Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures.

Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by α-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu-1-Ser1 bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu-12-Lys-11 bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 °C and retained approximately 90 % of its original activity after incubation at 70 °C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys32-Cys48 and Cys180-Cys183 cumulatively contributed to the thermostability of TS-GSE.

Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes.

Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75- and 45-kDa mature forms involves both hetero- and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations.

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and ß-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.