1-62: Isolation of saccharification enzymes-producing fungi and functional screening of novel genes from metagenome libraries derived from soils

In order to isolate functional saccharification enzyme, two approaches were carried out. One is the screening of filamentous fungi from decayed wood based on the weight-loss of softwoods unbleached kraft pulp (SUKP) and the productivity of saccharification enzymes in liquid culture. The other is the functional screening of novel saccharification enzymes from metagenome libraries which were constructed from soils.

One hundred twenty eight strains were isolated from decayed softwoods and 19 strains were selected based on weight-loss of SUKP. When the productivities of CMCase and FPase by selected fungi were compared with that by Trichoderma reesei in liquid culture, five fungal strains showed the higher productivity of each enzyme in liquid culture with avicel. The productivity was changed by carbon sources (Avicel, SUKP and wood powder), and concentrated and desalted extracellular fluids showed good saccharification activity for woody biomass. 

Two metagenome libraries were constructed from soils via short-term enrichment culture. The cultures for accumulation contained avicel or kraft pulp as carbon source and the cultures were incubated for a week. Bacterial cells were collected by centrifugation, and whole genomes were extracted. Partial digested (using Sau3AI) genomes were ligated into pUC19 plasmid, and then transformed into Escherichia coli DH5 a chemical competent cells. Recombinant cells were spread on LB agar medium containing RBBR-stained CMC or beech wood xylan. After incubation, CMCase- or Xylanase-positive clones appeared with clear halo against blue culture color. DNA sequence analysis of the transformed inserts revealed that the sequence contained novel CMCase and Xyalanase gene.