The cellulolytic enzyme system of Trichoderma reesei: Molecular cloning, characterization and expression of the cellobiohydrolase genes: Dissertation

In this study, gene technology has been used to investigate the cellulolytic enzymes of the filamentous fungus, Trichoderma reesei.A genomic library of T. reesei was constructed using a phage lambda vector and the chromosomal copies of the two cellobiohydrolase genes were isolated.An RNA isolation method, facilitating the synthesis of long cDNAs, was adapted for filamentous fungi.Efficient cDNA cloning method involving blunt end ligation to plasmid vectors was developed and used to construct a cDNA library of T. reesei.From this library the cDNA copies of the genes coding for two cellobiohydrolases and an endoglucanase were identified.Sequence analysis of the cellulolytic enzyme genes led to the identification of two separate domains in these enzymes.Comparison of the deduced primary structures to other carbohydrate degrading enzymes suggests that the glycosylated terminal domains have a role in substrate binding.Analysis of the regulatory regions of the cbh2 gene revealed multiple transcription initiation sites and comparison of the chromosomal and cDNA copies of the cellulolytic enzyme genes revealed the presence of two or more short introns.Expression of the cbhl cDNA was optimized in the heterologous host, Escherichia coli.The highest expression levels were obtained by fusing the mature CBH I to either the E. coli ß-galactosidase or a short N- terminal leader peptide of the bacteriophage lambda Cro protein.The Cro-CBH I fusion protein was produced as intracellular aggregates devoid of enzymatic activity and the CBH I activity was restored by renaturation.Computer aided molecular modeling was used to study the folding of the proposed active site region of CBH I.The model obtained does not conflict with the idea that this region in CBH I has a similar tertiary structure to the active site of the phage T4 lysozyme.The enzyme-substrate interactions, proposed on the basis of the model, can be tested using site directed mutagenesis.