三种典型的纤维素模式底物，研究了不同类型纤维素底物酶解反应性。通常纤维素的改性一般会增加纤维素的比表面积，并提高纤维素无定形区的比例，因此，在对酶解效率改变的结果进行分析时应测定比表面积与无定形区的相对比例。
2．建立了一个可准确测定外切纤维素酶(CBHI)活力的动态光谱法。
CBHI是纤维素酶系中对结晶纤维素降解起决定作用的组分，目前，广泛应用的测定CBHI的PNPC水解活力方法，对底物浓度以及反应时间的选择都是经验性的，因此其酶活力测定的结果具有很大的随机性，并不适用于动力学研究。本文根据CBHI水解PNPC过程中动态光谱的变化，基于等吸光度点建立的一阶导数光谱的测定方法更加灵敏，其结果比经验性的方法具有更高的可比性，此外，该方法也应用于分析整个反应过程，以得到反应过程的动态(dynamic)变化，为酶催化机理分析提供了新方法。
本文建立的测定方法其主要步骤为：
(1)以样条插值的方法平滑实验数据以便测定瞬时反应速度；
(2)应用基于导数光谱的等吸光度点为据，测定产物PNP的浓度；
(3)以340-400nm一阶导数光谱的AUC对CBHI浓度作图，选择合适的反应时间，确定在此时间内酶浓度与催化速率之间应呈线性相关；
(4)在选择的反应时间内，以一系列不同浓度的CBHI或者稀释的粗酶样品水解高浓度的底物PNPC。可以直接通过以CBHI的催化速率以及其瞬变速率对相应的CBHI浓度作图测定V_(max)与[E]_t，然后，通过简单计算即可以得到K_(cat)(反应速率常数)，可用于酶作用机理的分析。
该方法不仅能够很好的应用于CBHI纯酶催化活力的测定，而且对于含有内切酶和β-葡萄糖苷酶的酶制剂同样适用。另外，本方法对于多种邻／对硝基苯基类化合物酶水解活力的测定也具有普适性的意义。
3．深入分析了CBHI与PNPC的结合机理。分别以紫外、荧光与ITC的方法以瞬变速率为基础，测定了CBHI与PNPC作用的摩尔结合位点数，并在饱和结合条件下测定了CBHI与PNPC的结构变化。证实了纤维二糖抑制纤维素酶活力的机理为其可以与CBHI催化结构域附近的色氨酸结合形成位阻效应，从而阻止纤维素糖链进入活性中心。
通常的研究中，动力学分析主要集中于对一定条件下平衡状态的研究，习惯以可逆反应机理处理结合／吸附数据以及构建结合／吸附方程和进行机理分析，很少考虑饱和结合能力对结合结果的影响。我们的研究表明只有在饱和吸附点受体与配体结合的比例为一定值，游离的配体与受体所占比例最低，其光谱信号才能真正反映结合复合物的内禀的结构变化。在此基础上，本文以紫外光谱、荧光光谱以及ITC技术等三种方法，同时测定了CBHI与PNPC作用的饱和结合点，取得一致的结果；并提出可以通过求结合饱和度的瞬变速率V_(Inst)的方法，准确的测定结合的“摩尔结合位点数”，与传统方法中的最大结合量(B_(max))和达到1／2Bmax的底物浓度(K_d)等表征结合的参数比较，该参数可准确表征CBHI与PNPC之间的相互作用(Fig.2)。
Fig.2 Comparison of the V_(Inst) curves of PNPC converted to PNP, fluorescence quenching %, and the quantity of heat released of CBHI binding by different molar ratio of PNPC with normalization.
并在CBHI与PNPC的饱和结合点，以紫外光谱法观察到在结合复合物中PNPC的分子结构向PNP转变的构型变化，以荧光光谱法测定了结合诱导CBHI产生的构象变化。提出CBHI结合PNPC是受其结合的部分饱和度控制的不可逆过程，催化循环中，PNPC构型改变的热力学动力可能来自于结合过程中CBHI构象的可逆变化，在PNPC诱导CBHI发生构象变化的同时，CBHI也作用与PNPC使之产生构型的改变。另外，对荧光粹灭光谱的二阶导数谱进行分析表明，CBHI催化结构域内的色氨酸残基在PNPC的结合过程中发挥作用，这与CBHI结合结晶纤维素的方式有所差别。对本结果的普适性进行了讨论，提出：不可逆结合和底物在结合中会发生构型变化，在酶催化过程中具有普适性的看法，是对传统的“诱导—契合”理论的一个新的发展。
4．从反应动力学和热力学机理以及数学分析相结合的角度，指出经典研究关于温度影响酶催化活力的分析以及计算Arrhenius活化能的局限性。根据时间—温度叠加法则分别选择复合函数瞬时速率c-v_(inst)以及关于产物瞬时增量dp_(t，T)作为目标函数，表征温度和时间对催化的综合影响；提出水解过程中，酶构象反复发生构型的变化，酶是把环境中的热能转化为功的分子热能转换器，催化是一做功的过程，酶本身是参与反应的，酶催化实际为可逆和不可逆的复合过程，可逆平衡假说具有局限性。
温度是催化反应的重要变量。然而，在有／无底物存在的情况下，温度对酶的影响存在明显区别，因此，在存在底物的情况下讨论温度对酶催化特别是在酶的热稳性研究中的影响，在实际应用中具有更大的指导意义，但传统研究中均沿用无底物条件下升温的方法。
本文以AUC_(370-400nm)为指标，测定了不同温度下CBHI水解PNPC的时间过程中PNP的产量。其结果表明PNP的产量是水解时间(t)与反应温度(T)的复合函数。因此，本文提出在讨论温度对催化的影响时，必须结合相应的时间过程。而通过圆二色谱解链试验，证明在所选的反应温度下，CBHI的活力改变受控于催化和抑制作用，而不可逆变性的影响可以忽略不计。根据温度—时间叠加法则，本文分别以复合函数求导得到的瞬时速率(V_(inst))与以偏导数法计算产物的瞬时增量(dp_(T，t))的方法分析上述两个参量对温度和时间的依赖性(Fig．3)。
Fig. 3 Dependence of temperature and time course on the c-v_(inst) (A) and dp_(T,t) (B).
有数种模型可以用于描述正态和不对称曲线，然而，因为这些曲线都十分复杂(比如方向、对称性和尾部等)，很难构建一个模型，对所有曲线都具有高度适应性，而且在大多数情况下，模型的参数具有物理或者化学意义。本文尝试根据一阶和二阶导数速率的关系，基于两个曲线拐点和c-v_(sec)(瞬时速率的变化)曲线上0点的位置，将整个v_(inst)曲线可以分为四个阶段(Fig．4)：
Ⅰ．加速上升阶段。c-v_(inst)的加速度不断增加，从反应起始阶段至c-v_(sec)曲线达到最高值，在该点d~2v／dT~2达到最大值，而其位点大约为c-v_(inst)最大值的1／2。
Ⅱ．减速上升阶段。c-v_(inst)的加速度不断减小至0，从c-v_(sec)曲线最高点降至0点，在该点d~2v／dT~2=0，而c-v_(inst)达到最大值的。
Ⅲ．下降阶段。c-v_(inst)不断降低，从最高点降至大约1／2最大值的位置，此时d~2V／dT~2降至最小值位置。
Ⅳ．加速下降阶段。c-v_(inst)曲线从1／2最大值位置到终点不断加速下降。
Fig. 4 Estimation of the four phases of v_(inst) curve based on the relationship between v_(inst) and v_(sec) and there corresponding T_(acc), T_(opt-inst) and T_(inac-99).
