Construction Of Cellulase Mutagenesis Library And Screening Improvd Mutants

Cellulose is the most abundant renewable natural biological resource, and theproduction of biobased products and bioenergy from less costly renewablelignocellulosic materials is important for the sustainable development of humanbeings. A reduction in cellulase production cost, an improvement in cellulaseperformance, and an increase in sugar yields are all vital to reduce the processingcosts of biorefineries. Improvements in specific cellulase activities is usually donethrough directed evolution of enzymes.The directed evolution of enzyme iscomponent of protein mutant library construction and screening of the mutantlibrary,and protein screening of the mutant library is a very important step,and it isdifficult to complete the screening of a large mutant library. Used as cellulose enzymescreening of the substrate is divided into soluble and insoluble substrates,andsubstrates for cellulose enzyme of soluble and insoluble substrate no clear relationshipbetween the active function. Using soluble substrates of cellulose enzyme canimprove the thermal stability of enzyme, but is not necessarily can improve itshydrolysis ability of natural substrate, so the soluble substrate cannot be used as ascreening or choose the high activity of cellulase. Insoluble substrate can’t through thecell membrane into the cell and reaction, so this experiment adopts the cell surfacedisplay-release system, use insoluble substrate as the screening substrate.The directed evolution of CpCel5A from Clostridium phytofermentans used thecell surface display-releasing system. For the construction of a large mutant library,carried on the error-prone PCR,and the PCR products through the POE-PCRconnected to the the cell surface display release system(pET20b-INP-In-5A).For thescreening of the mutant library, using96well plates cultivate and clean zone method.Different from the traditional double enzyme method, this experiment adopts thePOE-PCR method, the results successfully built a more than2000mutant library.Theexperiment using the cell surface of controlled release system, use the insolublesubstrate as a screening of the substrate,CpCel5A was transported to the cell surfaceby ice nucleoprotein protein, and then spliced by intein, so Cel5A can digested RAC around the E.coli. This experiment adopts clean zone experiment to carry on thescreening of the mutant library, through the verification and BCA method to measureactivty, successfully screened a high activty5D4, and it is successfully builded to20bfor expressing, detection the activity difference between for the substrate andinsoluble substrate.This study used the cell surface display release system, using the insolublesubstrate as the substrate of high-throughput screening,and get a high activitymutant.So the cell surface display-release system can be used in directed evolution ofcellulose enzyme,and this study will have broad application prospects in the field ofbiology.