Research On The Mechanism Of Adsorption And Desorption Of Cellulase' Carbohydrate-binding Modules And Its Application

Cellulases consist of catalytic domain(CD)and carbohydrate-binding modules(CBMs)as well as linker which linked the two parts together.CBMs can recognize and adsorb cellulose.However,CBMs also can bind lignin non-productively.Thus,it is vital to reduce the adsorption of CBMs onto lignin for enhance the cellulases hydrolysis.There are two methods to improve the activity of cellulases.One choice is to enhance the specifical binding of cellulases onto cellulose,and the other method is to decrease the non-productive adsorption of cellulases onto lignin.The main results conclude in this paper include:1)A Tet-on and Cre-loxP based genetic engineering system for convenient recycling of selection markers in Penicillium oxalicumThe lack of selective markers has been a key problem for gene editing in filamentous fungi.Cre-loxP system has been used extensively to remove selection markers in many organisms.However,constitutive expression of Cre will affect the recycling of selection marker genes flanked by loxP.To resolve this problem,we constructed a novel genetic manipulation system with the Cre-loxP system and Tet-on system in filamentous fungi.Using this system,two genes,ligD and bgll,were sequentially disrupted by one selective marke-ptrA.The successful application of this procedure will provide a useful tool for genetic engineering in filamentous fungi.2)Effect of CBMs engineering on interaction of cellulase-lignin or celluloseWe fused CBM,green-florescent protein(GFP)with linker of Cel7A from Trichoderma reesei.The fused protein was named GLC.The negative charge of GLC was increased by saturation mutagenesis.We proved that the adsorption of GLC onto AICS-lignin was decreased as the negative charge of GLC increased.Moreover,the hydrolysis efficiency of Cel7A's variant was higher than that of wildtype when microcellulose was mixed with AICS-lignin.3)Enhance hydrolysis activity of Cel7A by added additives with positive chargeWe tried to decrease the adsorption of GLC onto AICS-lignin by adding additives with positive charge.We chose two additives with positive charge,DTAB and PolyDADMAC in this study.We proved that the addition of DTAB or PolyDADMAC can decrease greatly the adsorption of GLC onto AICS-lignin and enhance the hydrolysis of microcellulose by Cel7A when microcellulose was mixed with AICS-lignin.The hydrolysis efficiency of Cel7A was 34%improved with addition of 0.20%DTAB.Moreover,the hydrolysis efficiencies of Cel7A's variants also were enhanced after addition of DTAB or PolyDADMAC.The hydrolysis efficiency of Cel7A's variant(S14D)was 22.6%improved with addition of 0.15%DTAB.