从数学角度讲，c-v_(inst)最大值所在的温度为反应的最适反应温度T_(opt-inst)。而二阶导数曲线c-v_(sec)最小值对应的温度为T_(inac-99)—催化活力丧失99％的温度。结果表明尽管在同样温度范围内，测定T_(opt)与T_(inac-99)都随反应时间过程的不同而有所区别。时间过程对T_(inac-99)比对T_(opt)的影响更加显著。
同样根据PNP的瞬时增量计算得到的T_(opt-inc)比T_(opt-inst)高3-5K，这是由于T_(opt-inst)的测定是基于瞬时速率达到最大值的温度，而T_(opt-inst)则基于PNP累积量最大的温度，进一步还表明对于酶来讲，不存在具有内禀性质的T_(opt)，所有的T_(opt)都是关于如温度、时间、pH等特定环境条件的函数。
依据Zhang等提出的近抛物线模型并做出一些修改，本文提出对c-v_(inst)对温度曲线进行合理近似以得到净反应速率R_(net)的模型。并以以R_(net)作为独立变量，根据Arrhenius作图法计算反应过程中的表观活化能。结果表明Arrhemius活化能(E_a)并不是常数，而是依赖于温度和反应时间的复合函数。在一定的温度以上则出现负活化能。本实验室共进行了四组不同的试验，测定不同糖苷水解酶的表观活化能，其中两组为内切酶，一组为β-半乳糖苷酶，而另一组也为CBHI但底物为微晶纤维素粉。发现在高于最适反应温度时，总能观察到负的E_a。
对上述结果的分析，表明以Arrhrnius作图的方法测定酶催化反应的表观活化能的结果，(E_a)对于解释反应机理不能提供可靠证据，而酶催化反应中负E_a的出现也表明不能将其作为表征温度影响酶催化的标准。
根据化学亲和力假说和以净反应速率反映酶催化反应进行的程度，提出了一个可表征负反应自由能变化的方法，在CBHI、EGm、EGt等糖苷酶水解过程中得到实证(Fig．5)。
Fig. 5 Comparison of the effects of temperature and time course on the hydrolysis of PNPC by CBHI expressed in three different manners.
在以ITC技术分别测定三个温度下CBHI与PNPC结合和水解的结果后，进一步对以亲和力分析的方法作为确定催化和热力学关系的新途径进行了讨论。并详细讨论了传统研究中，基于可逆平衡的假设进行讨论的弊端，指出酶反应过程中，包括有不可逆反应和非平衡态，必须基于不可逆和非平衡的角度才能得到更加正确的分析结果。本文的前一部分的研究结果表明吸附过程中，底物即发生了不可逆的构型变化；而本部分的研究则证实水解过程中，酶构象反复发生构型的变化，并进一步提出，酶是把环境中的热能转化为功的分子热能转换器，催化是一做功的过程，酶本身是参与反应的。最后，本文还分析了ITC结果可信度不高的原因。
最后，对木质纤维材料生物转化利用的新策略做了一些探索性的实验。
本文以玉米秸秆的皮穰分离利用为例，展望了玉米秸芯为原料产酒精、多糖以及菌体蛋白的可行性，提出农业废弃物资源的利用应该本着“物尽其用”的原则，对不同结构状态的木质纤维，以最低成本为准，分别利用，要考虑工业成本、原材料全利用和环境影响等多个方面，而不能仅以纤维素的利用转化率为唯一目标。而工业中纤维质废弃物的运输与存储的成本是其大规模生产的限制性因素，其综合利用有利于降低对环境的污染。因此，尽量增加纤维质废弃物的用途，提高其利用率和降低生产成本是主要的研究方向。本文还以海带残渣为原料生产燃料乙醇为例进行了讨论，表明海带残渣比秸秆等木质纤维素资源更适合用作发酵产乙醇的原料。木素的存在是木质纤维材料生物转化中必须克服的障碍，目前物理／化学预处理工艺为其必不可少的前提，然而，又一直存在成本过高和环境污染等难题。我们试图探索在不进行木素去除的条件下对某些木质纤维材料进行酶解利用的途径。
Cellulose is a biomass having the most potential in utilization. Its can be used to replace fossil energy. The main problem in its utilization is the conversion of cellulose to fermentable reducing sugars.
Because of the insolubility and inhomogeneous structure of cellulose, and the complexity of cellulase system, it is difficult to design a rational quantitative assay using cellulose as substrate. However, The cornerstone of enzyme engineering is to achieve a direct correlation between the enzyme assays or screening approaches and the changes in enzyme functions in the desired application.
Information of the catalysis mechanism and the effect of catalytic condition is very important in improving the cellulase enzyme performance. And the binding of enzyme and substrate is the prerequisite step of catalytic reaction. So far, the analysis in binding/ adsorption studies assumes a one-step reversible interaction between the ligand and receptor. However, as criticized in chemistry textbooks elsewhere, the above simple assumption only adapted for the elementary reaction, and is limited for multi-component systems. In addition, temperature is a very important factor of the enzymatic catalysis reaction. However, the method and the conclusion of studies about the effect of temperature on enzymatic reactions in kinetics, thermodynamics, and bioengineering are conflicting.
The crude cellulase and the purified 1,4-β-D-glucan cellobiohydrolase I (CBHI) produced by a cellulolytic fungus Trichoderma pseudonkoningii S-38 were used as the material. A new approach to accurately estimating the potential capacity of a cellulase system for the hydrolysis of insoluble cellulosics is described, which can be used to evaluate the susceptibility of different cellulose. Based on the dynamic spectra of reaction, an assay method determining the activity of CBHI for p-nitrophenyl-D-cellobioside (PNPC) was established. The mechanism of CBHI binding to PNPC and the effect of temperature on catalysis in certain time courses were studied in detail. At last, the strategy in biomass utilization was discussed. New discoveries established during my Ph.D., were as follows:
1. Establish a new approach to accurately estimating the potential saccharifying activity of a cellulase system for the hydrolysis of insoluble cellulosics, which can be used to evaluate the susceptibility of different cellulose.
Because the classic cellulase assay methods do not take into account all of the effective factor, it prevents any accurate comparison to be made between the outcomes of different experiments.
