Cloning and sequencing of an exoglucanase gene from Streptomyces sp. M 23, and its expression in Streptomyces lividans TK-24.
A gene encoding exoglucanase (CBHII) of Streptomyces sp. M 23 was cloned and sequenced. The cbhII gene consisted of 1359 bp capable of encoding a polypeptide of 453 amino acids with a calculated molecular mass of 45,175 Da. The deduced amino acid sequence showed homology with those of cellulases belonging to family 6 of the glycosyl hydrolases. The cbhII gene was subcloned into the plasmid pSEV1 and expressed in Streptomyces lividans TK-24. The transformed cells were able to secrete the enzyme efficiently in an active form. The CBHII expressed by S. lividans TK-24 was purified to homogeneity by SDS-polyacrylamide gel and characterized. The recombinant CBHII was stable up to 50 degrees C and more than 30% of the original activity remained after heating at 100 degrees C for 10 min. The amino-terminal amino acid sequence of the recombinant CBHII was identified as GPAAPTARVD. These results agreed well with the properties of the authentic CBHII.
Park
Chang-Su
CS
Division of Applied Biological Chemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.
Kawaguchi
Takashi
T
Sumitani
Jun-Ichi
J
Takada
Goro
G
Izumori
Ken
K
Arai
Motoo
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D020539:Sequence Analysis, Protein; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases