Linder M, Teeri T T
VTT, Biotechnology and Food Research, Espoo, Finland.
Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12251-5. doi: 10.1073/pnas.93.22.12251.
Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.
纤维素结合结构域（CBDs）能特异性结合纤维素，并构成大多数纤维素降解酶的独特结构域。CBD介导的酶结合在固体纤维素底物的水解过程中起着重要作用。在本研究中，我们探究了里氏木霉纤维二糖水解酶I的CBD与微晶纤维素结合的可逆性及动力学。该CBD在大肠杆菌中产生，经纯化后通过用³H进行还原烷基化进行放射性标记。对标记的CBD的灵敏检测使得对其行为的分析比以往更加详细，并揭示了重要的新特性。发现CBD的结合对温度敏感，在较低温度下亲和力增加。CBD与纤维素的相互作用被证明是完全可逆的，并且通过简单稀释就可以从纤维素上洗脱CBD。所测得的CBD - 纤维素相互作用的交换速率与纤维二糖水解酶I的水解速率相当，这与其作为一种连续外切葡聚糖酶的作用模式相一致。