Specific Substrate conversion percentage (SSC) of cellulose is closely related to the saccharifying activity of cellulase. SSC was used to express the catalytic rate to overcome the influence cause by the concentration of enzyme and cellulose (Fig.1-A). The converting ability of cellulase for the cellulose can be expressed by the of d[SSC]/dt. For expressing the intrinsic saccharifying of cellulase, the total area under the curve (AUC) of d[SSC]/dt was used to reflect information of the total hydrolysis reaction (Fig.1-B). The Y-intercept of AUC-cellulase adding curve was linear related with the amount of cellulase added.
Fig. 1 (A) Time courses of SSC, during hydrolysis for filter paper by different concentrations of crude cellulase; (B) The relationship between cellulase concentration and its AUC calculated as d[SSC]/dt.
This method takes account into the influence of concentration of cellulase and cellulose added, and the hydrolysis time. Thus, it quantitatively reflects the saccharifying activity of cellulase, and reliable results were obtained that the method can also be used to determine the activity of cellulase for cotton fiber, Avicel PH101, and phosphate acid swollen cellulose (PASC). The comparison of morphology, structure, and the susceptibility of dilute and enzymatic hydrolysis between cellulose I and cellulose II showed that the susceptibility of cellulosics for enzymatic hydrolysis must be estimated by actual hydrolysis. The assay method can also be used for estimating the susceptibility of different cellulosics to evaluate the effect of cellulose treatment. The susceptibility of enzymatic catalysis of cotton fiber, Avicel PH101, and PASC was studied. The treatment of cellulose often increases the special area and the ratio of amorphous region, so they were estimated as well.
2. Establish an assay method of CBHI by numerical differentiation of dynamic UV spectroscopy
In the widely used assay method of CBHI, the reaction conditions such as concentrations of enzyme and PNPC, and the reaction time are empirically selected, thus the dynamic properties of catalytic reaction will not be estimated.
This method was more sensitive based on the analysis of numerical differentiation of dynamic UV spectroscopy, and the result of which is more comparable, furthermore it can be used to analyze the entire reaction process. The main step of the method is:
(1) Smoothing experimental data by spline interpolation method;
(2) Use of isosbestic points of derivative absorbance curves as an index to determine the concentration of PNP.
(3) From the plot of first derivative AUC_(340-400nm) versus concentrations of CBHI, evaluation of suitable reaction time, in which a linear relationship would be established between enzyme concentration and catalytic activity.
(4) Finally, at the selected reaction time, using higher concentration of PNPC as substrate hydrolyzed by a series of different concentration of CBHI or a series of diluted crude cellulase samples. The V_(max) and [E]_t can be directly observed from the plot of instantaneous rate of CBH I activities versus different concentration of CBH I. Then K_(cat) will be obtained by simple calculate.
The above assay procedure was very well not only for pure CBH I preparations but also for crude cellulase preparations. 3. Analyzed the mechanism of PNPC binding to CBHI in detail. The binding stoichiometry was determined base on the instantaneous rate by UV and fluorescence quenching spectra, and isothermal titration calorimetry (ITC) technique. And the structure changes of CBHI and PNPC were determined at SBP.
The behavior of specific bonds between CBHI and PNPC was expressed by the binding stoichiometry — molar binding sites that can accurately determined by its instantaneous rate. Fig.2 combines the results of the instantaneous rate of UV spectra, fluorescence quenching, and the quantity of heat released (Fig.2), from which the SBP can be easily determined.
The conformational changes in CBHI induced by binding of PNPC were analyzed by second-derivative fluorescence spectrometry at the saturation binding point (SBP). And the configurational changes of PNPC in binding process have been evidenced by UV spectra. The technique of isothermal titration calorimetry (ITC) was used to accurately determine the stoichiometry of binding (the molar binding sites) for CBHI to PNPC. All the results suggested that there are at lease two points on CBHI surface that can be bound by PNPC, and the configurationally changes in PNPC converted to PNP (p-nitrophenyl) were observed during binding process. Analysis by fluorescence quenching indicated that the hydrophobic interactions probably play an important role in the binding process, and two tryptophan residues located in catalysis domain contribute to the binding. The results also demonstrated that the binding process of PNPC to CBHI is not an equilibrium process controlled by binding and de-sorption, but an irreversible process controlled by the saturability — degree of fractional saturation. These new insights derived from the interaction between the CBHI and PNPC may provide a basis for further understandings of the binding mechanism for enzyme-substrate interaction.
4. Propose that the effect of temperature on the catalysis and Arrhenius E_a must be analyzed combining with time course; the instantaneous rate (c-v_(inst) and dp_(t,T) was used as a basis for analyzing the effect of temperature and time course on the dynamics; both temperature and time course have the effect on the character of thermodynamics, and temperature was more affective; and estimating the effect of temperature on the structure of CBHI. Suggest that the conformation of enzyme continuously changes during the hydrolysis, further more an enzyme is a "machine" that translates the energy into work, and the enzyme take part in the catalysis process.
Temperature is a important variable of catalytic reactions. However, the effect of temperature on enzyme differs greatly with the presence and absence of substrate. Thus, the result is more instructive for actual use when analyzed the temperature effect on the enzymatic catalysis. The time courses of PNPC hydrolyzed by CBHI under different temperatures were determined. The result showed that output of PNP is the compound function of hydrolysis time (t) and reaction temperature (T). Thus, the effect of temperature on the catalysis must be analyzed combining with time course. The instantaneous rate (V_(Inst)) obtained by compound function and instantaneous increment (dP_(T,t)) obtained from partial derivative are two different concepts.
The variation of CBHI activity was controlled only by both combination of catalysis and inhibition effects and the affect by the irreversible denaturation can then be neglected. This is supported by the melting studies of Circular Dichroism. In the paper, compound function method was used to evaluate the combining effects of temperature and time course on V_(inst) , and the instantaneous increments (V_(inc))(Fig. 3). Fig. 3 Dependence of temperature and time course on the c-V_(inst)(A) and dp_(T,t) (B).
Several models can be used to describe the characteristics of those normal and asymmetric curves. However, because of the complexity of these curves (fronting, symmetric, and tailing etc), it is so difficult to construct a highest flexibility model best fitting to all those different curves, and in most cases no physical or chemical meaning can be associated with the values taken by the parameters. We therefore tried to consider a new way to overcome this difficulty.
Based on the position of two turning points and one zero point on curve of c-v_(sec), the change of instantaneous rate, the c-V_(inst) during entire curve can be separated into four phases:
I. Accelerating increase phase. The c-V_(inst) accelerating increases from the initial up to the
maximum point on curve of c-v_(sec) at which d~2V/dT~2 reached the maximum value it as about the 1/2 maximum of the c-V_(inst) ,
II. Decelerating increase phase. The c-V_(inst) increases with decelerating rate from the maximum point to the zero point on curve of c-v_(sec) at which d~2V/dT~2 reaches zero value.
