Site-Directed Mutagenesis Study Of Halophilic Cellulase CelB Substrate Recognition Sites

Cellulases have a wide range of applications in biomedical science, food industry and chemical industry. Alkaliphilic and halophilic cellulase has excellent stability and high salinity reactivity. So it is a very promising industrial enzyme. Alkaliphilic and halophilic Bacillus sp. BG-CS10 can encode a high halophilic cellulase CelB which has a high activity under high salt concentration, while it shows low activity at low salt concentrations. Cellulase CelB has endoglucanase activity, not exo-glucanase or ?-glycoside hydrolase activity. Therefore, it can hydrolyze soluble cellulose substrates such as carboxymethyl cel ulose sodium.Alkaliphilic and halophilic cellulase CelB consists of a catalytic domain,DUF291 Domain and CBM-46 Domain at the C-terminal peptide chain shaped as the letter L. The Catalytic Domain contains a(?/?) 8TIM- barrel structure which is the same as the glycoside hydrolase family 5. Experiments of the relevant domain of halophilic cellulase CelB show that deletion of DUF291 Domain or CBM-46 Domain will result in loss of activity of the cellulase CelB. This illustrates that three Domains of cellulase CelB are indispensable and mutual synergy. Crystals of CelB/substrate complex and site-directed mutagenesis are used to study the amino acid sites for catalyzing and substrate binding. Site-directed mutagenesis studies found that CelB contains a catalytic module Glu149-Glu271 and the active sites His104 and His224 may be used as electron transfer intermediate for the Glu-Glu catalytic module. Further studies found that the aromatic residues Trp37?Trp229?Trp310and Trp476 are substrate recognition sites of cellulase CelB. Meanwhile, This study also found that His349 and His366 of DUF291 Domains can affect the interaction of CelB and substrates. The study show that three domains of halophilic cel ulase CelB can form entire substrate binding sites.