Cloning and Sequencing of an Original Gene Encoding a Maltogenic Amylase from Bacillus sp. US149 Strain and Characterization of the Recombinant Activity

Abstract

The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrPC from PrPSc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala, and Met, residues that are absent from the protease accessible aminoterminal region of PrPSc. Therefore, although thermolysin readily digests PrPC into small protein fragments, full-length PrPSc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrPSc species is produced, PrP27-30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant PrPSc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrPSc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrPSc to disease pathogenesis.

Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions

“…WPD616 (AMWPD616) (Liu et al, 2006), respectively. These three amylases have optimal activities of 55, 60 and 50°C respectively, whereas MAUS149 has an optimum activity at 40°C (Ben Mabrouk et al, 2008). The inspection of the primary structure alignment of the above mentioned enzymes (Fig.…”

“…The supernatant constituted the enzyme crude extract. The purification of MAUS149 and its mutants was achieved using a protocol previously described by the authors (Ben Mabrouk et al, 2008) wherein ammonium sulfate precipitation and fast-performance liquid chromatography (FPLC) were employed.…”

“…The V max and K m values were calculated from a Lineweaver and Burk plot using the hyper-32 program. The characterization of the starch hydrolysis products was performed as previously described (Ben Mabrouk et al, 2008). The wild-type and mutated MAUS149 were separately added to starch solutions and the reaction mixtures were incubated at 40°C for 24 h. The resulting products were analyzed by HPLC (High Performance Liquid Chromatography) using a Eurokat Ag 10 lm Column (KNAUER).…”

“…The wild-type and mutant enzymes were produced and purified as previously described for the wild-type MAUS149 (Ben Mabrouk et al, 2008). The mutant proteins had molecular weights that were similar to that of the wild-type protein, i.e., 67.5 kDa (Fig.…”

“…US149 (MAUS149) has sufficient pH stability, but its moderate thermostability is insufficient for use in baking applications (Ben Mabrouk et al, 2008). Therefore, we used site-directed mutagenesis to obtain variants of this enzyme with higher thermostability.…”