Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF.
A gene encoding a spore-associated subtilase, designated protease CDF, was cloned from Thermoactinomyces sp. CDF and expressed in Escherichia coli. The enzyme gene is translated as a proform consisting of a 94 aa propeptide and a 283 aa mature protease domain. Phylogenetic analysis revealed that this enzyme belonged to the subtilisin family, but could not be grouped into any of its six known subfamilies. The mature protease CDF has an unusually high content of charged residues, which are mainly distributed on the enzyme surface. The recombinant proform of protease CDF formed inclusion bodies, but could be efficiently converted to the mature enzyme when the inclusion bodies were dissolved in alkaline buffers. The proform underwent a two-step maturation process, wherein the N-terminal part (85 residues) of the propeptide was autoprocessed intramolecularly, and the remaining 9-residue peptide was further processed intermolecularly. Protease CDF exhibited optimal proteolytic activity at 50-55 degrees C and pH 10.5-11.0. The enzyme was stable under high-pH conditions (pH 11.0-12.0), and NaCl could stabilize the enzyme at lower pH values. In addition, the enzyme was not dependent on calcium for either maturation or stability. By immunoblot analysis, protease CDF was found to be associated with spores, and could be extracted from the spores with 2 M KCl and alkaline buffers without damaging the coat layer, demonstrating that the protease CDF is located on the surface of the spore coat.
Cheng
Guyue
G
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Zhao
Peiwei
P
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Tang
Xiao-Feng
XF
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Tang
Bing
B
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013171:Spores, Bacterial / Q000201:enzymology* / Q000235:genetics; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D056509:Thermoactinomyces / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D013381:Subtilisins