A thermostable alkaline protease from a new alkaliphilic Nesterenkonia sp
Abstract: This thesis concerns a novel alkaline protease produced by an extremophilic microbial specie, designated as AL20, isolated from a feather sample collected at the shore of the alkaline soda lake Abjata in the Ethiopian Rift Valley. The isolate is a heterotrophic, alkaliphilic, halotolerant gram-positive, strictly aerobic, non-motile, non-spore forming bacterium. The organism grew at 37°C, pH 10, and 1M NaCl. Comparison of the 16S rDNA sequence showed that the organism was phylogenetically closely related to Nesterenkonia species, although differences in G+C content and low DNA-DNA hybridisation demonstrated that the organism represents a new species, which was named Nesterenkonia abyssinica. The alkaline protease was produced by the organism in alkaline medium (pH 10), using feather as the only sole source of carbon and nitrogen. The enzyme has a molecular mass of 22 876±5 Da and isoelectric point of 4.2. The enzyme was optimally active at 70 °C and over a broad pH range (7-11). The amount of calcium bound to the AL20 protease was determined to be only about 0.14 mol/mol of protease. The thermal unfolding of the enzyme was consistent with the classical two-state model in which the enzyme unfolded at about 74 °C and pH 10. The midpoint of thermal unfolding (Tm) was unaltered upon addition of calcium as well as after treatment with chelating agents. The thermodynamic parameters were nearly the same over a pH range of 7-10. The secondary structure of the AL20 enzyme, apparently a mixture of a-helix and b-sheet, remained intact after incubation for 24 h at 50 ºC, and in the presence of 1 % SDS as observed by circular dichroism. The enzyme exhibited unusual stability in the presence of hydrogen peroxide and various sequestering agents used in detergents. The enzyme was found to be highly active against larger peptide substrates containing aromatic or hydrophobic residues e.g. Phe, Tyr, Leu at the P1 site and neutral amino acid e.g Pro, Val at the P2 site. With the oxidized insulin B-chain as substrate, the bonds initially cleaved by the enzyme were Tyr16-Leu17 and Tyr26-Thr27 followed by Gln4-His5, Phe25-Tyr26 and Leu15-Tyr16 bonds. An additional cleavage site at Ser9-His10 was found during hydrolysis for long time. The enzyme hydrolysed casein and hemoglobin efficiently. It also showed keratin hydrolysing activity and was able to hydrolyse elastin-orcein to a detectable level. Finally, the enzyme was subjected to crystallization, X-ray analysis of the triangular prism-shaped crystals diffracted beyond 1.5 Å. A complete data set to 1.39 Å resolution was collected.
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