III. Declining phase. The c-V_(inst) continuously decreases from zero point to about the 1/2 maximum of the decline step at which d~2V/dT~2 reached the minimum point.
IV. Accelerating declining phase. The c-V_(inst) accelerating decreases from 1/2 maximum point to the end.
Mathematically, the corresponding point of the maximum value of c-v_(inst) on temperature-axis indicates the optimum temperature, T_(opt-inst) And the minimum point c-v_(sec) curve is corresponding to the T_(inac-99)-the catalysis activity has lost 99% at this temperature. They showed even the assays under same temperature, however, the T_(opt-inst) and T_(inac-99) are all varied with the assay time course and increase of temperature can decreased the time course for reached the T_(opt-inst) and T_(inac-99), while those effect is more for
T_(inac-99) than T_(opt-inst)(Fig. 4).
Fig. 4 Estimation of the four phases of v_(inst) curve based on the relationship between v_(inst) and v_(sec) and there corresponding T_(acc) , T_(opt-inst) and T_(inac-99)
Following the method proposed by Zhang and Wang for quasi-parabola model, and gives some modified, a rational approximation to modeling the curve of c-V_(ins) versus temperature to get the R_(net) . And R_(net) was used as independent variable to calculate the apparent activation energy by Arrhenius plot. As suggested, the Arrhemius activation energy (E_a) is not a constant while is a compound function of temperature and assay time course dependence. The positive slope is equivalent to negative activation energy, which only appeared as the temperature is beyond certain point.
There are four experimental results performed in our Lab, for estimation of apparent activation energy, two of which belong to β-Endoglucanase, one is the β-Galactosidase and the another one is also CBHI but for hydrolysis of Microcrystalline cellulose powder. A negative Ea are always observed as the temperature beyond the T_(opt).
The studies in enzyme-catalysis, performed with Arrhenius plot to estimate the apparent activation energy, E_a that does not provide favor- able information for explain the reaction mechanism. And the existence of negative E_a during enzyme-catalysis reaction also suggested that there is no possibility for identification of the Ea as a criterion to express the temperature effect on enzyme-catalysis reaction. Thus in this paper, we suggest using the extent of reaction of enzyme-catalysis can be reflected by the net reaction rate.
According to the Chemical affinity theory, based on the calculation of net reaction rates, a method was proposed for expressing the negative free energy, and examined by the hydrolysis result of CBHI, EGm and EGt (Fig. 5).
The quantity of heat released, the Q, during the binding of PNPC to CBHI at 277, 279 and 281 K and hydrolysis at 309, 311 and 313 K were measured by ITC, and explored a new way for establishing the connection between catalysis and thermodynamics ?affinity analysis. And point out that there are irreversible and non-equilibrium processes in the whole catalysis process, and this character must be considered in the analysis so that we can obtain the more correct result. The above result suggested that the irreversible configurational changes of substrate has occurred during the binding process; while in this part, we found that the conformation of enzyme continuously changes during the hydrolysis, and furthermore, an enzyme is a machine that translate the energy in the circumstance into work, and it takes part in the catalysis process.
5. Discussed the strategy in biomass utilization
The main problem of biomass utilization is the high cost in conversion. The separation of cornstalk core and its further conversion was chosen as an example for discussing the feasibility of the production of alcohol, amylase, and protein feed. Propose that the utilization of agricultural waste should follow the fundamental of "make the best use of everything". While the transport and storage cost of industrial waste was very low compared to agricultural waste, on the contrary, its utilization is beneficial to reduce the pollution. Thus, developing the new use and increase the utilized ratio of industrial waste is main object. The industrial waste of kelp was selected as material in alcohol production, and the result suggested that it is more suitable than lignocellolose such as cornstalk.
