Here we report that only the G2677A polymorphism was associated to NS susceptibility and steroid resistance. The TAT haplotype was associated with NS susceptibility especially at an early age and with steroid resistance.
Pullulanase type I of Geobacillus thermoleovorans US105 strain (PUL US105) was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the open reading frame was connected downstream of the lipase A signal peptide of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction. In addition, pul US105 was fused to the alpha-amylase signal sequence of the Bacillus stearothermophilus US100 strain. The monitoring of the pullulanase activity and Western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. The PUL US105 was purified to homogeneity from the periplasmic fraction, using heat treatment, size exclusion, and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2.
The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the β-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37°C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19°C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed Vmax and Km values of 253 ± 36 μmol of β-cyclodextrin/mg/min and 0.36 ± 0.18 g/L, respectively.
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.
The two novel mutations detected in our study extend the spectrum of known AGXT gene mutations. The screen for the mutations identified in this study can provide a useful, cost-effective, and first-line investigation in Tunisian PH1 patients.
A new alpha‐amylase‐producing strain was assigned as Bacillus subtilis US586. The used statistical methodology indicated that amylase production was enhanced by 5.3 folds. The crude enzyme analysis proved the presence of three amylases isoforms Amy1, Amy2, and Amy3 called Amy586. The purified amylases had molecular masses of 48, 52, and 68 kDa with a total specific activity of 2,133 U/mg. Amy586 generated maltose, maltotriose, and maltopentaose as main final products after starch hydrolysis. It exhibited a large 4–6 optimal pH, a 60°C temperature activity, and a moderate thermostability. Amy586 displayed a high pH stability ranging from 3.5 to 6. The addition of Amy586 to weak wheat flour decreased its P/L ratio from 1.9 to 1.2 and increased its dough baking strength (W) from 138 × 10−4 to 172 × 10−4 J. Amy586 also improved the bread texture parameters by reducing its firmness and boosting the cohesion and elasticity values.
Practical applications
Bacterial alpha‐amylases with novel properties have been the major extent of recent research. In this paper, we managed to demonstrate that the addition of a purified amylolytic extract from the new isolated Bacillus subtilis strain US586 to weak local flour improves dough rheological proprieties and bread quality. Therefore, Amy586 can be considered as a bread making improver.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.