Characterization Of A Spore-associated Protease And An Extracellular Serine Protease From Thermoactinomyces Sp. CDF

From Thermoactinomyces sp. CDF, a gene encoding a spore-associated subtilase, designated protease CDF, was cloned and expressed in Escherichia coli BL21(DE3). The enzyme gene is translated as proform consisting of a 94-amino acid propeptide and a 283-amino acid 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 efficiently convert to 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, N1) of the propeptide was autoprocessed intramolecularly, and the remaining 9-residues peptide was further processed intermolecularly. The intact N-terminal part (94 residues, N2) could inhibit the enzymatic activity and slowed down the maturation of protease CDF. The maturation process of protease CDF speed up at higher pH and Glu(-7) might act as a pH sensor. When Glu(-7) was changed to Gln, Lys or Ala, the proform was inclined to convert into mature protease at neutral pH. In contrast, the wild type or Glu(-7)Asp remained as the proform. Protease CDF exhibited optimal proteolytic activity at 50-55℃and pH 10.5-11.0. The enzyme was stable at high pH conditions (pH 11.0-12.0), and NaCl could stabilize the enzyme at lower pH values, and the enzyme retained more than 60% activity in the presence of 4 M NaCl. In addition, the enzyme was independent of calcium for either maturation or stability. By immunoblot analysis, protease CDF was found to be associated with the 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. Inactivation of the spore-associated protease CDF did not block the germination of the endospores. Human lung fibroblast WI-38 cells exposed to the mycelium and spores of Themoactinomyces sp. CDF produced higher level IL-6 than control cells. To date, at least eight genes involved in spore formation were found either upstream or downstream of the gene cdf.An extracellular serine protease, designated protease C2 was purified from the fermentation supernate of Thermoactinomyces sp. CDF. The gene of this protease was cloned, which shows 100% identity with a thermostable alkaline protease from Thermoactinomyces sp. E79 [1], but its upstream sequence is different from that of the latter. The enzyme was expressed in E. coli both as the soluble proteins and inclusion bodies. By treatment at high temperatures or with alkaline buffers, mature enzyme with high-yield and purity could be recovered from the soluble cellular fraction and inclusion bodies of E. coli cells expressing protease C2. When Bacillus subtilis WB700 was used as the host for expression, the recombinant enzyme was able to secret from the cell. Protease C2 has the ability to digest a variety of soluble and insoluble substrates including keratin. With the stability at high alkaline pH and high temperatures, the tolerance towards surfactants and oxidants, as well as the compatibility with commercial detergents, protease C2 is of great interest for biotechnological processes, such as the recycling of keratinous wastes and food processing, and has the potential to be used as detergent additive.