Crystallization of Cellobiohydrolase in the Presence of Cellulose-Degraded Cellobiose: Analysis of Intermolecular Interactions and Association Dynamics

Kazuo Onuma,Yuki Ohnishi,Naoki Furubayashi,Sachiko Kaito,Fujiko Shibata,Koji Inaka,Yoshiko Kobayashi

doi:10.4236/jcpt.2014.41001

Kazuo Onuma, Yuki Ohnishi + Show 5 more

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https://doi.org/10.4236/jcpt.2014.41001

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Abstract

Crystallization of enzymes in presence of impurities is important for clarifying the role of enzymes in natural world. Although it is proposed that impurities inhibit nucleation of enzyme crystallization, details are unclear. In this study, crystallization of cellobiohydrolase from Aspergillus niger was investigated by dynamic and time-resolved static light scattering using cellobiose as an impurity. We aimed to clarify how cellobiose inhibits cellobiohydrolase crystallization and to crystallize cellobiohydrolase in concentrated cellobiose without using seeds. The contribution of attractive forces to total intermolecular interactions of cellobiohydrolase monomers increased with the molar ratio of cellobiose/cellobiohydrolase (R(cb/ce)). Association dynamics of cellobiohydrolase using lithium sulfate, however, showed that the initial aggregation rate decreased with an increase in R(cb/ce). Because binding sites of cellobioses to cellobiohydrolase molecules differed from those for the growth of protein crystals, the binding of cellobioses would increase the chemical potential of the cellobiohydrolase monomers, which gradually reduced supersaturation for growth as the aggregate size increased. This result was in contrast with the conventional idea that cellobiose inhibits the nucleation of cellobiohydrolase crystals. Gentle agitation of cellobiose-containing cellobiohydrolase solutions during sitting-drop vapor-diffusion growth resulted in the growth of cellobiohydrolase single crystals for all R(cb/ce) conditions without using seeds.

Highlights

Molecular structure analysis of catalytic enzymes is the key to elucidating roles of proteins and polysaccharides in living organisms
Consistent with the Dynamic light scattering (DLS) result, positive A2 proportional to the inclination of the correlation line indicates that a repulsive force is dominantly operated between cellobiohydrolase monomers
The interaction of cellobiohydrolase monomers shifted to be attractive when cellobiose molecules were bound to them owing to an increase in the Hamaker constant

Summary

Introduction

Molecular structure analysis of catalytic enzymes is the key to elucidating roles of proteins and polysaccharides in living organisms. Developing drugs that quickly degrade proteins related to fatal diseases would greatly contribute to healthcare, and reducing the costs of catalysis for biomass production would contribute to the solution of energy problems. The most powerful and reliable method to analyze the molecular structure of catalytic enzymes is to apply Xray diffraction to high-quality protein single crystals. Protein crystallization is still problematic, advances in crystallization techniques based on many trials and errors have increased the chances of successful crystallization in pure solutions. The presence of foreign materials such as impurities and protein-degraded products in the solution used for crystal growth, a common situation, makes crystallization more difficult and sometimes even impossible unless

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