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Cellobiohydrolase I (CBH I) from a filamentous fungus
Trichoderma koningii G-39 has been purified by SP- Sephadex
C-50 and DEAE-Sephadex A-50 ion-exchange chromatographies. It
showed a molecular weight of 68 k and pI of 4.0 upon sodium
dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)
and isoelectric focusing. This enzyme has a specific activity
of 1.4 unit/mg using p-nitrophenyl-b-D-cellobioside as a
substrate and prefers to interact toward crystalline
substrates. It exhibited both exocellobiohydrolytic and
cellulose binding activities, with different chemical and
physical characteristics. CBH I encoding gene cbh1 has been
cloned, including a 1.4 kb cDNA and 7.0 kb genomic DNA. The
sequence analysis reveals that part of the cloned sequence
encodes a 513 amino acids-protein. The coding region is split
by two introns with lengths of 67 and 63 base pairs.
Nucleotide sequence alignment to the putative promoter region
of cbh1 reveals significant homology to the carbon catabolite
repressor binding sites of Aspergillus nidulans CREA and
Saccharomyces cerevisiae MIG1 promoters which respond to a wide-
domain regulatory system of glucose repression. In addition,
several transcription factor binding site-like sequences are
identified in 5'' flanking region of cbh1 as well. The
electrokaryotype of T. koningii is obtained by pulsed field gel
electrophoresis (PFGE). The fungal chromosomes were separated
to five bands. The lowest band showed non-proportional
fluorescent intensity, it is possible that at least two similar
size chromosomes comigrated on this band. Accordingly, the
fungal genome is thought to be constituted of five or more
chromosomes. The sizes of chromosomes are estimated as 4.4,
4.7, 5.7, 6.7 and 7.2 Mb, respectively, and the genome size is
calculated to be about 28.7 Mb. The results of PFGE followed
by Southern hybridization indicates that cbh1 and xyl2, a gene
coding for an endoxylanase, are both mapped at the lowest band.
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