Efficient Expression Of Heterologous Heat β-glucosidase Gene In Aspergillus Niger

The full name of β-glucosidase(EC.3.2.1.21)is β-D-glucoside hydrolase,which is one of cellulases and known as rate-limiting enzyme of cellulase hydrolysis.The paper makes determination on its curve of enzyme production,standard curve of protein and glucose,analyzes amplification and sequence of bgl gene,knocks out original bgl gene through homologous recombination,constructs expression vector pPHT-bgl of heterogenetic heat-proof β-glucoside hydrolase,transforms Aspergillus niger 3.213 with expression plasmid and makes determination on transformed Aspergillus niger 3.213 by taking Aspergillus niger 3.213 as experiment object.Specific conclusion is shown as follows:1.The optimal time of enzyme production for Aspergillus niger 3.213 is 168 h,with 1.704 U/mL of highest enzyme activity,which is determined by drawing standard curve of its enzyme production.2.Fungus genomic extraction kit and CTAB are used to extract genomic DNA of Aspergillus niger 3.213,which is taken as template to design primer and amplify PCR to obtain target gene segment with 2 083 bp of the size.Make blastn sequence comparison,if the homology is up to 100%,which indicates that bgl gene of Aspergillus niger 3.213 was amplified successfully,and make sequence analysis and multiple sequence alignment for it.3.Three-phase PCR method and homologous recombination are used to knock out the original bgl gene of Aspergillus niger 3.213,and then import hygromycin resistant gene(PHT)and deicide to knock out original bgl and eliminate the interference of original gene for target gene through selective screening of hygromycin and PCR inspection.4.Make PCR for TtrpC to obtain medium plasmid,and the connection way of plasmid pUC19 and its production is double enzyme digestion of EcoRⅠ and HindⅢ;connect medium plasmid and PglaA double enzyme digestion,and then connect it with PHT through double enzyme digestion,and connect obtained production and bgl gene through double enzyme digestion to construct high-level expression vector pPHT-bgl of Aspergillus niger 3.213 finally.5.Defined amount of hypha shall be suspended by hydrolysis liquid of mixed enzyme for the shaking of enzymolysis for 3 h.Filter to collect protoplast and make the transformation of foreign DNA and transform the expression plasmid into aspergillus niger 3.213 through protoplast.Make PCR detection for it,and the size of target strip is about 2 300 bp,which can preliminarily consider the expression ofheterogenetic heat-proof β-glucoside hydrolase in Aspergillus niger 3.213.6.Compare the change of enzyme production ability of original bacterial strain with transformed bacterial strain,and its peak of enzyme production is increased by33.5% than original bacterial strain that is obtained through data processing,which preliminarily completes high-level expression of heterogenetic heat-proof β-glucoside hydrolase in Aspergillus niger 3.213.