Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF

Abstract

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 6C 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.

Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

“…The major difference between the maturations of the two halolysins is that the Nep precursor can be trans-processed into its mature form by active Nep, but the SptC precursor is degraded by active mature SptC. Generation of the mature enzyme via transprocessing of the N-terminal propeptide of the precursor or the proform, either auto-or heterocatalytically, has also been described for other subtilases (27,(35)(36)(37)(38). In the precursor of the WF146 protease from thermophilic Bacillus sp.…”

“…To prevent self-degradation of the enzymes during sample preparation (boiling) or electrophoresis, the proteins were precipitated with 20% (wt/vol) trichloroacetic acid (TCA), washed with acetone, solubilized in loading buffer containing 8 M urea, and then subjected to electrophoresis without prior heat treatment. An anti-His tag monoclonal antibody (Novagen) was used for immunoblot analysis, as described previously (27). For N-terminal sequencing, proteins separated by SDS-PAGE were electroblotted onto a polyvinylidene difluoride membrane and then stained with Coomassie brilliant blue R-250.…”

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons

“…To prevent self-degradation of the protease during sample preparation, proteins were precipitated with 20% (wt/vol) trichloroacetic acid, washed with acetone, solubilized in loading buffer containing 8 M urea, and then subjected to SDS-PAGE without prior heat treatment. An anti-His tag monoclonal antibody (Novagen) was used for immunoblot analysis as described previously (47).…”

Molecular cloning and characterization of a subtilisin-like protease from Arabidopsis thaliana

“…Protease activity was assessed according to the methods described previously (Cheng et al, 2009) with slight modifications. Briefly, azocasein (Sigma-Aldrich, USA) was used as a substrate for the purified MBP-AtSBT1.9 protein.…”