CHARACTERIZATION OF A THERMOMONOSPORA-FUSCA EXOCELLULASE

The exocellulase E3 gene was cloned on a 7.1 kb NotI fragment from The rmomonospora fusca genomic DNA into Escherichia coli and expressed in Streptomyces lividans. The E3 gene was sequenced and encoded a 596 res idue peptide. The molecular masses of the native and cloned E3s were d etermined by mass spectrometry, and the value for E. coli E3, 59 797 D a, agreed well with that predicted from the DNA sequence, 59 646 Da. T he value of 61 200 Da for T. fusca E3 is consistent with E3 being a gl ycoprotein. E3 is thermostable, retaining full activity after 16 h at 55 degrees C. It also has a broad pH optimum around 7-8, retaining 90% of its maximal activity between pH 6 and 10. The cloned E3s were iden tical to the native enzyme in their activity, cellulose binding, and t hermostability. Papain digestion produced a 45.7 kDa catalytic domain with 77% of the native activity on amorphous cellulose and 33% on crys talline cellulose. E3 belongs to cellulase family B and retains the re sidues that have been identified to be crucial for catalytic activity in Trichoderma reesei cellobiohydrolase II and T. fusca E2. The E3 gen e contains a 14 bp inverted repeat regulatory sequence 212 bp before t he translational start codon instead of the 30-70 bp found for the oth er T. fusca cellulase genes. An additional copy of this sequence with one base changed is 314 bp before the translational start codon. The t ranscriptional start site of the E3 gene was shown to be between these two inverted repeats.