Isolasi dan Karakterisasi Gen Penyandi Endo-1,4-β-glukanase dari Kapang Aspergillus fumigatus dan Aspergillus niger

Abstract

Agricultural waste biomass containing cellulose is the raw material for bioethanol production and is the cheapest and most abundant in nature. It can be hydrolyzed with the help of cellulase enzymes produced by fungus A. fumigatus and A. niger. These enzymes consist of three main activities, namely endo-1,4-β- glucanase, exo-1,4-β-glucanase, and β-glucosidase. The main purpose of this research is isolation and characterization of genes encoding endo-1,4-β-glucanase from A. fumigatus and A. niger. The hypothesis is the gene encoding endoglucanase can be isolated using specific primers designed based on gene sequences accessible from the gene bank. Genes encoding endoglucanase isolated using RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) techniques and inserted into the vector pGEM-T Easy. Endoglucanase activity measurements carried out on pellets and supernatant of the recombinant cells by DNS (dinitrosalicylic acid) method. Electrophoresis of the PCR products showed that the gene has a size of approximately 1300 bp. BLAST analysis of the DNA sekuen showed that these genes are homologous with genes encoding endoglucanase accessible from the gene bank. Expression test of the genes encoding endoglucanase of A. niger showed that this gene has been successfully expressed in the cells of Escherichia coli through pYES2/CT vector. The highest activity of endoglucanase was found in the supernatant of the recombinant cells carrying vector pYES2/CT (8.8309 U/mg).