A new kumamolisin-like protease fromAlicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

Abstract

A new serine-carboxyl proteinase, called kumamolisin-ac , was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45 kDa, active over a wide temperature range (5.0 Á708C) and extremely acidic pHs (1.0 Á4.0), showing maximal proteolytic activity at pH 2.0 and 608C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 58C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t 1/2 at 808C, 10 h, pH 2.0) and over a broad range of pH (2.0 Á7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the 'S53' family. From the high identity with kumamolisin and kumamolisin-As , known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the 'S53' family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine a-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.

Thermostable Bacterial Collagenolytic Proteases: A Review

“…Another S53 family collagenolytic protease, kumamolisin-Ac, was purified from thermoacidophilic bacterium Alicyclobacillus acidocaldarius . It can efficiently hydrolyze type I collagen at pH3.0 and 60°C [ 57 ].…”

Recombinant Expression of Archaeal Superoxide Dismutases in Plant Cell Cultures: A Sustainable Solution with Potential Application in the Food Industry

“…Most of the SODs belonging to mesophile organisms often have low stability and easily lose their antioxidant capacity when exposed to the environment [ 5 ]; thus, it has been proposed that the protection of the conformational structure and longer storage stability by either site-directed mutagenesis or by mixing with compatible additives would make them potentially useful for industrial applications [ 6 , 7 ]. In this context, extremophile microorganisms, which thrive in extreme habitats that encompass both physical and geochemical environments, represent an interesting source of stable, highly-active, and resistant SOD enzymes, able to work in harsh conditions such as high and low temperatures or pHs, elevated UV doses, and high levels of salt [ 6 , 8 , 9 , 10 , 11 , 12 ]. Among these microorganisms, the archaeons Aeropyrum pernix and Saccharolobus solfataricus have attracted much attention for their unusual habitats and extraordinary resistance properties, as the first can be found in harsh marine environments, such as hydrothermal vents on the deep seafloors [ 13 , 14 , 15 , 16 ], and the second in hot volcanic sulfuric ponds [ 17 ].…”