Heterologous Expression And Biochemical Property Of Thermomyces Lanuginosus ?-Glucanase

?-Glucanases play an increasingly important role in the food industry and also in animal feed production,alcohol fermentation and even waste management.However,the applications of most ?-glucanases were largely limited by their low activities and heat instability.In order to obtain a more thermostable ?-glucanase,thermophilic filamentous fungus Thermomyces lanuginosus was used for cloning genes encoding?-glucanases,and we conducted research work as follows:Based on the genome sequencing data of T.lanuginosus,two exo-?-glucanase genes(glnB and glnD)and one endo-?-glucanase gene(glnE)were cloned via RT-PCR.The complete ORF s of these genes are predicted to be 1260-,2694-and 1938 bp,respectively.Phylogenetic relationships were analyzed with MEGA 6.0 software,and the result indicates that these genes showed distant phylogenetic relationships with those beta-glucanases reported previously.According to Pichia Expression Kit,three ?-glucanase producing recombinant yeast strains(GS-glnB,GS-glnD,GS-glnE)were obtained.Laminarin was used as a substrate to assay the activity of GlnB and GlnD.The activity of recombinant enzymes GlnB and GlnD reached 11.5 U/mL and 3.25 U/mL,respectively.SDS-PAGE showed that the recombinant enzymes are 44.0 kD and a 93.4 kD,respectively,consistent with the-predicted sizes.GlnB exhibited optimum temperature at 65? and retained 95%of its activity at 50? after 2-hour incubation,the optimum pH was 5.0 and stable at the range of pH 3.0-9.5,while GlnD was optimally active at 70? and retained 90%of its activity at 55? after 2 hours.GlnD showed pH optimum at 2.5 and was stable over the pH 1.5-4.0.CMC-Na was used as a substrate for assaying GlnE by the method of viscometric.The relative activity of GlnE reached 202 U/ml.The endo-?-glucanase encoded by glnE was determined to be 68.7 kDa by SDS-PAGE,which was consistent with the expect size.