Characterization of the leader peptide of an endo-type cellulase produced by an alkalophilic Streptomyces strain.
An endo-type semi-alkaline cellulase (CMCase I) produced by an alkalophilic Streptomyces strain has an extraordinarily long leader peptide of about 70 amino acids (aa), which can be grouped into four distinct regions, an NH2-terminal region (13 aa), an Arg-cluster region (13 aa), a hydrophobic region (23 aa), and an Ala/Pro-repeat region (12 aa). For identification of the function of each part of the leader peptide for secretion of the enzyme, mutation in the leader peptide were generated by site-directed mutagenesis using the cloned gene, and the mutant genes were expressed in a cellulase-negative mutant strain, Streptomyces lividans HN1. Although all the alterations in the leader peptide, except for one case, decreased secretion to various extents, in S. lividans, the following conclusions were obtained. Comparison of the intra- and extra-cellular enzyme activities of the mutants suggested that the Arg-cluster region was essential in secretion of the cellulase. Deletion of 8 amino acids rich in threonine and proline in the NH2-terminal region enhanced the secretion to a small extent. Deletion of the Ala/Pro repeat region had almost no effect on secretion.
Department of Agricultural Chemistry, University of Tokyo, Japan.
J S
JS
Park
S
S
Horinouchi
T
T
Beppu
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D013302:Streptomyces / Q000201:enzymology*
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D006026:Glycoside Hydrolases; D002480:Cellulase; C019826:carboxymethylcellulase