与蛋白酶类、果胶酶类以及淀粉酶类相比较，纤维素酶(系)活力的准确测定十分困难，一直是理论和应用研究中的难题。由于纤维素的不溶性与结构异质性，以及纤维素酶系统的复杂性，建立以其为底物的实用且可靠的纤维素酶活力测定方法十分困难，而建立高效的活力测定或者筛选方法，以选择具有人们期望的特性的酶或者菌株，是酶工程学研究的基础。
阐明纤维素酶的催化机理与催化环境的影响是提高其转化率的基础。酶与底物结合是催化过程的首要阶段，目前对酶与底物的结合的研究仍然沿用可逆平衡的假说进行分析，但实际上该理论并不适用于如纤维素酶催化体系等多组分反应体系。另外，温度是影响酶催化效率的重要影响因素，在热力学、动力学与生物工程学中都有关于温度对酶催化影响的研究，但无论是思路还是方法都没有形成统一的认识，彼此之间存在很多矛盾。
山东大学微生物技术国家重点实验室从微生物学、生物化学、分子生物学、结构生物学等多学科出发对于解决上述问题进行了深入的研究。本文是从反应机理的角度，从反应动力学机理出发，探讨解决实际应用中的问题。本论文以纤维分解真菌Trichoderma pseudonkoningii S-38分泌的胞外纤维素酶以及从中分离到的外切纤维素酶1，4-β-D-glucan cellobiohydrolase I(CBHI)为材料，主要研究内容为：
●建立能够准确评价纤维素酶制剂对水解纤维材料的长时间总糖化能力，以及不同性质纤维材料酶解敏感性的测定方法；
●以反应过程的动态光谱变化为基础，建立CBHI酶活力的测定方法；
●深入的分析CBHI与其小分子底物对硝基酚纤维二糖苷(p-nitrophenyl-D-cellobioside,PNPC)的结合(Binding)过程的构象构型变化，以阐明结合在酶催化过程中的作用机理；
●研究了，温度和时间过程对CBHI催化PNPC反应的复合影响，以阐明动力学和热力学过程对酶催化的控制机理；
●对生物质资源的利用策略进行了探索和讨论。
研究工作取得具有一定创新性结果如下：
1．提出了一个可准确评价对纤维材料酶解糖化潜力的纤维素酶(系)催化活力的新方法，为应用研究中纤维素酶的准确定量测定这一难题，提供了解决方法。
2．建立了一个可准确测定外切纤维素酶(CBHI)活力的动态光谱法，为纤维素酶作用机理研究提供了技术支持。
3．证实OBHI结合底物对硝基苯纤维二糖(PNPO)时，OBHI的构象(Conformation)变化可导致PNPC构型(Configutation)的改变；证实了纤维二糖抑制纤酶活力的机理在于其能与OBHI活性中心附近的色氨酸残基结合发生位阻效应，是对酶催化机理的“诱导—契合”假说的进一步发展。
4．从反应动力学和热力学机理以及数学分析相结合的角度，指出传统的研究中温度影响酶催化能力(催化活力和热稳定性测试)以及Arrhenius活化能等方法的局限性。并指出以时间—温度叠加法则测定了温度对酶催化过程的作用。分别选择复合函数表征瞬时速率和以偏导数之和表征产物瞬时增量的方法，以分析温度和时间对催化的综合影响。
5．根据化学亲和力(Chemical affinity)并以净反应速率为工具，建立了一个可反映酶催化过程中自由能变化规律的方法。综合上述结果提出水解过程中，酶构象反复发生构型的变化，对以可逆平衡为基础等假说存在的问题进行分析的基础上，进一步提出酶是把环境中的热能转化为功的分子热能转换器，催化是一做功的过程，酶本身是参与反应的等关于酶反应机理的新假说。
最后，对木质纤维材料生物转化利用的新策略做了一些探索性的实验。
1．构建了能够准确评价纤维素酶制剂水解纤维材料的新方法，该方法可以用于评价不同性质纤维材料的酶解感受性。解决了目前应用CMC-Na方法和滤纸酶活法难以准确评价纤维材料被酶解糖化潜力的难题。
由于纤维素的不溶性与结构异质性，以及纤维素酶系统的复杂性，并且因为传统的酶活力测定方法由于没有考虑到酶、底物浓度以及反应时间的影响，限于经验性的结果，拟合而缺少反应动力学分析的支持。特别是酶解过程中，底物结构变化对结果的影响，使测定结果之间难以比较，且不能反应纤维素酶制剂的真实糖化能力和纤维底物可被糖化的潜力。
纤维素比底物转化率(Specific substrate conversion%,SSC)与纤维素酶的糖化能力密切相关，以其表征酶催化活力的变化，既能够反应纤维素酶制剂对底物的水解情况，又避免了酶与底物浓度对计算结果的影响，(Fig.1-A)。SSC的瞬时速率可以表征纤维素酶对底物的转化能力的变化，为了表征纤维素酶制剂对纤维素内禀的糖化能力，本文采用计算瞬时速率的曲线下总面积(The total area under the curve,AUC)的方法，该方法可以反映整个水解过程的信息(Fig.1-B)。在较大的纤维素酶与底物转化率范围之内，AUC-纤维素酶体积曲线在Y轴的截距与加入的酶浓度呈线性相关。
Fig.1 (A) Time courses of SSC, during hydrolysis for filter paper by different concentrations of crude cellulase; (B) The relationship between cellulase concentration and its AUC calculated as d[SSC]/dt.
本方法考虑到了纤维素酶浓度、纤维素底物浓度以及水解时间三个因素的共同影响，微分—积分方法，拟合数据合正确应用。所以能够准确测定纤维素酶粗酶对固体纤维素的糖化能力。经验证，本方法也适用于纤维素酶制剂对棉纤维、微晶纤维素PH101以及磷酸膨化纤维素等不同酶水解敏感度的纤维素底物水解活力的测定。经比较证实，本方法较国际纯粹和应用化学学会推荐的FPA(滤纸活力)法，能准确反映纤维材料被酶解糖化的潜力。
对纤维素Ⅰ比纤维素Ⅱ形态与结构测定以及两者对稀酸水解和酶水解的感受性的比较结果表明，必须结合纤维素的酶水解过程才能正确评价不同纤维素底物酶解感受性的变化。应用本文提出的测定方法同样可以评价不同纤维素底物对特定纤维素酶的水解感受性，并在实际应用中评价各种纤维素改性方法对酶水解效率的促进作用。本文选取具有不同的结晶度、聚合度，以及无定形区含量的棉纤维、微晶纤维素PH101以及磷酸膨化纤维素等三种典型的纤维素模式底物，研究了不同类型纤维素底物酶解反应性。通常纤维素的改性一般会增加纤维素的比表面积，并提高纤维素无定形区的比例，因此，在对酶解效率改变的结果进行分析时应测定比表面积与无定形区的相对比例。
2．建立了一个可准确测定外切纤维素酶(CBHI)活力的动态光谱法。
CBHI是纤维素酶系中对结晶纤维素降解起决定作用的组分，目前，广泛应用的测定CBHI的PNPC水解活力方法，对底物浓度以及反应时间的选择都是经验性的，因此其酶活力测定的结果具有很大的随机性，并不适用于动力学研究。本文根据CBHI水解PNPC过程中动态光谱的变化，基于等吸光度点建立的一阶导数光谱的测定方法更加灵敏，其结果比经验性的方法具有更高的可比性，此外，该方法也应用于分析整个反应过程，以得到反应过程的动态(dynamic)变化，为酶催化机理分析提供了新方法。
本文建立的测定方法其主要步骤为：
(1)以样条插值的方法平滑实验数据以便测定瞬时反应速度；
(2)应用基于导数光谱的等吸光度点为据，测定产物PNP的浓度；
(3)以340-400nm一阶导数光谱的AUC对CBHI浓度作图，选择合适的反应时间，确定在此时间内酶浓度与催化速率之间应呈线性相关；
(4)在选择的反应时间内，以一系列不同浓度的CBHI或者稀释的粗酶样品水解高浓度的底物PNPC。可以直接通过以CBHI的催化速率以及其瞬变速率对相应的CBHI浓度作图测定V_(max)与[E]_t，然后，通过简单计算即可以得到K_(cat)(反应速率常数)，可用于酶作用机理的分析。
该方法不仅能够很好的应用于CBHI纯酶催化活力的测定，而且对于含有内切酶和β-葡萄糖苷酶的酶制剂同样适用。另外，本方法对于多种邻／对硝基苯基类化合物酶水解活力的测定也具有普适性的意义。
3．深入分析了CBHI与PNPC的结合机理。分别以紫外、荧光与ITC的方法以瞬变速率为基础，测定了CBHI与PNPC作用的摩尔结合位点数，并在饱和结合条件下测定了CBHI与PNPC的结构变化。证实了纤维二糖抑制纤维素酶活力的机理为其可以与CBHI催化结构域附近的色氨酸结合形成位阻效应，从而阻止纤维素糖链进入活性中心。
通常的研究中，动力学分析主要集中于对一定条件下平衡状态的研究，习惯以可逆反应机理处理结合／吸附数据以及构建结合／吸附方程和进行机理分析，很少考虑饱和结合能力对结合结果的影响。我们的研究表明只有在饱和吸附点受体与配体结合的比例为一定值，游离的配体与受体所占比例最低，其光谱信号才能真正反映结合复合物的内禀的结构变化。在此基础上，本文以紫外光谱、荧光光谱以及ITC技术等三种方法，同时测定了CBHI与PNPC作用的饱和结合点，取得一致的结果；并提出可以通过求结合饱和度的瞬变速率V_(Inst)的方法，准确的测定结合的“摩尔结合位点数”，与传统方法中的最大结合量(B_(max))和达到1／2Bmax的底物浓度(K_d)等表征结合的参数比较，该参数可准确表征CBHI与PNPC之间的相互作用(Fig.2)。
Fig.2 Comparison of the V_(Inst) curves of PNPC converted to PNP, fluorescence quenching %, and the quantity of heat released of CBHI binding by different molar ratio of PNPC with normalization.
