氏名　Karim Nurul
学位の種類　博士（工学）
学位記番号　博甲第335号
学位授与の日付　平成17年3月25日
学位論文題目　Precise analysis of the cellulase-catalyzed hydrolysis of cello-oligosaccharides using isothermal titration calorimetry　（セルラーゼが触媒するセロオリゴ糖加水分解の等温滴定熱量測定法を用いた精密解析）
論文審査委員
　主査　助教授　城所　俊一
　副査　教授　曽田　邦嗣
　副査　教授　福田　雅夫
　副査　助教授　岡田　宏文
　副査　助教授　野中　孝昌

• 論文目次
• 論文要旨

［平成16（2004）年度博士論文題名一覧］ ［博士論文題名一覧］に戻る．

Contents

General Introduction　p.1

Chapter 1　Precise and continuous observation of cellulase-catalyzed hydrolysis of cello-oligosaccharides using isothermal titration calorimetry　p.12
　1.1 Introduction　p.12
　1.2 Materials and Methods　p.13
　1.3 Results　p.17
　1.3.1 Evaluation of the kinetics of the endoglucanase by ITC　p.17
　1.3.2 HPLC analysis of the hydrolysis of cello-oligosaccharides　p.25
　1.3.3 Inhibition of endoglucanase　p.30
　1.4 Discussion　p.37

Chapter 2　Calorimetric evaluation of the activity and the mechanism of cellulases for the hydrolysis of cello-oligosaccharides accompanied by the mutarotation reaction of the hydrolyzed products　p.39
　2.1 Introduction　p.39
　2.2 Materials and methods　p.40
　2.3 Results　p.42
　2.3.1 hydrolysis of cello-oligosaccharides catalyzed by retaining-type endoglucanase　p.42
　2.3.2 The pH dependence hydrolytic activity of inverting-type endoglucanase and mutarotation contributions　p.47
　2.3.3 Mutarotation contributions for retaining-type endoglucanase　p.49
　2.3.4 Substrate concentration dependence of mutarotation heats　p.49
　2.3.5 Estimation of mutarotation rate constant　p.56
　2.3.6 Thermal activation of hydrolysis and mutarotation rate　p.57
　2.4 Discussion　p.63

Chapter 3　The Catalytic activity of cellulase mutants designed to perturb the electrostatic potential at the active site　p.67

　3.1 Introduction　p.67
　3.2 Materials and methods　p.68
　3.3 Results　p.83
　3.3.1 Effect of mutation on three-dimensional structure　p.83
　3.3.2 Evaluation of thermal stability　p.83
　3.3.3 pH dependence of hydrolytic activity　p.87
　3.4 Discussion　p.100

General conclusion　p.103

References　p.107

Pubication list　p.116

Acknowledgements　p.117

Quantitative evaluation of the catalytic activity of enzymes is inevitably important in order to reveal the enzymes' reaction mechanisms and to design more effective catalysts. In this thesis, a new analysis method to evaluate kinetics of cellulase-catalyzed hydrolysis of cello-oligosaccharides was developed by using isothermal titration calorimetry （ITC）. Furthermore, the application of the new methodology to investigate the effect of electrostatic field perturbation around the active site of newly designed cellulases on the pH profile of their enzymatic parameters was studied.
Heat is a characteristic of all chemical reactions, and ITC provides a possibleway to continuously detect the heat from catalytic reactions with high sensitivity and reproducibility. In endoglucanase-catalyzed cello-oligosaccharides reaction, the hydrolysis heat of single β-1,4 glycosidic bond was successfully detected and confirmed by combined analysis of ITC and normal- phase HPLC, and the time course of the enzymatic reaction was monitored continuously by ITC.
The enzymatic parameters kcat and KM, obtained from calorimetric observables, clearly indicated that the reaction was well approximated by a simple Michaelis-Menten equation. Moreover, a strong substrate concentration dependence of endoglucanase activity was observed and from ITC observations it was confirmed that the inhibition was exerted not from the hydrolysis products but from the substrate itself.
In an attempt to evaluate the heat effect accompanying the mutarotation of the newly produced reducing end of the products, which becomes large and detectable in neutral pH the developed methodology was extended. By using this method, the activities of two endoglucanases-an inverting-type enzyme and a retaining-type enzyme-were successfully evaluated against cello-oligosaccharides at different pHs. While the observed heats at acidic pHs were explained by a simple hydrolysis reaction without the mutarotation effect, the ITC data in neutral pH, clearly showed the presence of post-hydrolysis reaction, and were explained very well by the new model considering the heat of the mutarotation of the products. The exothermic heat or the endothermic heat caused by the mutarotation reaction was obtained in the case of the inverting- or the retaining-type endoglucanase, respectively. These results indicate that the calorimetric evaluation of the activity of endoglucanases can be done even in cases in which the mutarotation was not negligible, and that the sign of the mutarotation heat can be used to distinguish the anomeric type of the newly produced reducing end of the hydrolyzed products. The mutarotation rates were suggested to be greatly effected by reaction temperature and pH.
Physical perturbation method based on the three dimensional （3D） structure, was applied as an effective strategy to design the cellulase mutants. It was shown that the design of the electrostatic potential perturbation at one of the active site of an endoglucanase can enhance the activity of the enzyme. The three-dimensional （3D） structure of the enzyme was estimated from the amino-acid sequence by homology modeling and molecular dynamics （MD） simulation. With the numerical calculation of the electrostatic potential based on the estimated structure, a single site, Gln138 was designed and selected where the introduced negative residue, glutamate or a positive residue among the lysine and arginine was found to be perturb the electrostatic potential around the general base, Asp10 of the enzyme. The circular dichroism （CD） spectra and differential scanning calorimetry （DSC） measurement indicated that the 3D structure of the mutants and the thermal stability were almost the same as those of the wild type.
The catalytic activities of wild type and the mutants were evaluated by ITC and biocinchoninic acid （BCA） methods using the substrates, cello-oligosaccharides and carboxymethyl cellulase, respectively. Both the methods clearly suggested that the negative or positive charge at that site increased or decreased the enzyme activity, respectively. The ITC observation clearly showed that the perturbation of the electrostatic potential led to change the kcat value whereas the binding affinity to the substrate was little affected by the mutation. A significant pH dependence of the mutation effect on the catalytic activity was also observed.