THE GENE ENCODING THE CELLULASE (AVICELASE) CEL1 FROM STREPTOMYCES-RETICULI AND ANALYSIS OF PROTEIN DOMAINS

Abstract

Streptomyces reticuli produces an unusual cellulase (Avicelase), with an apparent molecular weight of 82 kDa, which is solely sufficient to degrade crystalline cellulose. During cultivation the processing of the Avicelase to a truncated enzyme (42 kDa) and an inactive protein (40 kDa) correlated with the occurrence of an extracellular protease. After its purification this 36 kDa protease cleaved the S. reticuli Avicelase in vitro in the same manner. Using antibodies raised against the Avicelase and its truncated form (42 kDa) and gene libraries of S. reticuli DNA in the Escherichia coli phage vectors lambda gt11 and Charon 35, the Avicelase gene (cel1) was identified. Further subcloning and DNA-sequencing revealed a G+C rich (72%) reading frame of 2238bp encoding a protein of 746 amino acids. The transcriptional start site was mapped about 180bp upstream from the GTG start codon. A signal sequence of 29 amino acids was identified by aligning the deduced amino acids with the characterized N-terminus of the 82 kDa Avicelase. Comparison of the N-terminal amino acids from the purified proteins with the amino acid sequence derived from the Avicelase gene revealed that the truncated enzyme (42 kDa) corresponds to the C-terminal region whereas the inactive proteolytically derived protein (40 kDa) represents the N-terminal part of the 82 kDa Avicelase. Comparisons with amino acid sequences deduced from known cellulase genes indicated the presence of three putative protein domains: (i) an N-terminal part showing significant similarity with a repeat region of endoglucanase C from Cellulomonas fimi, recently shown to be a cellulose-binding domain; (ii) an adjoining region sharing homology with the N-terminal domains with unknown function of endoglucanase A from Pseudomonas fluorescens, endoglucanase D from Clostridium thermocellum and a cellodextrinase from Butyrivibrio fibrisolvens, and (iii) a C-terminal catalytic domain belonging to cellulase family E.