并在CBHI与PNPC的饱和结合点，以紫外光谱法观察到在结合复合物中PNPC的分子结构向PNP转变的构型变化，以荧光光谱法测定了结合诱导CBHI产生的构象变化。提出CBHI结合PNPC是受其结合的部分饱和度控制的不可逆过程，催化循环中，PNPC构型改变的热力学动力可能来自于结合过程中CBHI构象的可逆变化，在PNPC诱导CBHI发生构象变化的同时，CBHI也作用与PNPC使之产生构型的改变。另外，对荧光粹灭光谱的二阶导数谱进行分析表明，CBHI催化结构域内的色氨酸残基在PNPC的结合过程中发挥作用，这与CBHI结合结晶纤维素的方式有所差别。对本结果的普适性进行了讨论，提出：不可逆结合和底物在结合中会发生构型变化，在酶催化过程中具有普适性的看法，是对传统的“诱导—契合”理论的一个新的发展。
4．从反应动力学和热力学机理以及数学分析相结合的角度，指出经典研究关于温度影响酶催化活力的分析以及计算Arrhenius活化能的局限性。根据时间—温度叠加法则分别选择复合函数瞬时速率c-v_(inst)以及关于产物瞬时增量dp_(t，T)作为目标函数，表征温度和时间对催化的综合影响；提出水解过程中，酶构象反复发生构型的变化，酶是把环境中的热能转化为功的分子热能转换器，催化是一做功的过程，酶本身是参与反应的，酶催化实际为可逆和不可逆的复合过程，可逆平衡假说具有局限性。
温度是催化反应的重要变量。然而，在有／无底物存在的情况下，温度对酶的影响存在明显区别，因此，在存在底物的情况下讨论温度对酶催化特别是在酶的热稳性研究中的影响，在实际应用中具有更大的指导意义，但传统研究中均沿用无底物条件下升温的方法。
本文以AUC_(370-400nm)为指标，测定了不同温度下CBHI水解PNPC的时间过程中PNP的产量。其结果表明PNP的产量是水解时间(t)与反应温度(T)的复合函数。因此，本文提出在讨论温度对催化的影响时，必须结合相应的时间过程。而通过圆二色谱解链试验，证明在所选的反应温度下，CBHI的活力改变受控于催化和抑制作用，而不可逆变性的影响可以忽略不计。根据温度—时间叠加法则，本文分别以复合函数求导得到的瞬时速率(V_(inst))与以偏导数法计算产物的瞬时增量(dp_(T，t))的方法分析上述两个参量对温度和时间的依赖性(Fig．3)。
Fig. 3 Dependence of temperature and time course on the c-v_(inst) (A) and dp_(T,t) (B).
有数种模型可以用于描述正态和不对称曲线，然而，因为这些曲线都十分复杂(比如方向、对称性和尾部等)，很难构建一个模型，对所有曲线都具有高度适应性，而且在大多数情况下，模型的参数具有物理或者化学意义。本文尝试根据一阶和二阶导数速率的关系，基于两个曲线拐点和c-v_(sec)(瞬时速率的变化)曲线上0点的位置，将整个v_(inst)曲线可以分为四个阶段(Fig．4)：
Ⅰ．加速上升阶段。c-v_(inst)的加速度不断增加，从反应起始阶段至c-v_(sec)曲线达到最高值，在该点d~2v／dT~2达到最大值，而其位点大约为c-v_(inst)最大值的1／2。
Ⅱ．减速上升阶段。c-v_(inst)的加速度不断减小至0，从c-v_(sec)曲线最高点降至0点，在该点d~2v／dT~2=0，而c-v_(inst)达到最大值的。
Ⅲ．下降阶段。c-v_(inst)不断降低，从最高点降至大约1／2最大值的位置，此时d~2V／dT~2降至最小值位置。
Ⅳ．加速下降阶段。c-v_(inst)曲线从1／2最大值位置到终点不断加速下降。
Fig. 4 Estimation of the four phases of v_(inst) curve based on the relationship between v_(inst) and v_(sec) and there corresponding T_(acc), T_(opt-inst) and T_(inac-99).
从数学角度讲，c-v_(inst)最大值所在的温度为反应的最适反应温度T_(opt-inst)。而二阶导数曲线c-v_(sec)最小值对应的温度为T_(inac-99)—催化活力丧失99％的温度。结果表明尽管在同样温度范围内，测定T_(opt)与T_(inac-99)都随反应时间过程的不同而有所区别。时间过程对T_(inac-99)比对T_(opt)的影响更加显著。
同样根据PNP的瞬时增量计算得到的T_(opt-inc)比T_(opt-inst)高3-5K，这是由于T_(opt-inst)的测定是基于瞬时速率达到最大值的温度，而T_(opt-inst)则基于PNP累积量最大的温度，进一步还表明对于酶来讲，不存在具有内禀性质的T_(opt)，所有的T_(opt)都是关于如温度、时间、pH等特定环境条件的函数。
依据Zhang等提出的近抛物线模型并做出一些修改，本文提出对c-v_(inst)对温度曲线进行合理近似以得到净反应速率R_(net)的模型。并以以R_(net)作为独立变量，根据Arrhenius作图法计算反应过程中的表观活化能。结果表明Arrhemius活化能(E_a)并不是常数，而是依赖于温度和反应时间的复合函数。在一定的温度以上则出现负活化能。本实验室共进行了四组不同的试验，测定不同糖苷水解酶的表观活化能，其中两组为内切酶，一组为β-半乳糖苷酶，而另一组也为CBHI但底物为微晶纤维素粉。发现在高于最适反应温度时，总能观察到负的E_a。
对上述结果的分析，表明以Arrhrnius作图的方法测定酶催化反应的表观活化能的结果，(E_a)对于解释反应机理不能提供可靠证据，而酶催化反应中负E_a的出现也表明不能将其作为表征温度影响酶催化的标准。
根据化学亲和力假说和以净反应速率反映酶催化反应进行的程度，提出了一个可表征负反应自由能变化的方法，在CBHI、EGm、EGt等糖苷酶水解过程中得到实证(Fig．5)。
Fig. 5 Comparison of the effects of temperature and time course on the hydrolysis of PNPC by CBHI expressed in three different manners.
