Study On Endo-cellulase Binding Site Of Penicillium

Cellulase is a kind of multi-component enzyme system that can degrade cellulose into glucose.The synergy of them can hydrolyze cellulose into cellobiose and oligosaccharides,then finally to glucose.Structurally,endoglucanase is composed of the catalytic domain?CD?,cellulose binding domain?CBD?,and a linker peptide?Linker?to connect these two domains.In the hydrolysis process of cellulose by cellulase,cellulose can be adsorbed by the binding domain,and then hydrolyzed by catalytic domain.Previously,we isolated a cellulose-hydrolyzing strain Penicillium sp.601,and cloned its endoglucanse gene?EGL1?by reverse transcription.Subsequent studies have found that the CBD domain of EGL1 has a stronger binding ability to cellulose,and the CD domain has a weaker binding ability.This paper is intended to study the structureal and functional relationship between Penicillium cellulase catalytic domain?EGL1 CD?,a binding domain?EGL1 CBD?,and their cellulose catalytic activity and binding activity.Comparison to the reported amino acid sequences of cellulase binding domain,EGL1 CBD was found to have two aromatic amino acid-rich regions,M1 and M2.In order to determine the binding sites location,aromatic amino acids of two regions in EGL1 CBD were mutated into glycines to form four type of mutants.Four mutant genes,egfp-cbd and egfp were transforming to express in E.coli.The blank eGFP,control protein eGFP-CBD,mutant eGFP-CBD-2R?3 point mutation,M1 zone?,eGFP-CBD-3R?6 point mutation,three points in the M1 zone,three points located in M2 district?,eGFP-CBD-4F?3 point mutation,located district M2?,eGFP-CBD-5F?1 point mutation,located in M2 region?were purified and observed the binding situation to cellulose with a fluorescent microscope.The fluorescence signal intensity of these mutants determined was eGFP-CBD> eGFP-CBD-5F> eGFP-CBD-4F> eGFP-CBD-2R> eGFP-CBD-3R> eGFP.Isothermal titration calorimeter was detected adsorption heat of protein to cellopentose.The adsorption experimental results showed that the amino acid in M1 site contribute more than M2 site,which is consistent with the cellulose binding experiment.Homology modeling three-dimensional structure of EGL1 CBD,M1 and M2 forming a flat,cooperative involved in binding cellulose.This difference on binding of these aromatic amino acids may connected with the spatial conformation of the CBD domain and the connected linker region.According to the structural simulation of CD sequence,aromatic amino acids Tyr154 and Phe156 located in the bottom of CD domain,and Trp166 and Trp168 close to the active site were mutated to glycines.Four mutant genes and CD domain were transformed into the expression host Escherichia coli.The CD,CDM1(W₁₆₆,W₁₆₈),CDM2?F154,Y156?and CDM3(W₁₆₆,W₁₆₈,F154,Y156)were purified and the filter paper activities of these mutants were checked.The enzyme activity results was shown as that CD> CDM2> CDM1> CDM3.According to the results,we speculated that aromatic amino acids F154,Y156 at the bottom of CD domain may contribute to the binding of cellulose,aromatic amino acid W₁₆₆,W₁₆₈ near the active site may be involved in the transmission of cellulose into or out of active site of catalytic domain.These results generated from Penicillium cellulase might provide a theoretical basis for further study of cellulase.