Direction Envolution Of Cellulase By Cell Surface Display-releasing System

Lignocellulosic raw material is the most abundant renewable biomass on Earth.After pretreated by biological, physical or chemical process, biomasses degraded into fermentable carbohydrates and then produce alcohols, organic acids and other bulk industrial products by fermentation. The biofuels formed from renewable biomass is the alternative solution for non-renewable fossil energy and the raw material.Compared to physical and chemical pretreatment, the enzymatic hydrolysis showed several advantages including green and environmentally friendly. However, the hydrolysis efficiency of cellulases is one of the rate-limiting steps in the process.Directed evolution of enzyme is the widely used technique to obtain high activity cellulase. However, lignocellulosic biomass is a heterogeneous material that consists of highly crystalline and amorphous regions, which is a challenge for the directed evolution. So a cell surface display-releasing(CSDR) system was designed and constructed, which ice nucleation protein(INP) was used as a carrier protein, the N-terminal of Intein was inserted between target protein and INP, formed a sandwich structure of INP-IN-target protein. CSDR system was designed for the directed evolution of cellulases by using insoluble regenerated amorphous cellulose(RAC) as screening substrate.The endoglucanase Cp Cel5 A from Clostridium phytofermentans was cloned and constructed into CSDR system. Cp Cel5 A mutant library was generated by error-prone PCR, and the large-size random mutant library was developed by prolonged overlap extension polymerase chain reaction POE-PCR. The Congo red plate assay screening platform was also developed for selection of endo-cellulase variants. More than ten mutants were selected from 3,000 colonies. Combined cellulase activity assay, two mutants, 3A11 and 5D4, were finally obtained. The substitutions in 3A11 and 5D4 were A139T, A184 T, respectively. The specific activities of 3A11 against RAC andsoluble carboxymethyl cellulose(CMC) were 1.3-and 1.1-fold higher than that of the wild type, respectively, while the specific activities of 5D4 were decreased 1.3- and1.0-fold to RAC and CMC. The optimum p H of 3A11 and wild-type was no significant difference. The influencing mechanisms of mutation on activity were further investigated by time course hydrolysis of RAC and structure modeling.In this study, we generated the Cp Cel5 A mutant library of CSDR system,developed the Congo red plate assay screening platform. The activity improved mutants were successfully isolated. The result indicated that the CSDR system can be used in endo-cellulase directed evolution. Further more, CSDR system also shown high potential in other industrial enzymes directed evolution and enzyme immobilization.