在以ITC技术分别测定三个温度下CBHI与PNPC结合和水解的结果后，进一步对以亲和力分析的方法作为确定催化和热力学关系的新途径进行了讨论。并详细讨论了传统研究中，基于可逆平衡的假设进行讨论的弊端，指出酶反应过程中，包括有不可逆反应和非平衡态，必须基于不可逆和非平衡的角度才能得到更加正确的分析结果。本文的前一部分的研究结果表明吸附过程中，底物即发生了不可逆的构型变化；而本部分的研究则证实水解过程中，酶构象反复发生构型的变化，并进一步提出，酶是把环境中的热能转化为功的分子热能转换器，催化是一做功的过程，酶本身是参与反应的。最后，本文还分析了ITC结果可信度不高的原因。
最后，对木质纤维材料生物转化利用的新策略做了一些探索性的实验。
本文以玉米秸秆的皮穰分离利用为例，展望了玉米秸芯为原料产酒精、多糖以及菌体蛋白的可行性，提出农业废弃物资源的利用应该本着“物尽其用”的原则，对不同结构状态的木质纤维，以最低成本为准，分别利用，要考虑工业成本、原材料全利用和环境影响等多个方面，而不能仅以纤维素的利用转化率为唯一目标。而工业中纤维质废弃物的运输与存储的成本是其大规模生产的限制性因素，其综合利用有利于降低对环境的污染。因此，尽量增加纤维质废弃物的用途，提高其利用率和降低生产成本是主要的研究方向。本文还以海带残渣为原料生产燃料乙醇为例进行了讨论，表明海带残渣比秸秆等木质纤维素资源更适合用作发酵产乙醇的原料。木素的存在是木质纤维材料生物转化中必须克服的障碍，目前物理／化学预处理工艺为其必不可少的前提，然而，又一直存在成本过高和环境污染等难题。我们试图探索在不进行木素去除的条件下对某些木质纤维材料进行酶解利用的途径。
Cellulose is a biomass having the most potential in utilization. Its can be used to replace fossil energy. The main problem in its utilization is the conversion of cellulose to fermentable reducing sugars.
Because of the insolubility and inhomogeneous structure of cellulose, and the complexity of cellulase system, it is difficult to design a rational quantitative assay using cellulose as substrate. However, The cornerstone of enzyme engineering is to achieve a direct correlation between the enzyme assays or screening approaches and the changes in enzyme functions in the desired application.
Information of the catalysis mechanism and the effect of catalytic condition is very important in improving the cellulase enzyme performance. And the binding of enzyme and substrate is the prerequisite step of catalytic reaction. So far, the analysis in binding/ adsorption studies assumes a one-step reversible interaction between the ligand and receptor. However, as criticized in chemistry textbooks elsewhere, the above simple assumption only adapted for the elementary reaction, and is limited for multi-component systems. In addition, temperature is a very important factor of the enzymatic catalysis reaction. However, the method and the conclusion of studies about the effect of temperature on enzymatic reactions in kinetics, thermodynamics, and bioengineering are conflicting.
The crude cellulase and the purified 1,4-β-D-glucan cellobiohydrolase I (CBHI) produced by a cellulolytic fungus Trichoderma pseudonkoningii S-38 were used as the material. A new approach to accurately estimating the potential capacity of a cellulase system for the hydrolysis of insoluble cellulosics is described, which can be used to evaluate the susceptibility of different cellulose. Based on the dynamic spectra of reaction, an assay method determining the activity of CBHI for p-nitrophenyl-D-cellobioside (PNPC) was established. The mechanism of CBHI binding to PNPC and the effect of temperature on catalysis in certain time courses were studied in detail. At last, the strategy in biomass utilization was discussed. New discoveries established during my Ph.D., were as follows:
1. Establish a new approach to accurately estimating the potential saccharifying activity of a cellulase system for the hydrolysis of insoluble cellulosics, which can be used to evaluate the susceptibility of different cellulose.
Because the classic cellulase assay methods do not take into account all of the effective factor, it prevents any accurate comparison to be made between the outcomes of different experiments.
Specific Substrate conversion percentage (SSC) of cellulose is closely related to the saccharifying activity of cellulase. SSC was used to express the catalytic rate to overcome the influence cause by the concentration of enzyme and cellulose (Fig.1-A). The converting ability of cellulase for the cellulose can be expressed by the of d[SSC]/dt. For expressing the intrinsic saccharifying of cellulase, the total area under the curve (AUC) of d[SSC]/dt was used to reflect information of the total hydrolysis reaction (Fig.1-B). The Y-intercept of AUC-cellulase adding curve was linear related with the amount of cellulase added.
Fig. 1 (A) Time courses of SSC, during hydrolysis for filter paper by different concentrations of crude cellulase; (B) The relationship between cellulase concentration and its AUC calculated as d[SSC]/dt.
This method takes account into the influence of concentration of cellulase and cellulose added, and the hydrolysis time. Thus, it quantitatively reflects the saccharifying activity of cellulase, and reliable results were obtained that the method can also be used to determine the activity of cellulase for cotton fiber, Avicel PH101, and phosphate acid swollen cellulose (PASC). The comparison of morphology, structure, and the susceptibility of dilute and enzymatic hydrolysis between cellulose I and cellulose II showed that the susceptibility of cellulosics for enzymatic hydrolysis must be estimated by actual hydrolysis. The assay method can also be used for estimating the susceptibility of different cellulosics to evaluate the effect of cellulose treatment. The susceptibility of enzymatic catalysis of cotton fiber, Avicel PH101, and PASC was studied. The treatment of cellulose often increases the special area and the ratio of amorphous region, so they were estimated as well.
2. Establish an assay method of CBHI by numerical differentiation of dynamic UV spectroscopy
In the widely used assay method of CBHI, the reaction conditions such as concentrations of enzyme and PNPC, and the reaction time are empirically selected, thus the dynamic properties of catalytic reaction will not be estimated.
This method was more sensitive based on the analysis of numerical differentiation of dynamic UV spectroscopy, and the result of which is more comparable, furthermore it can be used to analyze the entire reaction process. The main step of the method is:
(1) Smoothing experimental data by spline interpolation method;
(2) Use of isosbestic points of derivative absorbance curves as an index to determine the concentration of PNP.
(3) From the plot of first derivative AUC_(340-400nm) versus concentrations of CBHI, evaluation of suitable reaction time, in which a linear relationship would be established between enzyme concentration and catalytic activity.
(4) Finally, at the selected reaction time, using higher concentration of PNPC as substrate hydrolyzed by a series of different concentration of CBHI or a series of diluted crude cellulase samples. The V_(max) and [E]_t can be directly observed from the plot of instantaneous rate of CBH I activities versus different concentration of CBH I. Then K_(cat) will be obtained by simple calculate.
The above assay procedure was very well not only for pure CBH I preparations but also for crude cellulase preparations. 3. Analyzed the mechanism of PNPC binding to CBHI in detail. The binding stoichiometry was determined base on the instantaneous rate by UV and fluorescence quenching spectra, and isothermal titration calorimetry (ITC) technique. And the structure changes of CBHI and PNPC were determined at SBP.
The behavior of specific bonds between CBHI and PNPC was expressed by the binding stoichiometry — molar binding sites that can accurately determined by its instantaneous rate. Fig.2 combines the results of the instantaneous rate of UV spectra, fluorescence quenching, and the quantity of heat released (Fig.2), from which the SBP can be easily determined.
The conformational changes in CBHI induced by binding of PNPC were analyzed by second-derivative fluorescence spectrometry at the saturation binding point (SBP). And the configurational changes of PNPC in binding process have been evidenced by UV spectra. The technique of isothermal titration calorimetry (ITC) was used to accurately determine the stoichiometry of binding (the molar binding sites) for CBHI to PNPC. All the results suggested that there are at lease two points on CBHI surface that can be bound by PNPC, and the configurationally changes in PNPC converted to PNP (p-nitrophenyl) were observed during binding process. Analysis by fluorescence quenching indicated that the hydrophobic interactions probably play an important role in the binding process, and two tryptophan residues located in catalysis domain contribute to the binding. The results also demonstrated that the binding process of PNPC to CBHI is not an equilibrium process controlled by binding and de-sorption, but an irreversible process controlled by the saturability — degree of fractional saturation. These new insights derived from the interaction between the CBHI and PNPC may provide a basis for further understandings of the binding mechanism for enzyme-substrate interaction.
4. Propose that the effect of temperature on the catalysis and Arrhenius E_a must be analyzed combining with time course; the instantaneous rate (c-v_(inst) and dp_(t,T) was used as a basis for analyzing the effect of temperature and time course on the dynamics; both temperature and time course have the effect on the character of thermodynamics, and temperature was more affective; and estimating the effect of temperature on the structure of CBHI. Suggest that the conformation of enzyme continuously changes during the hydrolysis, further more an enzyme is a "machine" that translates the energy into work, and the enzyme take part in the catalysis process.
Temperature is a important variable of catalytic reactions. However, the effect of temperature on enzyme differs greatly with the presence and absence of substrate. Thus, the result is more instructive for actual use when analyzed the temperature effect on the enzymatic catalysis. The time courses of PNPC hydrolyzed by CBHI under different temperatures were determined. The result showed that output of PNP is the compound function of hydrolysis time (t) and reaction temperature (T). Thus, the effect of temperature on the catalysis must be analyzed combining with time course. The instantaneous rate (V_(Inst)) obtained by compound function and instantaneous increment (dP_(T,t)) obtained from partial derivative are two different concepts.
The variation of CBHI activity was controlled only by both combination of catalysis and inhibition effects and the affect by the irreversible denaturation can then be neglected. This is supported by the melting studies of Circular Dichroism. In the paper, compound function method was used to evaluate the combining effects of temperature and time course on V_(inst) , and the instantaneous increments (V_(inc))(Fig. 3). Fig. 3 Dependence of temperature and time course on the c-V_(inst)(A) and dp_(T,t) (B).
Several models can be used to describe the characteristics of those normal and asymmetric curves. However, because of the complexity of these curves (fronting, symmetric, and tailing etc), it is so difficult to construct a highest flexibility model best fitting to all those different curves, and in most cases no physical or chemical meaning can be associated with the values taken by the parameters. We therefore tried to consider a new way to overcome this difficulty.
Based on the position of two turning points and one zero point on curve of c-v_(sec), the change of instantaneous rate, the c-V_(inst) during entire curve can be separated into four phases:
I. Accelerating increase phase. The c-V_(inst) accelerating increases from the initial up to the
maximum point on curve of c-v_(sec) at which d~2V/dT~2 reached the maximum value it as about the 1/2 maximum of the c-V_(inst) ,
II. Decelerating increase phase. The c-V_(inst) increases with decelerating rate from the maximum point to the zero point on curve of c-v_(sec) at which d~2V/dT~2 reaches zero value.
III. Declining phase. The c-V_(inst) continuously decreases from zero point to about the 1/2 maximum of the decline step at which d~2V/dT~2 reached the minimum point.
IV. Accelerating declining phase. The c-V_(inst) accelerating decreases from 1/2 maximum point to the end.
Mathematically, the corresponding point of the maximum value of c-v_(inst) on temperature-axis indicates the optimum temperature, T_(opt-inst) And the minimum point c-v_(sec) curve is corresponding to the T_(inac-99)-the catalysis activity has lost 99% at this temperature. They showed even the assays under same temperature, however, the T_(opt-inst) and T_(inac-99) are all varied with the assay time course and increase of temperature can decreased the time course for reached the T_(opt-inst) and T_(inac-99), while those effect is more for
T_(inac-99) than T_(opt-inst)(Fig. 4).
Fig. 4 Estimation of the four phases of v_(inst) curve based on the relationship between v_(inst) and v_(sec) and there corresponding T_(acc) , T_(opt-inst) and T_(inac-99)
Following the method proposed by Zhang and Wang for quasi-parabola model, and gives some modified, a rational approximation to modeling the curve of c-V_(ins) versus temperature to get the R_(net) . And R_(net) was used as independent variable to calculate the apparent activation energy by Arrhenius plot. As suggested, the Arrhemius activation energy (E_a) is not a constant while is a compound function of temperature and assay time course dependence. The positive slope is equivalent to negative activation energy, which only appeared as the temperature is beyond certain point.
There are four experimental results performed in our Lab, for estimation of apparent activation energy, two of which belong to β-Endoglucanase, one is the β-Galactosidase and the another one is also CBHI but for hydrolysis of Microcrystalline cellulose powder. A negative Ea are always observed as the temperature beyond the T_(opt).
The studies in enzyme-catalysis, performed with Arrhenius plot to estimate the apparent activation energy, E_a that does not provide favor- able information for explain the reaction mechanism. And the existence of negative E_a during enzyme-catalysis reaction also suggested that there is no possibility for identification of the Ea as a criterion to express the temperature effect on enzyme-catalysis reaction. Thus in this paper, we suggest using the extent of reaction of enzyme-catalysis can be reflected by the net reaction rate.
According to the Chemical affinity theory, based on the calculation of net reaction rates, a method was proposed for expressing the negative free energy, and examined by the hydrolysis result of CBHI, EGm and EGt (Fig. 5).
The quantity of heat released, the Q, during the binding of PNPC to CBHI at 277, 279 and 281 K and hydrolysis at 309, 311 and 313 K were measured by ITC, and explored a new way for establishing the connection between catalysis and thermodynamics ?affinity analysis. And point out that there are irreversible and non-equilibrium processes in the whole catalysis process, and this character must be considered in the analysis so that we can obtain the more correct result. The above result suggested that the irreversible configurational changes of substrate has occurred during the binding process; while in this part, we found that the conformation of enzyme continuously changes during the hydrolysis, and furthermore, an enzyme is a machine that translate the energy in the circumstance into work, and it takes part in the catalysis process.
5. Discussed the strategy in biomass utilization
The main problem of biomass utilization is the high cost in conversion. The separation of cornstalk core and its further conversion was chosen as an example for discussing the feasibility of the production of alcohol, amylase, and protein feed. Propose that the utilization of agricultural waste should follow the fundamental of "make the best use of everything". While the transport and storage cost of industrial waste was very low compared to agricultural waste, on the contrary, its utilization is beneficial to reduce the pollution. Thus, developing the new use and increase the utilized ratio of industrial waste is main object. The industrial waste of kelp was selected as material in alcohol production, and the result suggested that it is more suitable than lignocellolose such as cornstalk.
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